a case of family insomnia

8/3/2019 A Case of Family Insomnia

http://slidepdf.com/reader/full/a-case-of-family-insomnia 1/12

Brain (1999), 122, 5–16

Fatal familial insomnia: a new Austrian family

G. Almer,1,* J. A. Hainfellner,2,* T. Brucke,1,† K. Jellinger,3 R. Kleinert,4 G. Bayer,5 O. Windl,6

H. A. Kretzschmar,6 A. Hill,7 K. Sidle,7 J. Collinge7 and H. Budka2

1Clinic of Neurology, University of Vienna, 2 Austrian Correspondence to: Professor Herbert Budka, Institute of

Reference Centre for Human Prion Diseases and Institute Neurology, AKH, Wa hringer Gu rtel 18–20, POB 48,

of Neurology, University of Vienna, 3 Ludwig Boltzmann A-1097 Wien, Austria

Institute of Clinical Neurobiology, Hospital Lainz, Vienna, E-mail: H. [email protected] 4 Institute of Pathology, University of Graz, 5 Institute of

* Both authors contributed equally to this studyPathology, Hospital Oberwart, Austria, 6 Institute of

Neuropathology, University of Go ttingen, Germany and †Present address: Department of Neurology, Hospital7 Imperial College at St Mary’s, London, UK Wilhelminenspital, Vienna, Austria

SummaryWe present clinical, pathological and molecular features

of the first Austrian family with fatal familial insomnia.

Detailed clinical data are available in five patients and

autopsy in four patients. Age at onset of disease ranged

between 20 and 60 years, and disease duration between

8 and 20 months. Severe loss of weight was an early

symptom in all five patients. Four patients developed

insomnia and/or autonomic dysfunction, and all five

patients developed motor abnormalities. Analysis of the

prion protein (PrP) gene revealed the codon 178 point

mutation and methionine homozygosity at position 129.

In all brains, neuropathology showed widespread cortical

astrogliosis, widespread brainstem nuclei and tract

degeneration, and olivary ‘pseudohypertrophy’ with

Keywords: fatal familial insomnia; prion diseases; prion protein; transmissible spongiform encephalopathies

Abbreviations: BSE bovine spongiform encephalopathy; FFI fatal familial insomnia; GFAP glial fibrillary acidic

protein; HE haematoxylin–eosin; PCR polymerase chain reaction; PRNP PrP gene; PrP prion protein; PrPres

protease resistant form of PrP; SSCP single-strand conformational polymorphism

Introduction

Fatal familial insomnia (FFI) was first described in 1986 asan autosomal dominant heredopathy, clinically characterized

by progressive untreatable insomnia, dysautonomia and motor

signs (Lugaresi et al., 1986). Meanwhile, the disorder has

been recognized as a prion disease, thus enlarging the

spectrum of familial spongiform encephalopathies consisting

of familial Creutzfeldt–Jakob disease and Gerstmann–

Straussler–Scheinker disease (Goldfarb et al., 1992; Medori

et al., 1992b). The neuropathological hallmark of FFI is

predominance of lesions in the thalamus (Manetto et al.,

1992; Gambetti et al., 1995). Genetically, FFI is linked to a

GAC to AAC point mutation (aspartic acid to asparagine

substitution) at codon 178 of the prion protein (PrP) gene

(PRNP) on chromosome 20 in conjunction with methionine

© Oxford University Press 1999

vacuolated neurons, in addition to neuropathological

features described previously, such as thalamic and olivary

degeneration. Western blotting of one brain and

immunocytochemistry in four brains revealed quantitative

and regional dissociation between PrPres (the protease

resistant form of PrP) deposition and histopathology. In

the cerebellar cortex of one patient, PrPres deposits were

prominent in the molecular layer and displayed a peculiar

patchy and strip-like pattern with perpendicular

orientation to the surface. In another patient, a single

vacuolated neuron in the inferior olivary nuclei contained

prominent intravacuolar granular PrPres deposits,

resembling changes of brainstem neurons in bovine

spongiform encephalopathy.

at the polymorphic position 129 of the mutant allele (Goldfarbet al., 1992; Medori et al., 1992b).

We report here the first Austrian family with FFI in five

consecutive generations. We present detailed clinical features

of five patients, and neuropathological and molecular genetic

analysis of four patients. Data on this new family have been

published in part as abstracts (Almer et al., 1997; Budka

et al., 1997; Hainfellner et al., 1997a).

MethodsInformation on the pedigree was collected by reviewing all

pertinent medical and non-medical records, and notably by

interviews with family members. In patients III-5, III-13



6 G. Almer et al.

and IV-13, clinical data were retrieved retrospectively from

medical records. Patients IV-5 and IV-8 underwent personal

(G.A. and T.B.) neurological examination, and had several

EEGs, CT and MRI.Neuropathological and molecular genetic analysis were

performed in patients III-5, IV-5, IV-8 and IV-13. Autopsy

of patients IV-5 and IV-8 was done after 20 h post-mortem

and was restricted to the brain including the upper cervical

spinal cord. Numerous tissue blocks of cerebral cortex, basal

ganglia, brainstem and cerebellar cortex were sampled and

frozen. The remaining brain was immersion-fixed in 4%

formalin for 2 weeks and cut. Coronal slices of cerebrum,

an axial whole-mount slice of cerebellum and pons, and

numerous smaller tissue blocks including all major brain

regions were embedded in paraffin. Archival paraffin blocks

containing various brain regions of patient III-5, who

succumbed in 1991, and patient IV-13, who succumbed in1986, were retrieved from two municipal Austrian

(neuro)pathology laboratories. In addition, paraffin embedded

blocks of lungs, kidney, liver and spleen of patient IV-13

were available. Histological work-up was performed on 5 µm

thick sections with conventional and immunocytochemical

stains. Conventional stains comprised haematoxylin–eosin

(HE), luxol fast blue/nuclear fast red, Kanzler method and

Bielschowsky silver impregnation. Immunolabelling used a

polyclonal antibody against glial fibrillary acidic protein

(GFAP) (Dako, Glostrup, Denmark), and monoclonal

antibodies against neurofilament protein (NFP; clone NE14)

(Dako), microtubule associated protein-2 (MAP2; cloneAP20) (Boehringer, Mannheim, Germany), synaptophysin

(clone SY38) (Boehringer) and PrP (clone 3F4) (Dr R.

Kascsak, Staten Island, NY, USA). For anti-PrP

immunocytochemistry, sections were pre-treated with a three-

tiered protocol of hydrated autoclaving, concentrated formic

acid and guanidine isothiocyanate (Goodbrand et al., 1995).

Antibodies were followed by the avidin–biotin complex

method (for monoclonals) or the peroxidase–anti-peroxidase

technique (for polyclonals), with diaminobenzidine as

chromogen for visualization. Grey matter lesioning was

evaluated on HE- and GFAP-stained sections by

semiquantitative assessment of neuronal loss, spongiform

change and astrogliosis (Table 1). Lesioning of tracts and

white matter structures was evaluated by semiquantitative

assessment of regional nerve fibre degeneration on luxol fast

blue/nuclear fast red stained sections (Table 2).

Analysis of PRNP was performed on genomic DNA

isolated from the blood of patients IV-5 and IV-8 according

to standard procedures (Sambrook, 1989). One hundred

nanograms of DNA was used for PCR (polymerase chain

reaction) amplification of the coding region of PRNP using

the primers 895W and 896W (Kretzschmar et al., 1986;

Nicholl et al., 1995). The PCR product was inspected on a

1% agarose gel for potential insertion mutations and deletions.

Potential point mutations were screened by the single-strandconformational polymorphism (SSCP) technique (Orita et al.,

1989). For this purpose the coding region of PRNP was

reamplified in four overlapping fragments which were

analysed alongside the PRNP gene of patients with known

mutations (Windl et al., 1996). The genotypes of codons 129

and 178 were examined by digestion with the restrictionendonucleases NspI and Tth111I. The final sequence

confirmation was obtained by solid-phase direct sequencing

of the complete coding region of PRNP after reamplification

and purification of single-stranded PCR products using 5-

biotinylated primers 895W and 896W and streptavidin-

coupled Dynabeads M-280 (Dynal, Oslo, Norway). The

sequencing reactions were performed with the SequiTherm

EXCEL Long-Read Kit-LC (Epicentre Technologies,

Madison, Wis., USA), according to the manufacturer’s

recommendation, and 5-IRD-41 labelled oligonucleotides

5HUSEQ (5-TCTCCTCTTCATTTTGCAGAGC-3 ) or

3HUSEQ (5-GAAAGATGGTGAAAACAGGAAG-3). The

reaction products were loaded on a 4.3% Long-Rangergel (AT Biochem, Malvern, Pa., USA) and separated by

denaturing electrophoresis on an automated system (Model

4000L; LI-COR, Lincoln, Nev., USA).

DNA from paraffin embedded brain tissue of patients III-

5 and IV-13 was isolated using the QIAamp tissue kit

(QIAGEN, Hilden, Germany). The DNA from this material

was highly degraded. Therefore, two fragments of PRNP

were amplified with two sets of primers. Primers 5CEN

(5-AGGTGGCACCCACAGTCAGT-3) and 3CEN (5-AC-

GGTCCTCATAGTCACTGCCG-3) amplified a fragment

encompassing the codons 93–148 of PRNP, whereas primers

P33 (5-CATGGATGAGTACAGCAACCAG-3) and P34(5-TCTGGTAATAGGCCTGAGATTC-3 ) amplified a frag-

ment encompassing codons 166–228. PCR used identical

conditions to those used for the complete PRNP coding

region, but two successive rounds of 35 PCR cycles were

necessary for a sufficient yield of PCR product for further

examination. Codons 129 and 178 were examined by

digestion with restriction endonucleases NspI and Tth111I as

well as direct sequencing. Sequencing was performed as

outlined above, but the 5-biotinylated primers 5CEN and

P33 were used for purification of single-stranded PCR

products and the 5-IRD-41 labelled primers 3CEN and P34

for the sequencing reactions.

Western immunoblotting was performed with samples of

occipital and precentral cortex, thalamus, basal ganglia,

cerebellar cortex, brainstem and cervical spinal cord of patient

IV-5. The tissue samples were homogenized in 9 volumes of

lysis buffer (100 mM NaCl, 10 mM EDTA, 0.5% sodium

deoxycholate, 10 mM Tris pH 7.4) by repeated passage

through needles of decreasing diameter. The homogenates

were spun at 3000 r.p.m. for 5 min and the supernatant

removed to a fresh tube for analysis. Aliquots of the

homogenates were incubated at 37°C for 1 h with proteinase

K (at a final concentration ranging from 12.5–50 µg/ml).

The reactions were terminated by the addition of Pefabloc

(Boehringer) to 1 mM. Samples were electrophoresed on16% Tris–glycine acrylamide gels and blotted as described

previously (Collinge et al., 1996). The blots were developed



A new Austrian family with FFI 7

Table 1 Regional severity of brain tissue lesioning in four Austrian FFI patients

Patient

IV-5 IV-8 III-5 IV-13

NL GL SP NL GL SP NL GL SP NL GL SP

Cerebral cortexFrontal cortex 0 2 0 0 1 1† 0 2 0 0‡ 2‡ 0‡

Precentral gyrus 0 2 0 0 1 0 – – – – – –Temporal cortex 0 2 0 0 1 0 0 2 0 – – –Parietal cortex 0 2 0 0 1 0 0 2 0 – – –Occipital cortex 0 2 0 0 2 0 0 2 0 – – –Cingulate gyrus 0 2 0 0 1 0 – – – – – –Insular cortex 0 2 0 0 1 0 – – – – – –Dentate gyrus 0 1 0 0 1 0 0 1 0 – – –Hippocampus 0 2 0 0 1 0 0 2 0 – – –Subiculum 0 2 0 0 1 0 0 2 0 – – –

Pre/parasubiculum 0 2 1 0 2 0 0 2 1 – – –Basal gangliaPutamen 0 2 0 0 1 0 0 2 0 0 2 0Caudate nucleus 0 2 0 0 1 0 – – – – – –Globus pallidus 0 2 0 0 1 0 0 2 0 0 2 0Claustrum 0 2 0 0 1 0 – – – – – –Amygdaloid nucleus complex 0 2 0 – – – – – – – – –Basal nucleus 0 2 0 0 2 0 – – – – – –

ThalamusAnterior nucleus 3 3 1 2 3 1 – – – 2 ¶ 3 ¶ 1 ¶

Medial nucleus 3 3 1 2 3 1 2 3 1 2 ¶ 3 ¶ 1 ¶

Ventral lateral nucleus 1 2 1 1 1 1 1 2 1 – – –Pulvinar 2 3 1 – – – – – – – – –Lateral geniculate nucleus 0 1 0 – – – – – – – – –Subthalamic nucleus 0 1 0 0 1 0 0 0 0 – – –

HypothalamusParaventricular nucleus 0 1 0 – – – – – – – – –Supraoptic nucleus 0 1 0 – – – – – – – – –Lateral nucleus 1 2 0 – – – – – – – – –Anterior nucleus 1 1 0 – – – – – – – – –

Cerebellum*Neocerebellum 1 1 0 1 1 0 1 1 0 – – –Vermis 2 1 0 1 1 0 – – – 1# 1# 0#

Archicerebellum 1 1 0 1 1 0 – – – – – –Dentate nucleus 1 2 0 1 1 0 1 1 0 – – –

MidbrainNucleus ruber 1 3 0 1 2 0 – – – – – –Substantia nigra 1 2 0 1 1 0 1 1 0 – – –Central grey mesencephali 1 3 0 1 2 0 1 2 0 – – –Nucleus raphes dorsalis 2 3 0 – – – 1 2 0 – – –

Formatio reticularis 2 2 0 1 2 0 1 2 0 – – –Oculomotor nucleus – – – – – – 0 1 0 – – –Trochlear nucleus 0 1 0 – – – – – – – – –Mesencenphatic nucleus of trigeminal nerve 0 2 0 – – – 0 1 0 – – –Superior colliculus – – – 1 2 0 1 2 0 – – –Inferior colliculus 1 2 0 – – – – – – – – –

PonsLocus coeruleus 1 2 0 1 1 0 1 1 0 – – –Superior central nucleus 1 2 0 2 2 0 2 2 0 – – –Formatio reticularis 2 2 0 – – – 2 2 0 – – –Vestibular nuclei 1 2 0 – – – – – – – – –Pontine nuclei 0 1 0 0 1 0 0 1 0 0 1 0

Medulla oblongataVestibular nuclei 0 2 0 0 1 0 – – – – – –Nucleus cuneatus 0 1 0 – – – 1 1 0 – – –

Continued on next page



8 G. Almer et al.

Table 1 Continued

Patient


NL GL SP NL GL SP NL GL SP NL GL SP

Nucleus gracilis – – – – – – 1 1 0 0 1 0Hypoglossal nucleus 0 1 0 – – – 0 1 0 – – –Dorsal nucleus of vagus 0 1 0 – – – 1 2 0 – – –Spinal trigeminal nucleus 1 2 0 0 1 0 1 2 0 – – –Nucleus ambiguus 0 1 0 0 1 0 0 1 0 0 1 0Raphe nuclei 2 2 0 1 1 0 2 2 0 – –Formatio reticularis 2 2 0 1 2 0 2 2 0 2 3 0Inferior olivary nucleus 2 3 0 2 3 0 2 2 0 3 3 0Accessory olivary nuclei 2 3 0 2 3 0 3 3 0 – – –

Spinal cordPosterior horn 1 2 0 – – – – – – 1 2 0

Lateral horn – – – – – – – – – 1 2 0Intermediate grey 1 2 0 – – – – – – 1 2 0Anterior horn 1 2 0 – – – – – – 1 2 0

0, 1, 2, 3 no, slight, moderate, prominent neuronal loss, gliosis, spongiform change, respectively; – not available. NL neuronalloss; GL astrogliosis; SP spongiform change. *Loss of Purkinje cells; †focal SP, colocalizes with PrPres deposition; ‡two blocks of cerebral cortex, region unknown; ¶two fragments of thalamus, most likely of anterior and medial nucleus; #two blocks of cerebellarcortex, most likely vermis.

using an enhanced chemifluorescent substrate (Amersham,

UK), and analysed on a Storm 840 phosphoimager (Molecular

Dynamics, Sunnyvale, Calif., USA).

Results

Clinical findingsThe family pedigree is depicted in Fig. 1. The pedigree data

were collected after FFI was genetically diagnosed in a young

man in 1996 with clinical signs of a neurodegenerative

disorder (patient IV-5, see below). Among more than 50

members in five generations, probable (according to medical

and non-medical records) or definite (diagnosed by molecular

genetics and neuropathology) FFI was identified in 13 cases.

Patient IV-5At the age of 25 years, this male patient started suffering

from progressive tiredness and lethargy. Episodes of diplopia

and complex hallucinations followed (e.g. the patient

performed movements of sawing with a virtual saw and

stopped bewildered when told that there was no saw). Despite

increased appetite, he had continuous loss of weight (20 kg

within 6 months) and chronic therapy-resistant constipation.

Progressive change of personality with apathy became evident

and endogenous depression was diagnosed. Several clinical

check-ups including MRI and EEG were all inconclusive.

Four months after onset, dysarthria, hypophonia and reduction

of spontaneous speech developed. Speech disorder was

followed by gait ataxia and myoclonus with perioral andperiorbital predominance.

The patient was hospitalized at the Clinic of Neurology,

University of Vienna, 6 months after onset of disease. On

admission, he presented with markedly impaired vigilance,

lack of spontaneous speech, dysarthria, severe gait ataxia,

myoclonus and tremor. He had mild sleep disturbances. He

was fully oriented, but had severe deficiencies in short-term memory, and psychomotor speed was reduced. During

hospitalization, autonomic dysfunction manifested with

hyperhidrosis, hyperthermia, tachycardia, recurrent flushes,

dyspnoe and irregular breathing. Within several months,

marked insomnia with nocturnal motor unrest and stereotype

movements developed.

MRI showed mild supratentorial atrophy and discrete

hyperintense white matter lesions. EEG displayed mild to

moderate signs of diffuse non-specific parenchymal

dysfunction. In the late phase of disease, epileptiform

discharges were observed. Periodic or pseudoperiodic activity

was not recorded. Other clinical tests, such as electro-

oculography, visual evoked potentials and analysis of theCSF revealed no pathology. ACTH and cortisol levels were

within normal range. DNA analysis of blood leukocytes

detected the pathognomonic genotype of FFI (see PRNP

analysis below). The patient died from pneumonia 13 months

after onset of disease in a condition of severe cachexia

and stupor.

Patient IV-8An elder brother of patient IV-5 had onset of disease at the

age of 36 years. He had severe loss of weight and chronic

constipation, and developed later mild gait ataxia anddysphagia. Autonomic dysfunction followed, notably

prominent hypersalivation and hyperthermia.




Table 2 Nerve fibre degeneration in the CNS of four Austrian FFI patients

Patient


CerebrumFrontal white matter 0 1 1 1*Central white matter 2 1 – –Temporal white matter 1 1 1 –Parietal white matter 0 1 1 –Occipital white matter 0 1 1 –Corpus callosum 0 0 – –Internal capsule 2 1 1 1Anterior commissure 0 – 0 –Fornix 2 1 – –Mamillothalamic tract – – 2 –Optic chiasm 0 0 – –

Optic tract 0 0 – –Cerebellum

Neocerebellar white matter 1 1 1 –White matter of vermis 2 2 – 1†

Archicerebellar white matter 1 1 – –Brainstem/spinal cord

Crus cerebri 1 1 1 –Superior cerebellar peduncle 2 1 1 –Middle cerebellar peduncle 1 – 1 1Inferior cerebellar peduncle 1 2 0 –Medial longitudinal fasciculus 2 2 1 1Central tegmental tract 2 2 2 2Medial lemniscus 0 0 1 1Lateral lemniscus 1 0 1 –Fasciculus gracilis 1 – 1 1

Fasciculus cuneatus 1 – 1 1Pyramidal tract 0 1 1 1Posterior spinal nerve root 1 – – –

0, 1, 2 no, single, multiple nerve fibre degenerations, respectively,as indicated by myelin balls in luxol fast blue/nuclear fast redstain; – not available. *Two blocks of cerebral cortex, regionunknown; †two blocks of cerebellar cortex, most likely vermis.

On admission to the Clinic of Neurology, University of

Vienna, the patient reported a 2-week episode of severe sleep

disturbance, which had improved 6 months previously under

treatment with benzodiazepines. During hospitalization, the

patient had mild difficulty in falling asleep. Neurological

examination revealed myoclonus of face, tongue and upper

limbs, and mild ataxia of limbs and gait. Mild spastic

paraparesis was considered a residue of a car accident in

1983 which had resulted in a fracture of the lumbar spine

and affection of spinal cord. There was mild cognitive

impairment, and apathy and lethargy.

CSF analysis revealed no abnormalities. CT disclosed mild

diffuse brain atrophy. EEG displayed signs of diffuse non-

specific parenchymal dysfunction. Three months after

discharge from the clinic the patient died from pneumonia,11 months after onset of disease. No progression of sleep

disorder was reported by his family doctor.

Patient III-5This patient was the mother of patients IV-5 and IV-8.

Disease manifested with initial insomnia and nocturnal motor

unrest, memory impairment, and perioral and periorbitalmyoclonus. Severe loss of weight, progressive apathy,

dysarthria and episodic irregular breathing and inspiratory

stridor followed. EEG showed moderate to prominent signs

of diffuse non-specific parenchymal dysfunction. CT revealed

moderate brain atrophy with frontocerebellar accentuation.

The patient died at the age of 58 years from broncho-

pneumonia 8 months after onset of disease.

Patient IV-13Disease manifested in this second cousin of patients IV-5

and IV-8 at the age of 20 years with severe loss of weight.She then developed vertigo and ataxia. Diplopia, dysarthria,

tremor, autonomic dysfunction (hyperthermia and chronic

constipation), progressive apathy and amnestic deficiencies

followed. Sleep disturbances or insomnia have not been

recorded. EEG showed mild diffuse non-specific parenchymal

dysfunction. CT was normal. She died severely cachectic

from pneumonia 20 months after onset of disease.

Patient III-13The mother of patient IV-13 had onset of disease at the age

of 62 years. Initial symptoms comprised loss of weight,

tiredness and short-term memory impairment. Within several

months, nocturnal insomnia, dysarthria and episodes of

diplopia developed. Her family then noticed progressive

apathy and confusions, notably at night. In the late phase of

disease, she developed progressive gait ataxia and perioral

myoclonus. Dysautonomia has not been recorded.

Haematological and biochemical findings were normal. EEG

showed moderate signs of diffuse non-specific parenchymal

dysfunction. CT disclosed pronounced cerebral and cerebellar

atrophy. The patient died 18 months after onset of disease.

NeuropathologyThe fresh brain of patient IV-5 weighed 1360 g. Brain weights

of other autopsied patients are not on record. Grossly, the

brains of patients IV-5 and IV-8 showed diffuse oedema

with narrowing of external and internal CSF spaces. Brain

sectioning revealed thalamic atrophy with a marbled aspect

of the cut surface (Fig. 2).

Tables 1 and 2 summarize grey and white matter lesioning

in patients III-5, IV-5, IV-8 and IV-13. Regional

histopathology was similar in all patients. Cerebral cortex

showed no unequivocal neuronal loss. Neuronal loss was

moderate to conspicuous in thalamus (most prominent in

medial and anterior thalamic nuclei) and inferior olivarynuclei, and slight to moderate in dorsal raphes and superior

central nuclei, in hypothalamus, some brainstem nuclei and



10 G. Almer et al.

Fig. 1 The pedigree of the Austrian FFI family comprises more than 50 members in five generations.

spinal grey matter. Cerebellar cortex displayed slight to

moderate reduction of Purkinje cells and the granular layer

contained some torpedoes. Astrogliosis involved all grey

matter structures and was particularly conspicuous in thalamic

nuclei (in anterior and medial nuclei more than in ventral

lateral nuclei) (Fig. 3D), nucleus ruber, periaqueductal, tectal

and tegmental grey, and raphe and olivary nuclei (Fig. 4A

and B). Cerebral cortex showed bilaminar accentuation of

astrogliosis (Fig. 3B). Spongiform change was discrete and

detectable in thalamic nuclei (Fig. 3C) of all four brains, in

pre/parasubiculum of two brains and in a small focus of

frontal cortex of one brain (Fig. 3A). Deposits of protease

resistant PrP (PrPres) detected by immunocytochemistry were

discrete and occurred only in brains III-5, IV-5 and IV-8.

In brain III-5, patchy and strip-like PrPres deposits with

perpendicular orientation to the surface were localized in the

molecular layer in one out of three blocks of cerebellar cortex

(Fig. 5A and B); pre/parasubiculum harboured discrete, fine-

granular synaptic type deposits. In brain IV-5, a single

vacuolated neuron in the inferior olivary nuclei (see below)

contained prominent intravacuolar granular PrPres deposits(Fig. 5C). A few other neurons displayed discrete granular

PrPres deposits on the surface and/or in the vacuoles, but the

majority was negative. In brain IV-8, a small area of frontal

cerebral cortex showed spongiform change and synaptic

type PrPres deposits with perivacuolar accentuation were

detectable. A few patchy PrPres deposits were confined to a

small focus in the molecular layer of cerebellar cortex. A

few vacuolated neurons in the inferior olivary nuclei had

discrete granular PrPres deposits on the surface and/or in the

cytoplasmic vacuoles. Internal organs of patient III-13 were

devoid of detectable PrPres deposits.

White matter of cerebrum and cerebellum showed scattered

myelin balls indicating widespread Wallerian type of nerve

fibre degeneration (Table 2). The nerve fibre degenerations are

possibly the pathological substrate, as lesions accompanied by

oedema, of discrete supratentorial MRI findings in patient

IV-5 (see Clinical findings). Primary demyelination with

preserved axons was not detectable. Flourishing nerve fibre

breakdown was most conspicuous in brainstem tracts (Table 2,

Fig. 4E). Alveus and hilus of inferior olives, and hilus of

dentate nuclei showed prominent depletion of nerve fibres.

Residual olivary neurons showed signs of transneuronal

degeneration (Fig. 4C and D) with vacuolation of cell bodiesand hypertrophied antler-like dendrites (olivary

‘pseudohypertrophy’) in all brains.




Fig. 2 A coronal slice of brain IV-5 shows diffuse oedema with narrowing of external and internal CSFspaces. The thalami are atrophic and display a marbled aspect.

Western blottingAt a final proteinase K concentration of 50 µg/ml, PrPres was

not detectable in any of the seven investigated regions.

Reduction of proteinase K concentration to 12.5 µg/ml

resulted in a positive signal from the basal ganglia, precentral

region and thalamus (Fig. 6). Glycoform ratios are similar

to those previously reported for FFI, with the diglycosylated

PrPres band being the most abundant (Gambetti et al., 1995;

Parchi et al., 1995). In the basal ganglia the average ratios

for the three PrPres glycoforms are: high: 58.43%, low:

33.40%, unglycosylated: 8.17%, taken from an average of

four separate blots.

PRNP analysisPCR amplification of the complete coding region of PRNP

generated a single product of 874 bp, thus excluding an

insertion mutation or deletion. SSCP analysis revealed an

aberrant migration pattern indicating a point mutation in the

C-terminal half of the gene. Close inspection of this region

by digestion of the PCR product with enzyme Tth111I and

direct sequencing defined this mutation as aspartic acid

(GAC) to asparagine (AAC) substitution at codon 178 of

PRNP (D178N). SSCP analysis, NspI digestion and directsequencing revealed homozygous codon 129 for methionine

in all four patients.

DiscussionOnset of disease was insidious in our FFI family, with initial

or early loss of weight in all five patients with detailed

histories. The first neuropsychiatric symptoms were insomnia

in one patient, lethargy in one patient, cognitive impairment

in one patient and ataxia in two patients. In the course of

disease, four patients developed progressive insomnia, four

patients autonomic dysfunction and all five patients motor

abnormalities. Symptomatology of our patients is thus typical

for FFI (Lugaresi et al., 1986; Manetto etal., 1992; Nagayama

et al., 1996). According to medical records, patient IV-13

presented clinically with some unusual features. Age at onset

of disease was 20 years. Together with a recent FFI patientfrom Australia (Silburn et al., 1996), this patient is the

youngest reported so far. Insomnia was not recorded during

the whole course of disease. However, evaluation of sleep

patterns by polysomnography was not performed.

Molecular analysis of PRNP in our family revealed the

codon 178 point mutation and methionine homozygosity at

position 129 in all four patients examined. Codon 178

mutation in conjunction with methionine at position 129 of

the mutant allele is the diagnostic genotype for FFI (Goldfarb

et al., 1992; Medori et al., 1992b). It has been shown that

the genotype of polymorphic codon 129 associates in FFI

with characteristic neuropathological features (Gambettiet al., 1995; Parchi et al., 1995). Patients with homozygous

codon 129 have prominent thalamic pathology, whereas



12 G. Almer et al.

Fig. 3 Cerebral histopathology in the Austrian FFI family. Spongiform change (A) is detectable in a focus of frontal cortex (brain IV-8;HE; 110), and (C) is discrete in thalamic nuclei (brain IV-5; HE, 140). Anti-GFAP immunocytochemistry shows (B) corticalastrogliosis with bilaminar accentuation (brain IV-5; 25) and (D) prominent astrogliosis in the thalamus (brain IV-5; 170).

lesioning of cerebral cortex is minor or absent (Gambetti

et al., 1995; Parchi et al., 1995; Reder et al., 1995; Nagayama

et al., 1996; Silburn et al., 1996). However, two recent

patients of two different kindreds with homozygous codon

129 showed prominent lesioning of cerebral cortex (McLean

et al., 1997; Rossi et al., 1998). Patients with heterozygous

codon 129 have prominent lesioning of the cerebral cortex

in addition to thalamic pathology (Gambetti et al., 1995;

Parchi et al., 1995). Our patients were all homozygous atposition 129; neuropathology showed prominent thalamic

lesioning, whereas cerebral cortex displayed only minor

histopathology with spongiform change confined to small

areas. However, anti-GFAP immunocytochemistry detected

widespread laminar astrogliosis. Astrogliosis also involved

regions that are not supposed to receive thalamocortical

projections, i.e. the occipital cortex. Widespread cortical

astrogliosis in the absence of neuronal loss and spongiform

change has been observed in members of other FFI kindreds

(Manetto et al., 1992; Medori et al., 1992a). Cortical

astrogliosis indicates submicroscopical lesioning of brain

parenchyma. Possible targets of lesioning are neuronalsubpopulations of the cortex. In Creutzfeldt–Jakob disease

brains, subtotal loss of the parvalbumin positive subset of

GABAergic neurons has been observed despite ‘normal’

appearance of tissue (Guentchev et al., 1997).

Brainstem histopathology in FFI has been described as

variable in distribution and limited in extent. Neuronal loss

and gliosis are most conspicuous and frequent in inferior

olives and minor in other nuclei (Manetto et al., 1992;

Gambetti et al., 1995; Reder et al., 1995; Nagayama et al.,

1996; Silburn et al., 1996; McLean et al., 1997; Rossi et al.,

1998). In nine patients from five different FFI families, theperiaqueductal grey matter showed slight to moderate gliosis

(Parchi et al., 1995). In one out of four patients of another

kindred, brainstem lesioning involved locus coeruleus, raphe

nucleus and reticular formation (McLean et al., 1997). One

FFI patient had prominent neuronal loss in the tectum (Reder

et al., 1995). In contrast, all our neuropathologically analysed

patients consistently had widespread and conspicuous

brainstem histopathology, although disease duration was short

to moderate (8, 11, 13 and 20 months, respectively).

In bovine spongiform encephalopathy (BSE) and scrapie of

sheep, vacuolation of brainstem neurons is pathognomonic

(Wells and Wilesmith, 1995; DeArmond and Prusiner, 1997).In human prion disease, conspicuous neuronal vacuolation has

been observed only in kuru (Hadlow, 1959; Klatzo et al.,




Fig. 4 Brainstem pathology in the Austrian FFI family (brain IV-5). (A) In the pons, gliosis (dark blue colour) is conspicuous inperiaqueductal grey and raphe (Kanzler stain; 3). (B) In medulla oblongata, gliosis is prominent in inferior olivary nuclei (Kanzlerstain;4). Residual olivary neurons show signs of transneuronal degeneration with (C) neuronal vacuolation (HE; 400) and(D) hypertrophied antler-like dendrites (Bielschowsky; 260). (E) flourishing nerve fibre breakdown with myelin balls in olivocerebellartract (luxol fast blue/nuclear fast red; 430).

1959; Hainfellner et al., 1997b). The olives of our FFI patients

showed severe neuronal loss, and residual neurons were

hypertrophied and vacuolated. Neuronal vacuolation in our

patients is reminiscent of that in BSE andscrapie.However, we

interpret neuronal vacuolation in our patients as transneuronal

degeneration because vacuolation was confined to the olives,

central tegmental tracts showed conspicuous degeneration and

hypertrophied antler-like dendrites were found as well. Olivary

‘pseudohypertrophy’ with neuronal vacuolation is a well

known pattern of transneuronal degeneration which has been

described as a sequel of central tegmental tract lesioning, most

commonly due to ischaemic infarction within the brainstem(Gautier and Blackwood, 1961).

In our hands, immunocytochemistry on numerous blocks

detected PrPres deposits in only three out of four patients. In

patient III-3, conspicuous PrPres deposits accumulated in one

out of three blocks in the molecular layer of cerebellar

cortex, and minor PrPres deposits were detectable in pre/

parasubiculum. Patients IV-5 and IV-8 had discrete PrPres

deposits in the inferior olivary nuclei. Patient IV-8 had, in

addition, a small focus of PrPres deposition in frontal cortex

and scant deposits in the molecular layer of cerebellar cortex.

Thus, the diagnostic value of anti-PrP immunocytochemistry

is limited in FFI, and immunocytochemical PrPres detection

requires examination of numerous tissue blocks including

areas with minor histopathology. In contrast to immuno-cytochemistry, Western blot analysis of brain IV-5 detected

PrPres in three out of seven CNS regions after mild protease



14 G. Almer et al.

Fig. 5 PrPres deposition patterns in the Austrian FFI family. In patient III-5 ( A and B), patchy and strip-like PrPres deposits are confinedto the molecular layer and show perpendicular orientation to the surface (anti-PrP) (A, 50; B, 145). (C) A single vacuolated neuronin the inferior olivary nuclei of patient IV-5 shows prominent intravacuolar accumulation of granular PrPres deposits. Other neurons aredevoid of PrPres deposits (anti-PrP) (160; inset: 730).

K digestion. This confirms that Western blotting is superior

to immunocytochemistry in detecting PrPres in FFI.

Descriptions of the immunomorphology of PrPres in FFI

are scant. In two out of four patients of an Australian kindred,

a weak fine granular staining of the neutropil has been

observed (McLean et al., 1997). In one of our patients, focal

PrPres deposits in the frontal cortex showed a synaptic type

pattern with perivacuolar accentuation. In two out of four of

our patients, a few vacuolated neurons in the inferior olivary

nuclei had discrete fine granular PrPres deposits on the surface

and/or in the cytoplasmic vacuoles; a single neuron had

prominent intravacuolar deposits (Fig. 5C). This is a unique

observation in FFI, resembling changes of brainstem neurons

in BSE (Wells and Wilesmith, 1995). In another of our

patients, PrPres deposits in the molecular layer of cerebellar

cortex showed a peculiar strip-like pattern with perpendicular

orientation to the surface. A comparable pattern of PrPres

deposition has not been described in FFI so far and has

not been observed in sporadic Creutzfeldt–Jakob disease

(Hainfellner and Budka, 1996), kuru (Hainfellner et al.,1997b) or Gerstmann–Straussler–Scheinker syndrome

(Hainfellner et al., 1995), but has been observed in some

cases of familial Creutzfeldt–Jakob disease (J.A.H. and H.B.,

unpublished observation).

Western blot analysis of the regional distribution of PrPres

in FFI has shown that histopathology is confined to brain areas

with PrPres accumulation. Conversely, PrPres was detectable in

areas with and without histopathology. On the basis of this

observation, it has been hypothesized that tissue lesioning in

FFI develops only in the presence of critical amounts of

PrPres and that vulnerability of brain parenchyma to the

presence of PrPres is regionally variable (Parchi et al., 1995).

However, immunocytochemistry detected little PrPres, in spite

of severe and widespread histopathology. Moreover, in our

patient IV-5, Western blotting did not detect PrPres in the

severely damaged brainstem. Thus, PrPres accumulation in

our FFI patients dissociates not only quantitatively but also

topographically from histopathology. With regard to the

unresolved pathogenic role of PrP in prion diseases, this

dissociation supports a loss of function model (Whittington

et al., 1995) rather than neurotoxicity (Brown and

Kretzschmar, 1997). Experimental data suggest that loss of functional PrP impairs the maintenance and normal function

of synapses. Thus, synapses are a likely target of lesioning,




Fig. 6 Western blot analysis of a normal brain and of FFI brainIV-5. Positive signals are present in FFI in basal ganglia,precentral and thalamus regions. The diglycosylated PrPres band isthe most abundant. All samples were treated with proteinase K(PK) at a concentration of 12.5 µg/ml before electrophoresis.Molecular weight standards are shown on the left.

following loss of functional PrP in prion disease (Whittington

et al., 1995).

AcknowledgementsWe wish to thank Dr G. R. Trabattoni for helping with

neuropathological analysis and Mrs H. Flicker for excellent

technical assistance. This work is part of the European

Union Biomed-2 Concerted Action ‘Human transmissible

spongiform encephalopathies (prion diseases): neuro-

pathology and phenotypic variation’ (project leader:

H. Budka).

References

Almer G, Hainfellner JA, Budka H, Brucke T. Clinical, pathological

and molecular features of the first Austrian family with fatal familial

insomnia [abstract]. Eur J Neurol 1997; 4 Suppl 1: S1.

Brown DR, Kretzschmar HA. Microglia and prion disease: a review.

Histol Histopathol 1997; 12: 883–92.

Budka H, Hainfellner JA, Almer G, Brucke T, Windl O, Kretzschmar

HA, et al. A new Austrian family with fatal familial insomnia:

brain pathology without detectable PrPres [abstract]. Brain Pathol

1997; 7: 1267.

Collinge J, Sidle KC, Meads J, Ironside J, Hill AF. Molecularanalysis of prion strain variation and the aetiology of ‘new variant’

CJD. Nature 1996; 383: 685–90.

DeArmond SJ, Prusiner SB. Prion diseases. In: Graham, DI, Lantos

PL, editors. Greenfield’s neuropathology, Vol. 2. 6th ed. London:

Arnold; 1997. p. 235–80.

Gambetti P, Parchi P, Petersen RB, Chen SG, Lugaresi E. Fatalfamilial insomnia and familial Creutzfeldt-Jakob disease: clinical,

pathological and molecular features. Brain Pathol 1995; 5: 43–51.

Gautier JC, Blackwood W. Enlargement of the inferior olivary

nucleus in association with lesions of the central tegmental tract or

dentate nucleus. Brain 1961; 84, 341–61.

Goldfarb LG, Petersen RB, Tabaton M, Brown P, LeBlanc AC,

Montagna P, et al. Fatal familial insomnia and familial Creutzfeldt-

Jakob disease: disease phenotype determined by a DNA

polymorphism. Science 1992; 258: 806–8.

Goodbrand IA, Ironside JW, Nicolson D, Bell JE. Prion protein

accumulation in the spinal cords of patients with sporadic and

growth hormone associated Creutzfeldt-Jakob disease. Neurosci Lett1995; 183: 127–30.

Guentchev M, Hainfellner JA, Trabattoni GR, Budka H. Distribution

of parvalbumin-immunoreactive neurons in brain correlates with

hippocampal and temporal cortical pathology in Creutzfeldt-Jakob

disease. J Neuropathol Exp Neurol 1997; 56: 1119–24.

Hadlow WJ. Scrapie and kuru. Lancet 1959; 2: 289–90.

Hainfellner JA, Budka H. Immunomorphology of human prion

diseases. In: Court L, Dodet B, editors. Transmissible subacute

spongiform encephalopathies: prion diseases. Amsterdam: Elsevier;

1996. p. 75–80.

Hainfellner JA, Brantner-Inthaler S, Cervenakova L, Brown P,

Kitamoto T, Tateishi J, et al. The original Gerstmann-Straussler-Scheinker family of Austria: divergent clinicopathological

phenotypes but constant PrP genotype. Brain Pathol 1995; 5: 201–11.

Hainfellner JA, Almer G, Brucke T, Jellinger K, Kleinert R,

Bayer G, et al. Fatal familial insomnia in a new Austrian family:

dissociation of brain pathology from PrP deposition [abstract]. Clin

Neuropathol 1997a; 16: 261.

Hainfellner JA, Liberski PP, Guiroy DC, Cervenakova L, Brown P,

Gajdusek DC, et al. Pathology and immunocytochemistry of a kuru

brain. Brain Pathol 1997b; 7: 547–53.

Klatzo I, Gajdusek DC, Zigas V. Pathology of kuru. Lab Invest

1959; 8: 799–847.

Kretzschmar HA, Stowring LE, Westaway D, Stubblebine WH,

Prusiner SB, DeArmond SJ. Molecular cloning of a human prion

protein cDNA. DNA 1986; 5: 315–24.

Lugaresi E, Medori R, Montagna P, Baruzzi A, Cortelli P, Lugaresi

A, et al. Fatal familial insomnia and dysautonomia with selective

degeneration of thalamic nuclei. N Engl J Med 1986; 315: 997–1003.

Manetto V, Medori R, Cortelli P, Montagna P, Tinuper P, Baruzzi

A, et al. Fatal familial insomnia: clinical and pathologic study of

five new cases. Neurology 1992; 42: 312–9.

McLean CA, Storey E, Gardner RJ, Tannenberg AE, Cervenakova

L, Brown P. The D178N (cis-129M) ‘fatal familial insomnia’

mutation associated with diverse clinicopathologic phenotypes inan Australian kindred. Neurology 1997; 49: 552–8.

Medori R, Montagna P, Tritschler HJ, LeBlanc A, Cortelli P, Tinuper



16 G. Almer et al.

P, et al. Fatal familial insomnia: a second kindred with mutation of

prion protein gene at codon 178. Neurology 1992a; 42: 669–70.

Medori R, Tritschler HJ, LeBlanc A, Villare F, Manetto V, Chen

HY, et al. Fatal familial insomnia, a prion disease with a mutationat codon 178 of the prion protein gene. N Engl J Med 1992b; 326:

444–9.

Nagayama M, Shinohara Y, Furukawa H, Kitamoto T. Fatal familial

insomnia with a mutation at codon 178 of the prion protein gene:

first report from Japan. Neurology 1996; 47: 1313–6.

Nicholl D, Windl O, de Silva R, Sawcer S, Dempster M, Ironside

JW, et al. Inherited Creutzfeldt-Jakob disease in a British family

associated with a novel 144 base pair insertion of the prion protein

gene. J Neurol Neurosurg Psychiatry 1995; 58: 65–9.

Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T. Detection

of polymorphisms of human DNA by gel electrophoresis as single-

strand conformation polymorphisms. Proc Natl Acad Sci USA 1989;86: 2766–70.

Parchi P, Castellani R, Cortelli P, Montagna P, Chen SG, Petersen

RB, et al. Regional distribution of protease-resistant prion protein

in fatal familial insomnia. Ann Neurol 1995; 38: 21–9.

Reder AT, Mednick AS, Brown P, Spire JP, Van Cauter E, Wollmann

RL, et al. Clinical and genetic studies of fatal familial insomnia.

Neurology 1995; 45: 1068–75.

Rossi G, Macchi G, Porro M, Giaccone G, Bugiani M, Scarpini

E, et al. Fatal familial insomnia: genetic, neuropathologic, and

biochemical study of a patient from a new Italian kindred. Neurology

1998; 50: 688–92.

Sambrook J. Molecular cloning: a laboratory manual. 2nd ed. New

York: Cold Spring Harbor Laboratory Press; 1989.

Silburn P, Cervenakova L, Varghese P, Tannenberg A, Brown P,

Boyle R. Fatal familial insomnia: a seventh family. Neurology 1996;

47: 1326–8.

Wells GA, Wilesmith JW. The neuropathology and epidemiology

of bovine spongiform encephalopathy. Brain Pathol 1995; 5: 91–103.

Whittington MA, Sidle KC, Gowland I, Meads J, Hill AF, Palmer

MS, et al. Rescue of neurophysiological phenotype seen in PrP null

mice by transgene encoding human prion protein. Nat Genet 1995;

9: 197–201.

Windl O, Dempster M, Estibeiro JP, Lathe R, de Silva R, Esmonde

T, et al. Genetic basis of Creutzfeldt-Jakob disease in the United

Kingdom: a systematic analysis of predisposing mutations and

allelic variation in the PRNP gene. Hum Genet 1996; 98: 259–64.

Received May 21, 1998. Revised September 3, 1998.

Accepted September 7, 1998

a case of family insomnia

Documents