hereditary dementia, a clinical genetic perspective...prof.dr. g.a.m. widdershoven contents chapter...

Hereditary dementia, a clinical genetic perspective

Petra E. Cohn-Hokke

Cover photo and design: Petra E. Cohn-Hokke Lay-out: Petra E. Cohn-Hokke Printed by: Ipskamp Drukkers BV, Enschede ISBN: 978-94-028-0523-9 © 2017, Petra E. Cohn-Hokke All rights reserved. No parts of this publication may be reproduced in any form or by any means without permission of the author.

VRIJE UNIVERSITEIT

Hereditary dementia, a clinical genetic perspective

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad Doctor aan de Vrije Universiteit Amsterdam, op gezag van de rector magnificus

prof.dr. V. Subramaniam, in het openbaar te verdedigen

ten overstaan van de promotiecommissie van de Faculteit der Geneeskunde

op donderdag 13 april 2017 om 13.45 uur in de aula van de universiteit,

De Boelelaan 1105

door

Petra Elisabeth Cohn-Hokke

geboren te Heemstede

promotoren: prof.dr. E.J. Meijers-Heijboer prof.dr. J.C. van Swieten

copromotor: dr. Y.A.L. Pijnenburg

Overige leden promotiecommissie: prof.dr. F. Baas dr. J.A. Kievit

prof.dr. J.M. Rozemuller prof.dr. Ph. Scheltens dr. E.A. Sistermans prof.dr. E.M.A. Smets prof.dr. G.A.M. Widdershoven

CONTENTS Chapter 1 General introduction 9 Chapter 2 Genetic counselling and testing in dementia and related disorders

2.1 Genetics of dementia: update and guidelines for the clinician 25 2.2 The effect of predictive testing in adult onset 51 neurodegenerative diseases on social and personal life

Chapter 3 Prevalence and phenotypes of inherited dementia

3.1 The clinical and pathological phenotype of C9orf72 67 hexanucleotide repeat expansions 3.2 Mutation frequency of PRKAR1B and the major familial 95 dementia genes in a Dutch early onset dementia cohort

Chapter 4 Identification of new genetic causes of cognitive decline

4.1 A novel CCM2 variant in a family with non-progressive 111 cognitive complaints and cerebral microbleeds 4.2 Rare genetic variant in SORL1 may increase penetrance of 125

Alzheimer’s disease in a family with several generations of APOE-ε4 homozygosity

Chapter 5 Summary 145 Chapter 6 General discussion 151 Addendum Nederlandse samenvatting (Dutch summary) 163

List of publications 169 Dankwoord (Acknowledgements) 171 About the author 174

Chapter 1

General introduction

GENERAL INTRODUCTION Dementia denotes a decline in cognitive abilities severe enough to interfere with daily functioning. The prevalence of dementia is high, and increasing. According to the World Health Organization, 47.5 million people have dementia worldwide1. In the Netherlands, around 270.000 individuals have dementia and the life time risk is 1 in 5 (20%)2. Age is the most important risk factor: the prevalence increases from about 1% in persons aged 65-69 to almost 45% in persons aged 95 years and older (Figure 1), although dementia is not part of the common ageing process. Figure 1. Prevalence in percent of dementia by age in Europe.

Adapted from Alexander e.a. 2015 3.

According to international criteria for dementia, the cognitive impairment should involve at least two cognitive domains (memory, executive functioning, visuospatial functioning, language and behaviour) and the impairment should not be explained by delirium or a major psychiatric disorder4. Several primary neurodegenerative disorders cause dementia, such as Alzheimer’s disease (AD), frontotemporal dementia (FTD) and dementia with Lewy bodies (DLB). Dementia can also be the result of impaired cerebral circulation, in vascular dementia (VaD), or can be one of the several symptoms of a disease, as in Huntington’s disease (HD). The term young or early-onset dementia is mostly defined as dementia with an onset before the age of 65 years, and accounts for


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less than 5% of the cases2. The distribution of the causes is different in dementia with an early-onset than in the total group of dementia patients (Figure 2). Figure 2. Epidemiology of dementia.

A. in all dementia patients, and B. in patients with dementia with an onset between 45 and 65 years.5,6

Most diseases underlying dementia are considered to have a complex aetiology with multiple genetic and non-genetic factors contributing. In general, the younger the patient and the more affected family members, the more likely that genetic variants are involved. In families with multiple affected family members in consecutive generations, the disease is likely autosomal dominant inherited, caused by a single genetic mutation diseases causing dementia. Diseases causing dementia cannot be prevented cannot be prevented, treated or inhibited. However, patients may benefit temporarily from symptom reducing treatment. ALZHEIMER’S DISEASE AD is the most common cause of both late and young onset dementia: 60-70% of the dementia patients have AD1. Clinical criteria for the diagnosis AD were defined in 1984 by the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS) and the Alzheimer's Disease and Related Disorders Association (ARDRA, now known as the Alzheimer's Association)7. Updated diagnostic criteria incorporating biomarkers and genetic findings were proposed with the National Institute on Aging and the Alzheimer’s Association criteria in 20114. To diagnose probable AD dementia, the patient must be diagnosed with dementia by clinical and neuropsychological examination, the cognitive impairments have to be progressive and present in at least two cognitive domains, and dementia may not be caused by any other diseases or events. The 2011 criteria subdivide probable AD dementia into several categories based on the level of evidence of a pathophysiological process of AD, such

Chapter 1

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as positive biomarkers for amyloid-beta deposition or neuronal injury, characteristic cerebral amyloid plaques and tau tangles at autopsy, or a genetic causative mutation. Medication such as cholinesterase inhibitors delay symptoms in part of the patients but do not delay the disease process. In most patients, AD is a complex disorder with age as the most important risk factor, followed by female sex8. Also vascular risk factors, such as smoking, obesity, hypertension and increased cholesterol, have been associated with an increased risk9. Based on twin studies, the heritability in late onset AD (LOAD) is estimated to be around 60% (http://match.ctglab.nl). The most important and well known genetic risk factor is the ε4 allele of the APOE gene, which in heterozygous state more than doubles the risk on AD to about 25-50%, while homozygous APOE-ε4 carriers are considered to have a risk of AD of 50-90%10,11. Quite recently, carriers of the rare Arg47His variant in the TREM2 gene were found to have a comparable risk of AD as heterozygous APOE-ε4 carriers.12,13 Other genes associated with AD risk each have just a small effect14. Interestingly, specific variants in a few of the genes associated with an increased AD risk, such as the APP gene and ABCA7 gene, have been found to protect against AD15,16. Of the early-onset AD (EOAD) patients, 10-15% have a family history suggesting autosomal dominant inheritance17. The most commonly identified genetic causes of autosomal dominant AD are heterozygous mutations in the PSEN1 gene, encoding for presenilin-1 protein, the APP gene encoding for amyloid precursor protein, and the PSEN2 gene encoding for presenilin-2 protein. Mutations in the PSEN1 gene are the most prevalent and often result in AD with a remarkably young age at onset (<50 years). Besides mutations in the APP gene, also duplications of the whole gene cause AD. Although APP mutations in patients presenting with AD are quite rare, duplications are common when patients with Down syndrome are taken into account: since the APP gene is located at chromosome 21, all patients with Down syndrome have an APP duplication. Patients with APP mutations or duplications may develop AD, small bleeds visible on imaging of the brain caused by amyloid deposits in the cerebral vessels (cerebral amyloid angiopathy, CAA), or both. PSEN2 mutations are associated with a relatively late onset and could therefore be considered in most AD patients. However, mutations in this gene are extremely rare in the Netherlands. In 23-88% of the families with assumed autosomal dominant inherited AD, the genetic cause remains unclear18. The heritability factor of EOAD has been estimated in one study to be 92-100%19, however, only 5-10% of the EOAD patients have a mutation in one of the main AD genes18. Therefore, other genetic defects we cannot identify with the most


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commonly used techniques must be involved, for example epigenetic dysregulation, somatic mosaicism or a combined or synergic effect of genetic variants and non-genetic risk factors. In families without autosomal dominant inheritance, first degree relatives have a 2-3 fold risk of AD compared to individuals with a negative family history20. Especially offspring are at increased risk, while no significantly changed risk was found in siblings of AD patients21. FRONTOTEMPORAL DEMENTIA Frontotemporal dementia (FTD) is one of the most common causes of early-onset dementia, with an estimated prevalence world wide of 3-26 per 100.000 individuals aged 45-65 years22. The name refers to the two brain regions predominantly affected by this disease: the frontal and temporal lobes. FTD is characterized by changes in social and personal behaviour, apathy, blunting of emotions, and deficits in both expressive and receptive language. Based on the presenting symptoms, the disease is classified into three groups: the behavioural variant FTD (bvFTD) and the two language variants semantic dementia and progressive non-fluent aphasia. Each of these variants have their own diagnostic criteria23,24. To fulfil the criteria for possible bvFTD, at least three of the following characteristics must be met: I) early behavioural disinhibition; II) early apathy or inertia; III) early loss of sympathy or empathy; IV) perseverative, stereotyped or compulsive/ritualistic behaviour; V) hyperorality and dietary changes; VI) executive/general deficits on neuropsychological examination with relative sparing of memory and visuospatial functioning23. For the diagnosis of probable bvFTD, the decline must also be progressive and neuroimaging must be consistent with the diagnosis. The term “definite bvFTD” is reserved for patients with possible / probable FTD and either histopathological evidence of the disease or a known pathogenic mutation. Histopathology typically shows deposits of tau, TDP-43 or FUS protein. In familial cases, the precipitated proteins are strongly correlated with the genetic defect. Currently, no treatment is available to cure, delay or inhibit FTD. Around 40% of the FTD patients have a positive family history and 10-15% an autosomal dominant inheritance pattern25,26. Since studies on the risk of family members have been performed before the discovery of two of the three most important genes causing autosomal dominant FTD, no reliable risk estimates are available for relatives of assumed sporadic FTD patients.

Chapter 1

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The main genes involved in familial FTD are the genes MAPT, encoding for microtubule-associated protein tau, GRN encoding for (pro)granulin and C9orf72. The name of the latter gene describes the location of the gene (chromosome 9 open reading frame 72) since the exact function of the gene still has to be discovered. A hexanucleotide repeat expansion in a noncoding part of the gene predisposes to both FTD and amyotrophic lateral sclerosis (ALS). Therefore, individuals with a repeat expansion in this gene are at highly increased risk to develop FTD, ALS or both. Repeat expansions in this gene are also found in about 6% of the sporadic patients27. Since most C9orf72 repeat expansion mutation carriers have the same haplotype, C9orf72 repeat expansion mutation carriers are likely to have one or just a few common ancestors28,29. The relatively high mutation frequency of C9orf72 repeat expansions in sporadic patients is probably due to the fact that the common haplotypes are prone to expansion of the repeat, rather than harbouring the repeat expansion itself30,31. The phenotype of the C9orf72 repeat expansion is extremely variable, both in age at onset as in the course of the disease. In contrast, most MAPT mutations cause FTD with a strikingly early onset (<60 years). GRN mutations may be suspected in case of asymmetrical cerebral atrophy. Two third of the FTD patients with a strongly positive family history have a causative mutation in one of these three genes26,32. Therefore, other genetic factors, causative mutations and/or genetic risk factors, must be involved. HUNTINGTON’S DISEASE Huntington’s disease (HD) is a disease with an estimated prevalence of 5 per 100.000 in patients with a Western ancestry33. The disease is characterized by I. movement disorders, most characteristically involuntary movements (chorea), II. cognitive decline and/or changes in personality; III psychiatric problems such as depression, and IV. a positive family history in most patients. HD is caused by a CAG repeat expansion in the HTT gene encoding for huntingtin protein and is therefore by definition inherited. However, patients may have inherited the mutation from a parent with a shorter intermediate repeat prone to expand during reproduction, or with a repeat expansion in the range of reduced penetrance. Another possibility is that the affected parent passed away before manifestation of the disease. Siblings and offspring are at 50% risk of the disease. Chorea is a very characteristic feature of HD and often one of the main presenting symptoms. However, patients presenting with neuropsychiatric features may


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be suspected to have another disease such as FTD or depression, especially if the family history is negative or uninformative. GENETIC TESTING IN DEMENTIA While in the past genetic testing in dementia patients was mainly performed to confirm a diagnosis, as in HD, or to clarify the genetic cause in familial cases, nowadays genetic testing also used for diagnostic purposes or because the family wants to know whether the disease has a genetic cause. Previously, exomes of genes of interest were sequenced, often in serial, by Sanger sequencing. A few years ago, next generation sequencing (NGS) techniques became available in the Netherlands. With NGS, millions of small fragments of DNA are sequenced in parallel, enabling testing a large amount of genes simultaneously. In whole exome sequencing (WES), all protein encoding DNA, the exons, is analysed. Although the exome comprises just ~1% of the genome, the exome is thought to harbour the majority of the disease causing mutations. In WES, the exome is selected by enrichment techniques using capture probes for recognizing the exons, and then sequenced by a high-throughput DNA sequencing technology. Each nucleotide is aimed to be sequenced multiple times (coverage) to minimalize sequencing errors. In whole genome sequencing (WGS), also the non-coding DNA is sequenced. Because no enrichment is required for WGS and therefore no capture probes are used, WGS results in a more stable sequence coverage and more reliable detection of deletions and duplications (copy number variants).WES is currently the most commonly used NGS technique. However, when WGS costs decrease and data storage issues are solved, WGS is likely to be implemented to great extent in diagnostic and research laboratories. The advantage of NGS-based analysis over Sanger sequencing is the possibility to sequence a large amount of genes simultaneously. NGS-based testing is therefore extremely useful in identifying the genetic cause in diseases that can be caused by mutations in several genes (polygenic diseases) and in patients with a broad differential diagnoses. Comparing the exome or genome of the patient and their affected or healthy relatives may result in the identification of novel causative genetic defects. Although NGS techniques have many advantages, the use of these new techniques does not guarantee success in identifying the underlying genetic defect. For example when using WES, mutations in non-coding DNA are missed, and repeat expansions are difficult to capture. Moreover, mutations with reduced penetrance may be interpreted as

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rare polymorphisms when present in cohorts of healthy persons. Because variants identified by NGS may in fact be technical artefacts, variants often have to be validated by Sanger sequencing, especially when located in a region with low coverage. Furthermore, disturbed methylation, somatic mutations and epigenetic changes may be missed or misinterpreted. However, NGS studies have resulted in the identification of many novel associations between genes and diseases. When a causative genetic defect is identified in a patient, predictive testing becomes available for family members at risk of the disease. Although HD cannot be cured or prevented, 10-15% of the individuals at risk request DNA testing while asymptomatic34,35, in most cases to have more certainty about their future or because of reproductive options36. Due to the discovery of causative genes in many other diseases, especially in FTD, predictive testing for adult onset neurodegenerative disorders has become an option for an increasing number of family members at risk. In the Netherlands, neurologists may request DNA analysis in affected patients, but only clinical geneticists are permitted to perform predictive DNA tests in unaffected family members. This distinction is because of the difference in impact of a positive test results: in patients, the identification of a genetic defect confirms (or adjusts) the clinical diagnosis, but clinical consequences are rare. However, a positive test in an unaffected family member transforms this assumed healthy person into a patient awaiting a progressive, untreatable disease. In the Netherlands and most other countries, predictive testing for adult onset neurodegenerative diseases is performed according to the protocol designed for predictive testing of individuals at risk of HD37. According to this protocol, a predictive DNA test is preceded by multiple counselling sessions with both a genetic counsellor and a specialized social worker. Previous studies showed that predictive testing does not result in large psychological stress in carriers38, but studies on the consequences of predictive testing on social and family life are scarce. CLINICAL SETTING AND BACKGROUND OF THE THESIS The Alzheimer centres of the VU University Medical Centre in Amsterdam and the Erasmus MC in Rotterdam are both Dutch tertiary memory clinics with expertise on early-onset dementia39. Patients are screened according to an extensive protocol including neuropsychological testing, magnetic resonance imaging (MRI), cerebrospinal fluid (CSF) analysis, electroencephalography (EEG) or magnetoencephalography (MEG). Positron emission tomography (PET) imaging to identify cerebral amyloid


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deposits or hypometabolism is performed in selected cases. The extensive investigation enables the physicians and researchers to thoroughly study phenotypes in patients or cohorts. Close collaboration between the Alzheimer centres and the Departments of Clinical Genetics enabled us to perform genetic and phenotypic studies in well-defined clinical cohorts and in families with unexplained assumed autosomal dominant inherited dementia. The discovery of the C9orf72 gene created a need for new guidelines on genetic testing in dementia. Furthermore, it raised questions about the frequency of C9orf72 repeat expansions in Dutch FTD patients and the associated phenotype. We also wondered whether C9orf72 repeat expansions might cause an AD-like phenotype. As far as we know, studies on mutation frequencies in Dutch EOAD patients were lacking. Because of the need of patients and especially their offspring to be as certain as possible about the heritability of the disease, we performed a study with the aim to identify novel genetic causes of dementia. Furthermore, we performed a study on the effects of predictive testing for neurodegenerative disorders on personal and social life because of the need of counsellors to understand to what extent an unfavourable test result influence one’s life.

OUTLINE OF THE THESIS

In this thesis, we present our research on three different aspects of inherited dementia: I. genetic counselling and (predictive) testing, II. prevalence of mutations and associated phenotypes, and III. new genetic causes of cognitive decline. In Chapter 2,we discuss clinically relevant aspects of genetic testing in dementia and related disorders. The first part of the chapter is a state-of-the-art of the genetic background of the different types of dementia and recommendations for genetic testing in dementia patients. In the second part of the chapter, we describe the results of our study on the impact of predictive testing in asymptomatic individuals at risk of HD or FTD on personal and social life. Chapter 3 encompasses two observational studies on frequencies and phenotypes of causative mutations in Dutch dementia cohorts. This chapter starts with the first Dutch study on prevalence and clinical features of C9orf72 hexanucleotide repeat expansions. In the second part of the chapter, we describe the prevalence of mutations in the most common dementia genes and of PRKAR1B, a gene recently associated with a FTD-like

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phenotype, the first Dutch study on mutation frequency in AD which also included EOAD patients. Furthermore, we performed a study with the aim to identify new genes (GENDEM: Searching for GENes in families with frontotemporal dementia, dementia with Lewy bodies and early-onset Alzheimer DEMentia) using NGS. The WES results in two of the included families and the associated phenotype are reported in Chapter 4. In the first part of the chapter, we describe a family with three siblings with cognitive complaints and microbleeds and an assumed autosomal dominant family history for early-onset dementia. In the second part of the chapter, we describe a family in which we searched for genetic factor(s) co-occurring with APOE-ɛ4 homozygosity in an AD family. In both families, we found genetic variants likely to contribute to the phenotype.


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REFERENCES

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2. Alzheimer Nederland Fact sheet dementia 28-1-2016. https://www.alzheimer-nederland.nl/sites/ default/files/directupload/factsheet-dementie-algemeen.pdf, viewed 26-6-2016.

3. Alexander, M, Perera, G, Ford, L, et al. Age-Stratified Prevalence of Mild Cognitive Impairment and Dementia in European Populations: A Systematic Review. J Alzheimers Dis. 2015; 48:355-359.

4. McKhann, GM, Knopman, DS, Chertkow, H, et al. The diagnosis of dementia due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease. Alzheimers Dement. 2011; 7:263-269.

5. Alzheimer's Disease International. World Alzheimer Report 2009: The global prevalence of dementia. . https://www.alz.co.uk/research/world-report-2009.

6. Harvey, RJ, Skelton-Robinson, M, and Rossor, MN. The prevalence and causes of dementia in people under the age of 65 years. J Neurol Neurosurg Psychiatry. 2003; 74:1206-1209.

7. McKhann, G, Drachman, D, Folstein, M, et al. Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology. 1984; 34:939-944.

8. Tifratene, K, Robert, P, Metelkina, A, et al. Progression of mild cognitive impairment to dementia due to AD in clinical settings. Neurology. 2015; 85:331-338.

9. Tschanz, JT, Norton, MC, Zandi, PP, et al. The Cache County Study on Memory in Aging: factors affecting risk of Alzheimer's disease and its progression after onset. Int Rev Psychiatry. 2013; 25:673-685.

10. Genin, E, Hannequin, D, Wallon, D, et al. APOE and Alzheimer disease: a major gene with semi-dominant inheritance. Mol Psychiatry. 2011; 16:903-907.

11. Liu, CC, Kanekiyo, T, Xu, H, et al. Apolipoprotein E and Alzheimer disease: risk, mechanisms and therapy. Nat Rev Neurol. 2013; 9:106-118.

12. Guerreiro, R, Wojtas, A, Bras, J, et al. TREM2 variants in Alzheimer's disease. N Engl J Med. 2013; 368:117-127.

13. Jonsson, T, Stefansson, H, Steinberg, S, et al. Variant of TREM2 associated with the risk of Alzheimer's disease. N Engl J Med. 2013; 368:107-116.

14. Lambert, JC, Ibrahim-Verbaas, CA, Harold, D, et al. Meta-analysis of 74,046 individuals identifies 11 new susceptibility loci for Alzheimer's disease. Nat Genet. 2013; 45:1452-1458.

15. Jonsson, T, Atwal, JK, Steinberg, S, et al. A mutation in APP protects against Alzheimer's disease and age-related cognitive decline. Nature. 2012; 488:96-99.

16. Sassi, C, Nalls, MA, Ridge, PG, et al. ABCA7 p.G215S as potential protective factor for Alzheimer's disease. Neurobiol Aging. 2016; 235:e1-e9.

17. Cohn-Hokke, PE, Elting, MW, Pijnenburg, YA, et al. Genetics of dementia: update and guidelines for the clinician. Am J Med Genet B Neuropsychiatr Genet. 2012; 159B:628-643.

18. Cacace, R, Sleegers, K, and Van, BC. Molecular genetics of early-onset Alzheimer's disease revisited. Alzheimers Dement. 2016; 12:733-748.

19. Wingo, TS, Lah, JJ, Levey, AI, et al. Autosomal recessive causes likely in early-onset Alzheimer disease. Arch Neurol. 2012; 69:59-64.

20. Green, RC, Cupples, LA, Go, R, et al. Risk of dementia among white and African American relatives of patients with Alzheimer disease. JAMA. 2002; 287:329-336.

21. Scarabino, D, Gambina, G, Broggio, E, et al. Influence of family history of dementia in the development and progression of late-onset Alzheimer's disease. Am J Med Genet B Neuropsychiatr Genet. 2016; 171B:250-256.

22. Bang, J, Spina, S, and Miller, BL. Frontotemporal dementia. Lancet. 2015; 386:1672-1682. 23. Rascovsky, K, Hodges, J, Knopman, D, et al. Sensitivity of revised diagnostic criteria for the

behavioural variant of frontotemporal dementia. Brain. 2011; 134:2456-2477.

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24. Gorno-Tempini, ML, Hillis, AE, Weintraub, S, et al. Classification of primary progressive aphasia and its variants. Neurology. 2011; 76:1006-1014.

25. Stevens, M, van Duijn, CM, Kamphorst, W, et al. Familial aggregation in frontotemporal dementia. Neurology. 1998; 50:1541-1545.

26. Po, K, Leslie, FV, Gracia, N, et al. Heritability in frontotemporal dementia: more missing pieces? J Neurol. 2014; 261:2170-2177.

27. van der Zee, J, Gijselinck, I, Dillen, L, et al. A pan-European study of the C9orf72 repeat associated with FTLD: geographic prevalence, genomic instability, and intermediate repeats. Hum Mutat. 2013; 34:363-373.

28. Fahey, C, Byrne, S, McLaughlin, R, et al. Analysis of the hexanucleotide repeat expansion and founder haplotype at C9ORF72 in an Irish psychosis case-control sample. Neurobiol Aging. 2014; 35:1510-1515.

29. Chiang, HH, Forsell, C, Lindstrom, AK, et al. No common founder for C9orf72 expansion mutation in Sweden. J Hum Genet. 2016.

30. Fratta, P, Polke, JM, Newcombe, J, et al. Screening a UK amyotrophic lateral sclerosis cohort provides evidence of multiple origins of the C9orf72 expansion. Neurobiol Aging. 2015; 36:546-547.

31. Xi, Z, van, BM, Zhang, M, et al. Jump from pre-mutation to pathologic expansion in C9orf72. Am J Hum Genet. 2015; 96:962-970.

32. Wood, EM, Falcone, D, Suh, E, et al. Development and validation of pedigree classification criteria for frontotemporal lobar degeneration. JAMA Neurol. 2013; 70:1411-1417.

33. Pringsheim, T, Wiltshire, K, Day, L, et al. The incidence and prevalence of Huntington's disease: a systematic review and meta-analysis. Mov Disord. 2012; 27:1083-1091.

34. Tibben, A. Predictive testing for Huntington's disease. Brain Res Bull. 2007; 72:165-171. 35. Morrison, PJ, Harding-Lester, S, and Bradley, A. Uptake of Huntington disease predictive testing in a

complete population. Clin Genet. 2011; 80:281-286. 36. Paulsen, JS, Nance, M, Kim, JI, et al. A review of quality of life after predictive testing for and earlier

identification of neurodegenerative diseases. Prog Neurobiol. 2013; 110:2-28. 37. IHA and WFN. International Huntington Association and the World Federation of Neurology Research

Group on Huntington's Chorea. Guidelines for the molecular genetics predictive test in Huntington's disease. J Med Genet. 1994; 31:555-559.

38. Almqvist, EW, Brinkman, RR, Wiggins, S, et al. Psychological consequences and predictors of adverse events in the first 5 years after predictive testing for Huntington's disease. Clin Genet. 2003; 64:300-309.

39. van der Flier, WM, Pijnenburg, YA, Prins, N, et al. Optimizing patient care and research: the Amsterdam Dementia Cohort. J Alzheimers Dis. 2014; 41:313-327.


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Chapter 2

Genetic counselling and testing in dementia and related disorders

Chapter 2.1

Genetics of dementia: update and guidelines for the clinician

Petra E. Cohn-Hokke, Mariet W. Elting, Yolande A.L. Pijnenburg, John C. van Swieten

Am J Med Genet Part B 2012: 159B:628–643

ABSTRACT With increased frequency, clinical geneticists are asked for genetic advice on the heredity of dementia in families. Alzheimer's disease is in most cases a complex disease, but may be autosomal dominant inherited. Mutations in the PSEN1 gene are the most common genetic cause of early onset Alzheimer's disease, whereas APP and PSEN2 gene mutations are less frequent. Familial frontotemporal dementia may be associated with a mutation in the MAPT or GRN gene, or with a repeat expansion in the C9orf72 gene. All these genes show autosomal dominant inheritance with a high penetrance. Although Alzheimer's disease and frontotemporal dementia are clinically distinguishable entities, phenotypical overlap may occur. Rarely, dementia is caused by mutations in other autosomal dominant genes or by genetic defects with autosomal recessive, X-linked dominant or mitochondrial inheritance. The inherited forms of frontotemporal dementia and Alzheimer's disease show a large phenotypic variability also within families, resulting in many remaining uncertainties for mutation carriers. Therefore, genetic counselling before performing genetic testing is essential in both symptomatic individuals and healthy at risk relatives. This review provides an overview of the genetic causes of dementia and discusses all aspects relevant for genetic counselling and testing. Furthermore, based on current knowledge, we provide algorithms for genetic testing in patients with early onset Alzheimer's disease or frontotemporal dementia.

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INTRODUCTION Dementia is a major health problem with a prevalence of 35.6 million people affected worldwide in 2010, estimated to increase to 115.4 million by 20501. In the United States, the prevalence of Alzheimer's disease (AD), the most common type of dementia, is estimated on 13% in people aged over 65 years, increasing to >40% in people older than 85 years2. Although less common, dementia may also occur at a younger age, with a prevalence of 1–10 in 10,000 in persons aged 45–65 years3-5. Also in dementia with an younger than 65 years, AD is the most common cause, followed by frontotemporal dementia (FTD)3,4,6,7. Genetic factors are important in all forms of dementia but especially in early onset dementia, illustrated by an autosomal dominant family history in 10% of the patients. Early onset dementia most commonly refers to dementia with an onset before the age of 65 years, although this cut-off age is arguable. Due to the growing awareness of the genetic aspects of dementia among doctors, patients, relatives, and the public, clinical geneticists are consulted with increasing frequency about this disease. The identification of the causal gene defect enables us to make a better estimation of the risk of dementia for family members, and gives us the opportunity to offer genetic testing to relatives at-risk. Although a treatment to prevent or delay the disease is not available at this moment, subjects may request asymptomatic testing to prepare better for the future or in the light of reproductive choices. Genetic testing may also be helpful in establishing the correct diagnosis, which allows the patient and his or her family to anticipate on specific changes during the disease process and the physician to select patients with the same underlying pathogenesis for therapeutic trials. While most publications on the genetics of dementia discuss one specific cause of dementia, we aim to provide an up-to-date overview of all monogenetic causes of dementia, and to discuss the aspects of genetic counselling in dementia. Although dementia is a common feature in certain dysmorphological syndromes such as Down syndrome, these syndromes rarely present with isolated dementia and are therefore not discussed. An overview of the genetic causes of early onset dementia with its references is given in Table 1, background information on the different causes of dementia is stated below.

Genetics of dementia: update and guidelines

27

GENERAL INFORMATION ON ALZHEIMER’S DISEASE Clinically, AD is characterized by deficits in short-term memory, language, praxis, visuospatial and executive functioning, eventually resulting in global cognitive impairment, with a mean disease duration of 5–15 years8. The clinical presentation of early onset AD (EOAD) is frequently atypical with focal presentations, such as aphasia or visual agnosia, and less prominent memory deficits9. Neuropathology in AD patients is characterized by plaques formed by neurotoxic oligomeric aggregates of Aβ42 peptide, produced by cleavage of the amyloid-beta precursor protein by β- and γ-secretases instead of by α-secretase10, and tangles comprised of hyperphosphorylated tau protein. The cause of AD remains elusive, despite the genetic developments over the last two decades. The discovery of mutations in the APP gene (see below) has led to the amyloid cascade hypothesis. According to this hypothesis, plaques are the direct result of increased amyloid-β production, while hyperphosphorylation of tau protein is an effect of axonal damage caused by amyloid deposits11,12. However, amyloid deposits are neither sufficient nor necessary to develop AD13-15 and drug trials regulating amyloid levels have been disappointing16,17, suggesting a coexisting pathogenic pathway. The recently published criteria to establish the diagnosis of AD18 have replaced the long-time used NINDS-ADRDA criteria of 198419. The diagnosis probable or possible AD is based on clinical criteria and may be supported by reduced amyloid-β, increased total tau (t-tau) and elevated phosphorylated tau (p-tau) levels in the cerebrospinal fluid, hippocampal atrophy, reduced metabolism on FDG-PET imaging, or retention of amyloid on PiB-PET imaging. The term “pathophysiologically proved AD” is reserved for patients meeting both the clinical and the neuropathological criteria. GENES IN EARLY ONSET ALZHEIMER’S DISEASE Approximately 1–6% of all patients with AD have an early onset, of which 60% have at least one relative with dementia and 13% an autosomal dominant family history for EOAD20. Currently, three genes are known to cause autosomal dominant EOAD: the presenilin-1 gene (PSEN1), the amyloid beta A4 protein precursor gene (APP) and the presenilin-2 gene (PSEN2). All are involved in the amyloid-beta pathway. Table 1 summarizes the most relevant clinical characteristics associated with mutations in these genes.

Chapter 2.1

28

Tabl

e 1.

Ove

rvie

w o

f Men

delia

n di

sord

ers

with

dem

entia

as

a pr

esen

ting

sym

ptom

.

Diso

rder

Ge

ne

Inhe

ritan

ce*

Pene

tranc

e Mu

tatio

n fre

quen

cy in

EOA

D

or F

TD p

atien

ts

Mean

age a

t ons

et

dem

entia

Cl

inica

l cha

ract

erist

ics

EOAD

PS

EN1

AD

~90%

at 60

yrs,

>

95%

at 65

yrs44

18

-36%

in A

D EO

AD,

10-

25%

in fa

milia

l EOA

D,

0-5

% in

spor

adic

EOAD

45-4

7

35-6

0 yrs20

,29

Relat

ively

rapid

prog

ress

ive co

gnitiv

e

dec

line,

in so

me pr

eced

ed or

acco

mpan

ied

by f

ocal

neur

ologic

al sy

mptom

s20,29

-31 ;

may

cau

se F

TD-lik

e phe

notyp

e48-5

1 AP

P AD

>9

5%

6-26

% in

AD

EOAD

, ~

0% in

spor

adic

EOAD

20

,22,23

,40,41

,46,47

,52

40-6

0 yrs22

,23,53

-55

EOAD

and/o

r sev

ere C

AA56

PSEN

2 AD

>9

5% at

75 yr

s57

Rare

20,22

,23,47

45

-65 y

rs57

Alzh

eimer

’s dis

ease

with

early

or la

te on

set, i

n s

ome a

ccom

panie

d by s

eizur

es57

FT

D M

APT

AD

>95%

9-

21%

in fa

milia

l FTD

, ~

0% in

spor

adic

FTD58

-61

45-6

0 yrs60

In

most

case

s bvF

TD w

ith or

with

out

Par

kinso

nism,

in so

me pr

ogre

ssive

spee

ch

diffi

cultie

s, mi

ld lat

e-on

set P

arkin

sonis

m,

PSP

, CBD

, epil

eptic

seizu

res62

; may

caus

e E

OAD-

like p

heno

type63

-65

GRN

AD

~50%

at 60

yrs,

~

90%

at 70

yrs66

4-

23%

in fa

milia

l FTD

, 0

-4%

in sp

orad

ic FT

D58,60

,66-6

8 55

-65 y

rs69

bvFT

D, in

some

prog

ress

ive sp

eech

d

ifficu

lties,

memo

ry de

ficit,

Parki

nson

’s

dise

ase,

CBD,

PSP

or F

TD-M

ND68

,69; m

ay

cau

se A

D-lik

e or s

chizo

phre

nia-lik

e p

heno

type70

-73

C9or

f72

AD

Incom

plete74

,75

18-3

0% in

FTD

-MND

,

12-

18%

in fa

milia

l FTD

, 2

-4%

in sp

orad

ic FT

D74-7

7

55-6

0 yrs74

,75,77

bv

FTD,

PPA

, ALS

or F

TD-M

ND, in

some

m

emor

y defi

cits o

r psy

chos

is as

pres

entin

g fe

ature

74-7

8

FUS

AD

n/a

<3%

in fa

milia

l FTD

-MND

, r

are i

n iso

lated

FTD

79,80

n/a

MN

D or

FTD

-MND

79,80

TARD

BP

AD

n/a

1% in

fami

lial F

TD-M

ND,

rar

e in i

solat

ed F

TD81

-84

n/a

FTD-

MND

or M

ND, r

arely

FTD

81,85

,86

CHM

P2B

AD

n/a

Rare

in F

TD60

,61,87

n/a

MN

D, ra

rely

FTD88

-90


29

Tabl

e 1. O

verv

iew

of M

ende

lian

diso

rder

s w

ith d

emen

tia a

s a

pres

entin

g sy

mpt

om (c

ontin

ued)

. Di

sord

er

Gene

In

herit

ance

* Pe

netra

nce

Muta

tion

frequ

ency

in E

OAD

o

r FTD

pat

ients

Mean

age a

t o

nset

dem

entia

Cl

inica

l cha

ract

erist

ics

FTD

(

cont

inued

) UB

QLN2

X-

D Inc

omple

te in

f

emale

s91

<5%

in po

ssibl

e X-lin

ked f

amilia

l A

LS w

ith or

with

out F

TD91

n/a

FT

D-MN

D or

MND

91

VCP

AD

~100

% fo

r IBM

PFD,

~

30%

for F

TD92

Ex

treme

ly ra

re in

isola

ted F

TD,

>50

% in

IBMP

FD92

40

-55 y

rs92

Myop

athy,

Page

t dise

ase o

f bon

e and

/or

FTD

92, o

r fam

ilial A

LS93

CA

DASI

L NO

TCH3

AD

~1

00%

94

n/a

50-6

0 yrs95

Re

curre

nt str

okes

and/o

r cog

nitive

decli

ne,

ofte

n hist

ory o

f migr

aine,

invar

iably

c

hara

cteris

tic w

hite m

atter

lesio

ns in

te

mpor

al lob

es94

,95

fCJD

PR

NP

AD

Incom

plete

in so

me

muta

tions

96,97

n/a

55

-63 y

rs98,99

Cogn

itive d

eclin

e, ata

xia, m

yoclo

nus a

nd

hyp

okine

tic m

utism

, ofte

n pre

cede

d by

con

fusion

and m

emor

y defi

cits;

may c

ause

A

D- lik

e phe

notyp

e97,10

0,101

DR

PLA

ATN1

AD

>9

5%

n/a

30 yr

s102

Atax

ia, ch

oreo

atheto

sis an

d dem

entia

in

adu

lts, p

rogr

essiv

e inte

llectu

al

dete

riora

tion,

beha

viour

al ch

ange

s,

myo

clonu

s and

epile

psy i

n chil

dren

102

Metab

olic

d

isord

ers

Seve

ral g

enes

o

f nuc

lear a

nd

mito

chon

drial

D

NA

AR /

mate

rnal

n/a

n/a

n/a

Va

riety

of fea

tures

, inclu

ding c

ognit

ive

dec

line,

deafn

ess,

diabe

tes m

ellitu

s and

n

euro

logica

l featu

res,

espe

cially

o

phtal

mople

gia, p

tosis,

mus

cle w

eakn

ess,

a

taxia

and e

pilep

sy

n/a,

not

app

licab

le o

r not

ana

lyse

d. A

LS, a

myo

troph

ic la

tera

l scl

eros

is; b

vFTD

, beh

avio

ural

var

iant

FTD

; CA

A, c

ereb

ral a

myl

oid

angi

opat

hy; C

AD

AS

IL, c

ereb

ral a

utos

omal

do

min

ant a

rterio

path

y w

ith s

ubco

rtica

l inf

arct

s an

d le

ukoe

ncep

halo

path

y; C

BD

, cor

ticob

asal

deg

ener

atio

n; E

OA

D, e

arly

ons

et A

lzhe

imer

’s d

isea

se; f

CJD

, fam

ilial

C

reut

zfel

dt–J

akob

dis

ease

; FTD

, fro

ntot

empo

ral d

emen

tia; I

BM

PFD

, inc

lusi

on b

ody

myo

path

y as

soci

ated

with

Pag

et d

isea

se o

f bon

e an

d/or

fron

tote

mpo

ral d

emen

tia;

MN

D, m

otor

neu

ron

dise

ase;

PP

A, p

rimar

y pr

ogre

ssiv

e ap

hasi

a; P

SP

, pro

gres

sive

sup

ranu

clea

r pal

sy. *

Inhe

ritan

ce: A

D, a

utos

omal

dom

inan

t; A

R, a

utos

omal

rece

ssiv

e; X

-D,

X-lin

ked

dom

inan

t.

Chapter 2.1

30

PSEN1 gene The gene most frequently mutated in EOAD is the PSEN1 gene on chromosome 14q24.3, identified in 199521. While this gene is probable responsible for 18–36% in autosomal dominant EOAD when defined as AD with an onset before the age of 65 years, a much higher prevalence of 56–65% was found in studies with a cut-off age of 60 years20,22,23, probably due to the young mean age of onset of mutations in this gene. The penetrance is almost complete and de novo mutations are rare, with only few reports of proven cases24-28. Patients with PSEN1 mutations typically present with relatively rapid progressive AD manifesting between the age of 35 and 60 years20,29. Cognitive decline may be preceded or accompanied by focal neurological symptoms, such as spastic paraplegia, generalized seizures, and myoclonus30,31. Regional cerebral hypoperfusion and significantly lower CSF beta-amyloid-42 levels are found in asymptomatic carriers compared to controls32-

36. Characteristic cotton–wool amyloid plaques are typically found in the striatum insymptomatic, but also in asymptomatic, carriers36. The protein presenilin 1 is a component of the γ-secretase complex, and pathogenic mutations in the PSEN1 gene lead to an increase of the amounts of Aβ42. Most PSEN1 mutations in AD are missense mutations, whereas a few are small deletions or insertions

APP gene The APP gene on chromosome 21q21 was the first gene identified in autosomal dominant AD37. Since mutations in this gene are rare in non-autosomal dominant familial EOAD and have not been reported in sporadic EOAD, the penetrance is likely to be (close to) complete and de novo mutations rare. Patients with APP mutations present with EOAD, or with headache, focal neurological deficits and seizures, and often have asymptomatic, microbleeds suspective of cerebral amyloid angiopathy (CAA)38. CAA is the result of amyloid deposits in walls of arteries and arterioles in the brain leading to spontaneous, frequently recurrent, lobar cerebral haemorrhages. The pathological phenotype associated with APP mutations comprises amyloid plaques, neurofibrillary tangles, and CAA. The APP gene encodes for the amyloid protein, and mutations causing Alzheimer's disease are mainly missense mutations adjacent to the β and γ secretase cleavage sites or duplications of the whole gene, while familial CAA is in most cases caused by founder mutations within the Aβ region, encoded for by residues 21–23 and 34 (codons 692–694 and 705)39-42.


31

GENES IN LATE ONSET ALZHEIMER’S DISEASE

Late onset AD (LOAD) is considered a complex disease with genetic factors contributing for up to 80%103, explaining the commonness of familial LOAD. Monogenetic causes of LOAD have not been identified, with the exception of a few PSEN1 mutations associated with later onset AD29,104,105. About 25% of the LOAD patients have at least one relative with EOAD106. The ε4 allele of apolipoprotein E (APOE) is the major genetic risk factor for LOAD with a variable effect dependent of ethnicity and gender107-109. Over the last few years, genome-wide association studies have revealed several other genetic factors in AD, each with small effect110-113.

GENERAL INFORMATION ON FRONTOTEMPORAL DEMENTIA

Frontotemporal dementia denotes a disorder with neurodegeneration of frontal and temporal cortex leading to behavioural changes and language disturbances in the presence of intact memory and visuospatial functions. The disease was formerly called “Pick's disease,” after the Czech psychiatrist who first described the clinical picture in association with marked lobar atrophy. FTD is clinically divided into behavioural variant FTD (bvFTD) and primary progressive aphasia (PPA); the latter can be further subdivided into semantic dementia (SD), progressive non-fluent aphasia (PNFA), and logopenic progressive aphasia (LPA)114. BvFTD, the most common type115, manifests typically by disinhibition, compulsive or perseverative behaviour, and apathy with emotional bluntness116. Patients with PNFA have reduced speech production, while SD is characterized by difficulties in understanding words and recognizing objects. The core features of the recently added subtype LPA are word retrieval and sentence repetition deficits, but this type is in most cases caused by Alzheimer's disease pathology117. The age of onset of FTD is usually between 45 and 65 years, although 10% have an onset above the age of 70 years60. Parkinsonian signs are common and support the diagnosis of FTD, but typically emerge in a later stage of the disease118. Motor neuron disease (MND) develops in up to 13% of the FTD patients119, while 15% of the amyotrophic lateral sclerosis (ALS) patients have FTD120, supporting the hypothesis that FTD and ALS are part of a disease continuum. Frontotemporal lobar degeneration (FTLD) refers to the neuropathological disease116. FTLD pathology is classified by the accumulation of abnormal protein in neurons, the

Chapter 2.1

32

main categories being FTLD with tau pathology (FTLD-tau) and with ubiquitin positive inclusions (FTLD-U), the latter comprising FTLD with TDP-43 positive inclusions (FTLD-TDP) and the less frequent FTLD with inclusions staining positive for fused in sarcoma protein (FTLD-FUS)121,122. The clinical consensus criteria of Neary et al.118 are used to establish the diagnosis of FTD. Revised criteria for possible, probable and definite bvFTD, implementing brain imaging and pathologic findings, have become available in 2011123. Frontal and/or temporal lobar atrophy on neuroimaging, although often absent in the early stage of the disease, is a characteristic feature of FTD and is preceded by glucose hypometabolism on FDG-PET124. Cerebrospinal fluid analysis may help to distinguish AD from FTD, as increased tau and decreased beta-amyloid levels are typically lacking in FTD125. GENES IN FRONTOTEMPORAL DEMENTIA About 40% of the patients have at least one relative with dementia and in 10–15%, family history is consistent with an autosomal dominant inheritance87,126,127. The majority of families with autosomal dominant FTD have a mutation in one of the three most important genes for FTD; the microtubule-associated protein tau gene (MAPT), the progranulin gene (GRN) and the C9orf72 gene (see Table 1). MAPT gene The MAPT gene on chromosome 17q21.1 was identified first as a cause of FTD with Parkinsonism in 1998128,129, but was found to cause FTD without Parkinsonism as well. While this gene is an important cause in familial FTD, mutations were not found in Scandinavian cohorts130,131. Reduced penetrance has been suggested for some specific mutations132-135 and one case of a proven de novo mutation has been reported136. FTD with pathological-proven tau pathology is associated with MAPT mutations in 14% percent of all patients137 and in 33% of familial cases59. The most common clinical presentation is bvFTD, followed by progressive speech difficulties, mild late-onset parkinsonism, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and epileptic seizures62. The age of onset varies, also within families60. MRI scan shows grey matter loss particularly in the anterior and medial temporal lobes, with less involvement of the parietal lobes, and typically no involvement of cingulate gyrus or precuneus138. Neuropathology invariably shows neuronal deposition of hyperphosphorylated tau protein.


33

Microtubule-associated protein tau plays a role in maintaining neuronal integrity, and axonal transport. In the brain, the protein has six isoforms, differing in the translation of exon 2, 3, and 10. In adults, the isoforms with three (3R, without transcription of exon 10) and with four amino acid repeats (4R, with transcription of exon 10) are about equally present. Missense mutations in exons 9 through 13 reduce the microtubule-binding ability of tau protein, while intronic and some coding mutations in exon 10 alter the 3R/4R ratio by alternative splicing139. GRN gene The GRN gene on 17q21.31 was discovered in 2006140,141. Prevalence estimations suggest an overall mutation frequency of 2–8% in FTD patients, with, although less common, mutations in sporadic patients as well58,67,68,87,142. More than half of the patients with FTLD-U pathology and a positive family history have a GRN mutation66,142. The largest study thus far on the phenotype of GRN mutations, by an international collaboration, reported a positive family history in 75% of the patients, which might be explained by incomplete penetrance, insufficient information on family history, and perhaps by de novo mutations, although no proven de novo mutations have been reported. GRN mutations manifest most often as bvFTD, while an alternative clinical diagnosis is established in approximately 25 percent, including AD, Parkinson's disease, CBD and PSP, likely due to the presence of memory deficits, and Parkinsonism, language disorders, motor neuron disease or motor apraxia69. Brain imaging often shows remarkable asymmetric cerebral atrophy with, apart from frontotemporal atrophy, involvement of the parietal lobe68,143,144. Regional cerebral hypoperfusion is typically seen in frontal and temporal cortex, but may be also present in the hippocampal region, parietal lobe and posterior cingulate gyrus68. Neuropathological features consist of ubiquitin and TDP-43 positive “cat eye” or lentiform-shaped neuronal intranuclear inclusions (NII), and superficial laminar spongiosis with ubiquitin-positive neuritis and neuronal cytoplasmic inclusions (NCI) in the neocortex145. The GRN gene encodes for the progranulin protein, which is cleaved into peptides called granulins or epithelins. Both the peptides and the intact granulin protein are regulators of cell growth, for example in normal development, wound healing, inflammation and tumorigenesis146. Pathogenic mutations have been described throughout the entire GRN gene, and are in most cases nonsense, frameshift or splice site mutations resulting in haploinsufficiency. Pathogenic missense mutations are

Chapter 2.1

34

thought to cause protein degradation147,148 and reduced protein secretion148. Several groups have reported a significant decrease of plasma progranulin protein in both symptomatic and asymptomatic GRN mutation carriers149-152, and suggest that plasma measurement can be used as a screening procedure to detect GRN mutations. However, the best cut-off threshold has yet to be determined153 and to use plasma measurement in diagnostic setting, it is essential to be absolutely sure all pathogenic GRN mutations indeed cause haploinsufficiency. C9orf72 gene In September 2011, two international consortia independently identified a heterozygous expanded hexanucleotide repeat (GGGGCC) located between the non-coding exons 1a and 1b of the C9orf72 gene on chromosome 9p21 as a cause of FTD and/or ALS. A repeat length greater than 30 units is thought to be pathogenic78, although repeat lengths up to 35 have been found in a few healthy individuals as well75. The gene is probably involved in RNA metabolism, however, the exact function of the gene and the pathogenic mechanism are to be determined. C9orf72 repeat expansions are about as common as mutations in the MAPT gene and GRN gene (see Table 1). However, in a Finnish FTD cohort, the overall prevalence was much higher (29%)78, probably due to a founder effect in this isolated population. Repeat expansions seem to be an even more common cause of ALS, explaining 24–47% of the familial and 4–5% of the sporadic ALS cases74,77,78. The phenotype associated with C9orf72 repeat expansions is divers with a high intrafamilial variability and comprises FTD, ALS, and FTD-ALS. The FTD phenotype is most commonly bvFTD, with memory complaints reported at clinical presentation by half of the patients in one study75, and a third presenting with psychosis in another76. Of the FTD patients with a repeat expansion, 34–58% have a personal or family history of ALS74,75,78. Incomplete penetrance has been described in some families and is supported by the detection of repeat expansions in sporadic FTD and ALS patients, especially since most mutation carriers derive from a common ancestor154. The associated neuropathology of C9orf72 repeat expansions is FTLD-TDP, in a few cases combined with Alzheimer-type pathology75,155.


35

GENES IN FRONTOTEMPORAL DEMENTIA WITH MOTOR NEURON DISEASE (FTD-MND)

While the C9orf72 gene is the most important gene in FTD-MND, a few other genes, most commonly causing isolated ALS, account for a small proportion of the remaining FTD-MND cases. Mutations in the gene fused in sarcoma (FUS) may cause FTD-MND, but just one possibly pathogenic mutation in this gene has been found a FTD patient without MND156, even though FUS pathology is present in 3% of the isolated FTD patients157,158. Mutations in the TAR DNA binding protein (TARDBP or TDP-43) gene cause FTD-MND or isolated MND, but rarely isolated FTD. Mutations in the charged multivesicular body protein 2B (CHMP2B) gene, a rare cause of ALS, have only been described to cause FTD in individuals from Danish88 and Belgian159 ancestry. Mutations in the UBQLN2 gene cause X-linked familial ALS with or without FTD.

OVERLAP GENOTYPE-PHENOTYPE ALZHEIMER’S DISEASE AND FRONTOTEMPORAL DEMENTIA

A few PSEN1 mutations have been associated with a FTD phenotype, some of them are even associated with pathological Pick's bodies160,161. Vice versa, the mutation p.Arg406Trp in the MAPT gene is associated with slowly progressive episodic memoryloss with profound hippocampal atrophy, mimicking early onset AD, and several patients with mutations in the GRN gene received an initial clinical diagnosis of AD because of prominent memory deficit. C9orf72 repeat expansions are not a common cause of late onset AD162, whether they can cause an EOAD-like phenotype has not yet been determined.

OTHER CAUSES OF EARLY ONSET DEMENTIA

Vascular dementia Vascular dementia is mainly defined as dementia as a consequence of lacunar or cortical stroke, and is familial in only a small percentage of cases. The most common genetic causes with Mendelian inheritance are cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and familial cerebral amyloid angiopathy (CAA). CADASIL manifests in most cases with strokes or cognitive decline

Chapter 2.1

36

around the age of 50, although the extensive white matter lesions are consistently visible in the anterior temporal poles on cerebral MRI from the age of 21 years94. Mutations in the NOTCH3 gene are detected in up to 96% in individuals with well-defined or biopsy-proven CADASIL163,164. As mentioned before, mutations in the APP gene may cause the rare familial form of CAA. Dementia with Lewy bodies Dementia with Lewy bodies (DLB) is a common subtype of late-onset dementia, but may present at an early age as well. The disease is characterized by dementia, visual hallucinations, fluctuating cognition, and Parkinsonism165. DLB is a complex disease, with a doubled risk of the disease for siblings166. Probable DLB due to a pathogenic PSEN1 mutation has been described once167. Familial Creutzfeldt–Jakob disease Familial Creutzfeldt–Jakob disease (fCJD) is one of the three pathologically distinguishable though clinically overlapping inheritable prion diseases. The other two are Gerstmann–Sträussler–Scheinker (GSS) syndrome, primarily manifesting as progressive cerebellar ataxia, and fatal familial insomnia (FFI). Pathogenic mutations of the PRNP gene make the prion protein more prone to convert from the non-pathogenic isoform (PrPC) into the insoluble pathogenic isoform (PrPSc). The reported incidence of prion diseases is 6–11 per 10 million, with 15% caused by a genetic defect97,168. The penetrance is incomplete, at least for certain mutations, and both somatic mosaicism169 and de novo mutations170,171 have been described. fCJD typically presents with confusion and memory deficits and may therefore mimic AD, FTD, and other types of dementia. The variability is partly explained by homozygosity for methionin on codon 129 resulting in a younger age at onset172,173. Periodic synchronous wave complexes (PSWC) on the EEG, basal ganglia hyperintensities on MRI, and increased levels of 14-3-3 and tau protein in cerebrospinal fluid all support the diagnosis of prion disease97,99. DEMENTIA AS A FEATURE OF OTHER DISEASE Dementia may be a presenting symptom in several other diseases typically characterized by other features, such as Huntington's and Parkinson's disease. Since patients with these diseases may have an atypical presentation, a thorough neurological examination


37

and information on other (neurological) diseases in the family are essential in all patients with dementia, especially when the aetiology of the dementia is unknown or doubted. A comprehensive review has been published naming a long list of important causes of “dementia plus” syndromes174. Also less common diseases, such as inclusion body myopathy associated with Paget disease of bone and/or frontotemporal dementia (IBMPFD), dentatorubral-pallidoluysian atrophy (DRPLA), and metabolic disorders, occasionally present with dementia. IBMPFD is a disorder caused by mutations in the valosin containing protein (VCP) gene All three main features have a reduced penetrance, leading to a variable phenotype. FTD is present in 30% but is often preceded by other features, FTD as the sole feature of IBMPFD in a family has been described just once175. DRPLA manifests in adults as progressive ataxia, choreoathetosis, and dementia or character changes. This rare autosomal dominant disease, with its highest prevalence in Japan, is caused by a trinucleotide repeat expansion in the ATN1 gene, often expanding during transmission to offspring. The age of onset is highly variable (range 1–69 years) and depends on the length of the repeat expansion. If the ataxia is masked by the dementia or if psychosis is one of the presenting symptoms, the disease may mimic FTD or CJD. Metabolic disorders presenting with dementia in adulthood are rare, but are important to recognize because treatment is available for some. Deafness, diabetes mellitus and neurological features, especially ophtalmoplegia, ptosis, muscle weakness, ataxia, and epilepsy may be signs of mitochondrial or other metabolic defect, such as the lysosomal storage diseases Sanfilippo syndrome, Niemann–Pick type C, and the adult type of autosomal recessive neuronal ceroid lipofuscinosis. Some metabolic disorders are characterized by leukodystrophy with white matter intensities on T2-weighted and FLAIR sequences. Most metabolic diseases are autosomal recessive inherited, while mitochondrial disorders classically are maternally inherited, but may be autosomal recessive or, rarely, dominant. RECOMMENDATIONS FOR GENETIC TESTING Based on reviewed literature, we provide an algorithm for genetic testing in patients with EOAD (Figure 1) and with FTD (Figure 2). For this purpose, familial dementia is defined as at least one 1st or 2nd degree relative of the patient affected by dementia, and autosomal dominant dementia as at least three affected persons in two or more

Chapter 2.1

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successive generations. The proposed recommendations are to be used as directives, since each family is unique in its composition, expectations and requests. Since just part of the families with autosomal dominant dementia are explained by mutations in the currently known genes, genetic testing is preferably performed in affected individuals. In familial FTD, autopsy findings contain the most important clue which gene to test, however, these results are in most cases not (yet) available in diagnostic setting. Concomitant ALS or a relative with ALS suggests a repeat expansion in the C9orf72 gene, asymmetrical and/or parietal atrophy a GRN mutation, and a relatively young age of onset and a strongly affected family a MAPT mutation, but since the phenotypes overlap, we recommend to test all genes in familial FTD if no mutation is found in the most likely gene. A FTD phenotype may be caused by certain PSEN1 mutations, therefore, we suggest testing this gene as well in MAPT, GRN, and C9orf72 negative FTD families. In familial EOAD, mutations in the PSEN1 gene are the most common monogenetic defect. Mutations in the FTD genes may result in an AD-like phenotype, therefore, we recommend to test both the less frequent genes for EOAD and the GRN and MAPT genes in PSEN1 negative families with multiple affected individuals. Currently it is not clear whether repeat expansions in the C9orf72 gene may also lead to familial EOAD. Mutations may occasionally be identified in sporadic patients, especially in FTD patients and in AD patients with a strikingly young age at onset. This may be the case if the family history is not informative, for example because of a small family size or early deceased parents, but also because of incomplete penetrance or paternity issues. Genetic testing in sporadic patients can be useful in establishing the diagnosis when the aetiology of the dementia is unclear. In such cases, an extensive family history on all diseases and complaints associated with the different causes of dementia may help to identify a less obvious cause. Counselling before genetic testing is important in both patients and asymptomatic at risk family members. Although DNA-analysis may be requested to confirm the clinical diagnosis in a patient, the identification of a genetic cause has more consequences for the family than for the patient himself. The uptake of asymptomatic testing in 1st degree relatives is about 10%176,177. Relatives may request for asymptomatic testing because of the psychological burden of the uncertainty about their future178, but it is important to realize that mutation carriers still face many uncertainties, for example about the age of onset and the course of the disease. Guidelines and recommendations on genetic counselling procedures in (asymptomatic) AD and FTD have been published previously177,179.


39

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Chapter 2.1

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Although testing for APOE alleles in asymptomatic individuals does not cause significant short-term psychological risks180,181, several expert consensus statements recommend against APOE testing, mainly because the ε4 allele is neither necessary nor sufficient to cause AD and no prevention is available (which would justify testing moderate risk genes), and because individuals have difficulties with risk interpretation182,183. Relatives of a dementia patient have an increased risk on the disease, unless they have not inherited the familial mutation. Since not all genes for autosomal dominant dementia have been identified, this risk may be up to 50% for 1st degree relatives. Observational studies, in which no genetic testing was involved, found a threefold increased empiric lifetime risk on AD for 1st degree relatives of AD patients107,184, with the absolute risk strongly influenced by ethnicity and gender107,185,186, and a 3–4 times increased risk on dementia for 1st degree relatives of FTD patients187. CONCLUSIONS AND FUTURE PERSPECTIVES In this review, we summarize up-to-date genetic and phenotypic information needed in genetic counselling of possibly inheritable dementia, and we propose practical guidelines for genetic testing in early onset dementia. The use of next generation sequencing has led to the identification of the C9orf72 gene and is likely to result in the discovery of many other, mainly infrequent or reduced penetrance genes for dementia. Therefore, several important changes in genetic testing are to be expected. Firstly, while it is currently only possible to confirm an inheritable cause of dementia, the possibility of testing more genes would lower the post-test probability of autosomal dominant inheritance in patients without mutations. Secondly, implementing next generation sequencing in diagnostic setting enables parallel sequencing of all involved genes, although for the interpretation of the results, especially unclassified variants, knowledge of the genetic background of dementia remains extremely important. Furthermore, testing modifier genes may lead to a more accurate prediction of the age of onset or clinical phenotype in mutation carriers. In individuals with no mutations in the high-risk genes, testing a set of low and moderate risk genes may lead to a more reliable risk prediction. This may result in a change of mind about testing risk genes in asymptomatic individuals. Hopefully, treatment or prevention becomes available for dementia, in which case the interest in genetic testing in both affected and at risk individuals is likely to increase dramatically.

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141. Cruts, M, Gijselinck, I, van der Zee, J, et al. Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21. Nature. 2006; 442:920-924.

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142. Yu, CE, Bird, TD, Bekris, LM, et al. The spectrum of mutations in progranulin: a collaborative study screening 545 cases of neurodegeneration. Arch Neurol. 2010; 67:161-170.

143. Rohrer, JD, Warren, JD, Omar, R, et al. Parietal lobe deficits in frontotemporal lobar degeneration caused by a mutation in the progranulin gene. Arch Neurol. 2008; 65:506-513.

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153. Bird, TD. Progranulin plasma levels in the diagnosis of frontotemporal dementia. Brain. 2009; 132:568-569.

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164. Peters, N, Opherk, C, Bergmann, T, et al. Spectrum of mutations in biopsy-proven CADASIL: implications for diagnostic strategies. Arch Neurol. 2005; 62:1091-1094.


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165. McKeith, IG, Galasko, D, Kosaka, K, et al. Consensus guidelines for the clinical and pathologic diagnosis of dementia with Lewy bodies (DLB): report of the consortium on DLB international workshop. Neurology. 1996; 47:1113-1124.

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50

Chapter 2.2 The effect of predictive testing in adult onset neurodegenerative diseases on social and personal life Petra E. Cohn-Hokke, John C. van Swieten, Yolande A.L. Pijnenburg, Aad Tibben, Hanne Meijers-Heijboer, Anneke Kievit

Submitted

ABSTRACT Follow-up studies on predictive testing for hereditary neurodegenerative diseases mainly focussed on psychological outcome. We investigated whether the social and personal life of mutation carriers differ negatively from non-carriers and untested at risk individuals. Asymptomatic individuals (≥35 years) who received a genetic test result for Huntington’s disease, frontotemporal dementia or Alzheimer’s disease more than 2 years before the onset of the study and untested subjects at 50% risk were invited to complete a questionnaire and an additional questionnaire with extra or adjusted items. Of the 283 selected individuals, 115 returned a positive informed consent (response rate 39,6%). Of these, 17 carriers, 30 non-carriers and 27 untested persons fulfilled the criteria and completed both questionnaires. We found no significant differences in employment, financial situation and lifestyle or anxiety and depression between carriers and non-carriers or untested individuals at risk. Carriers were more often single and childless, though these differences were not significant. The findings of this study suggest that an unfavourable results of predictive testing on adult onset neurodegenerative diseases does not have a large negative effect on social and personal life, although these observations should be interpreted with caution because of the small number of participants and low response rate.

Chapter 2.2

52

INTRODUCTION Over the last decades, several genes have been identified causing autosomal dominant adult-onset neurodegenerative disease, such as HTT in Huntington’s disease (HD), PSEN1, PSEN2 and APP in (early onset) Alzheimer’s disease (AD) and MAPT, GRN and C9orf72 in frontotemporal dementia (FTD). Therefore, predictive testing has become an option for an increasing number of unaffected at-risk relatives. While a majority of the relatives seemed to be interested in predictive testing before genetic testing was available, the uptake of predictive testing for adult-onset neurodegenerative diseases is in general less than 15%1-4. Studies on the impact of predictive testing on neurodegenerative diseases mainly focussed on psychological aspects, such as anxiety and major depression and psychological adverse events5, or on perception of genetic discrimination6-9. The authors of a review article on quality of life after predictive testing on neurodegenerative diseases published in 2013, concluded that (a) extreme or catastrophic outcomes are rare; (b) depression and anxiety are common but mainly transient; (c) most participants report no regret; (d) many tested individuals report extensive benefits of knowing their genetic status, and (e) there is a need for regulations regarding genetic discrimination10. These findings suggest that predictive testing on adult onset neurodegenerative diseases is relatively safe and not harmful . Besides the need to decrease uncertainty, life planning including reproduction, work or retirement, finances and insurance, is one of the most cited reason for predictive testing in literature10. However, studies on the effect of predictive testing on adult onset neurodegenerative diseases on these non-psychological aspects of life are scarce. It seems plausible that awareness of being a carrier of a neurodegenerative disease or being at 50% risk does affect the course of life and decisions in family- and future planning in some degree. Furthermore, persons who know that they have not inherited the predisposition for the familial disease may benefit from the outcome of their genetic test. The aim of the study is to investigate whether mutation carriers of adult-onset neurodegenerative diseases are more often unemployed, have a lower income, experience more problems with insurances, live less healthy, and are more often single and childless compared to non-carriers and at risk relatives who have not been tested. In this manuscript, we describe the outcome of the exploratory study aimed to optimize the questionnaires and to identify trends for further investigation in a larger study.

Effect of predictive testing

53

SUBJECTS AND METHODS Participants We selected patients from 4 sources: I. patients who visited the Clinical Genetics Department of the Erasmus Medical Centre (EMC) in Rotterdam from January 2003 to December 2012 at risk of familial HD, FTD or AD (n=206); II. patients of the Clinical Genetics Department of the VU University Medical Centre (VUmc) in Amsterdam from January 2003 to December 2012 at risk of AD or FTD (n=9); III. participants of a genetic FTD research cohort on inherited FTD at the EMC11 (n=31); and IV. family members of study participants, who gave consent to be informed about our research project (n=37). Of the patients selected from the departments of clinical genetics, we checked in the national insurance database and hospital database whether they were alive and if they had changed addresses. Individuals of the FTD research cohort were asked permission to be contacted about this research project by their research physicians. The inclusion criteria of this exploratory study were a) age ≥35 years; b) tested while asymptomatic for HD, FTD or AD at least 2 years before the start of the study or at (presumed) 50% risk for one of these diseases c) sufficient knowledge of the Dutch language to understand the instructions and questionnaires. We chose the age cut-off because many important decisions and life events, such as building a career, having long-lasting relations and reproduction, are fulfilled at that age. We choose a minimum period since testing of two years to avoid including persons for whom the test had been too recently to result in any changes in their course of life. When we set up the study project, we decided not to exclude affected individuals who turned out to be able to fill in and return the questionnaire. However, since responses of participants diagnosed with the disease showed quite some inconsistencies, we decided to exclude them from the analysis to avoid involvement of the disease on the results and we therefore did not sent them the additional questionnaire. After completing the inclusion, we also decided to exclude the results of participants from AD families from the analysis, because their small number would not allow sub analysis of this group and would only increase the heterogeneity of the cohort. The medical ethics committees of the Erasmus MC and VU University Medical Centre approved the study.

Chapter 2.2

54

Data collection To eligible persons selected from the clinical genetics departments, we sent a package containing 1) a personal letter signed by the executive researcher and their own physician, 2) general information on the study, 3) an informed consent form, 4) a form to inform us about the contact details of relatives who gave them their consent to be contacted about this study, and 5) a return envelope. On the informed consent form participants could give their permission for being contacted on a short term with additional questions, being contacted on the long term for follow-up, and whether they wanted to be informed on the general results of the study. Persons who had agreed upon participation were sent a copy of the signed informed consent form and the questionnaire. Participants were reminded by phone or email if the questionnaire had not been returned within six weeks. Within a year of sending the first questionnaires, we sent the additional questionnaire. Questionnaires The primary questionnaire was developed by experience of the researchers, literature studies and consultation of a survey expert. The questionnaire comprised 70 questions on employment, financial issues, lifestyle, relations and family life, clustered in eight sections: I. general information on sex and date of completing the questionnaire, II. family history, III. health and lifestyle, IV. family and relations, V. education, employment and finance, VI, perception of the impact of the disease in the family, VII. perception of the impact of the personal risk, and VIII. the DNA-test (to be filled in only by tested participants). The validated Dutch version of the 12-Item Short Form Health Survey (SF-12)12 was embedded in the section on health and lifestyle. The SF-12 measures physical and mental health. The scoring system is influenced by age and other factors and therefore especially useful to compare results between or within groups. Most questions of the questionnaire were multiple choice items, with the option to elaborate or to choose not to answer the question or ‘not applicable’. Based on preliminary analysis of the results and comments made by the participants, an additional questionnaire was developed by the authors. The 47 items were either questions from the first questionnaire though slightly differently worded or with adjusted answer options, or additional questions. This questionnaire was structured in the following sections: I. general information on age and the date, II. family and relations, III well-being, IV. education, employment and finance, V. perception of the impact of the personal risk, and VI. the DNA-test. This questionnaire also encompassed the validated Hospital Anxiety and Depression Scale (HADS) questionnaire13-15, which


55

contains 7 items on depression and 7 on anxiety. A score of 8 to 10 on one of the subscales may reflect anxiety or a depressive state, a score of 11 or above is indicative of a clinically relevant level of distress. Data analyses Since the additional questionnaire also included rephrased questions of the first questionnaire, we only analysed the data of participants who returned both questionnaires. Survey data entry and analyses were conducted using the Statistical Package for the Social Sciences (SPSS) version 22 (Chicago, Ill., USA). Pearson chi-squared tests with asymptotic probability values were used for categorical association testing, and Kruskal-Wallis tests to compare medians. RESULTS Response and eligibility A total of 283 individuals received the information package (Figure 1). Of these, 193 were from HD families, 81 from FTD families and 9 from AD families. A positive informed consent form was returned by 115, an overall response rate of 39.5% (HD 33.2%, FTD 55.6% and AD 33.3%) Of the 171 non-participants, 17 returned a negative consent form or informed us about their decision by email, for reasons of current state of health (n=3), not wanting to be confronted with the disease, (n=2), being too young to participate (n=1), not being at risk of the disease (n=1), believing to be ineligible because being a non-carrier (n=1) and privacy or personal circumstances (n=1). Non-responders had a median age of 46.2 years (range 35-80), slightly though significantly younger than the positive responders, who had a median age of 49.4 years (range 29-75). No significant differences were detected between responders and non-responders in age, disease or carrier status (Table 1). Of note; of a few individuals, we had no prior information on age, carrier status or date of testing. Some of the responders turned out to be ineligible. Characteristics of the participants The characteristics of the participants are described in Table 2. Carriers were the youngest and untested individuals the eldest group. The majority of the responders were female (68.9%). The majority of carriers and non-carriers were from HD families (77.0%), while most untested individuals were at risk of FTD (74.1%). A relative high

Chapter 2.2

56

proportion (40.5%) of the participants was highly educated compared to 26.8% of the general population16. The average age of onset of the disease within in the family was, as far as known by the participants, comparable between carriers, non-carriers and untested individuals at risk. Most participants did not have an affected parent while under-age. None of the untested individuals but 15% of the tested participants were regular care givers for affected relatives. The carriers and non-carriers were comparable in age at testing and the time since receiving the test result. Figure 1. Selection and inclusion of the participants.

Table 1. Characteristics of responders and non-responders. Responders a

(n=112) Non-responders (n=171)

Responders vs non-responders (p value)

Age, median (range) 49.4 (29-75) 46.2 (35-80) 0.002 Gender male 44 (39.2%) 66 (38.8%)

ns

Familial disease Huntington’s disease Frontotemporal dementia Alzheimer’s disease

64 (57.1%) 45 (40.2%) 3 ( 2.7%)

129 (75.4%) 36 (21.1%) 6 ( 3.5%)

Carrier status (%) Carrier Non-carrier At risk Unknown

29 (25.9%) 40 (35.7%) 41 (36.6%) 2 ( 1.8%)

61 (35.7%) 49 (28.7%) 57 (33.3%) 0

ns: not significant a of some of the persons we informed about the study, characteristics were unknown. Some of the responders turned out to be ineligible, for example because of age.


57

Tabl

e 2.

Cha

ract

eris

tics

of th

e pa

rtici

pant

s.

Ca

rrier

s (n=

17)

Non-

carri

ers n

=30)

Un

test

ed at

risk

(n=2

7)

Tota

l (n=

74)

Carri

ers v

s oth

ers

Age

(rang

e)

43 (

37-7

2)

48 (

37-6

3)

52 (

37-6

9)

48 (

37-7

2)

ns

ns

ns

ns

ns

ns

ns

ns

Gend

er m

ale

5 (

29.4%

) 1 1

(36.7

%)

7 (

25.9%

) 23

(31

.1%)

Fami

lial d

iseas

e

Hun

tingto

n’s di

seas

e

Fro

ntotem

pora

l dem

entia

11 (

64.7%

) 6

(35

.3%)

25 (

83.3%

) 5

(16

.7%)

7 (

25.9%

) 20

(74

.1%)

43 (

58.1%

) 31

(41

.9%)

High

ly ed

ucate

d a 6

(35

.3%)

13 (

43.3%

) 11

(40

.7%)

30 (

40.5%

) Ag

e of o

nset

of the

dise

ase i

n the

fami

ly, m

edian

(ran

ge) b

55 (

35-7

0)

50 (

25-8

5)

60 (

43-6

6)

55 (

25-8

5)

Age a

t awa

rene

ss of

an in

herite

d dise

ase i

n the

fami

ly,

med

ian (r

ange

) c 27

(14

-67)

30

(14

-52)

38

(14

-55)

30

(14

-55)

Pare

nt aff

ected

durin

g par

ticipa

nts’ c

hildh

ood d

3 (

20.0%

) 10

(37

.0%)

4 (

17.4%

) 17

(26

.2%)

Regu

lar or

daily

care

for a

n affe

cted r

elativ

e e 4

(23

.5%)

3 (

10.0%

) 0

7

( 9

.6%)

p =

0.04

7 Ag

e at D

NA te

st, m

edian

(ran

ge)

35 (

23-6

1)

34 (

20-5

6)

na

na

ns

ns

Year

s sinc

e pre

dictiv

e tes

ting,

media

n (ra

nge)

9

( 3

-21)

11

( 3

-27)

na

na

a C

olle

ge o

r uni

vers

ity

b Unk

now

n or

una

nsw

ered

by

5 ca

rrie

rs, 5

non

-car

riers

and

4 u

ntes

ted

indi

vidu

als.

c U

nkno

wn

or u

nans

wer

ed b

y 3

non-

carr

iers

. d U

nkno

wn

or u

nans

wer

ed b

y 2

carr

iers

, 3 n

on-c

arrie

rs a

nd 4

unt

este

d pa

rtici

pant

e U

nans

wer

ed b

y 1

unte

sted

par

ticip

ant

na: n

ot a

pplic

able

ns

: not

sig

nific

ant

Chapter 2.2

58

Outcome The main outcomes of our study are described in Table 3. None of the differences were significant. In all groups, a small majority had a paid job for at least 20 hours a week. The proportion of participants that were retired was highest in the untested group (22.2%) and lowest in the non-carrier group (3.3%). Two carriers and one non-carrier had been declared unfit for work. Untested individuals more often had an income of over > € 66.000 than carriers (20.0% vs 0%). Life, disability or additional health insurances were rarely refused or offered at a higher premium in all groups. Two carriers and no non-carriers or untested participants had severe debts requiring professional help. There were no differences in life style between carriers, non-carriers an untested at risk participants. None of the participants declared to use any drugs. We found no difference in mental or physical health, although we found a wider range of the SF-12 scores in tested compared to untested individuals. Indications for depression or anxiety were slightly more often present in carriers. Although not significantly different, carriers lived slightly less often with a partner, were more often divorced and more often childless. Child wish at the moment of testing was reported by 8 carriers and 14 non-carriers. Of the carriers, 4 refrained from having any (more) children, 4 had prenatal testing or preimplantation genetic diagnostics performed and one decided to accept the risk to pass the mutation to a future child. None of the carriers adopted a child or chose for a donor parent. Six of the carriers and ten of the 14 non-carriers with child wish had a child after the test result. A mainly negative influence of their personal risk on the disease on their marital state was reported by 5.6% of the carriers and none of the non-carriers, and a mainly positive effect by 2.8% of the carriers and 5.6% of the non-carriers. Furthermore, 8.1% of the carriers and 5.5% of the non-carriers reported to have once ended a relationship (partly) because of their personal risk on the disease. None of the untested participants reported any influence of their personal disease risk on these items. DISCUSSION In this exploratory study, we found no large negative effects of predictive testing for FTD or HD on employment, salary, financial issues or health and well-being between carriers and non-carriers and untested individuals at risk.


59

Tabl

e 3.

Res

ults

of t

he s

tudy

.a

Ca

rrier

s (n=

17)

Non-

carri

ers (

n=30

) Un

test

ed at

risk

s (n=

27)

Tota

l (n=

74)

Emplo

ymen

t

Pay

ed w

ork >

20 hr

/wk

P

ayed

wor

k ≤ 20

hr/w

k

Dec

lared

unfit

for w

ork

Lo

oking

for a

job

R

etire

d

9 (

52.9%

) 2

(11

.8%)

3 (

17.6%

)b 0

2

(11

.8%)

24 (

80.0%

) 1

( 3

.3%)

1 (

3.3%

) 2

( 6

.7%)

2 (

6.7%

)

14 (

51.9%

) 7

(25

.9%)

0 0 6 (

22.2%

)

47

(63.5

%)

10

(13.5

%)

3

( 4.1%

) 2

( 2

.7%)

10

(13.5

%)

Salar

y (%

of p

erso

ns w

ith a

paye

d job

who

answ

ered

the q

uesti

on)

<

€ 33

.000 /

yr

€ 33

.000 –

66.00

0 /yr

>

€ 66

.000 /

yr

4 (

44.4%

) 5

(55

.6%)

0

9 (

39.1%

) 11

(47

.8%)

3 (

13.0%

)

5 (

33.3%

) 7

(46

.7%)

3 (

20.0%

)

18

(38.3

%)

23

(48.9

%)

6

(12.8

%)

Debts

Cur

rent

or fo

rmer

debts

requ

iring p

rofes

siona

l help

2

(11

.8%)

0 0

2

(2.7

%)

Insur

ance

refus

ed or

offer

ed at

an in

creas

ed fe

e

(% of

perso

ns w

ho ap

plied

for a

n as

sura

nce)

Life

insur

ance

Disa

bility

insu

ranc

e

Add

itiona

l hea

lth in

sura

nce

1 (

7.1%

) 1

(11

.1%)

0

2 (

7.7%

) 2

(11

.1%)

1 (

3.3%

)

0

1 (

7.7%

) 0

3

( 4.9

%)

4

( 9.8

%)

1

( 1.5

%)

Healt

h and

well

being

Smo

king

D

rinkin

g >3 u

nits a

lcoho

lic be

vera

ges a

day

U

sing s

oft dr

ugs o

r har

d dru

gs

M

ental

healt

h sco

re S

F-12

, med

ian (in

terqu

artile

rang

e)

P

hysic

al he

alth s

core

SF-

12, m

edian

(inter

quar

tile ra

nge)

HAD

S de

pres

sion s

core

≥8

H

ADS

anxie

ty sc

ore ≥

8

3 (

17.6%

) 1

( 5

.9%)

0 52.1

(15

.6)

54,0

( 8

.5)

2 (

11.8%

) 3

(17

.6%)

7 (

23.3%

) 2

( 6

.7%)

0 52.2

(10.4

) 52

.5 (

8.3)

2

( 6

.7%)

3 (

10.0%

)

7 (

25.9%

) 1

( 3

.7%)

0 54.9

( 5

.4)

54.0

( 5

.0)

2 (

7.4%

) 4

(14

.8%)

17

(23.0

%)

4

( 5.4

%)

0 53.0

(8

.8)

54.0

(8

.0)

5

( 8.1

%)

10

(13.5

%)

Chapter 2.2

60

Tabl

e 3.

Res

ults

of t

he s

tudy

(con

tinue

d). a

Fa

mily

life

Li

ving w

ith a

partn

er

M

arrie

d now

and/o

r in th

e pas

t

Divo

rced (

% of

perso

ns ev

er m

arrie

d)

H

aving

≥1 c

hild(

ren)

12

(70.6

%)

13

(76.5

%)

6

(46.2

%)

11

(64.7

%)

27 (

90.0%

) 26

(86

.7%)

7 (

26.9%

) 26

(86

.7%)

24 (

88.9%

) 21

(77

.8%)

3 (

14.3%

) 22

(81

.5%)

63

(85.1

%)

60

(81.1

%)

16

(26.7

%)

59

(79.7

%)

a Non

e of

the

diffe

renc

es b

etw

een

carr

iers

and

oth

ers

(non

-car

riers

and

unt

este

d at

risk

) wer

e si

gnifi

cant

. b O

ne o

f whi

ch d

ecla

red

parti

ally

unf

it fo

r wor

k


61

The only differences we found, although not significant, were in family life: carriers lived more often without a partner, were more often divorced despites their younger age and were more often childless. According to the national registration in the Netherlands in 2014, 40.1% of the marriages end in a divorce16. Therefore, carriers are probably not more often divorced, but the at risk untested participants of our study are less often divorced. Also according to this national database, 78% of the Dutch persons have one or more children16. The carriers in our study are more often childless than the other participants, but the non-carriers and untested at risks are less often childless than the general population. However, only tested individuals reported an influence of their personal risk on the disease on relations. Although caution is required when interpreting results of a study with small numbers of participants, the absence of differences in our study in most social aspect in life is encouraging. Our findings suggest that if a positive predictive test result has a significant impact on personal and social life, the impact is probably limited to a minority, or is relatively small or only temporarily. One possible explanation for the lack of significant differences between carriers and non-carriers or untested individuals at risk may be that all individuals are unaware of their genetic status for a certain period of time. The median ages at testing of the tested participants was 35 years, therefore, half of them lived at least the first 35 years of their lives without knowing their genotype. These first decades may be crucial in developing a personality, but also in building a career and creating a family. It would be interesting to analyse whether the outcome is different in patients who were tested at a young age. Because of the small numbers in our study and exclusion of individuals aged younger than 35 years, such a sub-analysis was not possible in this exploratory study. Another explanation is self-selection of participants who consented to participate and who remained in the study. Timman et al. reported on a long term follow-up study of individuals tested for HD and found that identified carriers, who were lost to follow-up after disclosure of test results, reported significantly more pretest distress than did retained carriers17. They speculated that studies that report few harmful effects may have underestimated the real impact. In general, we found fewer differences in outcomes between carriers and non-carriers, than between tested and untested individuals. Carriers and non-carriers seem more alike. The decision for predictive testing is probably influenced by many factors, including personality traits, the perception of the severity of the disease, the risk perception and the degree of involvement with affected relatives. We did not find a difference between tested and untested individuals in age of onset of the disease within the family or in

Chapter 2.2

62

having an affected parent during their childhood. However, only tested individuals had been involved in regularly care for an affected relative, and only tested individuals reported influence (negative or positive) of their disease risk on marital status and relations. The strengths of our study are that we included untested at risk individuals in our study, and that because of the design, we were able to adjust and supplement questions. However, the numbers of this study are small and we should be cautious when interpreting the outcomes. Furthermore, the response rate was low and a significant influence of a selection bias is likely. However, we did not find large differences in age, gender or carrier status. Carriers less often participated, probably partly due to a larger proportion already being affected. The response rate was higher in individuals of FTD families than in AD and HD families. This difference in probably caused by our methods of selecting patients: most individuals from FTD families were selected from the FTD research cohort and were requested permission to be informed in this study beforehand. In conclusion, our hypothesis that mutation carriers of adult-onset neurodegenerative diseases are more often unemployed, have a lower income, experience more problems with insurances, live less healthy, and are more often single and childless compared to non-carriers and at risk relatives who have not been tested was not confirmed. These findings suggest that in general, predictive testing on these diseases do not significantly influence the course of life. However, further (larger) studies are necessary before the results can be used in clinical genetic counselling. ACKNOWLEDGEMENTS

We thank Reinier Timman and Lidewij Henneman for their advice on the design of the study and the questionnaires.


63

REFERENCES

1. Steinbart, EJ, Smith, CO, Poorkaj, P, et al. Impact of DNA testing for early-onset familial Alzheimer disease and frontotemporal dementia. Arch Neurol. 2001; 58:1828-1831.

2. Riedijk, SR, Niermeijer, MFN, Dooijes, D, et al. A decade of genetic counseling in frontotemporal dementia affected families: few counseling requests and much familial opposition to testing. J Genet Couns. 2009; 18:350-356.

3. Tibben, A. Predictive testing for Huntington's disease. Brain Res Bull. 2007; 72:165-171. 4. Morrison, PJ, Harding-Lester, S, and Bradley, A. Uptake of Huntington disease predictive testing in a

complete population. Clin Genet. 2011; 80:281-286. 5. Almqvist, EW, Brinkman, RR, Wiggins, S, et al. Psychological consequences and predictors of

adverse events in the first 5 years after predictive testing for Huntington's disease. Clin Genet. 2003; 64:300-309.

6. Bombard, Y, Penziner, E, Suchowersky, O, et al. Engagement with genetic discrimination: concerns and experiences in the context of Huntington disease. Eur J Hum Genet. 2008; 16:279-289.

7. Penziner, E, Williams, JK, Erwin, C, et al. Perceptions of discrimination among persons who have undergone predictive testing for Huntington's disease. Am J Med Genet B Neuropsychiatr Genet. 2008; 147:320-325.

8. Williams, JK, Erwin, C, Juhl, AR, et al. In their own words: reports of stigma and genetic discrimination by people at risk for Huntington disease in the International RESPOND-HD study. Am J Med Genet B Neuropsychiatr Genet. 2010; 153B:1150-1159.

9. Erwin, C, Williams, JK, Juhl, AR, et al. Perception, experience, and response to genetic discrimination in Huntington disease: the international RESPOND-HD study. Am J Med Genet B Neuropsychiatr Genet. 2010; 153B:1081-1093.

10. Paulsen, JS, Nance, M, Kim, JI, et al. A review of quality of life after predictive testing for and earlier identification of neurodegenerative diseases. Prog Neurobiol. 2013; 110:2-28.

11. Dopper, EG, Rombouts, SA, Jiskoot, LC, et al. Structural and functional brain connectivity in presymptomatic familial frontotemporal dementia. Neurology. 2014; 83:e19-e26.

12. Gandek, B, Ware, JE, Aaronson, NK, et al. Cross-validation of item selection and scoring for the SF-12 Health Survey in nine countries: results from the IQOLA Project. International Quality of Life Assessment. J Clin Epidemiol. 1998; 51:1171-1178.

13. Zigmond, AS and Snaith, RP. The hospital anxiety and depression scale. Acta Psychiatr Scand. 1983; 67:361-370.

14. Spinhoven, P, Ormel, J, Sloekers, PP, et al. A validation study of the Hospital Anxiety and Depression Scale (HADS) in different groups of Dutch subjects. Psychol Med. 1997; 27:363-370.

15. Bjelland, I, Dahl, AA, Haug, TT, et al. The validity of the Hospital Anxiety and Depression Scale. An updated literature review. J Psychosom Res. 2002; 52:69-77.

16. Centraal Bureau voor de Statistiek Nederland. Statline. http://statline.cbs.nl/Statweb/?LA=en, viewed 17-5-2016.

17. Timman, R, Roos, R, Maat-Kievit, A, et al. Adverse effects of predictive testing for Huntington disease underestimated: long-term effects 7-10 years after the test. Health Psychol. 2004; 23:189-197.

Chapter 2.2

64

Chapter 3

Prevalence and phenotypes of inherited dementia

Chapter 3.1

The clinical and pathological phenotype of C9orf72 hexanucleotide repeat expansions

Javier Simón-Sánchez*, Elise G.P. Dopper*, Petra E. Cohn-Hokke, Renate K. Hukema, Nayia Nicolaou, Harro Seelaar, J. Roos A. de Graaf, Inge de Koning, Natasja M. van Schoor, Dorly J. H. Deeg, Marion Smits, Joost Raaphorst, Leonard H. van den Berg, Helenius J. Schelhaas, Christine E.M. de Die-Smulders, Danielle Majoor-Krakauer, Annemieke J. Rozemuller, Rob Willemsen, Yolande A.L. Pijnenburg, Peter Heutink, John C. van Swieten *equal contribution

Brain. 2012: 135:723-35

ABSTRACT

There is increasing evidence that frontotemporal dementia and amyotrophic lateral sclerosis are part of a disease continuum. Recently, a hexanucleotide repeat expansion in C9orf72 was identified as a major cause of both sporadic and familial frontotemporal dementia and amyotrophic lateral sclerosis. The aim of this study was to investigate clinical and neuropathological characteristics of hexanucleotide repeat expansions in C9orf72 in a large cohort of Dutch patients with frontotemporal dementia. Repeat expansions were successfully determined in a cohort of 353 patients with sporadic or familial frontotemporal dementia with or without amyotrophic lateral sclerosis, and 522 neurologically normal controls. Immunohistochemistry was performed in a series of 10 brains from patients carrying expanded repeats using a panel of antibodies. In addition, the presence of RNA containing GGGGCC repeats in paraffin-embedded sections of post-mortem brain tissue was investigated using fluorescence in situ hybridization with a locked nucleic acid probe targeting the GGGGCC repeat. Hexanucleotide repeat expansions in C9orf72 were found in 37 patients with familial (28.7%) and five with sporadic frontotemporal dementia (2.2%). The mean age at onset was 56.9 ± 8.3 years (range 39–76), and disease duration 7.6 ± 4.6 years (range 1–22). The clinical phenotype of these patients varied between the behavioural variant of frontotemporal dementia (n = 34) and primary progressive aphasia (n = 8), with concomitant amyotrophic lateral sclerosis in seven patients. Predominant temporal atrophy on neuroimaging was present in 13 of 32 patients. Pathological examination of the 10 brains from patients carrying expanded repeats revealed frontotemporal lobar degeneration with neuronal transactive response DNA binding protein-positive inclusions of variable type, size and morphology in all brains. Fluorescence in situ hybridization analysis of brain material from patients with the repeat expansion, a microtubule-associated protein tau or a progranulin mutation, and controls did not show RNA-positive inclusions specific for brains with the GGGGCC repeat expansion. The hexanucleotide repeat expansion in C9orf72 is an important cause of frontotemporal dementia with and without amyotrophic lateral sclerosis, and is sometimes associated with primary progressive aphasia. Neuropathological hallmarks include neuronal and glial inclusions, and dystrophic neurites containing transactive response DNA binding protein. Future studies are needed to explain the wide variation in clinical presentation.

Chapter 3.1

68

INTRODUCTION

Frontotemporal dementia (FTD) is the second most common type of presenile dementia and is characterized by behavioural changes, executive and language dysfunctions due to neurodegeneration of the frontal and temporal cortex1,2. Amyotrophic lateral sclerosis (ALS) is the most common type of motor neuron disease, characterized by rapidly progressive paralysis due to degeneration of upper and lower motor neurons leading to death within a few years3. There is increasing clinical, pathological and genetic evidence for the hypothesis that FTD and ALS are part of a disease continuum. First of all, patients with FTD frequently develop symptoms of motor neuron disease, and cognitive dysfunction is often seen in patients with ALS4. Secondly, the transactive response DNA binding protein of 43 kDa (TDP-43), an RNA binding protein, is the major pathological protein in FTD and ALS, with neuronal and glial TDP-43-positive inclusions in neocortex, basal ganglia and/or spinal cord5,6. Thirdly, the two disorders have been shown to share genetic aetiology, apart from the genetic defects distinctive for each. Microtubule-associated protein tau (MAPT) and progranulin (GRN) mutations are exclusively associated with FTD; the same is true for superoxide dismutase 1 and optineurin mutations in ALS, but fused in sarcoma, valosin-containing protein and TDP mutations are also occasionally found in patients with FTD7-19. Families in which affected members present with FTD, ALS or both have shown significant linkage to chromosome 9p21.320-25. Moreover, genome-wide association studies of both ALS and FTD have shown a significant association with the same chromosomal locus26-28. These findings indicate that this locus has a major genetic contribution to FTD and ALS. The associated risk haplotype appears to be the same for most chromosome 9p-linked families of European ancestry, suggesting a common founder29. In September 2011, we and others simultaneously identified a (GGGGCC)n repeat expansion in a non-coding region of C9orf72 on chromosome 9 in FTD and ALS30,31. Pathogenic expanded repeats were found in 30–50% of cases with familial ALS and FTD, and in 4–10% of sporadic cases30,31. Quantitative messenger RNA analysis has shown that the presence of the expanded repeats leads to reduced expression of one of the transcripts of C9orf72 encoding a protein with an unknown function, suggesting a (partial) loss-of-function disease mechanism30. However, a toxic gain-of-function of abnormal messenger RNA has been hypothesized as well, based on the discovery of multiple nuclear RNA foci in brain tissue from patients carrying the expanded repeats using fluorescence in situ hybridization experiments with a probe targeting the GGGGCC repeat30.

Phenotype of C9orf72 repeat expansions

69

As the clinical and pathological phenotype has been studied in few families with FTD + ALS so far, it is important to investigate the phenotypical variation of the repeat expansion in more detail in a larger cohort. In the present study, we investigated the clinical and pathological characteristics of the GGGGCC hexanucleotide repeat expansion in C9orf72 in a large Dutch cohort of patients with familial and sporadic FTD with and without ALS.

PATIENTS AND METHODS

Patients and controls The Dutch FTD series comprises 458 patients with FTD, ascertained in an on-going genetic–epidemiological study conducted in The Netherlands since 1994, including patients that were referred to Neurology departments of the Erasmus Medical Centre or the VU University Medical Centre, or that were ascertained by research physicians visiting nursing homes and psychogeriatric hospitals. The diagnosis of FTD was based on international consensus criteria32, and concomitant ALS was diagnosed when patients also met El Escorial criteria33. Pathological confirmation of frontotemporal lobar degeneration (FTLD) was obtained in 94 patients34. The study was approved by the Medical Ethical Committee of the Erasmus Medical Centre and VU University Medical Centre. Following receipt of informed consent, DNA samples were obtained from each patient. We excluded all patients with MAPT or GRN mutations (46 and 30 patients, respectively), or with tau-positive FTLD (19 patients). The remaining cohort to be screened for the repeat expansion in C9orf72 consisted of 363 patients with FTD, including 38 patients with concomitant ALS. The mean age at onset was 58.0 ± 8.3 years (range 28–76). The mean age at death in patients that died during follow-up (n = 208) was 66.3 ± 9.7 years (range 35–89), with mean disease duration of 8.2 ± 4.4 years (range 1–23) (Table 1). The most common clinical presentation was the behavioural variant of FTD (n = 262), followed by primary progressive aphasia (PPA) (n = 101). Family history was positive for dementia (n = 130), ALS (n = 25) or Parkinson's disease (n = 19) in at least one first-degree relative in 133 patients from 120 families. In two families, a relative with a pure ALS presentation was also genotyped. Our control group consisted of 564 neurologically normal subjects (269 males and 295 females) from the Longitudinal Aging Study Amsterdam (LASA, http://www.lasa-

Chapter 3.1

70

http://brain.oxfordjournals.org/content/135/3/723.long#T1

vu.nl/). The mean age at clinical examination for this group was 67.8 ± 6.0 years (range 60–81).

Table 1. Demographic features of the Dutch FTD Cohort.

FTD cohort (n=363) Female (%) 178 (49.0) Age at onset, years (range) 58.0±8.3 (28-76) Age at death (n=208), years (range) 66.3±9.7 (35-89) Disease duration (n=208), years (range) 8.2±4.4 (1-23) FTD Subtype bvFTD (%) PPA (%)

262 101

(72.2) (27.8) (10.5)

ALS 38 Family history Positive for dementia (%, no. of families) Positive for ALS (%, no. of families) Positive for PD (%, no. of families) Negative (%)

130 25 19 230

(35,8, 117) (6.9, 17) (5.2 ,16) (63.4)

Neuropathological examination (n= 51) FTLD-TDP FTLD-FUS FTLD-ni FTLD (subtype unknown)

45 4 1 1

(88.2) (7.8) (2.0) (2.0)

FTD, frontotemporal dementia; bvFTD, behavioural variant of FTD; PPA, primary progressive aphasia; ALS, amyotrophic lateral sclerosis; FTLD, frontotemporal lobar degeneration; TDP, transactive response DNA binding protein of 43 kDa; FUS, fused in sarcoma; ni, no inclusions.

Clinical data Detailed clinical history and family history were obtained for all patients by interviewing relatives and collecting data from medical records. We carried out a neurological examination of all patients and, when possible, patients underwent neuropsychological evaluation and neuroimaging (MRI or CT). Neuropsychological evaluation consisted of tests for language (e.g. Boston Naming Test, Semantic Association Test, word fluency), memory (e.g. Rey Auditory Verbal Learning Test, Visual Association Test), attention and concentration and executive functions (e.g. Trail Making Test, Stroop colour-word test, modified Wisconsin Card Sorting Test, Similarities and Proverbs of the Wechsler Adult Intelligence Scale) and visuospatial abilities (e.g. Clock drawing, Block Design of the Wechsler Adult Intelligence Scale). The presence and severity of frontal, temporal, parietal, occipital and cerebellar atrophy were reviewed by a neurologist (J.C.v.S.) and a radiologist (M.S.). Patients with signs suggestive of ALS, such as muscle weakness, atrophy or


71

fasciculations, underwent EMG. The age at onset was defined as the moment partners or other relatives noticed the first symptoms attributable to the disease. Three classes of family history were distinguished: (i) autosomal dominant, patients with at least two first-degree relatives with dementia or ALS; (ii) patients with only a single affected first-degree relative with dementia or ALS; and (iii) patients without affected relatives or an unknown family history.

Genotyping methods For the repeat-primed polymerase chain reaction 50 ng of genomic DNA from each patient, was mixed with FastStart Taq DNA polymerase PCR buffer (Roche Applied Science), 7-deaza-dGTP (New England Biolabs), Q-Solution (Qiagen Inc.), dimethylsulphoxide Hybri-Max (Sigma-Aldrich), MgCl2 (Roche Applied Science), reverse primer consisting of four GGGGCC repeats with an anchor tail, 6FAM™-fluorescent labelled forward primer located 128-bp telomeric to the repeat sequence, and an anchor primer corresponding to the anchor tail of the reverse primer, as described in our previous article31. Primer sequences are available upon request35,36. Fragment length analysis was performed on an ABI 3730xl genetic analyser (Applied Biosystems), and data analysed using Peak Scanner software version 1.0 (Applied Biosystems). Repeat expansions produce a characteristic saw tooth pattern with a 6-bp periodicity (Supplementary Fig. 1). We obtained hexanucleotide repeat lengths based on the repeat-primed polymerase chain reaction assay by successful genotyping in 353 cases and 522 controls (94.4% of the total cohort). To note, the repeat-primed polymerase chain reaction assay used for these experiments does not determine the actual number of repeats in a large pathogenic expansion. This technique only allows for testing whether a given sample carries a large pathogenic expansion or not. A cut-off value of 30 repeats was used to define expanded repeats, as previously described31.

Pathological examination Brain autopsy was carried out within 4 h of death according to the Legal and Ethical Code of Conduct of the Netherlands Brain Bank. Tissue blocks taken from all cortical areas, hippocampus, amygdala, basal ganglia, substantia nigra, pons, medulla oblongata, cerebellum and cervical spinal cord were embedded in paraffin blocks, and underwent routine staining with haematoxylin–eosin, Bodian, methenamine-silver and Congo red. Tissue blocks were taken from the right hemisphere in each case. Immunohistochemistry was performed using primary antibodies against

Chapter 3.1

72

http://brain.oxfordjournals.org/cgi/content/full/awr353/DC1

hyperphosphorylated tau (AT8, Innogenetics; 1:40), ubiquitin (anti-ubiquitin, Dako; 1:500, following 80°C antigen retrieval), β-amyloid protein (anti-beta amyloid, Dako;1:100, following formic acid pretreatment), α-synuclein (anti-α-synuclein, Zymed Laboratories; undiluted, following formic acid pretreatment), p62 (BD Biosciences Pharmingen; 1:200, following 80°C antigen retrieval), TDP-43 (Biotech; 1:100, following pressure cooking), TDP-43 phosphorylated at serine 409/410 (Cosmo Bio; 1:8000), fused in sarcoma (Sigma-Aldrich anti-fused in sarcoma; 1:25–200 with initial overnight incubation at room temperature, following pressure cooking) and C9orf72 (GeneTex; 1:200) and stained as previously described37. Primary antibodies were incubated overnight at 4°C. Endogenous peroxidase activity was inhibited by incubation in phosphate buffered saline–hydrogen peroxide–sodium azide solution (100 ml 0.1 M phosphate-buffered saline + 2 ml 30% H2O2 + 1 ml natriumazide) for 30 min. The Histostain-Plus broad-spectrum kit DAB (Zymed) was used, and slides were counterstained with Mayer's haematoxylin and mounted in Entellan®. The pathological diagnosis was made by a neuropathologist (A.J.M.R.). Brain autopsy performed in 51 out of the total cohort of 363 patients revealed TDP-43-positive pathology (FTLD-TDP) in 45 patients, FTLD with fused in sarcoma-positive pathology in five, FTLD with no inclusions in one and FTLD (subtype unknown) in one. The pattern of FTLD-TDP pathology was classified into the four following subtypes: type A is characterized by numerous short dystrophic neurites and crescentic or oval neuronal cytoplasmic inclusions, concentrated primarily in neocortical layer 2. Moderate numbers of lentiform neuronal intranuclear inclusions are also a common but inconsistent feature of this subtype; type B by moderate numbers of neuronal cytoplasmic inclusions, throughout all cortical layers, but very few dystrophic neurites; type C by a predominance of elongated dystrophic neurites in upper cortical layers, with very few neuronal cytoplasmatic inclusions; and type D by numerous short dystrophic neurites and frequent lentiform neuronal intranuclear inclusions34.

Fluorescence in situ hybridization The hypothesis of a toxic RNA gain-of-function mechanism for FTD/ALS suggests that RNA containing expanded non-coding hexanucleotide repeats accumulates in affected cells. To test this hypothesis, we examined paraffin-embedded sections of post-mortem temporal cortex and hippocampal tissue for the presence of RNA containing GGGGCC repeats using fluorescence in situ hybridization. For RNA-fluorescence in situ hybridization, brain sections were hybridized either with an oligonucleotide probe


73

(GGCCCC)3 5′ TYE563 or a CCCCGGCCCC 5′ TYE563 labelled locked nucleic acid oligonucleotide probe (both Exicon), which differs from the method used by DeJesus-Hernandez et al.30. After the RNA-fluorescence in situ hybridization protocol the slides were incubated with Hoechst stain in phosphate-buffered saline (1:15 000) and washed two times for 5 min with phosphate-buffered saline, followed by one wash in de-ionized water, before mounting in Mowiol®. Slides were examined using a confocal fluorescence microscope (Leica).

Statistical analysis Fisher's exact test was used to test for association between the presence of C9orf72 repeat expansion and both familial and sporadic FTD using the PLINK v1.07 toolset. Since presence of individuals from the same family could bias our association results, only one affected individual was included per family in this analysis. Independent samples t-tests to compare continuous variables between patients with the C9orf72 repeat expansion and patients with MAPT or GRN mutations were performed using SPSS 17.0 for windows (SPSS). A significance level of P < 0.05 was used.

RESULTS

Genotyping results A total of 353 cases with FTD and 522 controls were successfully genotyped with the repeat-primed polymerase chain reaction assay. Histograms of repeat lengths based on the repeat-primed polymerase chain reaction assay are shown for both cases and controls (Supplementary Fig. 2). The average repeat length in the control population was 9.1 ± 6.8 (range 2–35 repeats) and for the cases with FTD, the average repeat length was 13.9 ± 14.0 (range 1–64 repeats). A total of 42 cases from our cohort (11.9%) and three controls (0.6%) carried the expansion (Fisher's test P-value = 4.39 × 10−12; odds ratio = 19.22, 95% confidence interval = 5.89–62.66 after removal of cases belonging to the same family). Thirty-seven patients with FTD with the expansion had a positive family history (28.7% of genotyped patients with FTD with positive family history), with an autosomal dominant mode of inheritance in 25 patients (19 families, four of which are shown in Figure 1), including four families where reduced penetrance was observed (Family 3 in figure 1). Obligate carriers in two of the latter families died after the age of 70 without any signs of dementia or ALS. The remaining 12 patients with a positive family history had only one first-degree family member with either dementia or

Chapter 3.1

74

http://brain.oxfordjournals.org/cgi/content/full/awr353/DC1

http://brain.oxfordjournals.org/content/135/3/723.long#F1

Figu

re 1

. Ped

igre

es fo

r Fam

ilies

I–IV

.

Sub

ject

s th

at w

ere

incl

uded

in th

e se

quen

cing

coh

ort a

re in

dica

ted

with

( +

). M

ND

= m

otor

neu

ron

dise

ase;

FTD

= fr

onto

tem

pora

l dem

entia

.


75

Tabl

e 2.

Clin

ical

and

pat

holo

gica

l fea

ture

s of

FTD

pat

ient

s w

ith re

peat

exp

ansi

on.

No. o

f a

ffect

eds

(co

nfirm

ed

exp

ansio

n)

No. o

f f

amilie

s Ag

e at o

nset

(

rang

e)

Age a

t dea

th

(ra

nge)

Di

seas

e d

urat

ion

(

rang

e)

FTD

subt

ype

(no

. of p

atien

ts)

Imag

ing*

(

no. o

f pat

ients

) Pa

thol

ogy

(no

. of p

atien

ts)

Autos

omal

domi

nant

Fami

ly 1

10 (

3)

1 60

.8 (5

5-67

) 70

.0 (6

4-75

) 10

.0 (9

-12)

bv

FTD

(8),

ALS

(2)

T (1

), FT

(2),

G (1

) na

Fami

ly 2

6 (

2)

1 59

.2 (3

9-80

) 70

.2 (5

9-84

) 7.0

(1-

17)

bvFT

D (4

), bv

FTD+

ALS

(2)

N (1

), T

(1),

FT (1

) FT

LD-T

DP (1

)

Fami

ly 3

4 (

3)

1 54

.1 (4

5-62

) 59

.2 (5

2-64

) 8.0

(6-

9)

bvFT

D (4

) N

(1),

T (2

), G

(1)

FTLD

-TDP

(2)

Fa

mily

4 7

(1)

1

65.0

(64-

67)

69.3

(67-

73)

4.0 (

1-8)

bv

FTD

(5),

ALS

(1),

F

TD+A

LS (1

) T

(1)

na

Re

maini

ng au

tosom

al

d

omina

nt ca

ses

18 (

18)

15

57.1

(39-

67)

65.4

(46-

75)

7.2

(1-1

8)

bvFT

D (1

3), P

PA (1

),

ALS

(2),

bvFT

D+AL

S (1

), P

PA+A

LS (1

)

F (1

), T

(2),

FT (5

) N

(1),

G (1

) FT

LD-T

DP (6

)

Ca

ses w

ith on

ly on

e

affe

cted f

amily

mem

ber

12 (

12)

11

54.2

(39-

76)

62.7

(42-

78)

8.2

(2-2

2)

bvFT

D (6

), PP

A (3

),

bvF

TD+A

LS (3

) N

(2),

F (2

), T

(2),

F

T (1

), G

(1)

FTLD

-TDP

(2)

No fa

mily

histor

y 5

(5)

5

61.3

(55-

70)

67.9

(64-

72)

9.1 (

6-11

) bv

FTD

(2),

PPA

(2)

bvF

TD+A

LS (1

) T

(4),

F (1

) FT

LD-T

DP (2

)

bvFT

D, b

ehav

iour

al v

aria

nt o

f fro

ntot

empo

ral d

emen

tia; A

LS, a

myo

troph

ic la

tera

l scl

eros

is; P

PA

, prim

ary

prog

ress

ive

apha

sia;

FTL

D-T

DP

, fro

ntot

empo

ral l

obar

deg

ener

atio

n w

ith

TDP

43-p

ositi

ve in

clus

ions

; N, n

o at

roph

y; F

, fro

ntal

; T, t

empo

ral;

FT, f

ront

otem

pora

l; G

, gen

eral

ized

. na,

not

ava

ilabl

e *A

rea

of m

ost p

rom

inen

t atro

phy

is in

dica

ted.

Chapter 3.1

76

ALS (11 families) (Table 2). Fisher association analysis of familial FTD cases versus controls (individuals belonging to the same family were removed for this analysis) gave the following results: Fisher's test P-value = 1.76 × 10−20, odds ratio = 51.47, 95% confidence interval = 15.95–169.90. There was wide phenotypic variability within families with diagnoses of both FTD, ALS and FTD + ALS in 12 families. Furthermore three families included individuals with Parkinson's disease, however, we cannot be certain that this is also caused by the repeat expansion, since these individuals were not genotyped. In one family with autosomal dominant FTD + ALS the proband included in our clinical FTD cohort had a repeat length of 26 and was therefore assumed not to carry the repeat expansion. However, sequencing in family members revealed the repeat expansion in a so far unaffected person (age 42 years) and repeat length varying from 8 to 29 in affected persons. Therefore, we are uncertain about the pathogenicity of the repeats in this family, especially as it is not yet possible to determine exact repeat lengths. Moreover, in three of the families with the repeat expansion, there was one affected individual with a repeat length of 29. The repeat expansion in C9orf72 was found in 5 of the 224 genotyped patients with sporadic FTD (2.2%), which was not significantly different from healthy controls (Fisher's test P = 0.0569, odds ratio = 3.93, 95% confidence interval = 0.93–16.53). Of the remaining 311 patients in the cohort without the repeat expansion, 92 had a positive family history for dementia (89 families), with ALS in six families. Moreover, 31 of the patients with FTD without the repeat expansion had concomitant ALS. In an effort to investigate whether the expansion carriers in our cohort carry the recently identified risk haplotype on chromosome 9 (Mok et al., 2011), genotyping data from a parallel project in our laboratory was extracted for 20 of the 42 expansion carriers (Figure 2). Genotypes of 12 of these samples (60.0%) were concordant with the reported risk haplotype. Interestingly, all other samples shared the same core risk haplotype, differing from it only in the most distal positions. These results suggest that all Dutch C9orf72 mutation carriers derive from a common mutated ancestor.

Clinical features The mean age at onset in the 42 patients with FTD with the repeat expansion was 56.9 ± 8.3 years (range 39–76), mean age at death (n = 31) was 64.7 ± 8.6 years (range 42–78) and mean disease duration from onset till death was 7.6 ± 4.6 years (range 1–22). Behavioural variant FTD was the initial clinical presentation in 34 patients (apathy in 18, disinhibition in 11, obsessive–compulsive behaviour in five), and PPA in eight


77


http://brain.oxfordjournals.org/content/135/3/723.long#ref-29


Fig

ure

2. G

enot

ypin

g da

ta fo

r 20

expa

nsio

n ca

rrier

s fo

r whi

ch g

enom

e-w

ide

asso

ciat

ion

data

wer

e av

aila

ble.

Thos

e ge

noty

pes

that

are

con

cord

ant

with

the

alle

le i

n th

e ris

k ha

plot

ype

acco

rdin

g to

Mok

et

al.

(201

1) a

re s

hade

d in

gre

y. I

ndiv

idua

ls 0

6D03

315,

08D

0145

7, D

98.8

895,

D

9529

93 a

nd 0

7D01

783

did

not p

rese

nt w

ith f

amily

his

tory

of

FTD

or

ALS

. Fr

eq C

EU

= F

requ

ency

in C

EU

pop

ulat

ion

(Cep

h E

urop

eans

from

Uta

h);

SN

P =

sin

gle

nucl

eotid

e po

lym

orph

ism

.

Chapter 3.1

78

patients (Figure 3). Concomitant ALS was present in seven patients (bulbar onset in five, limb onset in two). Furthermore, we found the repeat expansion in two relatives of patients with FTD with pure limb onset ALS. Mean score at the Mini-Mental State Examination was 25.9 ± 3.4 (n = 19, range 17–30). Memory complaints were reported in 21 patients at clinical presentation. Six patients showed signs of Parkinsonism and apraxia was present in seven patients. Visual or auditory hallucinations were reported in two patients and delusions in none. Mean duration of follow-up from disease onset in patients with the repeat expansion was 5.2 ± 3.3 years (range 0.8–13.4).

Figure 3. Clinical features of C9orf72 expansion carriers.

(A–D) display C9orf72 expansion distribution depending on the clinical phenotype (behavioural variant FTD or PPA) and family history for FTD or ALS. (E) shows the clinical distribution of all 42 expansion carriers.

Of the eight patients with PPA, two showed fluent speech, anomia and single-word comprehension deficits at neuropsychological evaluation compatible with the diagnosis semantic dementia, supported by atrophy of the anterior temporal lobes (Figure 4).

Sporadic bvFTD (n=155)

expansionno expansion





3%




Sporadic bvFTD

Familial bvFTD

Sporadic PPA

Familial PPA

7% 14%

5%

2%

31%

21%

74%

A B

D C

E


79



Classification into one of the PPA variants was not possible in the remaining six patients due to lack of information. Four of them had fluent speech, anomia and impaired language comprehension according to their history, but extensive neuropsychological evaluation was not available or possible at the time of out-clinic visits. The other two had non-fluent speech with anomia and comprehension deficits in one. All patients with behavioural variant FTD who underwent extensive neuropsychological evaluation had executive dysfunctions, and 10 of them had severe language deficits on initial presentation. Neuroimaging was available for 32 patients with the repeat expansion. The pattern of cerebral atrophy was predominantly anterior temporal in 13 patients, frontal in four and frontotemporal in seven patients. In all patients with PPA, atrophy was most prominent in the temporal cortex. In four patients, the atrophy was generalized, and four patients (including a patient with pure ALS) had no atrophy. Atrophy was extended into the parietal cortex in 10 patients, and into the occipital cortex in one. Mild cerebellar atrophy was found in eight patients.

Figure 4. MRI scan of a patient with semantic dementia showing severe temporal atrophy.

L = left.

Neuropathological findings Brain autopsy was carried out by The Netherlands Brain Bank in 10 patients carrying the pathogenic repeat expansion and in the patient with a repeat length of 26 with an unaffected family member with the expansion (Table 3). The brain weight was reduced (mean 1112 g, range 886–1297). Macroscopy showed moderate to severe frontal and

Chapter 3.1

80


temporal atrophy in all except one brain. Hypopigmentation of the substantia nigra was found in three brains. Routine staining showed variable neuronal loss in the frontal and/or temporal cortex in all, except for two cases with FTD + ALS. In the PPA case, atrophy and neuronal loss was most severe in the temporal cortex. Mild neuronal loss in the substantia nigra was seen in seven cases and in the caudate nucleus and putamen in two. Immunohistochemistry with ubiquitin, p62 and TDP-43 antibodies revealed TDP-43 type B pathology in all brains34. Many neuronal cytoplasmatic inclusions of variable size (round, crescent, granular) and morphology (diffuse, dense) were seen, most abundant in the dentate gyrus of the hippocampus (Figure 5A), in superficial and deeper layers of the temporal, frontal and parietal cortex (Figure 5B), and with less density in the basal ganglia. Irregular-shaped aggregates were seen in many pyramidal cells of cornu ammonis 3 and 4. Many short, thin or swollen dystrophic neurites were seen in cortical areas in most cases, with the presence of long dystrophic neurites in the parietal cortex of three brains, and in the temporal cortex in only the PPA case (Figure 5C). Three brains showed a variable number of neuronal intranuclear inclusions in neocortex or basal ganglia (Figure 5D). A few irregular shaped or skein-like TDP-43- and p62-positive inclusions were found in the substantia nigra (Figure 5E) and brainstem (Figure 5F) of four brains. Small dense neuronal p62-positive inclusions and short neurites in the granular layer of the cerebellum were seen in 9 out of 11 brains (Figure 5G). Some p62- and TDP-43-positive glial inclusions (oligodendroglia like) were found in the subcortical white matter in a number of brains (Figure 5H). These glial inclusions did not stain with ubiquitin antibody. In one brain, many p62-, and TDP-43-positive glial inclusions of astrocytic nature were seen in the parietal and temporal cortex and the neostriatum (Figure 5I). TDP pathology was not more abundant in areas of atrophy. The p62 staining of inclusions was more intense than TDP-43 staining in all cases. Immunohistochemistry with AT8 and β-amyloid antibodies showed abundant neurofibrillary tangles and β-amyloid plaques in the temporal cortex in two brains (Braak stage 2C)38. Corticospinal tract degeneration was found in three brains. Immunohistochemistry with C9orf72 antibody shows that C9orf72 is a largely cytoplasmatic protein in neurons. Immunostaining with the antibody against C9orf72 protein showed a granular staining of the cytoplasm into the dendritic arborisations of neurons in cornu ammonis 3 and 4, but this was observed in FTD both with and without the repeat expansion.


81










Tabl

e 3.

Neu

ropa

thol

ogic

al fi

ndin

gs in

pat

ient

s w

ith a

repe

at e

xpan

sion

in C

9orf7

2.

Case

1 (F

amily

3 III:

5)

2 (Fam

ily 3

III:7)

3

4 5

6 7

8 9

10

11¶

Clini

cal

pres

entat

ion

bvFT

D bv

FTD

bvFT

D bv

FTD+

ALS

bvFT

D bv

FTD

PPA

bvFT

D bv

FTD

bvFT

D+AL

S bv

FTD+

ALS

Brain

weig

ht 10

65

1297

12

20

1096

10

95

1150

12

45

1146

88

6 95

8 10

76

Atro

phy

F, T

, P, S

N

F, T

, F,

T, S

N No

rmal

F, T

, P

F, T

, SN

T F,

T, P

F,

T

F, T

, P

F Ne

uron

al los

s F

ronta

l +

- -

- +

+ -

+ +

+ -

Tem

pora

l ++

++

++

+

++

++

++

++

+ +

++

Spin

al co

rd

- -

- +

- -

+ -

na

na

++

SN

+ -

+ ++

+

+ -

- -

+ ++

IH

C: T

DP43

/p62

Fro

ntal

++

++

++‡

+§ +

+ -

++

+ -

+ T

empo

ral

++

++

++

+ +

++

+† ++

+

++

++

Par

ietal

+†‡

+† +

++§

+ ++

+†

+ +

+ +

Hipp

ocam

pus

+ ++

++

+

++

++

++

++

++

++

++‡

Bas

al ga

nglia

+

+ ++

‡ +§

+ na

+

++

- -

+‡ C

ereb

ellum

++

++

+

++§

+ na

-

+ ++

-

++

Bra

instem

+

- na

+

+ -

- -

+ +

- S

pinal

cord

na

na

na

+

na

- na

-

na

na

+ IH

C, i

mm

unoh

isto

chem

istry

with

p62

and

/or T

DP

43 a

ntib

odie

s; F

, fro

ntal

; T, t

empo

ral;

P, p

arie

tal;

H, h

ippo

cam

pus;

SN

, sub

stan

tia n

igra

; -, n

one;

+, m

ild, +

+, m

oder

ate;

+++

, se

vere

; na,

not

ava

ilabl

e;

*p62

- or T

DP

43-p

ostiv

e ne

uron

al c

ytop

lasm

atic

incl

usio

ns a

nd/o

r dys

troph

ic n

eurit

es† Lo

ng d

ystro

phic

neu

rites

‡ Neu

rona

l int

ranu

clea

r inc

lusi

ons

§ Glia

l inc

lusi

ons

¶ The

repe

at e

xpan

sion

was

not

con

firm

ed in

this

cas

e, b

ut re

peat

leng

th w

as 2

6, a

nd a

so

far u

naffe

cted

rela

tive

did

carr

y th

e re

peat

exp

ansi

on.

Chapter 3.1

82

Of another five brains from patients with the pathogenic repeat expansion from other academic centres, the pathological diagnosis was FTLD with ubiquitin pathology. However, brain tissue was not available for extensive assessment using TDP-43 and p62 antibodies.

Figure 5. Immunohistochemistry with p62 and TDP-43 antibodies in brains of patients carrying the GGGGCC repeat expansion in C9orf72.

Many dense neuronal cytoplasmatic inclusions were present in the granular cells of the dentate gyrus (A). Dense or granular cytoplasmatic inclusions of variable size, and short dystrophic neurites were visible in the deep and superficial layers of the frontal and temporal cortex (B). Long dystrophic neurites were seen in the cortical areas of a few brains (C). The temporal and parietal cortex showed a number of TDP-43-positive neuronal intranuclear inclusions (D). Some skein-like or filamentous TDP-43-positive inclusions were found in neurons of the substantia nigra (E) and lower motor neurons in the brainstem (F). Abundant small cytoplasmatic p62-positive, TDP-43-negative inclusions and short neurites were seen in the granular layer of the cerebellum (G). Abundant p62-positive cytoplasmatic glial inclusions were present in white matter of the striatum (H). Several p62-positive inclusions in the cytoplasm and dendritic processes of glial (probably astrocytic) cells in the temporal cortex of one brain from a patient with FTD + ALS (I), TDP-43 antibody also has a positive, although weaker, staining of these inclusions (inset).

Fluorescence in situ hybridization The RNA-fluorescence in situ hybridization results with the locked nucleic acid probe were inconsistent, that is, we analysed post-mortem tissue of three patients with the expanded GGGGCC repeat, three patients with a GRN mutation, three patients with


83

MAPT mutations, one patient with fragile X-associated tremor/ataxia syndrome (CGG98 repeat expansion) and three non-demented controls for the presence of RNA foci. With the locked nucleic acid probe we did find RNA-positive inclusions in brains with GGGGCC repeat expansions, but also in three cases with MAPT mutations and in a non-demented control. The specificity of the staining observed with the locked nucleic acid probe remains to be determined. DeJesus-Hernandez et al. did not test cases with MAPT mutations30. Using the oligonucleotide probe (GGCCCC)3 5′ TYE563, no RNA foci could be detected in any of the samples studied.

Comparison with MAPT and GRN mutation carriers Whereas the MAPT and GRN mutation carriers in the Dutch cohort came from 12 and 6 large families, respectively; the carriers of the repeat expansions in C9orf72 came from 35 apparently unrelated families. There was no significant difference in age at onset, age at death or disease duration between repeat expansion carriers and MAPT or GRN mutation carriers (Table 4). The C9orf72 repeat expansion is associated with a wider phenotypic variability than MAPT and GRN mutations. In contrast to patients with MAPT and GRN mutations, concomitant ALS was a frequent finding in patients with the C9orf72 repeat expansion. The frequent finding of predominant temporal atrophy is in contrast to the imaging features in MAPT and GRN mutations, where predominant frontal atrophy was more common.

Table 4. Clinical features in FTD patients with a repeat expansion in C9orf72 compared to patients with MAPT and GRN mutations.

Repeat expansion in C9orf72 (n=42)

MAPT mutation (n=46)

GRN mutation (n=30)

No. of families 35 12 6 Female (%) 22 (52.4) 24 (52.2) 19 (63.3) Age at onset, years (range) 56.9±8.3 (39-76) 52.3±6.0 (39-65) 60.6±9.3 (45-79) Age at death, years (range) 64.7±8.6 (42-78) 61.2±7.7 (44-76) 69.4±10.2 (52-87) Duration of illness, years (range) 7.6±4.6 ( 1-22) 9.2±4.5 ( 3-20) 7.4±2.8 ( 2-13) Clinical subtype BvFTD (%) PPA (%)

34 (81.0) 8 (19.0)

45 (97.8) 1 ( 2.2)

28 (93.3) 2 ( 6.7)

ALS (%) 7 (16.7) 0 0 Imaging* No atrophy (%) Frontal (%) Temporal (%) Frontotemporal (%) Generalized (%)

3 ( 9.7) 4 (12.9) 13 (41.9) 7 (22.6) 4 (12.9)

0 23 (53.5) 15 (34.9) 4 (9.3) 1 (2.3)

0 23 (85.2) 1 (3.7) 1 (3.7) 2 (7.4)

bvFTD, behavioural variant of frontotemporal dementia; ALS, amyotrophic lateral sclerosis; PPA, primary progressive aphasia; MAPT, microtubule-associated protein tau; GRN, progranulin. *Area of predominant atrophy.

Chapter 3.1

84


DISCUSSION

The present study shows that the pathogenic hexanucleotide expansion in C9orf72 is one of the most common genetic causes of familial FTD in The Netherlands, and that the repeat expansion is associated with a wide variation in clinical phenotype (behavioural variant FTD, PPA, ALS) and with predominant temporal atrophy on neuroimaging. The percentage of the repeat expansion in the present series of familial FTD is higher than in the study by DeJesus-Hernandez et al. (28.7% versus 11.7%)30, which can be explained by the exclusion of patients with MAPT and GRN mutations in this study30. In this regard, if the whole population of screened patients with FTD and patients with MAPT and GRN mutations or tau-positive FTLD (n = 448) is accounted for, the C9orf72 hexanucleotide expansion accounts for 17.8% of familial and 9.4% of total FTD in The Netherlands. Of the total population of patients with FTD in The Netherlands, 26.3% is explained by mutations in GRN (6.7%), MAPT (10.3%) and the hexanucleotide repeat expansion in C9orf72 (9.4%). Considering only familial FTD, we can now explain up to 53.8% cases in The Netherlands (GRN: 13.9%, MAPT: 22.1% and C9orf72: 17.8%). The frequency of the repeat expansion in the present series of sporadic FTD is in line with the findings in the study by DeJesus-Hernandez et al.30. Although a larger proportion of sporadic FTD cases carry the expansion in comparison to controls, this difference was not statistically significant (P = 0.0569). A larger cohort of sporadic patients has to be screened to unveil the role of this expansion in the sporadic form of the disease. Genetic analysis of a subset of cases carrying this expansion showed that patients from apparently unrelated families carry the same risk haplotype, indicating that there is a common ancestor for all patients with expanded alleles of C9orf72. As this risk haplotype was also found in the five apparently sporadic cases, this indicates that these cases are in fact, cryptically related familial cases. The negative family history for these patients could be explained by early death of affected family members, non-paternity or a lack of medical information in previous generations. Another possible explanation of the occurrence of the repeat expansion in apparently sporadic cases in this and other studies, could be reduced penetrance of the repeat expansion in C9orf7230,31. This reduced penetrance is evident in at least two of the families in which unaffected obligate carriers lived long enough to develop the disease, although the possibility of non-paternity cannot be ruled out. The reduced penetrance is in line with previous reports on


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chromosome 9p-linked FTD, in which the repeat expansion has yet to be confirmed21. Another explanation may be that additional genetic and/or environmental factors may determine the wide inter- and intra-familial variation in age at onset and clinical presentation in the present and other chromosome 9p-linked families22-25. If reliable measurement of the exact length of the expanded repeats becomes possible in the future, further studies are needed to investigate the correlation between age at onset and repeat expansion length, and the possibility of anticipation, as observed in other repeat expansion disorders. The observed clinical heterogeneity within families, including behavioural variant FTD, PPA, ALS and Parkinsonism in the present study, is in line with previous observations in chromosome 9p-linked families. Concomitant ALS was a frequent finding (seven patients) in patients with FTD with the repeat expansion, and this frequency may even be an underestimation, since follow-up duration from disease onset was highly variable. PPA, defined as a prominent, isolated language deficit during the initial phase39, also frequently occurred in the present series (eight patients) and in a Finnish series of patients with FTD31, but not in previously reported chromosome 9p-linked families20,22,23. An association between PPA and ALS is further supported by a high frequency of a language-dominant presentation in patients with FTD + ALS40, which suggests a common cortical degenerative process for the language abnormalities in PPA, and tongue and bulbar muscle weakness in ALS. Two of the present patients with PPA with the pathogenic repeat expansion, without ALS symptoms, were classified as semantic dementia, which was supported by the presence of severe anomia and single-word comprehension deficits, and by atrophy of the anterior temporal lobes on neuroimaging39. The occurrence of semantic dementia was unexpected, as the association of semantic dementia and ALS has only been described in a few cases in a recent report40,41. Furthermore, patients with semantic dementia usually have a negative family history, which diminishes the likelihood of a genetic factor with a dominant effect in semantic dementia14,42,43. Therefore, further clinical studies of patients with the repeat expansion are needed to confirm our observation and to elucidate the genetic contribution in semantic dementia. In contrast to a study by Lillo et al.44 that reported that psychotic symptoms are a common feature in FTD + ALS, hallucinations were reported in only two patients with the repeat expansion in the present series and delusions in none. Predominant temporal atrophy on neuroimaging is a frequent (40.6%) finding in the present series of patients carrying the repeat expansion, especially in those with PPA. This clearly contrasts with the frontal or frontotemporal pattern of atrophy in

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chromosome 9p-linked families, and with the absence of a specific atrophy pattern for FTD + ALS in a correlative voxel-based morphometry study23-25,45. However, Coon et al.40 also found a trend towards more temporal atrophy in patients with language-dominant FTD + ALS than in those with behavioural-dominant FTD + ALS, and severe and circumscribed atrophy of the anterior temporal lobes has also been described in a case report of FTD + ALS46. Therefore, a voxel-based morphometry study in a large series of patients carrying the pathogenic repeat expansion is required to confirm our observation of temporal involvement. The neuropathological findings were consistent with type B FTLD-TDP pathology, with characteristic ubiquitin- and p62-pathology in the granular layer of the cerebellar cortex, in all except for two brains. This cerebellar pathology has been found in other families with FTD + ALS with the pathogenic repeat expansion24,30,31,47, and in a series of patients with FTD + ALS or FTLD-TDP48. Our observation confirms its strong association with the pathogenic repeat expansion, but it is not an absolute requisite for this disorder. Whether the cerebellar pathology is pathognomic for the repeat expansion has to be investigated in future studies. The TDP-43-negative staining probably indicates that the involvement of the TDP-43 protein is more downstream in the formation of these inclusions, whereas the p62 protein as a non-specific protein reflects the degradation of ubiquitinated proteins via the ubiquitin proteasome system48. In contrast to ubiquitin staining of neuronal inclusions, TDP-43- and p62-positive glial inclusions in the subcortical white matter found in several of the present brains did not stain with ubiquitin antibody, which is in accordance with the observations in other studies49,50. TDP-43- and p62-positive astrocytic inclusions in a single brain from our youngest patient with FTD + ALS were ubiquitin negative as well, as also mentioned by Zhang et al50. Perhaps, these lesions merely reflect that glial abnormalities are associated with a faster progression of the same disease process, instead of a different underlying pathophysiology. Gliebus et al.51 reported asymmetric TDP pathology in a PPA case, we could not confirm this in our patient with PPA, as only the right hemisphere was available for immunohistochemistry. Neuronal intranuclear inclusions were found in several of our patients with the pathogenic repeat expansion, as has previously been found in familial FTD + ALS37,52. The prediction of Bigio et al.52 that these inclusions might be associated with a repeat expansion disorder has been confirmed. Using a different method, we could not confirm the results from DeJesus-Hernandez et al.30 who demonstrated the specific presence of RNA-positive foci in the nucleus of frontal cortex neurons using a (GGCCCC)4 Cy3-labelled oligonucleotide probe. Nevertheless, the nuclear localization of the disease


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process was already suggested by the presence of TDP-43-positive intranuclear inclusions in a few of the present brains, and the absence of cytoplasmic abnormalities of C9orf72 protein in brain carrying the pathogenic repeat expansions. However, it is known from other non-coding expanded repeat disorders that repeat expansions in the transcripts may result in cellular toxicity and the formation of RNA foci of distinct morphology and size53. The mutant messenger RNA may interact with specific RNA-binding proteins, and sequestration of RNA-splicing factors and RNA binding proteins may lead to disruption of nuclear processes, including transcription, splicing or messenger RNA processing54. Another possible pathogenic mechanism underlying disease is loss-of-function, which is supported by reduced expression in one of the three transcripts of C9orf72, as demonstrated in the study of DeJesus-Hernandez et al.30. However, their findings have to be confirmed in future studies. Further studies to clarify these mechanisms may hopefully provide pharmacological target for preventing or delaying the disease. A few limitations of this study have to be addressed. First of all, our observations on the frequency and phenotype of the repeat expansion are confined to FTD, as patients with ALS were not included in this study. Secondly, the finding of predominant temporal atrophy in a subset of patients carrying the repeat expansion was semi-quantitatively assessed, and should be confirmed by voxel-based morphometry in another cohort. In conclusion, the hexanucleotide repeat expansion in C9orf72 is an important genetic cause of FTD and FTD + ALS. It will be a challenge to explain the wide variation in clinical phenotype of this genetic defect, including behavioural variant FTD, ALS and PPA. In addition, it would be interesting to determine whether the severe glial involvement upon the uniform TDP-43 pathology found in some cases has a pathophysiological significance or is just an epiphenomenon. Hopefully, revealing these underlying mechanisms of the repeat expansions will lead to the development of therapeutic interventions for this devastating disease.

ACKNOWLEDGEMENTS

The authors would like to thank Michiel Kooreman, Paul Evers and Afra van den Berg of the Netherlands Brain Bank and Anne Broekema for technical assistance. The authors would like to thank Guido Breedveld for technical support in sequencing C9orf72. The authors would like to thank Tom de Vries Lentsch for excellent

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photography, and Frans Verhey and Catharina Faber for their clinical evaluation of patients. This work was supported by Stichting Dioraphte Foundation (11 02 03 00); Nuts Ohra Foundation (0801-69), Hersenstichting Nederland (BG 2010-02). Research of the VU University Medical Centre’s Alzheimer Centre is part of the neurodegeneration research program of the Neuroscience Campus Amsterdam. The Alzheimer Centre is supported by Alzheimer Nederland and Stichting VUmc fonds. The clinical database structure was developed with funding from Stichting Dioraphte.


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24. Boxer, AL, Mackenzie, IR, Boeve, BF, et al. Clinical, neuroimaging and neuropathological features ofa new chromosome 9p-linked FTD-ALS family. J Neurol Neurosurg Psychiatry. 2011; 82:196-203.

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31. Renton, AE, Majounie, E, Waite, A, et al. A Hexanucleotide Repeat Expansion in C9ORF72 Is theCause of Chromosome 9p21-Linked ALS-FTD. Neuron. 2011; 72:257-268.


33. Brooks, BR. El Escorial World Federation of Neurology criteria for the diagnosis of amyotrophiclateral sclerosis. Subcommittee on Motor Neuron Diseases/Amyotrophic Lateral Sclerosis of the WorldFederation of Neurology Research Group on Neuromuscular Diseases and the El Escorial "Clinicallimits of amyotrophic lateral sclerosis" workshop contributors. J Neurol Sci. 1994; 124 Suppl:96-107.

34. Mackenzie, IRA, Neumann, M, Baborie, A, et al. A harmonized classification system for FTLD-TDP pathology. Acta Neuropathol. 2011; 122:111-113.

35. Warner, JP, Barron, LH, Goudie, D, et al. A general method for the detection of large CAG repeatexpansions by fluorescent PCR. J Med Genet. 1996; 33:1022-1026.

36. Kobayashi, H, Abe, K, Matsuura, T, et al. Expansion of intronic GGCCTG hexanucleotide repeat inNOP56 causes SCA36, a type of spinocerebellar ataxia accompanied by motor neuron involvement.Am J Hum Genet. 2011; 89:121-130.

37. Seelaar, H, Schelhaas, HJ, Azmani, A, et al. TDP-43 pathology in familial frontotemporal dementiaand motor neuron disease without Progranulin mutations. Brain. 2007; 130:1375-1385.

38. Thal, DR, Rub, U, Orantes, M, et al. Phases of A beta-deposition in the human brain and its relevancefor the development of AD. Neurology. 2002; 58:1791-1800.

39. Gorno-Tempini, ML, Hillis, AE, Weintraub, S, et al. Classification of primary progressive aphasia andits variants. Neurology. 2011; 76:1006-1014.

40. Coon, EA, Sorenson, EJ, Whitwell, JL, et al. Predicting survival in frontotemporal dementia withmotor neuron disease. Neurology. 2011; 76:1886-1892.

41. Kim, SH, Seo, SW, Go, SM, et al. Semantic dementia combined with motor neuron disease. J ClinNeurosci. 2009; 16:1683-1685.

42. Goldman, JS, Farmer, JM, Wood, EM, et al. Comparison of family histories in FTLD subtypes andrelated tauopathies. Neurology. 2005; 65:1817-1819.

43. Hodges, JR, Mitchell, J, Dawson, K, et al. Semantic dementia: demography, familial factors andsurvival in a consecutive series of 100 cases. Brain. 2010; 133:300-306.

44. Lillo, P, Garcin, B, Hornberger, M, et al. Neurobehavioral features in frontotemporal dementia withamyotrophic lateral sclerosis. Arch Neurol. 2010; 67:826-830.

45. Rohrer, JD, Geser, F, Zhou, J, et al. TDP-43 subtypes are associated with distinct atrophy patterns infrontotemporal dementia. Neurology. 2010; 75:2204-2211.

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48. King, A, Maekawa, S, Bodi, I, et al. Ubiquitinated, p62 immunopositive cerebellar cortical neuronalinclusions are evident across the spectrum of TDP-43 proteinopathies but are only rarely additionallyimmunopositive for phosphorylation-dependent TDP-43. Neuropathology. 2011; 31:239-249.

49. Hiji, M, Takahashi, T, Fukuba, H, et al. White matter lesions in the brain with frontotemporal lobardegeneration with motor neuron disease: TDP-43-immunopositive inclusions co-localize with p62, butnot ubiquitin. Acta Neuropathol. 2008; 116:183-191.

50. Zhang, H, Tan, CF, Mori, F, et al. TDP-43-immunoreactive neuronal and glial inclusions in theneostriatum in amyotrophic lateral sclerosis with and without dementia. Acta Neuropathol. 2008;115:115-122.

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SUPPLEMENTAL MATERIAL

Supplemental material available on publication (https://doi.org/10.1093/brain/awr353) and on request.

Table of contents Supplemental Figure 1: Capillary-based sequence traces of the repeat-primed PCR

assay. Supplemental Figure 2. Frequency distribution of GGGGCC hexanucleotide repeat

lengths in FTLD cases and control based on the repeat-primed PCR assay.


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Chapter 3.2 Mutation frequency of PRKAR1B and the major familial dementia genes in a Dutch early onset dementia cohort Petra E. Cohn-Hokke, Tsz Wong, Patrizia Rizzu, Guido Breedveld, Wiesje M. van der Flier, Philip Scheltens, Frank Baas, Peter Heutink, Hanne Meijers-Heijboer, John C. van Swieten, Yolande A.L. Pijnenburg J Neurol 2014: 261:2085–2092

ABSTRACT Genetic factors are important in all forms of dementia, especially in early onset dementia. The frequency of major gene defects in dementia has not been investigated in the Netherlands. Furthermore, whether the recently in a FTD family identified PRKAR1B gene is associated with an Alzheimer’s disease (AD) like phenotype, has not been studied. With this study, we aimed to investigate the mutation frequency of the major AD and FTD genes and the PRKAR1B gene in a well-defined Dutch cohort of patients with early onset dementia. Mutation analysis of the genes PSEN1, APP, MAPT, GRN, C9orf72 and PRKAR1B was performed on DNA of 229 patients with the clinical diagnosis AD and 74 patients with the clinical diagnosis FTD below the age of 70 years. PSEN1 and APP mutations were found in, respectively 3.5 and 0.4 % of AD patients, and none in FTD patients. C9orf72 repeat expansions were present in 0.4 % of AD and in 9.9 % of FTD patients, whereas MAPT and GRN mutations both were present in 0.4 % in AD patients, and in 1.4 % resp. 2.7 % in FTD patients. We did not find any pathogenic mutations in the PRKAR1B gene. PSEN1 mutations are the most common genetic cause in Dutch AD patients, whereas MAPT and GRN mutations were found in less than 5 percent. C9orf72 repeat expansions were the most common genetic defect in FTD patients. No pathogenic PRKAR1B mutations were found in the early onset AD and FTD patients of our study.

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INTRODUCTION Dementia is a major health problem with a world-wide prevalence of 44 million in 2013, estimated to increase to 135 million in 20501. Only a minority of the patients with dementia have an early onset, with a prevalence of dementia of 1–10 in 10,000 in persons aged 45–65 years2. Early onset dementia is usually defined as dementia with an onset before the age of 65 years, with Alzheimer’s disease (AD) and frontotemporal dementia (FTD) as most common subtypes 2,3. Genetic factors play an important role in all types of dementia, especially in early onset dementia. An autosomal dominant family history is found in 10–20 % of the patients with early onset dementia and can be explained by mutations in the presenilin 1 and 2 (PSEN1 and PSEN2) and amyloid-beta-protein precursor (APP) genes in up to 50 % of the early onset AD families, and in the microtubule-associated-protein-tau (MAPT) gene, (pro) granulin (GRN) gene and C9orf72 gene in most FTD families4. The frequency of the latter gene defects in Dutch FTD patients has been reported in previous studies5,6, which also showed a wide variation in the clinical presentation in mutation carriers, including an AD-like presentation. In contrast, mutation screening has not been carried out in Dutch early onset AD patients so far. Recently, a mutation in the PRKAR1B gene has been identified in a Dutch family with an autosomal dominant FTD-like presentation. Mutations in this gene are probably a rare cause of FTD, as no pathogenic variants were found in other patients with familial FTD7. Its phenotype encompasses behavioural and cognitive changes with mild parkinsonism. However, no pathogenic mutations were found in a cohort of familial Parkinson patients7. Whether mutations in the PRKAR1B gene may result in an AD-like phenotype, as is the case for GRN and some specific MAPT mutations, has not been studied. With this study, we aimed to estimate the mutation frequency of the most common autosomal dominant AD and FTD genes in Dutch early onset AD and FTD patients, and to investigate whether mutations in the newly identified PRKAR1B gene are a frequent cause of early onset AD.

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PATIENTS AND METHODS Subjects We included all patients with a clinical diagnosis FTD (n = 74) or AD (n = 229) below the age of 70 years from the memory clinic-based Amsterdam Dementia Cohort who visited the Alzheimer centre of the VU University Medical Centre in Amsterdam between October 2000 and November 2008 of whom DNA was available for research purposes. All patients were evaluated following a standard protocol including neurological examination, neuropsychological testing, biochemical analysis of blood, neuroimaging, and EEG. The clinical diagnosis was made by consensus in a multidisciplinary team. The diagnosis “possible” or “probable” AD was made according to the NINCDS-ADRDA criteria8, FTD according to the criteria of Neary et al.9. In all subjects with possible or probable AD, cerebrospinal fluid (CSF) was collected for scientific purposes, however, the CSF results were not included in the clinical decision making. Our laboratory applies reference values of Aβ1-42 ≤ 550 pg/ml and t-tau > 375 pg/ml10. The age at onset was defined as the age at which the first symptom, compatible with cognitive decline or with the diagnosis FTD, was observed by the spouse or a close relative. Family history was obtained from the medical records and considered positive in case of at least one 1st degree with dementia or, in FTD patients, with dementia or amyotrophic lateral sclerosis (ALS). Autosomal dominant inheritance was defined as the occurrence of at least three affected persons in at least two generations of one family. The characteristics of the patients can be found in Table 1. Of the patients with FTD, 68 were also included in our previous study on the mutation frequency of C9orf72 repeat expansions6. Table 1. Characteristics of the patients.

FTD patients (n=74) AD patients (n=229) Male (percentage) 45 (60.8) 104 (45.4) Mean age at diagnosis, years (range) 61.2 (46 – 69) 61.0 (35 – 69) Mean disease duration at diagnosis (range) 3.9 (0 - 18) 3.5 (0 – 13) Mean age at death, years (range)* 63.2 (46 - 71) 63.9 (39 – 74) Positive family history (percentage)** 28 (37.8) 80 (34.9) *Information on age at death available of 24 FTD and 47 AD patients **Positive family history in case of at least one first degree relative with dementia or, in FTD patients, with ALS.

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Genetic analysis Genomic DNA was extracted from peripheral-blood leukocytes according to standard procedures. All exons and exon–intron boundaries of the genes PSEN1, MAPT,GRN and PRKAR1B and exons 16 and 17 of the APP gene were amplified from genomic DNA (conditions and primer sequences available upon request). Direct sequencing was performed using BigDye terminator chemistry (Applied Biosystems, Foster City, CA), and sequencing products were processed on an Applied Biosystems 3730 automated DNA sequencer and analysed using SeqScape software version 2.7 (Applied Biosystems). Nucleotides are numbered according to Genbank accession numbers NM_007318 (PSEN1), NM_201414 (APP), NM_005910 (MAPT), NM_002087 (GRN) and NM_002735 (PRKAR1B) with A of initiator ATG numbered as +1. In 181 AD patients, analysis of the PRKAR1B gene was performed on exome sequencing data. Whole exome capture and sequencing were performed by Human Genomics Facility at Rotterdam. Exomes were captured by Nimblegen seqcap EZ human exome v3, and were sequenced with 2 × 100 paired-end sequencing on the Illumina HiSeq 2000 platform, according to the manufacturer’s protocol. Reads were mapped to the human reference genome sequence (UCSC hg19) using the Burrows-Wheeler Alignment Tool11. Duplicate read removal, local sequence realignment and variant filtering to minimize base calling and mapping errors were performed by Samtools12, Picard (http://picard.sourceforge.net) and Genome analysis Tool Kit (GATK)13. The identified variants per individual were called using GATK and annotated by ANNOVAR14. Variants with quality score <50, quality over depth <5.0, Strand bias >0.75 and depth <5.0 were filtered out. The average read depth for the PRKAR1B gene was 402×, with a range of 11 (exon 2) to 832. Variants in the PRKAR1B were examined on their frequency in dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP, build 138), the 1,000 genome project (www.1000genomes.org), the National Heart Lung Blood Institute Exome Variant Server (EVS) (https://evs.gs.washington.edu/EVS) and the Genome of the Netherlands (GoNL) (http://www.nlgenome.nl). Predicted functional effects of all protein-coding PRKAR1B were assessed by Polyphen-2 (http://genetics.bwh.harvard.edu/pph2), Sorting Intolerant from Tolerant (SIFT) (http://sift.jcvi.org/www/SIFT_enst_submit.html), PROVEAN (http://provean.jcvi.org/seq_submit.php) and Mutation Taster (www.mutationtaster.org). Splice site prediction was performed by Alamut 2.0 (Interactive Biosoftware). Possible pathogenic variants were confirmed by Sanger sequencing.


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http://picard.sourceforge.net/

http://www.ncbi.nlm.nih.gov/projects/SNP

http://www.1000genomes.org/

https://evs.gs.washington.edu/EVS

http://www.nlgenome.nl/

http://provean.jcvi.org/seq_submit.php

http://www.mutationtaster.org/

Patients were screened for APP gene deletions and duplications using the multiplex ligation-dependent probe amplification (MLPA) method and SALSA kit P170 APP probes mix according to the manufacturer’s instructions (MRC-Holland b.v.) Results were analyzed using GeneMarker Software (version 1.75, SofGenetics, LLC). Dosage ratio values of ≤0.7 and ≥1.3 were used as boundaries for deletions and duplications, respectively. At least one negative control was used, no positive controls were available. The hexanucleotide repeat expansion in the C9orf72 gene was determined by repeat-primed polymerase chain reaction as described previously6,15. A cut-off value of 30 repeats was used to define expanded repeats16. The C9orf72 analysis was successful in 218 AD and 72 FTD patients. Statistical analysis Means and percentages were calculated with SPSS, version 20.0 (Chicago, Ill., USA). RESULTS No pathogenic PRKAR1B mutations were identified in AD or FTD patients. Nonetheless, several variants were found, mainly synonymous variants (Table 2). In two AD patients, a non-synonymous variant of unknown significance was found. The variants p.Thr85Asn and p.Ala139Thr are predicted to be benign by at least 3 out of 4 software programs. The variant c.708+6T>C, possibly affecting a splice site, was found in one AD patient. This variant is a known SNP with a prevalence of 0.6 % in the Dutch population and predicted to be benign by 4 out of 5 splice site prediction algorithms, and therefore, unlikely to be pathogenic. Mutations in the PSEN1 gene were found in 8 out of the 229 patients with AD (3.5 %), and a duplication of the APP gene, a MAPT mutation, a GRN mutation and a C9orf72 repeat expansion were each identified in one AD patient (0.4 %). All mutations found in AD patients have been described previously and are considered to be pathogenic (www.molgen.vib-ua.be/ADMutations). The mutations and a description of the presenting phenotype of the patients can be found in Table 3. The overall mutation frequency in AD patients was 11 % in familial and 2 % in sporadic cases. The parents of the sporadic patients with PSEN1 mutations died at an age older than 64 years. Information on the current age or age of death of the parents of the patient with the C9orf72 repeat expansion is lacking. The CSF analysis in the patients with an AD-like clinical presentation and a MAPT mutation or C9orf72 repeat expansion showed a

Chapter 3.2

100

Tab

le 2

. Var

iant

s de

tect

ed in

the

PR

KA

R1B

gen

e.a

Loca

tion

Nucle

otid

e cha

nge

Prot

ein ch

ange

db

SNPb

Freq

FTDc

Freq

ADc

MAF

dbSN

Pd MA

F EV

Se MA

FGoN

Lf

Exon

2 c.6

C>T

p.Ala2

Ala

- 0.0

000

0.002

3 -

- -

Exon

3 c.2

54C>

A p.T

hr85

Asn

- 0.0

082

0.000

0 -

- -

Exon

4 c.4

15G>

A p.A

la139

Thr

rs185

6411

79

0.000

0 0.0

023

0,000

5 -

0.001

0 Ex

on 5

c.477

C>T

p.Ile1

59Ile

-

0.000

0 0.0

023

- -

- Ex

on7

c.642

C>T

p.Thr

214T

hr

rs760

6146

9 0.0

000

0.002

3 0.0

009

0,001

7 0.0

030

Intro

n 7

c.708

+6T>

C n/a

rs7

4939

612

0.000

0 0.0

023

0.001

8 0.0

017

0.006

0 Ex

on 9

c.810

G>A

p.Ala2

70Al

a rs7

7809

618

0.000

0 0.0

023

0.000

9 0.0

009

0.001

0 a E

xoni

c an

d sp

lice

site

var

iant

s in

the

PR

KA

R1B

-gen

e w

ith a

MA

F <0

.01

(dbS

NP

). R

efer

ence

s fo

r ann

otat

ion

of v

aria

nts:

Gen

Ban

k n.

NM

_002

735.

2 an

d N

P_0

0272

6.1.

b S

NP

refe

renc

e nu

mbe

r in

dbS

NP

137

c Min

or a

llele

freq

uenc

y in

our

coh

ort o

f AD

and

FTD

pat

ient

s d M

inor

alle

le fr

eque

ncy

in d

bSN

P

e Min

or a

llele

freq

uenc

y in

the

Nat

iona

l Hea

rt Lu

ng B

lood

Inst

itute

Exo

me

Var

iant

Ser

ver (

EV

S)

f Min

or a

llele

freq

uenc

y in

Gen

ome

of th

e N

ethe

rland

s (G

oNL)


101

Tabl

e 3.

Cha

ract

eris

tics

of A

D p

atie

nts

with

a g

enet

ic d

efec

t. Ca

se

Gene

Nu

cleot

ide

cha

nge

Amin

o ac

id

cha

nge

Age a

t o

nset

Ag

e at

diag

nosis

Ag

e at

d

eath

Fa

mily

h

istor

y1 Pr

esen

ting

phen

otyp

e Im

agin

g at

p

rese

ntat

ion

CSF

analy

sis

(pg

/ml)

AD01

PS

EN1

c.236

C>T

p.Ala7

9Val

53

57

64

Posit

ive

Memo

ry los

s, mi

ld ex

ecuti

ve

func

tion a

nd la

ngua

ge

diso

rder

s, ap

athy

Parie

tal at

roph

y, no

h

ippoc

ampa

l atro

phy

Aβ1-

42 4

18 (

↓)

Tau

836

(↑)

AD02

PS

EN1

c.236

C>T

p.Ala7

9Val

51

53

57

Posit

ive

Memo

ry los

s, ex

ecuti

ve an

d v

isuo-

spati

al fun

ction

d

isord

ers

Glob

al atr

ophy

in

cludin

g the

h

ippoc

ampi

Aβ1-

42 1

75 (

↓)

Tau

44

3 (↑

)

AD03

PS

EN1

c.344

A>G

p.Tyr1

15Cy

s 40

41

45

Au

tosom

al

dom

inant

Memo

ry los

s, ex

ecuti

ve an

d v

isuo-

spati

al fun

ction

d

isord

ers

Parie

tal at

roph

y, no

h

ippoc

ampa

l atro

phy

Aβ1-

42 3

77 (

↓)

Tau

432

(↑)

AD04

PS

EN1

c.367

G>A

p.Glu1

23Ly

s 64

67

70

Ne

gativ

e Me

mory

loss,

exec

utive

and

visu

o-sp

atial

functi

on

diso

rder

s, lan

guag

e d

isord

ers

Glob

al co

rtical

atrop

hy

Aβ1-

42 4

87 (

↓)

Tau

491

(↑)

AD05

PS

EN1

c.617

G>A

p.Gly2

06As

p 33

35

39

Au

tosom

al

dom

inant

Memo

ry los

s, ex

ecuti

ve an

d v

isuo-

spati

al fun

ction

d

isord

ers,

langu

age

diso

rder

s

Glob

al co

rtical

atrop

hy

Aβ1-

42 4

52 (

↓)

Tau

164

7 (↑)

AD06

PS

EN1

c.806

G>A

p.Arg

269H

is 66

68

n/a

Ne

gativ

e Me

mory

loss a

nd la

ngua

ge

diso

rder

s Mi

ld pa

rietal

and

hipp

ocam

pal a

troph

y,

vas

cular

whit

e matt

er

dise

ase

Aβ1-

42

337

(↓)

Tau

62

2 (↑

)

AD07

PS

EN1

c.806

G>A

p.Arg

269H

is 57

62

n/a

Po

sitive

Me

mory

loss,

exec

utive

and

visu

o-sp

atial

functi

on

diso

rder

s

Glob

al, pr

edom

inantl

y p

ariet

al atr

ophy

, no

h

ippoc

ampa

l atro

phy

Aβ1-

42 3

78 (

↓)

Tau

315

0 (↑)

AD08

PS

EN1

c.113

0G>T

p.A

rg37

7Met

51

56

n/a

Posit

ive

Memo

ry los

s Pa

rietal

atro

phy

Aβ1-

42 3

11 (

↓)

Tau

51

9 (↑

) AD

09

APP

Who

le ge

ne

dup

licati

on

n/a

47

52

59

Posit

ive

Memo

ry los

s, ex

ecuti

ve

functi

on di

sord

ers

Norm

al Aβ

1-42

35

9 (↓

) Ta

u

594

(↑)

Chapter 3.2

102

Tabl

e 3.

Cha

ract

eris

tics

of A

D p

atie

nts

with

a g

enet

ic d

efec

t (co

ntin

ued)

. Ca

se

Gene

Nu

cleot

ide

cha

nge

Amin

o ac

id

cha

nge

Age a

t o

nset

Ag

e at

diag

nosis

Ag

e at

d

eath

Fa

mily

h

istor

y1 Pr

esen

ting

phen

otyp

e Im

agin

g at

p

rese

ntat

ion

CSF

analy

sis

(pg

/ml)

AD10

GR

N c.3

88_3

91

delC

AGT

p.Gln1

30fs

42

47

n/a

Posit

ive

Memo

ry los

s, ap

athy,

m

ild la

ngua

ge di

sord

er

Parie

tal at

roph

y, no

h

ippoc

ampa

l atro

phy

Aβ1-

42

477

(↓)

Tau

57

3 (↑

) AD

11

MAP

T c.2

221C

>T

p.Arg

406T

rp

55

59

n/a

Autos

omal

d

omina

nt Me

mory

loss

Hipp

ocam

pal a

troph

y Aβ

1-42

102

6 Ta

u

442

(↑)

AD12

C9

orf72

Re

peat

e

xpan

sion

n/a

64

69

70

Nega

tive

Memo

ry los

s, ex

ecuti

ve

func

tion d

isord

ers,

mild

v

isuo-

spati

al fun

ction

d

isord

ers,

apath

y,

disi

nhibi

tion

Fron

tal an

d med

ial

temp

oral

atrop

hy,

mild

cere

bella

r a

troph

y

Aβ1-

42 9

45

Tau

60

7 (↑

)

n/a:

not

app

licab

le


103

normal amyloid-beta and increased tau level, while increased tau and decreased amyloid-beta levels were found in the patient with the AD-like presentation and the GRN mutation. Analysis of 74 FTD patients showed one MAPT mutation (1.4 %), two GRN mutations (2.7 %), seven C9orf72 hexanucleotide repeat expansions (9.9 %) and no mutations in the genes PSEN1 and APP. A description of the presenting phenotype of the patients with mutations can be found in Table 4. The p.Asn296Asp mutation in the exon 10 of MAPT gene is likely to be pathogenic as several other pathogenic mutations have been reported involving this amino acid, although this mutation has not been reported earlier. Both mutations in GRN gene have been described before as pathogenic mutations (www.molgen.vib-ua.be/ADMutations). All FTD patients with mutations had a positive family history for dementia or ALS, the mutation frequency in familial cases was, therefore 36 %, and zero in sporadic cases. DISCUSSION The main finding of our study is that we found no evidence for pathogenic PRKAR1B mutations in a large cohort of patients with early onset dementia. We found an overall mutation frequency of 5 % in AD patients, with PSEN1 being the most commonly mutated gene and a low frequency of mutations in other genes. In FTD patients, the mutation frequency was 14 %, mostly consisting of hexanucleotide repeat expansions in the C9orf72 gene. We identified MAPT and GRN gene mutations and a repeat expansion in the C9orf72 gene in three patients with clinically AD. The negative results of the PRKAR1B analysis suggest that mutations in this gene are rare in early onset dementia patients. Although it is likely that mutations in this gene are not associated with an AD-like phenotype, this needs to be studied in a larger cohort. The observed mutation frequency of common genes in both early onset AD and FTD patients is relatively low compared to other studies4. Most of our FTD patients were previously included in a Dutch study, in which a total mutation frequency of 26.3 % was found in FTD patients6. However, this latter cohort was enriched by a large proportion of patients with an autosomal dominant family history, referred to our collaborating centre with special expertise in hereditary FTD. The frequency of C9orf72 repeat expansions is comparable between the past and present study.

Chapter 3.2

104

Tabl

e 4.

Cha

ract

eris

tics

of F

TD p

atie

nts

with

a g

enet

ic d

efec

t. Ca

se

Gene

Nu

cleot

ide

cha

nge

Amin

o ac

id

cha

nge*

Ag

e at

ons

et

Age a

t d

iagno

sis

Age a

t d

eath

Fa

mily

h

istor

y for

d

emen

tia

Fam

ily

hist

ory

for

ALS

Pres

entin

g ph

enot

ype

Imag

ing

at

pre

sent

atio

n

F01

MAP

T c.8

86A>

G p.A

sn29

6Asp

49

62

62

Au

tosom

al

dom

inant

Nega

tive

Disin

hibitio

n, st

ereo

type /

c

ompu

lsive

beha

viour

, la

ngua

ge di

sord

ers,

memo

ry lo

ss

Temp

oral

atrop

hy

F02

GRN

c.380

_381

d

elCT

p.Pro

127fs

48

52

56

Po

sitive

Ne

gativ

e Ap

athy,

exec

utive

func

tion

diso

rder

s Fr

ontal

and

tempo

ral

atro

phy

F03

GRN

c.388

_391

d

elCAG

T p.G

ln130

fs 56

59

n/a

Po

sitive

Ne

gativ

e Me

mory

loss,

mild

exec

utive

fu

nctio

n diso

rder

s, ap

athy

Righ

t temp

oral

atrop

hy

F04

C9or

f72

Repe

at

exp

ansio

n n/a

60

61

61

Po

sitive

Ne

gativ

e La

ngua

ge di

sord

ers,

mild

d

isinh

ibitio

n As

ymme

tric fr

ontal

an

d tem

pora

l atro

phy

F05

C9or

f72

Repe

at

exp

ansio

n n/a

61

64

70

Au

tosom

al

dom

inant

Posit

ive

Apath

y, lan

guag

e diso

rder

s Gl

obal

atrop

hy

F06

C9or

f72

Repe

at

exp

ansio

n n/a

55

65

60

Ne

gativ

e Po

sitive

Di

sinhib

ition,

mild

stere

otype

/ c

ompu

lsive

beha

viour

, la

ngua

ge di

sord

ers,

mild

m

emor

y los

s, m

ild v

isuo-

s

patia

l func

tion d

isord

ers

Norm

al

F07

C9or

f72

Repe

at

exp

ansio

n n/a

49

59

64

Po

sitive

Po

sitive

Di

sinhib

ition,

mild

stere

otype

/ c

ompu

lsive

beha

viour

, m

emor

y los

s

Norm

al

F08

C9or

f72

Repe

at

exp

ansio

n n/a

41

46

46

Po

sitive

Ne

gativ

e Ap

athy,

langu

age d

isord

ers,

m

emor

y los

s, ex

ecuti

ve an

d v

isuo-

spati

al fun

ction

diso

rder

s

Glob

al atr

ophy

F09

C9or

f72

Repe

at

exp

ansio

n n/a

58

61

64

Po

sitive

Ne

gativ

e Mu

tism,

apath

y, ste

reoty

pe /

com

pulsi

ve be

havio

ur,

hall

ucina

tions

Glob

al atr

ophy

F10

C9or

f72

Repe

at

exp

ansio

n n/a

50

55

60

Au

tosom

al

dom

inant

Nega

tive

Disin

hibitio

n, ap

athy,

mild

stere

otype

/ com

pulsi

ve be

havio

ur

Fron

tal at

roph

y

n/a:

not

app

licab

le


105

The age at onset and phenotype of the patients with mutations are in most cases in concordance with previously described associated phenotypes. Interestingly, mutations were found in three AD patients with a negative family history. Furthermore, one of the patients with the PSEN1 mutation p.Arg269His had his first manifestations at the age of 66 years, a late onset according to the most commonly used definition. This mutation was previously found in several families and is most often associated with early onset AD17-19. In one family, however, the mean age at onset was above 65 years20. These findings suggest that a late onset of AD and a negative family history do not by definition exclude an identifiable genetic cause. Interestingly, 13 % (3/13) of the mutations in patients with the clinical diagnosis of AD were found in a FTD gene. An Alzheimer-like phenotype has been previously described in patients with the mutation p.Arg406Trp in the MAPT gene (www.molgen.vib-ua.be/ADMutations), and this specific case has been described before21. A phenotype with predominant memory loss resembling AD is frequently seen in GRN mutations22. Although low CSF amyloid-beta levels have been described in GRN mutation carriers23, a low amyloid-beta and high tau level in the CSF in this patient strongly suggests a concomitant AD. Furthermore, the clinical diagnosis frontal variant of AD in the patient with a C9orf72 repeat expansion could retrospectively be considered as a misdiagnosis by the presence of a normal CSF amyloid-beta and increased tau level. This is further supported by frontal, temporal and cerebellar atrophy on MRI compatible with FTD, especially a C9orf72 repeat expansion. One of the strengths of our study is the well-defined large cohort of early onset dementia patients. The extensive investigations performed on patients with dementia are likely to have resulted in a correct diagnosis in most patients, especially if the CSF results had been included in the clinical decision making. This is confirmed by the low frequency of mutations in the FTD genes in EOAD patients with an AD CSF profile, and no mutations in the AD genes in FTD patients. One major limitation of our study is that we performed genetic analysis of common genes associated with AD and FTD only. As the frequency of mutations in other genes, like PSEN2, CHMP2B, VCP and TDP-43, is very low, we did not expect to find any mutations in these genes. Furthermore, the sensitivity and specificity of the method used for the analysis of the C9orf72 repeat expansion differs by performing laboratory24. Since unreliable results of the C9orf72 analysis were not included in the outcome, the actual mutation frequency may be slightly higher than reported. Also, since the depth of the exome sequencing was quite low for some exons of the PRKAR1B gene, it is possible that mutations in this gene have been missed.

Chapter 3.2

106

In conclusion, no pathogenic PRKAR1B mutations were found in our Dutch cohort of early onset AD and FTD patients. Furthermore, mutations in AD patients are quite rare, but are occasionally also found in patients with a late onset dementia or lacking a positive family history. Therefore, patients and relatives with questions about the heritability of the dementia should be referred to a clinical geneticist for counselling independently of age at onset and family history. Also, mutations in genes associated with FTD may cause an AD-like phenotype. In FTD patients, we only found mutations in patients with a positive family history for dementia and/or ALS. However, previous studies have shown that especially repeat expansions in the C9orf72 gene may be present in sporadic cases6,16. Therefore, also all FTD patients with questions about a genetic cause should be offered genetic counselling. ACKNOWLEDGEMENTS The authors thank all participating patients. We acknowledge W. Kudrop for reviewing the clinical phenotype of mutation carriers. Research of the VU University Medical Centre’s Alzheimer Centre is part of the neurodegeneration research program of the Neuroscience Campus Amsterdam. The Alzheimer Centre is supported by Alzheimer Nederland and Stichting VUmc fonds. The clinical database structure was developed with funding from Stichting Dioraphte.


107

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22. van Swieten, JC and Heutink, P. Mutations in progranulin (GRN) within the spectrum of clinical and pathological phenotypes of frontotemporal dementia. Lancet Neurol. 2008; 7:965-974.

23. Carecchio, M, Fenoglio, C, Cortini, F, et al. Cerebrospinal fluid biomarkers in Progranulin mutations carriers. J Alzheimers Dis. 2011; 27:781-790.

24. Akimoto, C, Volk, AE, van, BM, et al. A blinded international study on the reliability of genetic testing for GGGGCC-repeat expansions in C9orf72 reveals marked differences in results among 14 laboratories. J Med Genet. 2014; 51:419-4.

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Chapter 4

Identification of new genetic causes of cognitive decline

Chapter 4.1 A novel CCM2 variant in a family with non-progressive cognitive complaints and cerebral microbleeds Petra E. Cohn-Hokke, Henne Holstege, Marjan M. Weiss, Wiesje M. van der Flier, Frederik Barkhof, Erik A. Sistermans, Yolande A.L. Pijnenburg, John C. van Swieten, Hanne Meijers-Heijboer, Philip Scheltens Am J Med Genet B Neuropsychiatr Genet. 2016 June [Epub ahead of print].

ABSTRACT Lobar cerebral microbleeds are most often sporadic and associated with Alzheimer's disease. The aim of our study was to identify the underlying genetic defect in a family with cognitive complaints and multiple lobar microbleeds and a positive family history for early onset Alzheimer's disease. We performed exome sequencing followed by Sanger sequencing for validation purposes on genomic DNA of three siblings with cognitive complaints, reduced amyloid-beta-42 in CSF and multiple cerebral lobar microbleeds. We checked for the occurrence of the variant in a cohort of 363 patients with early onset dementia and/or microbleeds. A novel frameshift variant (c.236_237delAC) generating a premature stop codon in the CCM2 gene shared by all three siblings was identified. Pathogenicity of the variant was supported by the presence of cerebral cavernous malformations in two of the siblings and by the absence of the variant exome variant databases. Two siblings were homozygous for APOE-ϵ4; one heterozygous. The cognitive complaints, reduced amyloid-beta-42 in CSF and microbleeds suggest preclinical Alzheimer's disease, but the stability of the cognitive complaints does not. We hypothesize that the phenotype in this family may be due to a combination of the CCM2 variant and the APOE status.

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INTRODUCTION Cerebral microbleeds (CMBs), small round hypointense lesions on hemosiderin sensitive MR sequences, are common in Alzheimer's disease patients but also occur in the general population1,2. Based on clinical and epidemiological studies, CMBs with a lobar location presumably represent cerebral amyloid angiopathy (CAA), while CMBs with a deep location may represent hypertensive vasculopathy2-4. CAA is most often sporadic, with age being the most important risk factor5. Specific (founder) mutations in the amyloid-precursor protein (APP) gene, the cystatin 3 (CST3) gene, and the integral membrane protein 2B (ITM2B) gene are associated with hereditary CAA6-8. Mutations in presenilin 1 (PSEN1) and presenilin 2 (PSEN2) genes are associated with familial early onset Alzheimer's disease with CAA9-11. The APOE-ϵ4 allele is the strongest known genetic risk factor for Alzheimer's disease12,13, but is also associated with the incidence and severity of CAA14 and with the prevalence of CMBs in Alzheimer's disease patients15,16. We present a family with CMBs and cognitive complaints with no known genetic predisposition for CAA or Alzheimer's disease, in whom we performed whole exome sequencing in three affected siblings. We detected a deletion leading to a frameshift in the CCM2 gene, a gene associated with familial cerebral cavernous malformations. MATERIALS AND METHODS Clinical Ascertainment We selected a family of which two family members were known at our clinic, the Alzheimer centre of the VU University Medical Center, because of cognitive complaints and microbleeds and an autosomal dominant family history for Alzheimer's disease. All patients visiting the Alzheimer centre are offered an extensive standardized dementia assessment including medical history, informant-based history, a physical examination, routine blood (including glucose) and cerebrospinal fluid (CSF) laboratory tests, neuropsychological testing, electroencephalogram (EEG), and magnetic resonance imaging (MRI) of the brain including susceptibility-weighted T2* images17. The clinical diagnosis Alzheimer's disease is made by consensus in a multidisciplinary team based on the NINCDS-ADRDA criteria for Alzheimer's disease18, and a clinical diagnosis mild cognitive impairment (MCI) based on the Petersen criteria19. Patients are labelled as having subjective complaints when reporting cognitive complaints while cognitive

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and laboratory investigations are normal and criteria for MCI, dementia or any other neurological or psychiatric disorder associated with cognitive complaints are not met. All patients who give consent for research are included in the Amsterdam Dementia Cohort17. A subset of this cohort, consisting of 363 patients with early onset AD and/or multiple microbleeds, was selected for whole exome sequencing for other research purposes. All participants of this study had at least two MRI's performed on a 3.0 T GE scanner (type HDXT) with SWI sequence. Analysis of small vessel disease was performed according to STRIVE20. The three participating subjects of the described family gave written informed consent for genetic research specifically prior to inclusion. The research protocol was approved by the ethical review board of our hospital. Genetic Analysis DNA of the described participants and of the 363 selected patients of the Amsterdam dementia cohort was derived from peripheral blood. Exomes were captured by the Nimblegen human exome v3 capture kit, and were sequenced with 2 × 100 paired-end sequencing on the Illumina HiSeq 2000 platform, according to the manufacturer's protocol. Reads were mapped to the human reference genome sequence (UCSC hg19) using the Burrows-Wheeler Alignment Tool (http://bio-bwa.sourceforge.net)21. Duplicate read removal, local sequence realignment, and base quality recalibration were performed by Picard (http://picard.sourceforge.net) and Genome analysis Tool Kit (GATK, (https://www.broadinstitute.org/gatk/)22. Variants were called using the GATK HaplotypeCaller, and filtered using the variant filtration tool. For each variant, we set the filter to PASS if the variant complied with (i) GATK quality score ≥50; (ii) quality over depth ≥1.5; (iii) Strand bias ≤60; (iv) total read depth ≥5.0. Variants were annotated and analysed with Cartagenia (http://www.cartagenia.com/) filter tree specifically designed to detect variants causative for a trait with an autosomal dominant inheritance pattern. In the described family, variants were selected if (i) absent in the following databases: dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP, build 138), the 1.000 genome project (www.1000genomes.org) or the National Heart Lung Blood Institute Exome Variant Server (EVS) (https://evs.gs.washington.edu/EVS); (ii) prevalent ≤5% in the 363 patients of the Amsterdam Dementia Cohort17; (iii) heterozygote in all three affected individuals; (iv) potential causative based on the possible effects of the variants on the expression or function of the protein and in a morbid OMIM gene.

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To find out whether a predisposition for CCMs is common in patients with presumed microbleeds, we searched the exomes of 363 patients of the Amsterdam Dementia Cohort17, including 68 patients with microbleeds, for occurrence of exonic and splice site variants in the genes KRIT1, CCM2, and PDCD10. Nucleotides were numbered according to Genbank accession number NM_004912.3 (KRIT1), NM_001029835.2 (CCM2), and NM_007217.3 (PDCD10) with A of initiator ATG numbered as +1. Variants in the CCM genes were analysed by using the Combined Annotation Dependent Deletion (CADD) scoring tool (http://cadd.gs.washington.edu/) v1.2.We used Sanger sequencing to confirm the novel variant we found with exome sequencing. We submitted the variant in the CCM2 gene to the Leiden Open Variant Database (http://ccm2.lovd.nl). APOE genotyping was performed by Sanger sequencing of codons 112 and 158 of the APOE gene. For this, a 428 bp fragment was generated from genomic DNA by PCR, checked for size (Fast DNA analysis with QIAxcel), and sequenced (BigDye Terminator v3.1 Cycle Sequencing kit followed by ABI 3130XL Genetic Analyser). RESULTS Characteristics of the Participants The pedigree is shown in Figure 1. Individuals III-1, III-2, and III-3 participated in our study. They reported early-onset dementia in one parent (II-6) and one grandparent (I-1). Three siblings of the affected parent had symptoms of dementia before the age of 65 years (II-1, II-2, and II-3), another relative (II-4) died of a stroke of unknown aetiology. Of these reported affected relatives, neither detailed medical information nor DNA was available. Case III-1 This patient visited our centre at the age of 58 years with memory complaints for over 5 years. The MMSE score was 27 out of 30. Physical examination, routine blood tests, neuropsychological testing, and EEG were all normal. Cerebral MRI showed nine lobar CMBs in the absence of cortical atrophy (global cortical atrophy [GCA] score 0) and hippocampal atrophy (medial temporal lobe atrophy [MTA] score 0) (Figure 2A), a few punctiform white matter lesions (Fazekas 1) and a large T2*-hypointensity in the pons consistent with a macroscopic haemorrhage. CSF analysis showed decreased amyloid-beta-42 and increased tau levels (Aβ: 529 ng/L, reference > 550 ng/L; t-tau: 673 ng/L,


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reference ≤ 375 ng/L; ptau-181: 86 ng/L, reference ≤ 52 ng/L). The patient was homozygous for the APOE-ϵ4 allele. Diagnostic DNA-analysis revealed no mutations in the genes APP (Sanger sequencing and copy number variant analysis), PSEN1, PSEN2, and MAPT. Figure 1. Pedigree of the described family.

The sex of each individual is masked and the pedigree scrambled to protect the privacy of the participants. The diamonds indicate individuals with cognitive complaints and microbleeds (filled symbols), dementia (filled upper right quadrant), and stroke (filled lower right quadrant). CCM+ indicates that the subject is positive for the c.236_237delAC variant in the CCM2 gene. E3 indicates an APOE-ϵ3 allele, E4 an APOE-ϵ4 allele. “n” in a diamond indicates more than one individual. Consequently, the ID under the symbols with an “n” refers to multiple individuals as a group.

Since the clinical and neuropsychological exam were within the normal range, the cognitive complaints were labelled as subjective cognitive decline. CMBs were interpreted as suggestive of underlying CAA and the CSF biomarkers indicative of preclinical AD. During the following 7 years, the patient reported only mild progression of her cognitive dysfunction, and neuropsychological testing remained normal. Repeat MR imaging showed a few new CMBs but still no signs suggestive of neurodegeneration such as cortical atrophy.

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Figure 2. Cerebral MR imaging.

A: Microbleed (arrow) in individual III-1 visible on T2-weighted image. B: Left cerebellar cavernous malformation (arrow) in individual III-2 on T2-weighted image. C: Left frontal cavernous malformation (arrow) in individual III-3 with characteristic central high signal on T2 weighted image and hypointense outer rim.

Case III-2 This patient visited our centre at the age of 54 years with complaints of long lasting mild memory loss. Physical examination and routine blood tests showed no relevant abnormalities. The patient scored 29 out of 30 on the MMSE, and neuropsychological testing revealed only mild language disturbances. T2*-weighted MR imaging revealed three lobar CMBs, two larger hematomas (cerebellar and parietal) with a hyperintense centre on FLAIR and T1 consistent with a cavernous malformation (Figure 2B), and a few punctiform vascular white matter lesions (Fazekas 1); cerebral cortical or hippocampal atrophy was absent (GCA 0, MTA 0). The EEG was normal. CSF analysis showed decreased amyloid-beta-42 but normal t-tau and borderline p-tau levels (Aβ: 418 ng/L, t-tau: 343 ng/L, ptau-181: 56 ng/L). The patient was homozygous for the APOE-ϵ4 allele. No mutations were identified in the genes PSEN1, PSEN2, and MAPT. The cognitive complaints were labelled as subjective cognitive decline. The CMBs were interpreted as suggestive for underlying CAA. During the following 7 years, the patient reported a slight worsening of cognitive complaints, which could not be confirmed by neuropsychological testing (criteria for MCI not fulfilled). Repeat MRI showed mild increase of white matter hyperintensities and a few new lobar CMBs. Case III-3 The youngest sibling presented with progressive memory loss and executive dysfunction at another hospital at the age of 51 years and was diagnosed with


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Alzheimer's disease. Detailed information on the test results was not available. No mutations, duplications, or deletions were found in the genes APP and PSEN1. The patient visited our clinic for a second opinion 4 years later, as symptoms had not worsened. At this time, MMSE was 25/30, and neuropsychological assessment revealed executive dysfunction and mild impairment of memory and naming. MRI showed more than 20 supra- and infratentorial lobar CMBs, a few punctiform vascular white matter lesions (Fazekas 1), and a cavernous malformation in the left frontal lobe (Figure 2C). There was no cortical atrophy (GCA 0) or relevant hippocampal atrophy (MTA 0 on the left and grade 1 on the right). The patient had an APOE ϵ3/ϵ4 genotype. A lumbar puncture was refused by the participant. The patient did not fulfil the criteria for Alzheimer's disease and was diagnosed with MCI. The CMBs were interpreted as probably due to CAA. Two-year follow up showed no further clinical deterioration. Genetic Findings With exome sequencing, we identified a heterozygous two-base pair deletion in exon 3, c.236_237delAC, in the CCM2 gene in all three siblings. This deletion creates a frameshift starting at codon Tyr79 resulting in a premature stop codon. Based on the location of the new stop, the transcript is likely to be targeted by nonsense-mediated mRNA decay, resulting in haploinsufficiency. The variant has not been reported before in literature or in the Dutch genetic biobank GoNL (http://www.nlgenome.nl/) and was not found in 363 patients with early onset Alzheimer's disease and/or multiple microbleeds. Based on the predicted effect of variants and known function of the associated genes, we found no other variants of interest in the family. To investigate whether mutations in the CCM2-gene or related genes are common in patients with CMBs, we analysed the cohort of 363 patients with early onset Alzheimer’s disease and/or microbleeds for rare variants in the genes KRIT1, CCM2, and PDCD10. We detected two missense variants of unknown significance in the KRIT1 gene with a minor allele frequency of less than 0.5%: One protein modifying variant, c.1882A > C, p.Asn628His was found in an Alzheimer's disease patient without vascular abnormalities on brain imaging. This variant has a CADD score of 23.1 and is found in the Dutch genomic biobank GoNL with an allele frequency of 0.1%. A synonymous variant, c.1752C > T, p.Ile584 = with a CADD score of 17.5 was found in an Alzheimer's disease patient with moderately severe vascular white matter lesions but no CMBs or cavernous malformations. The GoNL biobank reports an allele frequency of close to 0.5% of this variant.

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DISCUSSION We describe a novel variant in the CCM2 gene identified by whole exome sequencing in a family with non-progressive cognitive symptoms in three siblings at relatively young age, with CCMs in two and multiple lobar CMBs in all three on cerebral MRI. In addition, decreased amyloid beta-42 levels in CSF were found in both tested individuals. Mutations in the genes APP, PSEN1, and PSEN2 were absent, and at least one APOE-ϵ4 allele was present in all three. The novel frameshift variant generates a premature stop codon in the CCM2 gene, probably resulting in haploinsufficiency by nonsense mediated mRNA decay. Loss of function mutations in the CCM2 gene and in the genes KRIT1 and PDCD10 are associated with familial cerebral cavernous malformations (FCCM)23-26. Cerebral cavernous malformations (CCMs) are enlarged, thin walled capillaries in the brain and spinal cord without fibrous support tissue. In familial cases, a combination of a germ line mutation (first hit) and a somatic mutation (second hit) in one of the FCCM genes are associated with CCMs27,28. The KRIT1-CCM2-PDCD10-complex is considered to interact with the PI3 K/Akt signaling pathway associated with metabolism, growth, proliferation, survival, transcription, and protein synthesis mechanisms29. Most symptomatic FCCM patients present between the age of 10 and 40 years with seizures, focal neurologic deficits, non-specific headaches, or acute cerebral hemorrhage30. Up to 50% of the patients with FCCM remain asymptomatic, although most asymptomatic mutation carriers do have at least one CCM on MRI30. The presence of CCMs in two of the three siblings (individual III-1 and III-2) supports the diagnosis of FCCM in this family. The absence of a CCM in the third individual does not contradict this diagnosis, since FCCM is known to have an incomplete penetrance. However, decreased CSF amyloid beta-42 has not been reported in FCCM and cognitive complaints and CMBs are not common symptoms of this disease30. The (subjective) cognitive decline may reflect a concomitant preclinical Alzheimer disease. The abnormal amyloid beta levels in CSF and the presence of APOE-ϵ4 in homozygous or heterozygous state in all three affected siblings would support this. However, the stable character of the cognitive complaints over 7 years does not. Poor cognitive function has also been associated with APOE-ϵ4 status in the absence of Alzheimer's disease31 as well as the presence of lobar CMBs32,33. Reduced amyloid beta-42 levels in CSF are strongly correlated with Alzheimer's disease, but have been associated with the APOE-ϵ4 genotype regardless of the presence of Alzheimer's disease34, and with several other brain diseases, such as CADASIL,


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neuroinflammation, and Creutzfeldt-Jakob Disease35-37. No studies have been published on CSF profiles in FCCM patients38. An intriguing question is whether the occurrence of CMBs in this family is due to FCCM, or whether it should be attributed to a co-existent disease such as preclinical Alzheimer's disease or isolated CAA. CMBs as such have not been reported in (F)CCM38. The mean prevalence of CMBs in women aged <70 years is about 5% in the general population39,40, therefore, normal ageing does not seem a plausible cause in these siblings. Based on the CSF findings, also isolated CAA is unlikely. The aspects and location of the bleeds do not fit with other causes of small bleeds such as vasculitis, hypertensive encephalopathy, or coagulopathy. Another possibility, however, is that the CMBs are in fact small CCMs. While larger CCMs typically show signs of stagnant blood in the sinusoidal lumen, extravasated blood at varying stages of degradation and a characteristic hemosiderin rim on MRI41, the small Zambinski's classification type 4 CCM lesions42 are more difficult to distinguish from CMBs. The presence of variants in the FCCM genes in other patients with presumed CMBs would support this hypothesis. We did not find any other variants predicted to result in a loss of function in a cohort of patients with multiple CMBs, however, the number of tested individuals was small. It is interesting to hypothesize whether ApoE and CCM2 interact. No common pathway has been described. However, APOE-ϵ4 has been reported to increase the susceptibility to blood-brain-barrier injury43 and, therefore theoretically, this genotype may result in an increased bleeding risk of CCMs. Taken together, the non-progressive cognitive complaints, the lobar hypointense lesions on cerebral MRI, and the reduced amyloid beta-42 levels in CSF may be due to the combination of the CCM2 variant and the APOE-ϵ4 genotype, although an early stage of Alzheimer's disease cannot be ruled out. Unfortunately, no other relatives were available for segregation analysis. Further studies on CSF profiles in FCCM patients and mutations in the FCCM genes in patients with multiple CMBs could give more insight into the pathogenic mechanism in this family.

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ACKNOWLEGDEMENTS The authors thank the participating patients. Research of the VU University Medical Centre’s Alzheimer Centre is part of the neurodegeneration research program of the Neuroscience Campus Amsterdam. The Alzheimer Centre is supported by Alzheimer Nederland and Stichting VUmc fonds.


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19. Petersen, RC. Mild cognitive impairment as a diagnostic entity. J Intern Med. 2004; 256:183-194. 20. Wardlaw, JM, Smith, EE, Biessels, GJ, et al. Neuroimaging standards for research into small vessel

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analyzing next-generation DNA sequencing

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Chapter 4.2 Rare genetic variant in SORL1 may increase penetrance of Alzheimer’s disease in a family with several generations of APOE-ε4 homozygosity Petra E. Cohn-Hokke*, Eva Louwersheimer*, Yolande A.L. Pijnenburg, Marjan M. Weiss, Erik A. Sistermans, Annemieke J. Rozemuller, Marc Hulsman, John C. van Swieten, Cock M. van Duijn, Frederik Barkhof, Teddy Koene, Philip Scheltens, Wiesje M. van der Flier, Henne Holstege *equal contribution J Alzheimers Dis. 2017;56:63-74

ABSTRACT The major genetic risk factor for late onset Alzheimer’s disease (AD) is the APOE-ε4 allele. However, APOE-ε4 homozygosity is not fully penetrant, suggesting co-occurrence of additional genetic variants. We identified a family with nine AD patients spanning four generations, with an inheritance pattern suggestive of autosomal dominant AD, with no variants in PSEN1, PSEN2, or APP. We collected DNA from four affected and seven unaffected family members and found that all affected family members were homozygous for the APOE-ε4 allele. We performed exome sequencing on DNA from three affected and one unaffected APOE-ε4 homozygous family members and segregation analysis in eleven family members. Statistical analysis revealed that AD onset in this family was significantly earlier than could be expected based on APOE genotype and gender. Next to APOE-ε4 homozygosity, we found that all four affected family members carried a rare variant in the VPS10 domain of the SORL1 gene, associated with APP processing and AD risk. Furthermore, three of four affected family members carried a rare variant in the TSHZ3 gene, also associated with APP processing. Affected family members presented between 61 and 74 years, with variable presence of microbleeds/CAA and electroencephalographic abnormalities. We hypothesize that next to APOE-ε4 homozygosity, impaired SORL1 protein function, and possibly impaired TSHZ3 function, further disturbed Aβ processing. The convergence of these genetic factors over several generations might clarify the increased AD penetrance and the autosomal dominant-like inheritance pattern of AD as observed in this family.

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INTRODUCTION Alzheimer’s disease (AD) is a complex and heterogeneous neurodegenerative disease. AD incidence increases with age, and about one third of the population aged 85 years and older is estimated to have AD1. AD is typically characterized by deficits in short-term memory, language, praxis, visuospatial and executive functioning, eventually resulting in global cognitive decline. Despite intense research during past decades, the exact causes of AD are not yet understood. The leading hypothesis of AD pathogenesis is the amyloid cascade hypothesis, which proposes that aberrant processing of the amyloid precursor protein APP leads to increased production of amyloid-beta (Aβ) peptide in the brain cells (reviewed in 2). In turn, Aβ peptides are misfolded and accumulate into protein aggregates, ultimately leading to the formation of neurotoxic amyloid plaques that disrupt normal cellular processes. Genetic mutations in autosomal dominant AD are detected in genes involved in Aβ processing: the amyloid precursor protein (APP), which is the source of Aβ, and the presenilins (PSEN1 and PSEN2) involved in APP-processing3-5. Twin studies estimated that ~60-80% of late onset AD risk is heritable with the remainder being environmental (LOAD, age at onset >65 years)6. By far the most important susceptibility gene for late onset AD is the Apolipoprotein E (APOE) gene7. Next to functions related with lipid and cholesterol processing, the protein product of the APOE gene, ApoE, is suggested to be involved in the clearance of Aβ from the brain (reviewed in 2). The APOE gene contains three common allelic variants (APOE-ε2, APOE-ε3, APOE-ε4), which encode the ApoE2, ApoE3, and ApoE4 protein isoforms. Optimal Aβ clearance efficiency has been suggested to explain the neuroprotective nature of the ApoE2 isoform relative to the most common ApoE3 isoform, whereas presumably, the impaired Aβ clearance by the ApoE4 isoform explains the increased AD risk for APOE-ε4 allele carriers2. In fact, more than 30% of the AD cases in the population can be attributed to the APOE-ε4 allele (population attributable fraction), whereas at most 8% of AD cases can be attributed to any of the genes detected in GWAS studies8. Carrying the APOE-ε4 allele predisposes for AD in a dose-dependent manner: compared to non-APOE-ε4 carriers, AD risk is increased 3-5 fold for heterozygous APOE-ε4 carriers, and 10-15-fold for homozygous APOE-ε4 carriers9. However, despite this large effect size, the penetrance of APOE-ε4 homozygosity is incomplete. The chance that APOE-ε4 homozygotes develop AD before the age of 85 years is 50% for males and 60% for females10. Some APOE-ε4 homozygotes reach ages over 100 years

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while retaining their cognitive health11. This suggests that next to being homozygous for the APOE-ε4 allele, additional genetic modifiers are necessary for the development of AD. To our knowledge, it has never been investigated whether other genetic variants co-occur with APOE-ε4 homozygosity in AD patients. We identified a family with AD patients with a relatively early onset of disease, spanning at least four generations, with an inheritance pattern that suggests autosomal dominant AD. DNA was available from four affected and seven unaffected family members, from the two youngest generations. We found that all four genetically tested affected family members were homozygous for the APOE-ε4 allele. Therefore, this family provided the unique opportunity to investigate additional genetic variants next to APOE-ε4 homozygosity, that might have contributed to AD. We describe the pedigree, the phenotype of the affected family members, the outcome of whole exome sequencing, and the segregation of the genetic variants. MATERIALS AND METHODS Pedigree and participants We describe a family comprising nine individuals with AD symptoms who span four generations within one pedigree (Figure 1, Table S1A): eight were diagnosed with AD, or were reported to have symptoms of AD (0.2, I.1, I.3, I.4, II.1, II.3, II.4 and II.6) and one individual had preclinical AD (III.1). Multiple individuals did not present AD symptoms at the time of observation (youngest individuals were merged into III.4-III.5 to avoid recognition). Four family members with (preclinical) AD and seven of the unaffected family members (aged ≤ 60 years) consented to participate in this study (II.3 and II.4 by consent of their proxies). Affected family member II.1 consented to the use of his clinical data for research purposes. Detailed clinical data were not available for the affected family members of the first generations (0.2, I.1, I.3 and I.4). Two family members with AD (II.1 and II.6) and the family member with preclinical AD (III.1) visited the Alzheimer centre at the VU University medical centre (VUmc) in the Netherlands and underwent extensive standardized diagnostic work-up12. The other affected family members (II.3 and II.4) were diagnosed elsewhere in the Netherlands. All diagnoses of AD were based on the NINCDS-ADRDA criteria as described by McKhann et al13. Post-mortem autopsy results of individual II.4 were reviewed by our

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neuropathologist. The local medical ethics committee of the VUmc approved the study. We did not obtain consent to reveal disease history of family member II.8. Figure 1. Pedigree of family with four generations of AD patients.

Black diamonds: Family members affected with AD; White diamonds: non-affected family members at time of death or last screening; Grey diamond: no consent to reveal disease history; “W” in diamond: family member included in whole exome sequencing; ‘n’ in a diamond: multiple family members merged and represented as one; AD [number]: Alzheimer’s disease with age at diagnosis; d [number]: age at death. E4/E4: APOE-ε4 homozygosity; SORL1+ : subject is positive for the variant c.2012A>G in SORL1; TSHZ3+ subject is positive for the variant c.707C>T in TSHZ3; Grey text: DNA was not available for these family members, we estimated the chances that an individual has a given APOE genotype, based on (i) frequency of genotype combinations in the Dutch population, (ii) Mendelian inheritance patterns given the genotype distribution within the family structure and (iii) disease status. The chances for APOE genotypes do not add up to 100% when (smaller) chances for other APOE genotypes remain (Table S1). Inferred chances of carrying of SORL1 and TSHZ3 genes are based on normal Mendelian inheritance patterns; Sex is not indicated and the order of siblings is rearranged to avoid recognition of this family and individual family members.. See Table S1 for list of genotype/phenotype data per-family member.


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DNA availability Using common procedures, DNA was isolated from peripheral blood from three affected family members (II.3, II.6, II.4), the family member with preclinical AD (III.1), and the seven unaffected participating family members (III.2, III.3, four family members merged into III.4-5, and III.6). APOE genotyping and imputation For all participants with DNA available, APOE genotyping was performed after genomic DNA isolation from 7-10 mL EDTA blood, using a QIAxcel DNA Fast Analysis kit (Qiagen, Venlo, The Netherlands). For the individuals comprising generation I and two individuals from generation II, blood samples could not be collected. For individuals I.1, I.2, II.1 and II.8 APOE genotypes were determined in retrospect by estimating the posterior probability of the possible APOE genotypes. To this end, we applied Bayes theorem based on (i) the known APOE genotype combinations in generations II and III, (ii) the population frequencies of APOE genotype combinations in the Dutch population published by LASA14 (Table S1B), and (iii) the chances for developing dementia for all APOE genotype combinations by age and gender published by Genin et al10 (Table S1C). Analysis of AD penetrance in this family In a Caucasian sample comprising >17,500 cases and controls, Genin et al evaluated the AD incidence per APOE-genotype, across age at onset and gender relative to baseline AD incidence10. These AD incidence distributions across age allowed us to determine the a priori chance for any individual to develop AD at a certain age given their APOE genotype and gender. For each member of our family, the age at AD onset can be seen as a p-value w.r.t. to empirical the incidence distributions extracted from the Caucasian cohort. Persons who had not yet reached 60 years at last check-up were excluded, as the chance to develop AD before this age is very low. To account for the unknown age at AD onset for several family members, we determined a p-value by repeated sampling from a uniform distribution of p-values (truncated p-value approach15. We used both Fisher’s approach and Stouffer’s approach to combine p-values of all family members. Apart from their genetic dependency (which we are testing), we assume that development of AD is independent between family members.

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Variant detection: exome sequencing of four family members DNA of three affected family members (II.3, II.6 and III.1) and the oldest unaffected family member (III.2) was exome sequenced in parallel with 400 AD patients from the Amsterdam Dementia cohort (ADC)12 who had been diagnosed with early onset dementia. The exomes were captured by the Nimblegen human exome v3 capture kit, and 100bp paired-end sequencing reads were generated on the Illumina HiSeq 2000 platform, according to the manufacturer’s protocol. We sequenced to at least 40x mean coverage to ensure sufficient read depth for variant calling. Reads were mapped to the human reference genome sequence (UCSC hg19) using Burrows-Wheeler alignment16. Duplicate read removal, local sequence realignment and base quality recalibration were performed by Picard (http://picard.sourceforge.net) and with GATK (Genome analysis tool kit)17. Variants were called using GATK haplotype caller, and filtered using the variant filtration tool. For each variant we set the filter to PASS if the variant complied with (I) GATK quality score ≥50, (II) quality over depth ≥1.5, (III) Strand bias ≤60, (IV) total variant read depth ≥5.0. Variants were annotated and analysed with Cartagenia (http://www.cartagenia.com/) using a filter tree specifically designed to detect variants causative for a trait with an autosomal dominant inheritance pattern. Since we aimed to identify rare pathogenic variants, we selected variants that (i) were absent in the following databases: dbSNP138 (http://www.ncbi.nlm.nih.gov/projects/SNP, build 138), the 1,000 genome project (www.1000genomes.org) or the National Heart Lung Blood Institute Exome Variant Server (EVS) (http://evs.gs.washington.edu/EVS/); (ii) had a prevalence of ≤5% in the whole Amsterdam Dementia cohort; (iii) were heterozygote in the 3 affected family members; and (iv) were localized in a gene listed in the OMIM database (www.omim.org). The predicted functional effects of the selected sequence variants were assessed by PolyPhen2 (genetics.bwh.harvard.edu/pph2/), SIFT (sift.jcvi.org/), Mutation Taster (www.mutationtaster.org), and the combined annotation dependent deletion (CADD) score18. Information about localization and conservation of the selected variants was assessed by Uniprot (www.uniprot.org/uniprot/Q92673), and Alamut visual (www.interactive-biosoftware.com/alamut-visual/). Detected variants were confirmed by Sanger sequencing, Also, loci were genotyped in the all family members for whom DNA was available.


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RESULTS Clinical description Family member II.1 presented with complaints of memory decline over the previous three years at the age of 67. As a child, the family member had suffered a skull fracture. The mini-mental state examination (MMSE)19 score was 25/30, and neuropsychological assessment showed impairment of episodic memory. Routine blood analysis was normal, except for increased serum cholesterol levels. Magnetic resonance imaging (MRI) showed mild bilateral hippocampal atrophy (medial temporal lobe atrophy (MTA) grade 120), mild white matter hyperintensities (WMH) (Fazekas grade 121), and no microbleeds. Electroencephalogram (EEG) revealed a discordant low background rhythm of 6 to 7 Hz with increased amounts of intermitting delta activity in the frontotemporal regions. Cerebrospinal fluid (CSF) was not obtained. Based on these findings, the diagnosis was mild cognitive impairment (MCI)22. At the age of 69, the MMSE was 21/30. Repeated neuropsychological assessment showed progression of memory impairment and impaired executive functions. At this time, MRI showed biparietal atrophy, with no progression of the hippocampal atrophy or WMH. The clinical diagnosis was probable AD13. Disease progression was characterized by further deterioration in all cognitive domains, including the development of behavioural disturbances (loss of initiative and increased irritability). This family member was admitted into a nursing home, suffered from episodes of focal neurological deficits probably due to recurrent strokes and died at the age of 76 years. This family member gave consent to his physician to use his medical data for research purposes, but DNA was not available. Family member II.3 visited a geriatrician at a local hospital at the age of 72 because of memory complaints and fatigue for two years. This family member had diabetes mellitus type 2, hypertension and dyslipidaemia, and had been treated for depression with amitriptyline for over 20 years. MMSE was 26/30 with disorientation in time, and neuropsychological testing showed impaired recall on memory tests. Computed tomography (CT) imaging showed mild diffuse cortical atrophy, aspecific hypodensities in the brainstem and in the basal ganglia. No formal diagnosis was made at that time. A second opinion by another neurologist was obtained at the age of 74. At this point, the family member reported the occurrence of headaches, and scored 23/30 on the MMSE. Routine blood analysis and EEG were normal. MRI and CSF analysis were not performed. The family member was diagnosed with probable AD13. Diagnostic DNA

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testing revealed no variants in PSEN1, PSEN2, or APP. The family member died at the age of 78 years (cause unspecified). Family member II.4 visited a local memory clinic at the age of 70, because of progressive memory complaints, which initiated at the age of 59 after a head trauma. The family member had suffered from bacterial meningitis at the age of 69. Neuropsychological testing showed impairments in concentration and memory, disorientation in place, and dyscalculia. No additional investigations were performed. The family member was diagnosed with probable AD [13]. The patient died at the age of 74 years most likely due to a heart attack. Post mortem examination of the brain confirmed the diagnosis of severe AD (Braak stage 6/6 for tau and Thal phase 5/5 for amyloid-beta (Aβ) with extensive CAA type 123 (Figure 2). Figure 2. Immunohistochemistry in temporal cortex of subject II.4.

a) Immunohistochemical staining for Aβ reveals cerebral Aβ angiopathy (black arrow), classical plaques (arrow head) and diffuse plaques (white arrow) in the temporal cortex (10x obj.); b) Immunohistochemical staining for tau (mab AT8) reveals neuropil threads, (pre)tangles (arrow) and neuritic plaques (arrow head) in the temporal cortex (10x obj.).

Family member II.6 was evaluated at our memory clinic at the age of 70 years because of the positive family history of dementia. At this visit, this family member reported no cognitive complaints, MMSE was 30/30 and neuropsychological testing showed no abnormalities except for some difficulties with concentration. Routine blood analysis showed no abnormalities. MR imaging displayed no hippocampal atrophy (MTA grade 0), mild WMH (Fazekas grade 1), but a high number of 47 microbleeds, suggestive of CAA (Figure 3). EEG showed a remarkably decreased background pattern with reactive alpha-theta activity till 8 Hz. CSF analysis showed a decreased Aβ level of 232 ng/L (reference >550 ng/L), an increased total tau level of 993 ng/L (reference ≤ 375ng/L), and an increased level of tau phosphorylated at threonine-181 (ptau) of 123 ng/L


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(reference ≤ 52 ng/L)). Based on the clinical examination, the family member was diagnosed with subjective cognitive decline (SCD)24. During the following years, the family member developed memory complaints, loss of initiative and sleeping problems. At the age of 74 years, MMSE was 29/30, and neuropsychological testing showed disturbances in episodic memory. MRI showed no progression of WMH, but the number of microbleeds had increased to 58. Repeated EEG displayed progressive slowing with theta activity of 7 Hz next to dominant posterior rhythms of 8 Hz. The family member was diagnosed with MCI. The family member was diagnosed with probable AD by a local geriatrician at the age of 82, with a MMSE score of 21 out of 30. Figure 3. Baseline MR imaging of subject II.6.

Cerebral MRI imaging of subject II.6 at age 70 showing several microbleeds (arrows). T2 weighted image.

Family member III.1 presented at our memory clinic at 58 years with memory complaints, and self-reported difficulties with organizing and planning. This family member was treated for diabetes mellitus, hypertension and dyslipidaemia. MMSE score was 29/30, performance on neuropsychological testing was normal, and MRI showed no abnormalities. EEG was disturbed with a normal alpha background pattern of 9 Hz, but with early intermitting left predominant temporal theta activity. CSF concentrations showed a mildly decreased Aβ level of 549 ng/L, increased tau level of 435 ng/L and ptau level of 68 ng/L. Pittsburgh compound (PiB)-PET showed increased Aβ binding in all cortical areas. F18-fluorodeoxyglucose (FDG)-PET showed a normal pattern of glucose metabolism. Based on clinical evaluations, the diagnosis was SCD.

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The abnormal AD biomarkers indicated preclinical AD, with a high likelihood of underlying Alzheimer pathophysiology25. Over the next four years, the family member remained clinically stable. Generation 0 and I: Family member I.2 died of cancer at age 55. Family member 1.3, a sibling of I.2, was reported to have dementia onset at 61. Family member 1.4, also a sibling of I.2, was reported to have cognitive decline prior to death at age 73. One of their parents (generation 0) was reported to have dementia symptoms suggestive of Alzheimer’s disease at age 80, Family member I.1 (partner from I.2) died at the age of 85 years from a heart attack and was reported to have had dementia with AD characteristics. For these family members, no formal diagnosis was available at the time and no DNA was secured. The unaffected family members in generation III for whom DNA was available (seven individuals merged into III.2, III.3, III.4-5 and III.6) were aged 47 to 60 years and self-reported no cognitive complaints. Their partner and/or a close relative confirmed absence of signs of cognitive impairment. No formal cognitive tests were performed in these family members. APOE genotype distribution in family structure All affected family members with for whom DNA was available (II.3, II.4, II.6, III.1) were homozygous for the APOE-ɛ4 allele. Of the seven unaffected family members from generation III for whom DNA was available, two were genotyped APOE-ɛ4/ɛ4, four were APOE-ɛ3/ɛ4, and one was APOE-ɛ3/ɛ3 (Table S1A, Figure 1). Given the frequency of all APOE genotype combinations in the Dutch population, disease status of each family member by age, and the APOE genotypes for all individuals in generations II and III, we estimated a 65% chance that individuals I.1 and I.2 are APOE-ɛ3/ɛ4 and APOE-ɛ4/ɛ4; and we estimated a 29% chance that individuals are both APOE-ɛ3/ɛ4 (grey text in Figure 1). Chances for other possible genotypes were negligible (Table S1D). Likewise, we estimated a 76% chance that the APOE genotype of individual II.1, who was diagnosed with AD at 69, was APOE-ɛ4/ɛ4: and a 22% that it was APOE-ɛ3/ɛ4; chances for other possible genotypes were negligible (Table S1E). Individual II.8 has a 87% chance of being APOE-ɛ3/ɛ4 and a 12% chance of being APOE-ɛ3/ɛ3 (Table S1F). Together, this provides support that this family included at least three generations of APOE-ɛ4 homozygotes.


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Chances of developing AD in this family: a statistical analysis. We evaluated the AD incidence in this family w.r.t. empirical age at onset distributions extracted from a Caucasian cohort, given APOE genotype and gender [10] (Table S1C). The age of AD onset in this family was significantly earlier than expected: p=0.0001 and p=0.00006 using respectively Fisher’s method and Stouffer’s method of combining p-values. P-values remain significant when we use the age at diagnosis, suggesting that the finding is robust: p=0.0290 and p= 0.0094 using Fisher’s method and Stouffer’s method respectively (Table S1A). Of note, the high incidence of AD may have stimulated family members to visit our hospital for cognitive testing. This may have introduced a bias at the age at AD onset level that we cannot account for. However, further family research revealed that I.3 and I.4 (two siblings of I.2, who died of cancer at age 55) were reported to have dementia at relatively early ages at onset irrespective of family bias (61 and 73 years respectively). Exome sequencing outcome To investigate whether additional genetic variants occurred next to APOE-ɛ4 homozygosity, we performed exome sequencing in two siblings with AD (II.3, II.6), in one family member with preclinical AD (III.1) and in the eldest participant without cognitive complaints hitherto (III.2), all homozygous for the APOE-ɛ4 allele. Exome sequencing revealed no mutations in the PSEN1, PSEN2 and APP genes in any of the family members. We detected 16 variants that passed filtering, several of which were rare and predicted to have a deleterious effect on protein function (Table S2). Two of these occurred in a gene that might be functionally associated with amyloid processing or Alzheimer’s disease: (i) the missense variant c.2021A>G, p.Asn674Ser, in exon 14 of the sortilin related receptor 1 (SORL1) gene (NM.003105.5), and (ii) c.707C>T, p.Thr236Met, in exon 2 of the teashirt zinc finger homeobox 3 (TSHZ3) gene (NM.020856.2). The coverage of SORL1 and TSHZ3 captured with the exome kit was similar to the median read depth over the whole exome. This SORL1 variant was present in all three affected family members and the family member with preclinical AD, and in four unaffected family members (aged <60 years). The variant was not detected in any other subject in the Amsterdam Dementia cohort. One study detected the variant in a 63-year old healthy female (control group n=1938, MAF <0.001)26, and the ExAC database reports only one heterozygous carrier of this variant (MAF < 0.00001). The variant locus is at a highly conserved glycosylation site in the VPS10 domain of SORL1 (Figure 4), it has a CADD score of 23.6, and it is

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considered deleterious by PolyPhen and Mutation Taster (although not by SIFT). The p.Thr236Met missense variant in TSHZ3 was detected in two of the three family members with AD, in the family member with preclinical AD and in one unaffected family member, who was also homozygous for APOE-ε4 but did not carry the SORL1 variant. This TSHZ3 variant is located in a highly conserved amino acid, has a CADD score of 27.0 and is predicted to be deleterious by PolyPhen, Mutation Taster and SIFT. The variant was reported in 47 individuals in the ExAC database (MAF <0.001). Figure 4. p.Asn674Ser in the SORL1 protein.

SORL1 is located on chromosome 11q23.2-q24.2 and codes for a 250-kDa membrane protein with seven distinct domains. Black: extracellular domains; grey: intracellular domain; arrow: the p.Asn674Ser variant we identified in this family; P: Pro-peptide; VPS10: vacuolar protein sorting domain 10; LDLR-B: LDL-receptor class B repeats; EGF: epidermal growth factor precursor type repeat; LDLR-A: LDL-receptor class A repeats; FN-III: fibronectin type-III repeats; IC: intracellular component; LDL: low density lipoprotein. The figure is based upon information from Uniprot (http://www.uniprot.org/uniprot/Q92673), transcript NM_003105. DISCUSSION We describe a family with an inheritance pattern suggestive of autosomal dominant AD of which all affected family members tested were homozygous for the APOE-ε4 allele. The age at AD onset was significantly earlier than expected, based on the APOE genotypes and gender of family-members, suggesting that next to a high load of APOE-ɛ4, this family is relatively enriched with other AD-associated elements. Whole exome sequencing revealed two additional variants co-inherited with APOE-ε4 homozygosity that might disturb Aβ processing: a rare missense variant leading to p.Asn674Ser in the SORL1 protein and a rare missense variant leading to p.Thr236Met in the TSHZ3 protein. We speculate these SORL1 and TSHZ3 variants increased the penetrance of AD in this family. Without APOE-ε4 homozygosity, neither variants may reach full AD penetrance, as can be observed in the pedigree where several APOE-ε4 heterozygous or APOE-ε4 negative family members carry the SORL1 and/or TSHZ3 gene variants without having the disease. It should be noted however, that these family members are


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still young, and may develop AD at a later age, such that we cannot exclude the possibility that either variant confers full AD penetrance. For the same reason we cannot (yet) identify the effect of APOE-ε4 gene dosage, by analysing to what extent impaired SORL1 and/or TSHZ3 modulates AD penetrance in a background of heterozygous APOE-ε4. Moreover, this family carried several additional rare genetic variants, some of which were predicted to have a deleterious effect on protein function. Although these variants map in genes that are currently not associated with AD, we cannot a priori rule out that they modulate AD susceptibility in this family. TSHZ3 may modulate Aβ processing The rare variant detected in TSHZ3 is predicted to have a damaging effect on protein function by all effect predictor algorithms, and has a relatively high CADD score of 27. Although evidence is limited, this rare variant might complicate Aβ processing since the TSHZ3 protein has been found to bind to FE65, an adaptor protein that can modulate APP trafficking and/or processing27. TSHZ3 downregulates Caspase 4, which is involved cell death induced by cytotoxic APP peptides28-30. However, more evidence is needed to link disturbed TSHZ3 protein function to AD. Impaired SORL1 increases Aβ production In sharp contrast with TSHZ3, evidence has accumulated that impaired SORL1 function associates with AD: common genetic polymorphisms in the SORL1 locus were associated with AD in GWAS studies31, disruptive variants were only detected in AD cases and not in controls26 and rare pathogenic SORL1 variants were found to increase the risk for EOAD by five-fold32. Functional studies suggested that the SORL1 sorting receptor has a dual function: (i) SORL1 binds the amyloid precursor protein APP and prevents it from processing into Aβ33, and (ii) SORL1 binds newly synthesized extracellular Aβ and targets it to the lysosome for degradation34. To exert these functions, SORL1 has two important protein domains: an APP-binding complement type repeat domain, and an Aβ-binding VPS-10 domain33. The p.Asn674Ser variant in SORL1 that we detected in this family affects a highly conserved N-glycosylation site in the VPS10 domain, which is important for proper protein folding and for protein-protein interaction33. Previously, variants that map in the VPS10 domain (p.Glu270Lys, p.Ala528Thr) were associated with impaired retrograde sorting of APP and enhanced Aβ production when expressed in cells35, suggesting that a wild-type VPS10 domain is essential for proper Aβ processing. We speculate that the

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p.Asn674Ser change detected in this family may at disrupt the Aβ-binding capacity of SORL1, resulting in less efficient lysosomal degradation of Aβ34. Combined effect of APOE-ε4 homozygosity, impaired SORL1 and impaired TSHZ3 Experimental studies suggest that the proteins encoded by the APOE and SORL1 genes functionally interact36. By binding to the complement type repeats of the SORL1 protein, ApoE4 reduced the APP-binding-capacity of SORL137. Furthermore, overexpression of SORL1 increases the uptake of extracellular Aβ in an ApoE-isoform-dependent manner, most efficiently in the presence of the ε4 isoform36. Therefore, the clearance of Aβ is expected to be more dependent on SORL1 expression in APOE-ε4 carriers than in individuals with no APOE-ε4 alleles. A combination of homozygous or heterozygous ApoE4 and dysfunctional SORL1 may therefore lead to abnormal increases in extracellular Aβ loads, which may underlie the neurodegenerative processes in this family. Effect of the genotype on phenotype Homozygous APOE-ε4 carriers typically present with an amnestic phenotype, however the AD phenotype of the five affected family members for whom detailed clinical data were available, was heterogeneous. The age at onset differed between affected family members and ranged between 61 and 74 years, which fits with the relatively early age of disease onset associated with APOE-ε4 homozygosity38. Homozygous APOE-ε4 and disrupted SORL1 are both associated with cerebral amyloid angiopathy (CAA), presumably as a result of the less effective Aβ clearance39-41. Indeed, two family members with AD had extensive microbleeds and CAA, while two others remained free of microbleeds. Both APOE and SORL1 are involved in cholesterol metabolism/transport, and APOE-ε4 carriers have been found to have increased cholesterol levels42,43. Indeed, three affected family members were diagnosed with hypercholesterolemia in this family. Likewise, the EEG pattern of APOE-ε4 allele carrier-patients shows a greater decrease of alpha activity than non-APOE-ε4 carrier-patients44,45. However, in this family, neither microbleeds/CAA, hypercholesterolemia or EEG patterns cosegregated with APOE-ε4 homozygosity and/or the SORL1 variant.


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CONCLUSION We hypothesize that the convergence of multiple genetic factors that disturb the Aβ processing pathways over several generations results in an autosomal dominant like inheritance pattern of AD in this family. Extracellular Aβ load might be abnormally increased as a result of the concerted effects of (i) ineffective clearance of extracellular Aβ from the brain by the ApoE4 protein isoform, and (ii) impaired uptake of extracellular Aβ for lysosomal degradation due to a disturbed VPS10 domain of the SORL1 protein. It is possible that a disturbed TSHZ3 function might have further contributed to impaired APP regulation, but compared to ApoE4 and SORL1, the evidence for an association of disrupted TSHZ3 with AD is currently very limited. Moreover, other genetic variants that were left undetected with our analysis strategy might have further influenced disease penetrance. Given these findings, the currently unaffected family member who is homozygous for APOE-ε4 and who carries the SORL1 variant may be at the highest risk to develop AD. Follow-up of this family in the future will resolve these speculations. We expect that this polygenic model, possibly involving other genetic variants, might also explain autosomal dominant inheritance patterns in other APOE-ε4 positive families. ACKNOWLEDGEMENTS The authors thank the participating patients and relatives. Research of the VU University Medical Centre’s Alzheimer Centre is part of the neurodegeneration research program of the Neuroscience Campus Amsterdam. The Alzheimer Centre is supported by Alzheimer Nederland and Stichting VUmc.

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19. Folstein, MF, Folstein, SE, and McHugh, PR. "Mini-mental state". A practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res. 1975; 12:189-198.

20. Scheltens, P, Launer, LJ, Barkhof, F, et al. Visual assessment of medial temporal lobe atrophy on magnetic resonance imaging: interobserver reliability. J Neurol. 1995; 242:557-560.

21. Fazekas, F, Chawluk, JB, Alavi, A, et al. MR signal abnormalities at 1.5 T in Alzheimer's dementia and normal aging. AJR Am J Roentgenol. 1987; 149:351-356.

22. Albert, MS, DeKosky, ST, Dickson, D, et al. The diagnosis of mild cognitive impairment due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease. Alzheimers Dement. 2011; 7:270-279.

23. Thal, DR, Ghebremedhin, E, Rub, U, et al. Two types of sporadic cerebral amyloid angiopathy. J Neuropathol Exp Neurol. 2002; 61:282-293.


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24. Jessen, F, Amariglio, RE, van, BM, et al. A conceptual framework for research on subjective cognitive

decline in preclinical Alzheimer's disease. Alzheimers Dement. 2014; 10:844-852. 25. Sperling, RA, Aisen, PS, Beckett, LA, et al. Toward defining the preclinical stages of Alzheimer's

disease: Recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease. Alzheimers Dement. 2011; 7:280-292.

26. Verheijen, J, Van den Bossche, T, van der Zee, J, et al. A comprehensive study of the genetic impact of rare variants in SORL1 in European early-onset Alzheimer's disease. Acta Neuropathol. 2016; 132:213-224.

27. Kajiwara, Y, Akram, A, Katsel, P, et al. FE65 binds Teashirt, inhibiting expression of the primate-specific caspase-4. PLoS One. 2009; 4:e5071.

28. Hitomi, J, Katayama, T, Eguchi, Y, et al. Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Abeta-induced cell death. J Cell Biol. 2004; 165:347-356.

29. Sollberger, G, Strittmatter, GE, Kistowska, M, et al. Caspase-4 is required for activation of inflammasomes. J Immunol. 2012; 188:1992-2000.

30. Li, C, Wei, J, Li, Y, et al. Transmembrane Protein 214 (TMEM214) mediates endoplasmic reticulum stress-induced caspase 4 enzyme activation and apoptosis. J Biol Chem. 2013; 288:17908-17917.

31. Lambert, JC, Ibrahim-Verbaas, CA, Harold, D, et al. Meta-analysis of 74,046 individuals identifies 11 new susceptibility loci for Alzheimer's disease. Nat Genet. 2013; 45:1452-1458.

32. Nicolas, G, Charbonnier, C, Wallon, D, et al. SORL1 rare variants: a major risk factor for familial early-onset Alzheimer's disease. Mol Psychiatry. 2016; 21:831-836.

33. Willnow, TE and Andersen, OM. Sorting receptor SORLA--a trafficking path to avoid Alzheimer disease. J Cell Sci. 2013; 126:2751-2760.

34. Caglayan, S, Takagi-Niidome, S, Liao, F, et al. Lysosomal sorting of amyloid-beta by the SORLA receptor is impaired by a familial Alzheimer's disease mutation. Sci Transl Med. 2014; 6:223ra20.

35. Vardarajan, BN, Zhang, Y, Lee, JH, et al. Coding mutations in SORL1 and Alzheimer disease. Ann Neurol. 2015; 77:215-227.

36. Yajima, R, Tokutake, T, Koyama, A, et al. ApoE-isoform-dependent cellular uptake of amyloid-beta is mediated by lipoprotein receptor LR11/SorLA. Biochem Biophys Res Commun. 2015; 456:482-488.

37. Perneczky, R, Alexopoulos, P, Eisele, T, et al. Does the Apolipoprotein E Genotype Influence Amyloid Precursor Protein Sorting by Sortilin-Related Receptor: Implications for Alzheimer's disease? Med Hypotheses Res. 2010; 6:19-24.

38. van der Flier, WM, Pijnenburg, YA, Fox, NC, et al. Early-onset versus late-onset Alzheimer's disease: the case of the missing APOE varepsilon4 allele. Lancet Neurol. 2011; 10:280-288.

39. Rannikmae, K, Samarasekera, N, Martinez-Gonzalez, NA, et al. Genetics of cerebral amyloid angiopathy: systematic review and meta-analysis. J Neurol Neurosurg Psychiatry. 2013; 84:901-908.

40. Bu, G. Apolipoprotein E and its receptors in Alzheimer's disease: pathways, pathogenesis and therapy. Nat Rev Neurosci. 2009; 10:333-344.

41. Schuur, M, van Swieten, JC, Schol-Gelok, S, et al. Genetic risk factors for cerebral small-vessel disease in hypertensive patients from a genetically isolated population. J Neurol Neurosurg Psychiatry. 2011; 82:41-44.

42. van, VP. Cholesterol and late-life cognitive decline. J Alzheimers Dis. 2012; 30 Suppl 2:S147-S162. 43. Reitz, C. Dyslipidemia and the risk of Alzheimer's disease. Curr Atheroscler Rep. 2013; 15:307. 44. Ponomareva, NV, Korovaitseva, GI, and Rogaev, EI. EEG alterations in non-demented individuals

related to apolipoprotein E genotype and to risk of Alzheimer disease. Neurobiol Aging. 2008; 29:819-827.

45. de, WH, Stam, CJ, Blankenstein, MA, et al. EEG abnormalities in early and late onset Alzheimer's disease: understanding heterogeneity. J Neurol Neurosurg Psychiatry. 2011; 82:67-71.


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SUPPLEMENTAL MATERIAL Supplemental material available on publication (http://content.iospress.com/articles /journal-of-alzheimers-disease/jad160091#S1) and on request.

Table of contents Supplemental Table 1: APOE genotype estimates. Supplemental Table 1A: Pedigree annotation. Supplemental Table 1B: APOE genotype population frequency (LASA). Supplemental Table 1C: Lifetime AD risk by age, per APOE genotype and gender. Supplemental Table 1D: Probabilities of APOE genotypes for individuals I.1 and I.2. Supplemental Table 1E: Probabilities of APOE genotypes for II.1. Supplemental Table 1F: Probabilities of APOE genotypes for II.8, who has an

APOE-ε3/ε3 child. Supplemental Table 2: Variants detected by whole exome sequencing.


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Chapter 5

Summary

SUMMARY

In the first part of this thesis, we focus on the clinical aspects of inherited dementia. Chapter 2.1 provides an overview of genetic causes of dementia. We explained that although dementia is in most cases a complex disease with multiple factors contributing, dementia is Mendelian inherited in a small percentage of the cases. We described that mutations in the presenilin 1 (PSEN1) gene are the most common genetic cause of early onset Alzheimer’s disease (AD), whereas amyloid precursor protein (APP) and presenilin 2 (PSEN2) mutations are less frequent. Frontotemporal dementia (FTD) may be caused by mutations in the microtubule-associated protein tau (MAPT) or (pro)granulin (GRN) gene, or by a hexanucleotide repeat expansion in the chromosome 9 open reading frame 72 (C9orf72) gene. All these genes show autosomal dominant inheritance with a high penetrance. We describe genotype-phenotype correlations and conclude that phenotypes overlap. Furthermore, we provide algorithms for genetic testing in patients with early onset Alzheimer’s disease or frontotemporal dementia, with the most important recommendation to consider offering genetic testing to all patients with bvFTD, both familial and sporadic.

In chapter 2.2, we describe our study on the effect of predictive testing of individuals at risk of Huntington’s disease (HD) or familial FTD. Since most follow-up studies on predictive testing for neurodegenerative diseases focussed on psychological outcome, we aimed to investigate whether the life of mutation carriers of adult-onset neurodegenerative diseases differs negatively from non-carriers and untested at risk individuals. We invited individuals aged ≥35 years, tested while asymptomatic for HD, FTD or AD more than 2 years before the start of the study or at 50% risk for one of these diseases, to complete a questionnaire of 70 items and an additional questionnaire of 47 items sent within a year afterwards. Of the selected individuals, 115 (39,6%) were willing to participate. Of these, 17 carriers, 30 non-carriers and 27 untested persons fulfilled the criteria and completed both questionnaires. We found no significant differences between carriers and non-carriers or untested individuals at risk in employment, financial situation and lifestyle or anxiety and depression. Carriers were more often single and childless, though these differences were not significant. Although the outcome of this study is likely to be influenced by a response bias, these findings suggest that in most individuals, an unfavourable outcome of predictive testing on adult onset neurodegenerative diseases does not have a large negative effect on social and personal life.

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In chapter 3, we investigated the frequency of causative mutations in patients with dementia and the associated phenotype. In chapter 3.1, we describe the clinical and neuropathological characteristics of hexanucleotide repeat expansions in the C9orf72 gene in a large cohort of Dutch patients with FTD. We determined the repeat length in a cohort of 353 patients with sporadic or familial FTD with or without amyotrophic lateral sclerosis (ALS), and 522 neurologically normal controls, We identified hexanucleotide repeat expansions in 37 (28.7%) of the individuals with familial FTD and 5 (2.2%) of the sporadic FTD patients. In the repeat expansion carriers, the mean age at onset of the FTD was 56.9 ± 8.3 years (range 39-76), and the mean disease duration 7.6 ± 4.6 years (range 1-22). Most patients with the C9orf72 repeat expansion had behavioural variant FTD (bvFTD) (n = 34), and 7 patients had concomitant amyotrophic lateral sclerosis (ALS). Neuroimaging was characterized by predominant temporal atrophy in 13 of 32 patients. Pathological examination showed frontotemporal lobar degeneration with neuronal transactive response DNA binding protein (TDP)-positive inclusions of variable type, size and morphology in all 10 investigated brains. In chapter 3.2, we investigated the frequency of causative mutations in the common dementia genes in a cohort of patients with early onset dementia. Furthermore, we investigated whether mutations in the recently identified gene PRKAR1B were present in this cohort. We performed mutation analysis of the genes PSEN1, APP, MAPT, GRN, C9orf72 and PRKAR1B on DNA of 229 patients with the clinical diagnosis AD and 74 patients with the clinical diagnosis FTD below the age of 70 years. We found PSEN1 and APP mutations in respectively 3.5% and 0.4% of AD patients, and none in FTD patients. C9orf72 repeat expansions were present in 0.4% of AD and in 9.9% of FTD patients, whereas MAPT and GRN mutations were both present in 0.4% in AD patients and in 1.4% resp. 2.7% in FTD patients. We did not find any pathogenic mutations in the PRKAR1B gene. We concluded that in Dutch patients with early onset dementia, PSEN1 mutations are the most common genetic cause, though rare, in AD and C9orf72 repeat expansions the most common mutation in FTD patients. PRKAR1B mutations are probably rare in Dutch patients with a clinical diagnosis of early onset AD or FTD.

Chapter 4 describes the genetic defects and the associated phenotype of two families with assumed autosomal dominant dementia. In chapter 4.1, we present three siblings with cognitive complaints, reduced amyloid-beta-42 in CSF and multiple cerebral lobar microbleeds, and a positive family history for autosomal dominant early onset dementia. With whole exome sequencing, we identified in all three siblings a novel frame shift variant generating a premature stop codon in the CCM2 gene, one of the genes

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associated with familial cerebral cavernous malformations. We did not find this variant in a cohort of 363 patients with early onset dementia, with or without multiple lobar microbleeds, or in control databases including the Dutch genetic biobank GoNL. Furthermore, two siblings were homozygous for APOE-ε4 and one heterozygous. Two of the patients had cerebral cavernous malformations, confirming the diagnosis familial cerebral cavernous malformations. The observed microbleeds could be due to the APOE-ε4, but could also be in fact bleeds from very small cavernous malformations. The cognitive complaints, reduced amyloid-beta-42 in CSF and microbleeds suggest preclinical AD, but the stability of the cognitive complaints does not. We hypothesized that both the CCM2 variant and the APOE status may have contributed to the phenotype. In chapter 4.2, we describe a family with clinically heterogeneous AD and an assumed autosomal dominant family history, in which all four genotyped affected family members were homozygous for the APOE-ε4 allele. Affected family members presented with a mean age of symptom onset of between 61 and 74 years, with variable presence of microbleeds on cerebral imaging and electroencephalographic abnormalities. We performed exome sequencing on three affected and one unaffected family and identified a rare pathogenic variant in the VPS10 domain of the AD-related SORL1 gene. Segregation analysis showed that his variant was present in all four affected and one unaffected family member. Furthermore, three affected family members and one unaffected family member carried a rare pathogenic variant in the TSHZ3 gene. Both SORL1 and TSHZ3 are involved in the amyloid pathway, and SORL1 variants are associated with an increased risk of AD. We hypothesized a combined effect of Apoe4, the SORL1 variant of possibly also the TSHZ3 variant on AD development. Furthermore, we speculated that the convergence of multiple genetic factors over several generations might also clarify the autosomal dominant-like inheritance pattern of AD in other families.

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Chapter 6

General discussion

GENERAL DISCUSSION

In the previous chapters, we described our research on different aspects of inherited dementia: genetic counselling and testing in patients and unaffected family members, the prevalence of mutations and the associated phenotypes, and our search for new genetic causes of cognitive decline. In this section, the results of these research projects and future perspectives are discussed.

Genetic counselling and testing in patients with adult onset neurodegenerative diseases Chapter 2.1 is a state-of-the-art of the genetic background of the different types of dementia and recommendations for genetic testing in dementia patients. This manuscript was published just shortly after the identification of the C9orf72 gene and therefore one of the first articles including information on this gene. New genetic causes of dementia have been identified since the publication in 2012, however most of them are rare and explain the disease in just one or a few families. The most important new finding in AD is a rare variant in the triggering receptor expressed on myeloid cells 2 gene (TREM2). Mutations in this gene were initially discovered in patients with Nasu–Hakola disease, a very rare autosomal recessive early-onset dementia syndrome. The risk of AD in patients with the Arg47His variant in this gene is estimated to be increased two to four fold,1,2 comparable to the increase in risk associated with APOE-ε4 heterozygosity. In FTD, mutations the TANK-binding kinase 1 gene (TBK1) have been described by a Belgian research group as the most common cause of unexplained familial FTD in their country 3 and have since been found in other FTD and ALS cohorts as well.4-7 The associated phenotype is ALS, bvFTD but also language variant FTD, and FTD-MND.8 It would be interesting to study the prevalence of mutations in this gene and the associated phenotype in our Dutch FTD and ALS cohorts. In the Netherlands, genetic testing by sequential Sanger analysis has been replaced to a great extent by whole exome sequencing (WES) based gene panel analysis, allowing parallel testing of a large amount of genes. Since most gene panels encompasses genes rarely mutated or associated with diseases with overlapping symptoms, causative variants may be found in genes less obvious. These variants would probably been missed by serial Sanger sequencing. Since the use of WES based gene panels is likely to result in broadening of the disease spectrum of genetic defects and in an increase of the mutation detection rate, these gene panels are preferable above sequential Sanger sequencing. However, not all countries have NGS facilities. Therefore, the algorithms

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proposed in chapter 2.1 for sequential sequencing in dementia patients are still useful abroad. Because of the poor coverage of repeat expansions by exome sequencing, WES-based gene panels should be supplemented in combination with repeat length analysis, most importantly of the C9orf72 hexanucleotide repeat. Since co-occurrence of a C9orf72 repeat expansion and another pathogenic mutation has occurred in several patients,9 C9orf72 analysis should be performed regardless the results of the WES-based analysis. When no mutations are found in patients with strong indications of a genetic cause, such as multiple affected family members or an strikingly young age at onset, an open WES without filtering for dementia related genes may be considered. Identifying (all) mutations in a family is extremely important in order to be able to exclude family members from an increased risk. While genetic testing has been common practice in patients suspected of HD for decades, in FTD the presence of a pathogenic mutation was included in the diagnostic criteria just a few years ago. Moreover, C9orf72 repeat expansions are not only found in familial but also in 6% of the sporadic patients. Therefore, genetic testing should be offered to all FTD patients, regardless their family history. To make sure all eligible patients are offered genetic testing and/or counselling, clinical geneticists should be involved in the selection: directly by involvement in patient care, or indirectly by contributing to the development of protocols for genetic testing. Of note, counselling about the incidental findings in genetic testing needs less attention in dementia patients than in patients with other disorders: an unexpected predisposition for an untreatable late onset disorder is feared most, however, finding such a predisposition is exactly the aim of the test in dementia patients. As mentioned in the introduction, both neurologists and clinical geneticist are allowed to request DNA analysis in affected individuals in the Netherlands. While previously testing was mainly offered to patients suspected of having a disease that is by definition inherited, such as Huntington, or with a strongly affected family history, nowadays genetic testing is also offered to patients less likely to have a causative mutation. This is partly due to the decreased costs, the shorter turnaround time of the genetic tests and the increased focus on genetics in the medical field. Because of this shift in the selection of patients offered testing, a need has arisen for special attention to the individual circumstances of a patient: while in autosomal dominant dementia families the offspring often expect to have an increased risk on the disease, the identification of a causal mutation in sporadic patients may have a huge impact on the relatives, and on family ties. Therefore, proper counselling by a clinical geneticist or trained neurologist and

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informed consent of patients and their relatives before requesting DNA testing is extremely important. This requires a new educating role for clinical geneticists. Predictive testing for untreatable adult onset neurodegenerative diseases We investigated the impact on social and personal life of predictive testing in FTD and HD and found no large effect of an unfavourable test result (chapter 2.2). Previous studies showed that most family members at risk are able to cope with an unfavourable result of predictive testing on adult onset neurodegenerative disorders. However, genetic counsellors are unable to inform these at risk family members about the expected influence of a unfavourable test result on their lives. We designed the study as an exploratory study, with the aim to optimize the questionnaires and to identify trends that may be interesting to further investigate in a larger cohort. In our small cohort, we found no significant differences between carriers as a group compared to non-carriers and untested individuals at risk, and only a possible, non-significant, difference in personal life, especially in relations. The study needs to be validated in a larger cohort before any results can be implemented in counselling sessions in the clinical setting. Performing this study in a larger cohort may also enable subgroup analysis, for example in carriers tested at a young age. If such a subgroup analysis would identify a subgroup of patients at increased risk of negative effects of the predictive testing, special care could be offered to patients of this subgroup. Ideally, we would perform a prospective study, in order to have information on both pre- and post-test state, which would also give more background information on the non-responders. Because of the (close to) complete penetrance of mutations in the included families, testing of asymptomatic family members was indeed predictive testing in our study. Patients with C9orf72 repeat expansions were not included because no individuals had been tested more than 2 years before the onset due to the recent discovery of this gene. In contrast to the extreme high penetrance of MAPT and GRN mutations, the penetrance of the C9orf72 repeat expansion is reduced, although exact risk estimates are not available. Moreover, carriers of C9orf72 repeat expansions are not only at risk of FTD but also of ALS. Therefore, testing of asymptomatic family members at risk of dementia is strictly speaking not predictive testing when it concerns the C9orf72 gene. In order to be able to offer carriers a better prediction about their future, it would be interesting to further investigate the penetrance curve of C9orf72 repeat expansions by testing extended families with C9orf72 repeat expansions and clinically follow-up family members at risk. Moreover, further studies on the impact of an unfavourable test result should evaluate the effect of the uncertainties for C9orf72 repeat expansion carriers.

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Another issue in ‘predictive’ testing is the variability of the phenotype associated with a mutated gene. Especially in families with C9orf72 repeat expansions, the age at onset and course of the disease may differ, also within families.10,11 Therefore, carriers have to deal with many uncertainties. Since this variability may be explained by both genetic and non-genetic factors, future studies should not only focus on genetic modifying factors, but also on other factors associated with an increased risk of neurological diseases, such as vascular risk factors and auto-immune disorders. Prevalence and phenotypes of inherited dementia The prevalence of mutations and the phenotype associated with a genetic defect are relevant when estimating the probability of a genetic cause in a patient. In chapter 3.1, we describe that C9orf72 repeat expansions are the most common genetic defect in Dutch FTD patients. For this study, we selected patients by their diagnosis of FTD. More recent studies showed that the FTD in C9orf72 repeat expansion carriers may be preceded by psychiatric disorders, mainly psychosis 12, and that part of the patients do not fulfil the clinical criteria of bvFTD, often because their symptoms are hardly progressive13. Therefore, a selection bias may have resulted in an underestimate of the prevalence of C9orf72 repeat expansions. As mentioned before, the phenotype of C9orf72 repeat expansions is extremely variable. Although bvFTD or ALS are the most common manifestations, the C9orf72 repeat expansion is also found in patients with the language variants of FTD and in patients fulfilling the clinical criteria for AD or DLB14. The only known genetic modifier in C9orf72, protecting against FTD, is the TMEM106B gene,15,16 also known as a modifier in GRN-related FTD and as a genetic risk factor for sporadic FTLD.17,18 Because of the limited and yet uncertain effect size, this gene is not included in most gene panels for neurodegenerative disorders. Further research on co-occuring of genetic variants or polymorphisms may result in a more reliable prediction about the course of the disease in both patients and unaffected mutation carriers. In chapter 3.2, we investigated the frequency of mutations in patients of our tertiary memory clinic with early onset dementia, and found a mutation frequency in the AD patients of 5%, low compared to other studies19. The mutation frequency was 14% in FTD patients, which is also relatively low but explained by enrichment of previously studied cohorts by patients with an autosomal dominant family history. We used Sanger sequencing, because WES was not available when we started this study. We chose not to test the cohort on mutations in the PSEN2 gene, as this gene defect is extremely rare

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and has never been found in the Netherlands. Therefore, we still do not know whether PSEN2 mutations occur in the Dutch population. In both studies of chapter 3, the C9orf72 hexanucleotide repeat length was determined by repeat-primed polymerase chain reaction (rpPCR), a commonly used method but unsuitable to determine the exact repeat length of expanded alleles >70-80 units. We used the a cut-off value of 30, commonly used though controversial. This is partly due to the limited knowledge on the effect of short repeat expansions: while one study found no evidence with functional studies of pathogenicity of a repeat of 70,20 another research group identified repeats of 47 – 80 units in FTD and ALS patients.21 The repeat length may differ between leukocytes and brain tissue due to somatic mosaicism, therefore testing in leukocytes may result in false positive, and may be also in false negative results. Another possible explanation of the variability in pathogenicity of short repeat is a reduced penetrance of these alleles. Autopsy studies and large cohort studies using Southern blot hybridization may solve this issue. In both studies of chapter 3, we did not take into account the possibility of two mutations in one patient: FTD patients with known pathogenic mutations in GNR or MAPT were not tested on C9orf72 repeat expansions in the first study, and in the second study, we ended the genetic analysis as soon as we identified a pathogenic mutation. Although double mutations are rare, we may have missed them, especially since C9orf72 is often one of the mutated genes in de described cases with multiple mutations9 and the rpPCR was our final test. Identification of new genetic causes of cognitive decline In the last chapter, we describe the genetic findings and the phenotype in two families. These families participated in a study we set up with the aim to identify novel dementia genes (GENDEM: Searching for GENes in families with frontotemporal dementia, dementia with Lewy bodies and early-onset Alzheimer DEMentia). In the first part of this chapter (chapter 4.1), we describe a family with cognitive complaints, multiple microbleeds and a positive family history for early onset AD. However, the diagnosis AD was reconsidered in the affected sibling during the study and in the other two siblings, the cognitive complaints did not progress over time. The identified CCM2 mutation and the APOE-ε4 alleles together probably explain the phenotype in this family. Autopsy may support (or reject) this hypothesis. A study on the prevalence of mutations in the CCM genes in patients with multiple microbleeds and no or just one cavernous malformation may answer the question whether FCCM should be considered in patients with this phenotype.

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In chapter 4.2, we report on the findings in a family with presumed autosomal dominant Alzheimer’s disease, in which a few family members had striking electroencephalographic abnormalities and/or microbleeds. All affected family members of this family turned out be homozygous for the APOE-ɛ4 allele. Although this may have had restrained us from selecting this family for our research purpose of identifying novel dementia genes if we had known the APOE genotype prior to performing the WES, this family actually gave us a nice opportunity to search for genetic factors co-occurring with the APOE-ɛ4 allele. We found two variants in genes involved in the amyloid pathway: SORL1 and TSHZ3. The best strategy would be to test the functional effect of the combined effect of SORL1, ApoE4 and perhaps also TSHZ3 in a cell or animal model. However, since we hypothesize an APOE-ɛ4 dose-dependent effect on the SORL1 function, such functional studies are probably complex. While SORL1 is a known modifying factor in AD, and perhaps also a cause of the AD in a few families, our manuscript is unique in describing the phenotype of a family with both APOE-ε4 and a SORL1 variant. Although we found very interesting results in these two families, we did not succeed in our aim to identify novel genetic causes of autosomal dominant dementia. The main difficulty was to find families with multiple affected family members of whom DNA was available. Since hardly any novel autosomal dominant inheriting causes have been identified during the last decades, the question arises whether the unexplained familial AD cases are indeed caused by autosomal dominant inheriting mutations. More likely, familial AD is in most cases an oligogenic disease, as we hypothesize in our secondly described family. Moreover, environmental factors may be involved in the assumed heritability: monozygotic twins do not only share their DNA, but often also their environment and lifestyle to a greater extent than dizygotic twins. Further studies on the aetiology of familial dementia should therefore not only focus on rare single mutations, but also on variants with reduced penetrance, combinations of genetic variants and on epigenetic changes and non-genetic risk factors. Other future directions Because of the increasing incidence and thereby growing burden of dementia on society, the greatest gap is the lack of a cure or prevention for dementia. Since genetic studies may help to understand the aetiology, the identification of genetic variants involved in the aetiology is important. As mentioned in the previous paragraph, future genetic studies should not only focus on rare single mutations, but also on variants with

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reduced penetrance, combinations of genetic variants, epigenetic changes and the combination of genetic variants and non-genetic risk factors. Families with autosomal dominant inherited dementia are extremely useful in research on both the course of the disease and treatment, because future dementia patients can be identified and recruited in trials prior to the first symptoms. A large international study of the Dominantly Inherited Alzheimer Network (DIAN) indeed investigates the pathophysiological process of AD in carriers by monitoring biomarkers and cognitive function. The prospects for Dutch carriers on the possibility to participate in one of the drug trials of this study are promising. A much larger cohort of individuals at an extremely high risk of AD are persons with Down syndrome. Due to their duplication of chromosome 21, which harbours the APP gene, all individuals with Down syndrome are carriers of autosomal dominant AD. Their intellectual disability and comorbidity complicate follow-up studies, such as neuropsychological testing and lumbar puncture for CSF analysis. However, when selecting Down syndrome patients with a relatively high intelligence and low morbidity, these patients may be very valuable for research on the neuropathology and effect of treatment of AD. Many patients and especially their offspring have questions about the heritability and the risk for asymptomatic family members on dementia. The development of a prediction model based on a polygenetic risk score combined with the effect of other risk factors, such as smoking and hypertension, may be useful to comply (partly) with their request. A recent study on designing a polygenetic risk score showed quite promising results.22 However, since such predictions are only of use when extreme high or low risks can be generated, more knowledge on rare or common variants and their relation with dementia is necessary before such a prediction model can be used in the diagnostic setting. This requires a large cohort of dementia patients and therefore (international) collaboration. In conclusion, recent developments in techniques of genetic testing require a change in the flow of diagnostic genetic testing, but also creates an opportunity to expand our knowledge of genetic causes, modifiers and associated phenotypes rapidly. Hopefully in the near future, this knowledge helps us to develop treatment, cure or even prevention of the devastating diseases causing dementia.

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14. Gomez-Tortosa, E, Prieto-Jurczynska, C, Serrano, S, et al. Diversity of Cognitive Phenotypes Associated with C9ORF72 Hexanucleotide Expansion. J Alzheimers Dis. 2016; 52:25-31.

15. Gallagher, MD, Suh, E, Grossman, M, et al. TMEM106B is a genetic modifier of frontotemporal lobar degeneration with C9orf72 hexanucleotide repeat expansions. Acta Neuropathol. 2014; 127:407-418.

16. Van Blitterswijk M., Mullen, B, Nicholson, AM, et al. TMEM106B protects C9ORF72 expansion carriers against frontotemporal dementia. Acta Neuropathol. 2014; 127:397-406.

17. Cruchaga, C, Graff, C, Chiang, HH, et al. Association of TMEM106B Gene Polymorphism With Age at Onset in Granulin Mutation Carriers and Plasma Granulin Protein Levels. Arch Neurol. 2011; 68:581-586.

18. Van Deerlin, VM, Sleiman, PM, Martinez-Lage, M, et al. Common variants at 7p21 are associated with frontotemporal lobar degeneration with TDP-43 inclusions. Nat Genet. 2010; 42:234-239.

19. Cohn-Hokke, PE, Elting, MW, Pijnenburg, YA, et al. Genetics of dementia: update and guidelines for the clinician. Am J Med Genet B Neuropsychiatr Genet. 2012; 159B:628-643.

20. Xi, Z, van, BM, Zhang, M, et al. Jump from pre-mutation to pathologic expansion in C9orf72. Am J Hum Genet. 2015; 96:962-970. 21. Gijselinck, I, Van, MS, van der Zee, J, et al. The C9orf72 repeat size correlates with onset age of

disease, DNA methylation and transcriptional downregulation of the promoter. Mol Psychiatry. 2016; 21:1112-1124.

22. Escott-Price, V, Sims, R, Bannister, C, et al. Common polygenic variation enhances risk prediction for Alzheimer's disease. Brain. 2015; 138:3673-3684.

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Addendum

NEDERLANDSE SAMENVATTING

INTRODUCTIE

Dementie komt veel voor, met name op oudere leeftijd. Dementie is een term die wordt gebruikt wanneer hersenfuncties zodanig zijn aangetast dat iemands dagelijks functioneren wordt beïnvloed. Er zijn vele aandoeningen die dementie kunnen veroorzaken. De ziekte van Alzheimer is de meest voorkomende oorzaak van dementie. Van de meeste aandoeningen die leiden tot dementie, is de precieze oorzaak onduidelijk. Vermoedelijk is bij de meeste mensen met dementie de oorzaak multifactorieel bepaald. Dit betekent dat vele factoren, zowel genetische als niet-genetische, bijdragen aan het ontstaan. In een deel van de patiënten is de dementie erfelijk. Hiermee wordt bedoeld dat de ziekte die de dementie veroorzaakt (grotendeels) toe te schrijven is aan één verandering (mutatie) in het DNA. Aanwijzingen voor zo’n erfelijke oorzaak zijn onder andere een (zeer) jonge leeftijd bij het ontstaan van de klachten en het voorkomen van de aandoening bij meerdere personen in één familie. Hoe groot de kans op een erfelijke oorzaak van dementie is, hangt af van de onderliggende aandoening. De ziekte van Alzheimer uit zich vaak met geheugenverlies en oriëntatieproblemen. Bij patiënten bij wie de ziekte op jonge leeftijd ontstaat, staan vaak andere klachten op de voorgrond, zoals problemen met waarnemen, handelen of spreken. Wanneer de ziekte van Alzheimer op oudere leeftijd tot uiting komt (ouder dan 65 jaar), wordt zelden een verklarende mutatie in het DNA aangetoond. Wanneer iemand de ziekte al op jonge leeftijd ontwikkelt, is de kans op een mutatie afhankelijk van zijn familieanamnese: wanneer er geen andere familieleden zijn met dementie is de kans op een erfelijke oorzaak klein, maar wanneer ook één van de ouders op jonge leeftijd dementie had, is de kans op een erfelijke oorzaak juist groot. In bijna de helft van de families waarin het zeer waarschijnlijk is dat er een erfelijke aanleg voor de ziekte van Alzheimer speelt, kan met de huidige kennis en technieken de erfelijke oorzaak niet worden aangetoond. Hierdoor is het momenteel niet mogelijk om met DNA-onderzoek een erfelijke aanleg voor de ziekte van Alzheimer uit te sluiten. Frontotemporale dementie (FTD) is een ziekte die vaak al op jonge leeftijd (jonger dan 65 jaar) tot uiting komt. FTD wordt gekenmerkt door gedrags- en karakterveranderingen, en/of taalstoornissen. Ook bij FTD is de kans op een aantoonbare erfelijke oorzaak afhankelijk van de familiegeschiedenis. Echter, bij FTD

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wordt in een klein deel van de patiënten die geen belaste familiegeschiedenis hebben toch een erfelijke aanleg gevonden. Alhoewel nog niet alle erfelijke oorzaken bekend zijn, kan een groot deel van de families met FTD inmiddels verklaard worden door een mutatie in één van de op dit moment bekende genen. Sommige erfelijke oorzaken van FTD, waaronder de in Nederland meest voorkomende, geven niet alleen een verhoogd risico op FTD maar ook op amyotrofische lateraalsclerose (ALS), een ruggenmergaandoening die leidt tot snel progressieve spierzwakte. De ziekte van Huntington is een aandoening die vooral bekend is vanwege de bewegingsstoornissen, zoals willekeurige dansbewegingen (chorea). De ziekte kan zich ook presenteren met gedragsverandering en stemmingswisselingen, zoals ook wordt gezien bij FTD. De ziekte van Huntington wordt altijd veroorzaakt door een te lange herhaling van een drietal bouwstenen (nucleotiden) op een specifieke locatie in het DNA. Daarom is deze ziekte per definitie erfelijk. De erfelijke aandoeningen die dementie veroorzaken erven vrijwel altijd autosomaal dominant over. Dit betekent dat zonen en dochters van iemand met zo’n erfelijke ziekte ieder 50% kans hebben de aanleg te hebben geërfd. De penetrantie van de erfelijke oorzaken van dementie is hoog. Dit betekent dat iemand die een aanleg voor dementie bij zich draagt een zeer grote kans heeft om ziek te worden. De meeste ziekten die dementie veroorzaken kunnen niet genezen, geremd of voorkomen worden. Toch willen sommige familieleden zonder verschijnselen weten of zij drager zijn van de erfelijke aanleg. De reden van deze wens kan bijvoorbeeld zijn dat zij meer duidelijkheid willen over hun toekomst, of omdat zij willen voorkomen de aanleg door te geven aan hun toekomstige kinderen.

HET PROEFSCHRIFT

Het proefschrift is opgedeeld in drie delen, die elk een aspect van erfelijke dementie omvatten. Het eerste gedeelte (hoofdstuk 2) geeft een overzicht van de verschillende erfelijke oorzaken van dementie, de genetische counseling bij dementiepatiënten en de consequenties van voorspellend DNA-onderzoek. In het tweede gedeelte (hoofdstuk 3) wordt ingegaan op de vraag hoe vaak dementie een erfelijke oorzaak heeft en wat de verschijnselen zijn van de erfelijke vormen van dementie. In het laatste gedeelte (hoofdstuk 4) worden de uitkomsten besproken van onderzoek naar nieuwe erfelijke oorzaken van dementie.

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Hoofdstuk 2.1 bevat een overzicht van de verschillende oorzaken van erfelijke dementie. Mutaties in het presenilin 1 gen (PSEN1) zijn de meest voorkomende oorzaak van erfelijke ziekte van Alzheimer, mutaties in de genen amyloid precursor protein (APP) en presenilin 2 (PSEN2) komen minder vaak voor. FTD kan worden veroorzaakt door mutaties in de genen microtubul- associated protein tau (MAPT) of (pro)granulin (GRN), of door een verlenging van een specifieke herhaling van een zestal nucleotiden (bouwstenen) van chromosome 9 open reading frame 72 (C9orf72). In dit hoofdstuk bespreken we de symptomen die voorkomen bij de verschillende erfelijke oorzaken, en merken we op dat er overlap is tussen de genetische afwijkingen en de ziekteverschijnselen. Tot slot doen wij aanbevelingen aan welke patiënten met dementie genetisch onderzoek zou moeten worden aangeboden, en welk onderzoek dan geïndiceerd is. De meest opvallende aanbeveling die wij doen is dat aan alle patiënten met FTD genetisch onderzoek aangeboden wordt, ongeacht de familiegeschiedenis. In hoofdstuk 2.2 bespreken we de bevindingen van ons verkennend onderzoek naar het effect op de levensloop van voorspellend DNA-onderzoek naar de ziekte van Huntington of FTD bij gezonde mensen. Eerdere onderzoeken waren vooral gericht op het psychologische effect. Ons onderzoek richtte zich juist op de meetbare feitelijke verschillen in levensloop. Aan dit onderzoek hebben personen uit families met de ziekte van Huntington of erfelijke FTD meegewerkt: i) dragers van de ziekte die nog geen klachten hadden, ii) personen bij wie met DNA-onderzoek was aangetoond dat zij de aanleg níet hebben geërfd, en iii) personen met 50% kans op de aanleg die zich (nog) niet hebben laten testen. De deelnemers moesten 35 jaar of ouder zijn, en dragers en niet-dragers moesten minstens 2 jaar vóór de start van het onderzoek zijn getest op de aanleg. Aan de deelnemers zijn vele vragen over privéleven en werk gesteld middels twee vragenlijsten. Er waren 17 dragers, 30 niet-dragers en 27 niet geteste risicodragers die voldeden aan de criteria en beide vragenlijsten hadden ingevuld. We vonden geen significante verschillen tussen de drie groepen in werk, financiële situatie, levensstijl of angst of depressie. Dragers waren iets vaker alleenstaand en kinderloos, maar dit verschil was niet significant. Deze uitkomsten suggereren dat voorspellend DNA-onderzoek bij gezonde familieleden geen grote nadelige gevolgen heeft, of nadelige gevolgen bij slechts een deel van de personen. Echter, aangezien het aantal deelnemers aan het onderzoek beperkt is en omdat we alleen de gegevens hebben van mensen die deel wilden nemen aan het onderzoek (response bias), is aanvullend onderzoek nodig voordat deze uitkomsten gebruikt kunnen worden in de patiëntenzorg.


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Hoofdstuk 3 omvat twee onderzoeksprojecten naar de frequentie van mutaties in patiënten met dementie en naar symptomen en verschijnselen bij erfelijke dementie. Hoofdstuk 3.1 gaat specifiek over het C9orf72 gen. Kort na de ontdekking van deze erfelijke oorzaak van FTD hebben wij in een Nederlands cohort van 353 patiënten met FTD en 522 gezonde personen de frequentie bepaald van een te groot aantal herhalingen van zes specifieke bouwstenen in dit gen (hexanucleotide repeat expansion). Deze mutatie werd gevonden in 28,7% van de patiënten met FTD die een familielid met FTD of ALS hadden, en in 2.2% van de FTD-patiënten zonder belaste familiegeschiedenis. De gemiddelde beginleeftijd van FTD bij de C9orf72 repeat expansion was 56,9 jaar (variërend van 39 tot 76 jaar), en de gemiddelde ziekteduur was 7,6 jaar (1 tot 22 jaar). De meeste patiënten met de C9orf72 hexanucleotide repeat expansion hadden voornamelijk karakterveranderingen, en sommigen hadden naast de FTD ook ALS. De beeldvorming van de hersenen vertoonde wisselende afwijkingen; meest voorkomend was atrofie van voornamelijk de slaapkwabben. Bij weefselonderzoek van de hersenen van 10 overleden personen met een C9orf72 hexanuclotide repeat expansie werden, naast verval van de voor- en slaapkwabben ook ophopingen gezien van het neuronal transactive response DNA binding protein (TDP) eiwit. In hoofdstuk 3.2. bespreken we het onderzoek naar de frequentie van een aantoonbare erfelijke aanleg voor dementie bij Nederlandse patiënten met dementie op jonge leeftijd. Ook hebben we gekeken of in deze patiëntengroep mutaties voorkomen in het PRKAR1B- gen, een gen waarin kort ervoor in een familie met FTD-achtige verschijnselen een mutatie was gevonden. In een groep van 229 patiënten met de ziekte van Alzheimer gediagnosticeerd vóór het 70e jaar, vonden we in 3,5% van de patiënten een PSEN1 mutatie en in 0,4% een APP mutatie. Het PSEN2 gen werd niet onderzocht. Van de FTD-patiënten had 9,9% een C9orf72 hexanucleotide repeat expansie, 2,7% een GRN mutatie en 1,4% een mutatie in het MAPT-gen. Ook werden mutaties in C9orf72, GRN en MAPT teruggevonden in patiënten met de ziekte van Alzheimer: ieder met een frequentie van 0,4%. We vonden geen mutaties in het PRKAR1B gen. We concludeerden dat in ons Nederlandse patiëntengroep met de ziekte van Alzheimer op jonge leeftijd mutaties in het PSEN1 gen het meest voorkomen, maar alsnog zeldzaam zijn, en dat de C9orf72 hexanucleotide repeat expansie de meest voorkomende erfelijke oorzaak van FTD is. Ook concludeerden we dat mutaties in het PRKAR1B gen waarschijnlijk zeldzaam zijn in Nederlandse patiënten met de klinische diagnose FTD of ziekte van Alzheimer.

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In hoofdstuk 4 bespreken we de genetische achtergrond en de verschijnselen in twee families met een vermoedelijke erfelijke aanleg voor dementie. In hoofdstuk 4.1 bespreken we een familie waarin drie personen cognitieve klachten hadden, een verlaagd amyloid-beta-42 eiwit niveau in de hersenvloeistof en meerdere microbloedingen in de hersenen. In de familie zou de ziekte van Alzheimer voorkomen, passend bij een autosomaal dominante overerving. In alle drie de familieleden werd met whole exome sequencing, een methode waarbij al het DNA dat codeert voor eiwitten wordt geanalyseerd, een variant aangetoond in het CCM2 gen leidend tot de aanmaak van een verkort transcript. Deze variant werd niet gevonden in 363 patiënten met dementie op jonge leeftijd, al dan niet met microbloedingen, en is eveneens niet beschreven als bekende variant in databases. Daarbij bleken twee van de drie familieleden drager te zijn van tweemaal (homozygoot) het APOE-ε4 allel, het derde familielid had eenmaal dit allel (heterozygoot). Mutaties in het CCM2 gen geven familiaire cerebrale caverneuze malformaties, een aandoening die gepaard gaat met een sterk verhoogd risico op kleine vaatkluwen in hersenen en ruggenmerg (cavernomen). Het APOE-ε4 allel is een erfelijke risicofactor voor de ziekte van Alzheimer en leidt tot een vroegere beginleeftijd van de ziekte, en geeft tevens een verhoogd risico op microbloedingen. Twee van de drie familieleden bleken inderdaad één of enkele cavernomen te hebben. De microbloedingen zouden veroorzaakt kunnen worden door het ApoE eiwit, maar zouden ook bloedingen uit kleine cavernomen kunnen zijn. De cognitieve klachten, het lage niveau van het amyloid-beta-42 eiwit in het hersenvocht en de microbloedingen passen bij een voorstadium van de ziekte van Alzheimer, maar het feit dat de cognitieve klachten van de drie familielieden nauwelijks toenamen past hier niet bij. Vermoedelijk hebben zowel de variant in het CCM2 gen als het APOE-ε4 allel bijgedragen aan de klachten en verschijnselen bij de patiënten. In hoofdstuk 4.2 bespreken we een familie waarin meerdere personen de ziekte van Alzheimer hebben, passend bij een autosomaal dominante overerving. De aangedane familieleden hadden variabele klachten en verschijnselen. Alle vier aangedane personen van wie DNA beschikbaar was bleken homozygoot voor het APOE-ε4 allel. De beginleeftijd van de klachten varieerde tussen de 61 en 74 jaar, waarbij sommigen microbloedingen hadden bij beeldvorming van de hersenen en sommigen opvallende bevindingen bij electroencephalographisch onderzoek (EEG). Met whole exome sequencing werd een zeldzame variant in het VPS10 domein van het sortilin-related receptor gen (SORL1) gevonden, die wel aanwezig was bij drie aangedane familielid maar niet bij het geteste niet-aangedane familielid . Dit gen, en specifiek veranderingen in dit domein van dit gen, zijn eerder gevonden in patiënten met de ziekte van


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Alzheimer. Met segregatieonderzoek werd de variant ook aangetoond bij het vierde aangedane familielid en bij één jong familielid zonder klachten.. Daarnaast werd een zeldzame variant in het TSHZ3 gene gevonden in drie aangedane en één jong niet aangedaan. Zowel het SORL1 als het TSHZ3 eiwit zijn betrokken in de amyloid pathway, welke betrokken is bij het ontstaan van de ziekte van Alzheimer. Vermoedelijk heeft de combinatie van de APOE-ε4 homozygotie en de aanwezigheid van de SORL1variant, en mogelijk ook de TSHZ3 variant, gezorgd voor het ontstaan van de ziekte van Alzheimer in deze familie. Mogelijk spelen ook in andere families waarin de ziekte van Alzheimer een autosomaal dominante oorzaak lijkt te hebben, meerdere factoren een rol bij het ontstaan.

CONCLUSIE

Met dit proefschrift hebben wij een bijdrage geleverd aan verschillende aspecten van erfelijke dementie. Allereerst hebben wij voor klinisch genetici een overzichtsartikel opgesteld met alle informatie die nodig is voor het counselen van een patiënt met dementie of zijn familie, inclusief aanbevelingen voor genetisch onderzoek. Ook hebben wij een eerste stap gezet in het onderzoeken van de consequenties van voorspellend DNA-onderzoek naar ziekten die dementie veroorzaken op de levensloop. Wij hebben bijgedragen aan de kennis over het C9orf72 gen, en het eerste onderzoek verricht naar het voorkomen van de ziekte van Alzheimer op jonge leeftijd in Nederland. Tot slot hebben wij getracht nieuwe erfelijke oorzaken van dementie te vinden, waarbij wij zijn gestuit op een andere aandoening die mogelijk een deel van de klachten verklaard in één familie, en op een vermoedelijk samenspel van meerdere genetische factoren in een tweede familie. Alhoewel er veel onderzoek naar dementie is verricht, is de kennis over dementie nog zeer beperkt. De grootste behoefte is aan een goede behandeling, liefst preventief of genezend, of anders in elk geval het ziekteproces remmend. Genetisch onderzoek heeft reeds bewezen van groot belang te kunnen zijn in het ontrafelen van ziekteproces en daarmee in het dichterbij brengen van mogelijke oplossingen. Onderzoek naar nieuwe erfelijke oorzaken van ziekten zoals de ziekte van Alzheimer en FTD en naar het effect van deze erfelijke oorzaken op processen in het lichaam is daarom van groot belang.

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LIST OF PUBLICATIONS Thesis Cohn-Hokke PE, Van Swieten JC, Pijnenburg YAL, Tibben A, Meijers-Heijboer EJ, Kievit AJ. The effect of predictive testing in adult onset neurodegenerative diseases on social and personal life. Submitted.

Louwersheimer E*, Cohn-Hokke PE*, Pijnenburg YAL, Weiss MM, Sistermans EA,. Rozemuller AJ, Hulsman M, Van Swieten JC, Van Duijn CM, Barkhof F, Koene T, Scheltens P, Van der Flier WM, Holstege H. Rare genetic variant in SORL1 may increase penetrance of Alzheimer’s disease in a family with several generations of APOE-ε4 homozygosity. J Alzheimers Dis. 2017;56:63-74. doi: 10.3233/JAD-160091. (*equal contribution)

Cohn-Hokke PE, Holstege H, Weiss MM, Van der Flier WM, Barkhof F, Sistermans E, Pijnenburg YAL, Van Swieten JC, Meijers-Heijboer H, Scheltens P. A novel CCM2 variant in a family with non-progressive cognitive complaints and cerebral microbleeds. Am J Med Genet B Neuropsychiatr Genet. 2016 Jun 8 [Epub ahead of print]. doi 10.1002/ajmg.b.32468.

Cohn-Hokke PE, Rizzu P, Breedveld G, Wong TH, van der Flier WM, Scheltens P, Baas F, Heutink P, Meijers-Heijboer EJ, Van Swieten JC, Pijnenburg YAL. Mutation frequency of PRKAR1B and the major familial dementia genes in a Dutch early onset dementia cohort. J Neurol. 2014 Nov;261(11):2085-92. doi: 10.1007/s00415-014- 7456-y.

Cohn-Hokke PE, Elting MW, Pijnenburg YA, van Swieten JC. Genetics of dementia: Update and guidelines for the clinician. Am J Med Genet B Neuropsychiatr Genet. 2012; 159B:628–643. doi: 10.1002/ajmg.b.32080.

Simón-Sánchez J*, Dopper EG*, Cohn-Hokke PE, Hukema RK, Nicolaou N, Seelaar H, de Graaf JR, de Koning I, van Schoor NM, Deeg DJ, Smits M, Raaphorst J, van den Berg LH, Schelhaas HJ, De Die-Smulders CE, Majoor-Krakauer D, Rozemuller AJ, Willemsen R, Pijnenburg YA, Heutink P, van Swieten JC. The clinical and pathological phenotype of C9ORF72 hexanucleotide repeat expansions. Brain. 2012 Mar;135 (Pt 3):723-35. doi: 10.1093/brain/awr353. (*equal contribution)

List of publications

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Other publications

Wentink M, Nellist M, Hoogeveen-Westerveld M, Zonnenberg B, van der Kolk D, van Essen T, Park SM, Woods G, Cohn-Hokke P, Brussel W, Smeets E, Brooks A, Halley D, van den Ouweland A, Maat-Kievit A. Functional characterization of the TSC2 c3598C>T (pR1200W) missense mutation that co-segregates with tuberous sclerosis complex in mildly affected kindreds. Clin Genet. 2012 May;81(5):453-61.

Willemsen MH, Vulto-van Silfhout AT, Nillesen WM, Wissink-Lindhout WM, van Bokhoven H, Philip N, Berry-Kravis EM, Kini U, van Ravenswaaij-Arts CM, Delle Chiaie B, Innes AM, Houge G, Kosonen T, Cremer K, Fannemel M, Stray-Pedersen A, Reardon W, Ignatius J, Lachlan K, Mircher C, Helderman van den Enden PT, Mastebroek M, Cohn-Hokke PE, Yntema HG, Drunat S, Kleefstra T. Update on Kleefstra Syndrome. Mol Syndromol. 2012 Apr;2(3-5):202-212.

Zylicz SA, Cohn-Hokke PE, van Swieten JC. Frontotemporale dementie: een overzicht. Tijdschr Neurol Neurochir 2011;113:66-73.

Doubal FN, de Haan R, MacGillivray TJ, Cohn-Hokke PE, Dhillon B, Dennis MS, Wardlaw JM. Retinal arteriolar geometry is associated with cerebral white matter hyperintensities on magnetic resonance imaging. Int J Stroke. 2010 Dec;5(6):434-9.

Doubal FN, MacGillivray TJ, Hokke PE, Dhillon B, Dennis MS, Wardlaw JM. Differences in retinal vessels support a distinct vasculopathy causing lacunar stroke. Neurology. 2009 May 19;72(20):1773-8.

Doubal FN, Hokke PE, Wardlaw JM. Retinal microvascular abnormalities and stroke: a systematic review. J Neurol Neurosurg Psychiatry. 2009 Feb;80(2):158-65.

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DANKWOORD Dit proefschrift had niet tot stand kunnen komen zonder de bijdrage en steun van velen. Bij deze wil ik alle deelnemers hartelijk danken voor hun medewerking. Ik besef mij goed dat het voor sommigen behoorlijk confronterend was benaderd te worden over dit onderwerp en deel te nemen aan het onderzoek. Ook gaat mijn dank uit naar de vele patiënten van het Alzheimer centrum die, door toestemming te geven voor onderzoek in het algemeen en hiervoor ook DNA hebben afgestaan, hebben bijgedragen aan dit proefschrift. Promotoren en copromotor Prof. Meijers-Heijboer, beste Hanne, dank voor het vertrouwen dat je van begin af aan in mij hebt gehad. Behalve een pragmatische promotor ben je ook een inspirerend opleider voor mij geweest. Ik waardeer het zeer dat je mij altijd ruimte en vrijheid hebt gegeven voor mijn persoonlijke ideeën en ontwikkeling. Prof. Van Swieten, John, we moesten even wennen aan elkaars werkwijze maar het is gelukt, het proefschrift is af. Dank voor je kritische blik bij het schrijven van de artikelen. Dr. Pijnenburg, beste Yolande, dank voor de fijne samenwerking, zowel in het onderzoek als in de patiëntenzorg. Het was heel fijn dat je in de begeleiding van het onderzoek niet alleen hebt gewaakt over de inhoud maar ook over de praktische haalbaarheid. Commissieleden Veel dank aan de leescommissie voor het kritisch lezen en beoordelen van mijn proefschrift. Prof.dr. Scheltens, beste Philip, dank voor de samenwerking afgelopen jaren in zowel patiëntenzorg als onderzoek. Ik waardeer het dat je mij afgelopen jaar de mogelijkheid hebt geboden de genetica binnen de dementiezorg van het Alzheimercentrum vorm te geven. Dr. Kievit, beste Anneke, dank voor de prettige samenwerking en voor je vertrouwen en geduld bij het vragenlijstonderzoek. Dr. Sistermans, beste Erik, dank voor de prettige samenwerking afgelopen jaren, ik waardeer het dat je zo goed kan luisteren en open staat voor nieuwe ideeën.

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Prof.dr. Rozemuller, beste Annemieke, jouw betrokkenheid en enthousiasme voor de neurodegeneratie is zeer motiverend, dank voor het fijne overleg over bijzondere patiënten en families. Prof.dr. Frank Baas, prof.dr. Ellen Smets en prof.dr. Guy Widdershoven: hartelijk dank dat jullie aan de oppositie deel willen nemen. Paranimfen Lieve Christel, fijn dat jij mij (alweer!) bij wil staan op een belangrijk moment! Lieve Margot, lotgenoot in het opleidings- en promotietraject en een heel fijne collega, heel veel succes met de afronding van je eigen proefschrift! Medeauteurs en -onderzoekers Ik dank de vele medeauteurs voor hun onmisbare bijdragen aan de onderzoeken en artikelen in dit proefschrift. In het bijzonder Peter, hartelijk dank voor het ‘onderdak’ dat je mij hebt geboden op je afdeling tijdens de aftrap van mijn onderzoek, en de tijd die je altijd nam om uitleg te geven. Patrizia, dank voor het monnikenwerk dat je hebt verricht met het sequencen van al die losse genen (hoeveel tijd had het wel niet gescheeld als we al WES tot onze beschikking hadden gehad…). Henne, ik heb ongelooflijk veel respect voor hoe je al je werkzaamheden en je drukke privéleven weet te combineren, en voor je gedrevenheid bij te dragen aan de wetenschap. Eva, ik vond het fijn samenwerken met je, succes met je opleiding. Wiesje, dankjewel dat ik als gast mee mocht draaien met de scholing van de promovendi van het Alzheimercentrum, ik heb er een hoop van opgestoken. Janneke, ik waardeer het dat je ondanks de enorme werkdruk door de vele projecten waar je aan werkt toch altijd tijd vrij maakt ook voor kleinere onderzoeksprojecten. Ook hartelijk dank aan alle promovendi van John uit het Erasmus MC voor jullie bijdrage bij het verzamelen van patiënten, en aan alle anderen die op een of andere wijze aan de onderzoeksprojecten hebben bijgedragen. Collega’s Graag wil ik al mijn collega’s van de poli en het lab van de klinische genetica en van het Alzheimercentrum bedanken voor de fijne samenwerking. In het bijzonder Mariet, dank voor je fijne begeleiding bij de vele patiënten die ik onder jouw supervisie heb gezien en die mij achtergrond voor dit proefschrift hebben gegeven. Kyra, lange tijd hebben we in hetzelfde schuitje gezeten op het gebied van aanstelling en onderzoek, heel fijn dat het ook bij jou uiteindelijk zo goed heeft uitgepakt. Heel veel succes met de afronding van je proefschrift!

Addendum

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Familie en vrienden Lieve paps, dank voor je onvoorwaardelijke steun en vertrouwen. Lieve mams, fijn dat ik altijd stoom bij je kan afblazen. Dank aan Caren, Irina, Sanne en alle andere vrienden en vriendinnen die mij regelmatig afleiding hebben gegeven in de vorm van etentjes, feestjes en gezamenlijke vakanties. Lieve Danny, je bent echt een fantastische steun en toeverlaat. Bedankt dat je mij zoveel (vrije) tijd hebt gegund voor dit promotietraject. En sorry voor m’n knorrige buien als het me even te veel werd. Lieve Amelie en Nora, jullie prachtige liedjes, vele grapjes en warme knuffels maken mij altijd vrolijk, dank jullie wel daarvoor!

Dankwoord

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ABOUT THE AUTHOR Petra Cohn-Hokke was born on the 20th of May in 1981 in Heemstede as the youngest of three. She grew up in Haarlem and graduated high school (gymnasium) at the Kennemer Lyceum in Overveen in 1999. After obtaining a propaedeutic certificate in Industrial Design Engineering at the Technical University of Delft, she studied Behaviour and Society at the University of Amsterdam. Realizing that her main interest was in patient care, she switched again in 2002 and started Medical School at the Academic Medical Centre of Amsterdam, of the University of Amsterdam. In 2006, she performed a research internship on the topic of retinal abnormalities in lacunar stroke at the Department of Clinical Neurosciences of the University of Edinburgh under supervision of professor Joanna Wardlaw. She obtained her medical degree in November 2008 and started her residency in Clinical Genetics at the VU University Medical Centre in Amsterdam a month later. Since 2010, she combined this residency with a PhD research project on the topic of inherited dementia under supervision of professor Hanne Meijers-Heijboer, professor John van Swieten and dr. Yolande Pijnenburg, resulting in this thesis. In October 2015 she was registered as a clinical geneticist and continued her activities at the VU University Medical Centre in the field of neurogenetics. In 2017, she started working at the Familial Cancer Clinic of the Antoni van Leeuwenhoek Hospital in Amsterdam. She lives in Amstelveen with her husband Danny and her two daughters Amelie and Nora.

Addendum

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hereditary dementia, a clinical genetic perspective...prof.dr. g.a.m. widdershoven contents chapter...

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