ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2012

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 830

Genetic and ClinicalInvestigation of NoonanSpectrum Disorders

SARA EKVALL

ISSN 1651-6206ISBN 978-91-554-8511-5urn:nbn:se:uu:diva-183325

Dissertation presented at Uppsala University to be publicly examined in Rudbecksalen,Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, Friday, December 7, 2012 at09:15 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will beconducted in English.

AbstractEkvall, S. 2012. Genetic and Clinical Investigation of Noonan Spectrum Disorders. ActaUniversitatis Upsaliensis. Digital Comprehensive Summaries of Uppsala Dissertations fromthe Faculty of Medicine 830. 73 pp. Uppsala. ISBN 978-91-554-8511-5.

Noonan spectrum disorders belong to the RASopathies, a group of clinically relateddevelopmental disorders caused by dysregulation of the RAS-MAPK pathway. This thesisdescribes genetic and clinical investigations of six families with Noonan spectrum disorders.

In the first family, the index patient presented with severe Noonan syndrome (NS) andmultiple café-au-lait (CAL) spots, while four additional family members displayed multipleCAL spots only. Genetic analysis of four RAS-MAPK genes revealed a de novo PTPN11mutation and a paternally inherited NF1 mutation, which could explain the atypically severeNS, but not the CAL spots trait in the family. The co-occurrence of two mutations was alsopresent in another patient with a severe/complex NS-like phenotype. Genetic analysis of nineRASopathy-associated genes identified a de novo SHOC2 mutation and a maternally inheritedPTPN11 mutation. The latter was also identified in her brother. Both the mother and the brotherdisplayed mild phenotypes of NS. The results from these studies suggest that an additive effectof co-occurring mutations contributes to severe/complex NS phenotypes.

The inherent difficulty in diagnosing Noonan spectrum disorders is evident in familieswith neurofibromatosis-Noonan syndrome (NFNS). An analysis of nine RASopathy-associatedgenes in a five-generation family with NFNS revealed a novel NF1 mutation in all affectedfamily members. Notably, this family was initially diagnosed with NS and CAL spots. Theclinical overlap between NS and NFNS was further demonstrated in three additional NFNSfamilies. An analysis of twelve RASopathy-associated genes revealed three different NF1mutations, all segregating with the disorder in each family. These mutations have been reportedin patients with NF1, but have, to our knowledge, not been associated with NFNS previously.Together, these findings support the notion that NFNS is a variant of NF1. Due to the clinicaloverlap between NS and NFNS, we propose screening for NF1 mutations in NS patients negativefor mutations in NS-associated genes, preferentially when CAL spots are present.

In conclusion, this thesis suggests that co-occurrence of mutations or modifying loci in theRAS-MAPK pathway contributes to the clinical variability observed within Noonan spectrumdisorders and further demonstrates the importance of accurate genetic diagnosis.

Keywords: RASopathies, Noonan syndrome, neurofibromatosis type 1, neurofibromatosis-Noonan syndrome, RAS-MAPK pathway, mutation

Sara Ekvall, Uppsala University, Department of Immunology, Genetics and Pathology,Rudbecklaboratoriet, SE-751 85 Uppsala, Sweden.

© Sara Ekvall 2012

ISSN 1651-6206ISBN 978-91-554-8511-5urn:nbn:se:uu:diva-183325 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-183325)

Till min underbara familj

Supervisors Marie-Louise Bondeson, Associate Professor Göran Annerén, Professor, M.D. Dept. of Immunology, Genetics and Pathology Uppsala University Uppsala, Sweden Faculty opponent Göran Andersson, Professor Dept. of Animal Breeding and Genetics

Swedish University of Agricultural Sciences Uppsala, Sweden Review board Tobias Sjöblom, Associate Professor Dept. of Immunology, Genetics and Pathology

Uppsala University Uppsala, Sweden Jovanna Dahlgren, Associate Professor, M.D. Dept. of Paediatrics

The Queen Silvia Children’s Hospital University of Gothenburg Gothenburg, Sweden Margareta Dahl, Associate Professor, M.D. Dept. of Women’s and Children’s Health

Uppsala University Children’s Hospital Uppsala University

Uppsala, Sweden Chairman Berivan Baskin, Ph.D., FACMG, FCCMG Dept. of Immunology, Genetics and Pathology

Uppsala University Uppsala, Sweden

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Nyström A.M., Ekvall S., Strömberg B., Holmström G.,

Thuresson A.C., Annerén G., Bondeson M.L. (2009) A severe form of Noonan syndrome and autosomal dominant café-au-lait spots – evidence for different genetic origins. Acta Paediatr, 98(4):693–8

II Ekvall S., Hagenäs L., Allanson J., Annerén G., Bondeson M.L. (2011) Co-occurring SHOC2 and PTPN11 mutations in a patient with severe/complex Noonan syndrome-like phenotype. Am J Med Genet A, 155A(6):1217-24

III Nyström A.M.*, Ekvall S.*, Allanson J., Edeby C., Elinder M.,

Holmström G., Bondeson M.L., Annerén G. (2009) Noonan syndrome and neurofibromatosis type I in a family with a novel mutation in NF1. Clin Genet, 76(6):524-34

IV Ekvall S., Sjörs K., Jonzon A., Annerén G., Bondeson M.L.

Mutations in NF1 in families with neurofibromatosis type I and neurofibromatosis-Noonan syndrome. Manuscript

*Equal first authors

Reprints were made with permission from the respective publishers.

Additional publications by the author

1. Nyström A.M., Ekvall S., Berglund E., Björkqvist M., Braathen G., Duchen K., Enell H., Holmberg E., Holmlund U., Olsson-Engman M., Annerén G., Bondeson M.L. (2008) Noonan and cardio-facio-cutaneous syndromes: two clinically and genetically overlapping disorders. J Med Genet, 45(8):500-6

2. Nyström A.M., Ekvall S., Thuresson A.C., Denayer E., Legius E.,

Kamali-Moghaddam M., Westermark B., Annerén G., Bondeson M.L. (2010) Investigation of gene dosage imbalances in patients with Noonan syndrome using multiplex ligation-dependent probe amplifi-cation analysis. Eur J Med Genet, 53(3):117-21

3. Wittström E., Ekvall S., Schatz P., Bondeson M.L., Ponjavic V.,

Andréasson S. (2011) Morphological and functional changes in mul-tifocal vitelliform retinopathy and biallelic mutations in BEST1. Ophthalmic Genet, 32(2):83-96

Contents

Introduction ................................................................................................... 15 The human genome .................................................................................. 15 Human genetic variation .......................................................................... 15 Disease-causing variants .......................................................................... 17 Human genetic disorders .......................................................................... 17

Monogenic disorders ........................................................................... 17 Methods in disease-gene identification .................................................... 19

Linkage analysis .................................................................................. 19 Sanger sequencing ............................................................................... 19 Restriction fragment length polymorphism (RFLP) ............................ 20 Multiplex ligation-dependent probe amplification (MLPA) ................ 20 SNP arrays ........................................................................................... 21 Next-generation sequencing ................................................................ 21

The RAS-MAPK pathway ............................................................................ 22 Activation of the RAS-MAPK pathway ................................................... 22 Regulation of the RAS-MAPK pathway .................................................. 24

Phosphorylation and dephosphorylation .............................................. 24 Scaffolding proteins, phosphatases and inhibitors ............................... 25 Internalization and degradation of receptors ....................................... 25 Histone modifications .......................................................................... 25 Post-transcriptional regulation ............................................................. 25

Determination of signal specificity of the RAS-MAPK pathway ............ 26 Signal strength and duration ................................................................ 26 Cross-talk with other pathways ........................................................... 27 Subcellular localization of components of the pathway ...................... 27

Cancer and the RAS-MAPK pathway ...................................................... 27 Drug development ............................................................................... 28

RASopathies ................................................................................................. 29 Noonan and Noonan-like syndromes ....................................................... 30

Clinical description .............................................................................. 30 Genetic description .............................................................................. 31 Genotype-phenotype correlations ........................................................ 35

Neurofibromatosis type 1 ......................................................................... 37 Clinical description .............................................................................. 37

Genetic description .............................................................................. 38 Genotype-phenotype correlations ........................................................ 40

Neurofibromatosis-Noonan syndrome ..................................................... 41 Clinical description .............................................................................. 41 Genetic description .............................................................................. 41 Genotype-phenotype correlations ........................................................ 42

Animal models and future treatments ...................................................... 42

Present investigations .................................................................................... 44 Background .............................................................................................. 44 Aims ......................................................................................................... 45 Paper I ...................................................................................................... 45 Paper II ..................................................................................................... 47 Paper III .................................................................................................... 50 Paper IV ................................................................................................... 52

Concluding remarks and future perspectives ................................................ 54

Populärvetenskaplig svensk sammanfattning ............................................... 58

Acknowledgements ....................................................................................... 60

References ..................................................................................................... 62

Abbreviations

A Adenine or alanine AKT v-akt murine thymoma viral oncogene ARAF v-raf murine sarcoma 3611 viral oncogene homolog BRAF v-raf murine sarcoma viral oncogene homolog B1 C Cytosine or cysteine CAL Café-au-lait cAMP Cyclic adenosine monophosphate CBL Casitas B-lineage Lymphoma protein/gene Cdc25 Cell division cycle 25 protein cDNA Complementary deoxyribonucleic acid CFCS Cardio-facio-cutaneous syndrome c-Fos v-fos FBJ murine osteosarcoma viral oncogene CNV Copy number variant CR1-3 Conserved region 1-3 CS Costello syndrome CSRD Cysteine/serine-rich domain DECIPHER DatabasE of Chromosomal Imbalance and Phenotype in

Humans using Ensembl Resources DH Dbl homology DNA Deoxyribonucleic acid DUSP Dual-specificity phosphatase EGF Epidermal growth factor EGFR Epidermal growth factor receptor Elk1 E twenty-six-like transcription factor 1 EMH Extramedullary hematopoiesis ENCODE Encyclopaedia of DNA Elements ERK Extracellular signal regulated kinase EVI2A Ecotropic viral integration site 2A EVI2B Ecotropic viral integration site 2B F Phenylalanine FPPS Farnesyl diphosphate synthetase G Guanine or glycine GAP GTPase-activating protein GDP Guanosine diphosphate GEF Guanine-nucleotide-exchange factor GH Growth hormone

Grb2 Growth factor receptor-bound protein 2 GRD GAP-related domain GTP Guanosine triphosphate HD Histone domain HGMD Human Gene Mutation Database HRAS v-Ha-ras Harvey RAS homolog HuR Human antigen R I Isoleucine JMML Juvenile myelomonocytic leukaemia JNK c-Jun N-terminal kinases KRAS v-Ki-ras2 Kirsten RAS homolog KSR Kinase suppressor of RAS L Leucine LCRs Low copy repeats let-7 Lethal-7 LOD Logarithm of the odds LOVD Leiden Open Variation Database LS LEOPARD syndrome MAP2K1/2 Mitogen-activated protein kinase kinase 1/2 MAPK Mitogen-activated protein kinase Mb Mega bases MEK1/2 Mitogen-activated protein kinase kinase 1/2 miRNA Micro ribonucleic acid MKP-1 MAPK phosphatase 1 MLPA Multiplex ligation-dependent probe amplification MPNSTs Malignant peripheral nerve sheath tumours mRNA Messenger ribonucleic acid mTOR Mammalian target of rapamycin MYST4/KAT6B K(lysine) acetyltransferase 6B NCBI National Center for Biotechnology Information NCFCs Neuro-cardio-facio-cutaneous syndromes NF1 Neurofibromatosis type 1 or neurofibromin gene NFNS Neurofibromatosis-Noonan syndrome NF-κB Nuclear factor kappa-light-chain-enhancer of activated B

cells NGF Nerve growth factor NIH National Institutes of Health NRAS Neuroblastoma RAS viral (v-ras) oncogene homolog NS Noonan syndrome NS/LAH Noonan syndrome with loose anagen hair OMIM Online Mendelian Inheritance in Man OMGP Oligodendrocyte myelin glycoprotein OPG Optic pathway glioma ORF Open reading frame

PC12 Pheochromocytoma cell line 12 PCR Polymerase chain reaction PDGF Platelet-derived growth factor PH Pleckstrin homology PI3K-AKT Phosphatidylinositol 3-kinase-v-akt murine thymoma viral

oncogene PKA Protein kinase A PLA2 Phospholipase A2 PP1C Catalytic protein phosphatase 1 subunit PTP Protein tyrosine phosphatase PTPN11 Protein tyrosine phosphatase, non-receptor type 11 gene PUM2 Pumilio homolog 2 R Arginine RAC1 RAS-related C3 botulinum toxin substrate 1 RAF1 v-raf-1 murine leukaemia viral oncogene homolog 1 RAS Rat sarcoma viral oncogene; HRAS, KRAS and NRAS RAS-MAPK RAS-induced mitogen-activated protein kinase Rem RAS exchanger motif RFLP Restriction fragment length polymorphism RT-PCR Reverse transcription-polymerase chain reaction S Serine SAPK Stress-activated protein kinase Ser Serine SH2 Src-homology 2 domain SHOC2 Soc-2 suppressor of clear homolog SHP2 Protein tyrosine phosphatase, non-receptor type 11 SNP Single nucleotide polymorphism SOLiD Supported oligonucleotide ligation and detection SOS1/2 Son of sevenless homolog 1/2 SPRED1/2 Sprouty-related, EVH1 domain-containing protein 1/2 SPRY Sprouty homolog, antagonist of FGF signaling T Thymine or Threonine Tyr Tyrosine UTR Untranslated region

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Introduction

The human genome Almost 60 years have passed since Watson and Crick discovered the struc-ture of deoxyribonucleic acid, DNA, in 1953. [1] They put forward the prin-ciple that the four nucleotide bases, adenine (A), cytosine (C), thymine (T) and guanine (G), pair up with each other in a specific manner, A to T and C to G, and form a complementary double helix; thereby, explaining how an organism is able to copy its DNA.

In 2004, another important step in understanding the human genome was taken, when a near complete sequence of the human genome was published. [2] From this, we learned that the human genome is approximately three billion base pairs long and contains 20,000-25,000 protein-coding genes.

Recently, the Encyclopaedia of DNA Elements (ENCODE) project re-leased a number of publications, where regions of transcription, transcription factor association, chromatin structure and histone modification were sys-tematically mapped. Together, these researchers could assign biochemical functions for 80% of the genome, providing new insights into the organiza-tion of our genome and the mechanisms of gene regulation. This demon-strates that much more than just the protein-coding genes are of great im-portance for us. [3]

Human genetic variation Although the genomes between two randomly selected humans resemble each other, a considerable number of differences exist between them. [4, 5] These differences are called genetic variations and can vary in type and size, ranging from differences in single nucleotides to duplications of large seg-ments. A genetic variant present in more than 1% of the population is con-sidered to be a polymorphism. [6]

Two large projects, the International HapMap project and the 1000 Ge-nomes project, have been initiated to identify genetic similarities and differ-ences in humans. The International HapMap project started in 2002, with the aim of genotyping and characterizing single nucleotide polymorphisms (SNPs) and structural variations in large groups of individuals from different geographical origins. [7] In 2007, the 1000 Genomes project was launched,

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with the purpose of sequencing 1000 individuals from different populations using high-throughput sequencing technologies. [8]

One of the most common types of variation is SNPs. As the name suggests, a SNP is a difference, e.g. deletion, insertion or substitution, of one single nucleotide. They occur on average once every 100 to 300 bases, although the density can vary throughout the genome. [9] In June 2012, the NCBI’s SNP database made a new release containing approximately 38 million validated reference SNPs. (ncbi.nlm.nih.gov/projects/SNP/snp_summary.cgi)

Another type of variant is repeat sequences, either tandemly repeated or interspersed, and they account for more than 50% of the genome. [10] The tandem repeats can be subdivided into satellites, minisatellites, microsatel-lites and mononucleic tracts, depending on the length of the total repeat tract (more than 105 base pairs for satellites).

Structural variations are the largest variation with regards to the length of the involved DNA segment. They are defined as genomic alterations involv-ing segments of DNA larger than 1kb and can be divided into five subcate-gories: copy number variants (CNVs), segmental duplications, inversions, translocations and segmental uniparental disomy. [11]

CNVs are segments of DNA present at different copy numbers compared to a reference sequence and represent insertions, deletions or duplications. The total number of CNVs collected within the Database of Genomic Vari-ants has now reached approximately 67,000 (projects.tcag.ca/variation/). A recent study revealed that less than 5% of the human genome is affected by large CNVs [12], rather than the 12% first estimated [13].

The second category of structural variations, segmental duplications, is DNA segments occurring in two or more copies and with a sequence identity larger than 90%. They can vary in copy number; hence, segmental duplica-tions can also be CNVs. About 5% of the human genome is constituted of segmental duplications and they can be both inter- and intra-chromosomal. [2]

A DNA segment with a reversed orientation in reference to the rest of the chromosome is called an inversion, whereas a change in position of a DNA segment within the genome without a change in the total DNA content is called a translocation. [11] Translocations can also be either intra- or inter-chromosomal.

The final type of structural variation, segmental uniparental disomy, is a phenomenon where a pair of homologous chromosomes in one individual is derived from a single parent.

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Disease-causing variants Changes in the nucleotide sequence are often referred to as variants; some of them are responsible for causing human genetic disorders and are commonly denoted as mutations. There are many types of mutations and they can be subdivided based on either the type and size of the mutation, or the effect of the aberration on a molecular level, i.e. a loss-of-function or a gain-of-function mutation.

A point mutation is a substitution, a deletion or an insertion of a single nu-cleotide and it can be located in either coding regions or non-coding regions. In the coding region, point mutations can be classified as missense, non-sense, frameshift, splice site or silent mutations, all depending on the out-come of the protein. Point mutations in non-coding regions can be positioned in a promoter, a splice site or another regulatory sequence and still affect the protein outcome and cause disease. [14]

Larger aberrations, such as duplications, deletions, inversions, insertions and translocations, can range from only a few nucleotides up to several Mb and the effects are similar to point mutations, but here more than one gene can be affected.

Another type of mutation is trinucleotide repeat expansions, particularly associated with neurodegenerative disorders, e.g. Huntington’s disease. [15]

There are several databases collecting mutations associated with human dis-orders, such as the DatabasE of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources (DECIPHER; decipher.sanger.ac.uk), the Human Gene Mutation Database (HGMD; www.hgmd.org), the Online Mendelian Inheritance in Man (OMIM; ncbi.nlm.nih.gov/omim) and the Leiden Open Variation Database (LOVD; www.lovd.nl).

Human genetic disorders Genetic disorders can be classified into four different categories, depending on the type of mutation associated with the disorder and the possible in-volvement of the environment. The categories are monogenic (also called Mendelian) disorders, complex (also called multifactorial) disorders, chro-mosomal disorders and mitochondrial disorders. The present thesis will fo-cus on disorders belonging to the group of monogenic disorders.

Monogenic disorders Monogenic disorders are rare disorders, caused by mutations in one single gene or its regulatory sequences. However, allelic heterogeneity is usually

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present, i.e. when different mutations in the same gene cause the same disor-der. In some monogenic disorders, mutations at different loci cause the same phenotype, called locus heterogeneity. A third type of heterogeneity is clini-cal heterogeneity, where mutations in the same gene are associated with different disorders, often denoted as allelic disorders. [16]

Five different inheritance patterns exist for monogenic disorders, namely:

Autosomal dominant, in which affected individuals are heterozygous for the mutated allele, located on one of the autosomes. A disorder with this inheritance pattern can affect both males and females, and can be trans-mitted by either sex.

Autosomal recessive, where affected individuals are homozygous or compound heterozygous for the mutated allele, located on one of the au-tosomes. An autosomal recessive disorder can also affect both males and females and parents of affected individuals are usually heterozygous for the mutated allele.

X-linked dominant. Here, the mutated allele is located on the X-chromosome; thus, the only X-chromosome of affected males harbours the mutated allele and affected females are heterozygous. Both sexes can transmit this type of disorder, but females are more often affected, since an affected male will always transmit the disorder to his daughters.

X-linked recessive, in which affected females are homozygous for the mutated allele and the only X-chromosome of affected males harbours the mutated allele. Mainly males are affected by X-linked recessive dis-orders, since they carry only one X-chromosome. The mother of an af-fected male is usually heterozygous for the mutated allele, whereas the status of the father is of no importance. However, an affected male will always transmit the mutated allele to his daughters, but they will only be carriers of the disorder, not affected, unless they inherit a mutated allele from their mother as well.

Y-linked, where the mutated allele is located on the Y-chromosome; thus, only affecting males and also only transmitted by males. This type of disorder is extremely rare.

Reduced penetrance is a complicating factor when studying the inheritance of a disorder, meaning that not all individuals harbouring a mutation will express the disorder. Another complicating factor is the clinical variability, where individuals with the same disorder and even the same mutation ex-press different features or different severity of features. Furthermore, the age of onset of features and the changing of features with age are additional complicating factors.

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Both reduced penetrance and clinical variability could in some cases be explained by different genetic components that modify the phenotype, such as modifier genes, allelic or locus heterogeneity, or environmental factors.

Methods in disease-gene identification

Many different strategies exist, which aim to identify the genetic defects causing a disorder. A few examples, most of them used in the present thesis, will be discussed here.

Linkage analysis Linkage analysis identifies the disease gene by its position in the genome, with no assumptions about its function. This type of approach is most suita-ble for studies of large families, where the clinical diagnosis is well-defined.

The basic principle is that genetic markers, e.g. SNPs or microsatellites, close to the mutation will be inherited together with the mutation as a block (haplotype) more often than is expected by random segregation and are, thus, said to be linked to the disease locus. This linkage is due to the fact that re-combination is less likely to occur between closely positioned loci. By using linkage analysis, genomic regions associated with the disease can be identi-fied by studying the inheritance of these genetic markers in affected and unaffected family members, and statistically calculating the so-called LOD (Logarithm of the ODds) score, which is a measure of the likelihood of ge-netic linkage between two loci and a function of the recombination fraction. For a monogenic trait, a LOD score >3 (>2 for X-linked) is required for sig-nificant evidence of linkage, which means that the odds of the two loci being linked is 1000 times greater than the odds of them being unlinked. [17]

Linkage analysis can be performed in either a candidate-gene manner or a genome-wide approach.

Sanger sequencing Once linkage analysis has revealed a genomic region that is linked to a cer-tain disease, candidate genes can be selected by literature searches in public databases or by the use of interaction network programs, e.g. Ingenuity pathway analysis. A number of candidate genes can then be sequenced by Sanger sequencing to possibly find the disease-causing variant.

If the disorder of a patient has been previously associated with certain genes, the genomic region of interest is already defined; thus, Sanger se-quencing can be performed directly on that particular gene or those particu-lar genes of interest.

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Sanger sequencing is similar to the well-known technique polymerase chain reaction (PCR), but only one primer is used in each reaction instead of two. Furthermore, some of the nucleotides are fluorescently labelled terminators, which will stop the synthesis once incorporated into the PCR fragment. Thus, different sizes of fragments ending with a fluorescently labelled nu-cleotide terminator will exist in one reaction. These fragments are then sepa-rated according to size by capillary gel electrophoresis and the fluorophores are detected by a laser. Since each of the four types of nucleotide terminators has a different colour depending on the fluorophore, the sequence of the fragment can then be read and analysed by certain computer software. [18]

Restriction fragment length polymorphism (RFLP) When a variant is found by Sanger sequencing, there is usually a need for screening of additional family members or unrelated controls, in order to be able to determine whether it is the causative variant or not. This can be per-formed by Sanger sequencing, but another method of choice is RFLP.

This method is based on the fact that restriction enzymes recognize spe-cific sequence motifs and cleave a DNA fragment at these recognition sites. A mutation can either introduce or delete such a site through its change in DNA sequence, which will then give a different cleavage pattern when com-pared to the cleavage pattern of a sequence without that specific mutation. After the cleavage has been performed, the different cleavage patterns can be analysed by gel electrophoresis. [19]

Multiplex ligation-dependent probe amplification (MLPA) Several methods exist for detecting gains and losses of regions in the ge-nome. One such method is MLPA, which allows for simultaneous detection of several different targets. Each target has two probes, designed to bind adjacent to each other. The probes contain one target-specific hybridization sequence and one universal PCR primer recognition sequence. The length of the two probes together is unique for each target. After hybridization of the probes to the targets, the probes are ligated and then denatured. The next step is amplification of the ligated probes with a fluorescently labelled primer pair. Since all probes contain universal PCR primer recognition sequences, the amplification can be performed simultaneously for all probes. The ampli-fication products are then separated according to size by capillary gel elec-trophoresis and the unique length of each ligated probe pair makes it possi-ble to directly relate the amount of amplification product to the amount of initial target. By calculating the ratio of the amplification product in patients and controls, possible gains or losses for each target can be determined. A ratio of 1.5 indicates a gain and 0.5 a loss, whereas a ratio of 1 is considered as normal. [20]

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SNP arrays Another method to identify gains and losses in the genome is the use of SNP arrays, which are high-density synthetic oligonucleotide microarrays, requir-ing only a small amount of DNA to genotype hundreds of thousands or even millions of SNPs simultaneously. [12] This technique can also be used for linkage analysis (described earlier) and autozygosity mapping, which is the search for chromosomal regions where affected individuals are homozygous for an allele identical by descent. Autosomal recessive disease-causing genes are usually identified by autozygosity mapping of consanguineous families or individuals originating from the same geographical area.

The procedure starts with digestion of genomic DNA by a restriction en-zyme and ligation of adaptors to the digested fragments. The fragments are then amplified simultaneously by PCR, using primers recognizing the adap-tor sequences. This is followed by fragmentation of the amplified fragments, labelling and finally hybridization to the SNP array. The resulting hybridiza-tion pattern can then be interpreted by computer analysis, to identify the genotype of each SNP and evaluate possible copy number variations or linked regions by linkage analysis. (www.affymetrix.com)

Next-generation sequencing New technologies for sequencing have quite recently been developed, with the possibility of sequencing whole genomes in a significantly shorter period of time than traditional Sanger sequencing. These technologies, such as the Solexa sequencing-by-synthesis (Illumina), the 454 pyrosequencing (Roche 454) and the Supported oligonucleotide ligation and detection platform tech-nology (SOLiD; Applied Biosystems, now Life Technologies), will probably make sequencing of the whole genome or at least the exome, i.e. the 1-2% of the genome consisting of exons, a standard component of biomedical re-search and patient care in the future. [21] Already, they have been used suc-cessfully for screening of patients with unknown genetic causes and where there is no history of the disorder in the family or the family size is small, making linkage analysis very difficult. [22] Targeted re-sequencing of larger regions, for example those identified by linkage analysis, is also possible with these new techniques, and is much more time- and cost-efficient com-pared to using regular Sanger sequencing. [23] Sequencing of the exons of the X-chromosome in patients with mental retardation and control individu-als has also been performed with these types of technologies. [24]

The basic principle of these technologies is fragmentation of genomic DNA into short fragments, which are then amplified, either by emulsion or solid phase PCR, and sequenced by different techniques depending on the platform used. [25]

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The RAS-MAPK pathway

In order for an organism to function, the cells building up the organism must be able to communicate with each other and with the extracellular surround-ings. That is, the cells need to be able to respond to external signals either from other cells or from the environment, such as drugs, light or different kinds of antigens. This cellular communication is mediated by signalling pathways, in which receptors on the cell surface sense different molecules, such as growth factors, cytokines or hormones, and initiate a signalling cas-cade into the nucleus of the cell. In the nucleus, the expression of different genes can be regulated in a specific manner, depending on the external stim-ulus and the desired outcome in different cellular processes, such as differen-tiation, proliferation, apoptosis, cell survival or stress response.

Numerous signalling pathways exist in humans, but one central group is the MAPK signalling pathways. At least six different MAPK pathways exist, each named after their terminal kinases: ERK1/2, JNK1/2/3 or SAPKs, p38 MAPK, ERK3/4, ERK5 and ERK7/8, where the RAS-ERK1/2 (also denoted RAS-MAPK) pathway is one of the best characterized signalling pathways. This pathway (Figure 1) is involved in many cellular processes, such as pro-liferation, differentiation, motility and survival, and is activated by almost all growth factors and cytokines.

Activation of the RAS-MAPK pathway A number of different receptors, such as receptor Tyr kinases, G protein-coupled receptors and ion channels, can initiate the activation of the RAS-MAPK pathway. Upon stimulation of the extracellular domain of these re-ceptors, kinase activity in the cytoplasmic domain of the receptors is in-duced. This kinase activation phosphorylates C-terminal tyrosine residues of the receptors, providing docking sites for a complex of molecules, including enzymes, adaptors and docking proteins. The adaptor proteins, e.g. Grb2, further interact with guanine-nucleotide exchange factor SOS (SOS1 and SOS2) and recruit SOS to the plasma membrane, where small GTP-binding proteins, the RAS proteins (KRAS, NRAS and HRAS), are localized. SOS then catalyses the conversion of inactive guanosine-diphosphate-bound RAS (RAS-GDP) to active guanosine-triphosphate-bound RAS (RAS-GTP). Once RAS is activated, it activates the RAF family of kinases (ARAF, BRAF and

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Figure 1. A simplified overview of the RAS-MAPK pathway. (Updated and adapted from Ekvall et al. [26])

RAF1) by phosphorylation, causing them to further phosphorylate two serine residues of the MAP2 kinases (also known as MEKs; MEK1 and MEK2). The MEKs are dual-specificity kinases that when activated can phosphory-late two conserved threonine and tyrosine residues of ERK (ERK1 and ERK2), resulting in a conformational change in ERK and increased catalytic activity. [27-33] See Figure 1 for an overview.

Both RAF and MEK have restricted substrate specificity, whereas ERK has a wide range of different cytosolic and nuclear substrates. To date, ap-proximately 200 distinct substrates of ERK1/2 have been identified, where cytoplasmic PLA2, different cytoskeletal elements and intracellular domains of membrane receptors were the first identified substrates. Later identified

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substrates include the nuclear transcription factors Elk1, c-Fos and c-Jun. [30, 34]

Regulation of the RAS-MAPK pathway Regulation of the RAS-MAPK pathway occurs by a large variety of different mechanisms, some of which will be discussed in further detail below.

Phosphorylation and dephosphorylation ERK1/2 can phosphorylate RAF, which inhibits its phosphorylation of MEK, or SOS1/2 can be phosphorylated by ERK1/2, causing SOS1/2 to dissociate from the adaptor protein, Grb2, and preventing its activation of RAS. Specific phosphorylation of some receptors, e.g. EGFR, by ERK1/2 is also possible, which then inhibits the signal output. Another example of sim-ilar feedback controls is ERK1/2-dependent expression of dual-specificity phosphatases (DUSPs), which can dephosphorylate ERK1/2, making them inactive. See Figure 2. [35]

Figure 2. Regulation of the pathway by ERK. Phosphorylation (P) at specific protein residues by ERK inhibits the signals of the pathway. Furthermore, dephosphoryla-tion of ERK by ERK-dependent DUSPs also inhibits the signalling.

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Scaffolding proteins, phosphatases and inhibitors The regulation of the RAS-MAPK pathway is also modulated by a number of different scaffolding proteins, phosphatases and inhibitors. An example of a scaffolding protein is KSR, which coordinates assembly of the RAF-MEK complex, and catalyses the phosphorylation of MEK. [36] SHOC2 is another scaffolding protein, which link RAS to downstream signal transducers. [36] Negative regulation is the effect of the inhibitors SPRED1/2, which have their targets located between RAS and RAF and prevent phosphorylation and activation of RAF. [37] The GTPase activating protein, neurofibromin, is another example of a negative regulator, which accelerates the hydrolysis of active RAS-GTP to inactive RAS-GDP. [38] Furthermore, the phospha-tase SHP2 has been shown to regulate the RAS-MAPK pathway in several different aspects; first, it can act as a scaffolding protein and recruit the Grb2/SOS complex to the membrane. Second, it has been demonstrated to dephosphorylate and inactivate SPRY, an inhibitor binding to Grb2, and third, SHP2 can also dephosphorylate several other targets, which in turn promote activation of RAS. [39] See Figure 1 for an overview.

Internalization and degradation of receptors Another mechanism regulating this pathway is internalization and degrada-tion of active receptor tyrosine kinases. This is possible through recruitment of ubiquitin ligases, e.g. CBL (Figure 1), which connect ubiquitin to the receptors, making them prone to degradation. [40]

Histone modifications Recently, histone acetyltransferase MYST4/KAT6B (Figure 1) was found to primarily regulate MAPK signalling pathways, including the RAS-MAPK pathway, via H3 acetylation. MYST4/KAT6B does not interact directly with genes in the RAS-MAPK pathway, but affects the expression of genes, which interact with members of the RAS-MAPK pathway. [41]

Post-transcriptional regulation Besides post-translational regulations, the RAS-MAPK pathway is also sub-jected to post-transcriptional regulation. One example is binding of PUM2 to 3’UTR regulatory elements in ERK mRNA, which represses translation and stability of the mRNA. PUM2 can also regulate the pathway indirectly by targeting DUSP6, an inhibitor of ERK1/2. (Figure 3A) Another RNA-binding protein is HuR, which binds to the 3’UTR of MEK1 mRNA and makes it more stable, thereby promoting translation. Like PUM2, HuR can affect the regulation in a negative manner as well, by binding to MKP-1

26

mRNA, which then can dephosphorylate and inactivate members of the MAPK family. (Figure 3B) Moreover, the 3’UTR of RAS possesses several conserved and presumed binding sites for miRNA let-7, which has been shown to repress expression upon binding to its targets (Figure 3C). [42]

Figure 3. Post-transcriptional regulation of different compo-nents of the RAS-MAPK path-way. A) PUM2 inhibits transla-tion of ERK directly, but can also indirectly function as a positive regulator of ERK sig-nalling. B) HuR promotes MEK1 translation directly, but can also negatively regulate the pathway indirectly by promot-ing MKP-1 translation. C) miRNA let-7 can directly re-press expression of RAS by binding to the 3’UTR of RAS.

Determination of signal specificity of the RAS-MAPK pathway As mentioned, the RAS-MAPK pathway is involved in a number of different cellular processes, which raises the question as to what determines the speci-ficity of the signals within this pathway.

Signal strength and duration Differences in strength and duration of the signal have previously been found to have an impact on the biological outcome in response to extracellu-lar stimulation. PC12 cells were stimulated with either EGF or NGF, two growth factors that strongly induce ERK1/2 activation. In EGF-stimulated cells, a transient activation of ERK was detected, which promoted prolifera-tion of the cells, whereas in NGF-stimulated cells, ERK activation was sus-tained and cells were differentiating. An explanation for this difference was the effect of immediate early genes, which induce different cellular process-es depending on the duration of the signal. [34] However, sustained ERK signalling does not always lead to differentiation. In fibroblasts, sustained ERK signalling by PDGF has been shown to induce proliferation, whereas transient ERK signalling by EGF could not. [43] Despite these differences, one can conclude that the duration of the signal is of importance for the bio-

27

logical output, but different types of cellular systems can result in different outcomes.

Cross-talk with other pathways Furthermore, the RAS-MAPK pathway is not an independent pathway oper-ating alone, but part of a multi-dimensional signalling network, and can cross-talk with several other signalling pathways within this network. This, in turn, can influence and modulate the biological outcome. Members of this multi-dimensional network include other MAPK pathways, but also the PI3K-AKT pathway and the NF-κB pathway, among others. [34] In fact, the RAS-MAPK pathway and the PI3K-AKT pathway interact at multiple points with different outcomes, but in general, it seems as though members of the PI3K-AKT pathway have a positive impact on the RAS-MAPK pathway, which is most effective at low doses of growth factors, whereas RAS-MAPK negatively regulates the PI3K-AKT pathway, but at high doses of growth factors. [32]

Subcellular localization of components of the pathway A final mechanism in determining the specificity of signals in the RAS-MAPK pathway is localization of its components to specific subcellular compartments. In most resting cells, all components of the pathway are pri-marily localized in the cytosol, due to interaction with specific scaffolding proteins. Upon stimulation, RAF is recruited to the plasma membrane to interact with active RAS and start a phosphorylation cascade. Once MEK and ERK are activated, they are released from their anchors within the cyto-sol and can translocate to the nucleus or other organelles in the cell to per-form further interactions. However, a portion of the components stay at-tached to their anchors upon stimulation, to be directed to other specific tar-gets in the cytoplasm. Together, these specific localizations in the cell influ-ence the biological outcome in a distinct manner. [30, 34]

Cancer and the RAS-MAPK pathway The hallmarks of a cancer cell include increased or inappropriate prolifera-tion, motility and survival; all processes where the RAS-MAPK pathway is of great importance, making this pathway a hot target for many human can-cers. [35] In fact, several components within the pathway have been associ-ated with different types of cancers. Mutations in RAS genes have been iden-tified in ~30% of human cancers, where mutations in KRAS are by far the most common type (~85%). [31] Furthermore, BRAF and different types of receptors within the pathway, such as EGFR, are frequently mutated in dif-

28

ferent cancer-types, whereas the other two RAF genes as well as the MEK genes are rarely mutated in cancer. [33] In addition, mutations in PTPN11, the gene encoding SHP2, and CBL have been found to cause juvenile mye-lomonocytic leukaemia (JMML) and mutations in NF1, encoding neurofi-bromin, contribute mainly to solid tumours and myeloid leukaemias. [44-46]

Drug development Being a hot target for many cancers also makes the RAS-MAPK pathway an attractive and important target for development of new cancer therapeutics. Several inhibitors with direct or indirect effect on different components of the pathway have been developed with varying success. Promising develop-ment of drugs inhibiting farnesyltransferases, which localize RAS to the membrane, turned out to be unsuccessful, due to the ability of RAS to use an alternative transferase for this localization after inhibition.

Drugs targeting the receptors in patients with oncogenic receptor signal-ling have been more successful. However, these drugs have no effect in pa-tients with oncogenic mutations further downstream of the pathway. [47] To overcome this problem, several different inhibitors targeting RAF or MEK have been developed with successful results.

Then again, tumours develop resistance over time. For instance, cancer cells targeted with inhibitors of BRAF adapt and gain resistance by switch-ing from BRAF to RAF1, which then maintains ERK1/2 activation. [35] Another way for tumours to develop escape mechanisms is by activation of other signalling pathways that cross-talk with the RAS-MAPK pathway, such as the PI3K-AKT pathway. Indeed, increased signalling of the PI3K-AKT pathway has been detected in breast cancer cells, with almost complete blockade of the RAS-MAPK pathway. Therefore, an efficient strategy of inhibiting tumour growth has been shown to be the use of a combination of inhibitors targeting both the RAS-MAPK and the PI3K-AKT pathway. [33]

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RASopathies

The RASopathies are a group of clinically and genetically related develop-mental disorders, including Noonan syndrome (NS) and NS-like syndromes, cardio-facio-cutaneous syndrome (CFCS), LEOPARD syndrome (LS), Cos-tello syndrome (CS), Legius syndrome, neurofibromatosis type 1 (NF1) and neurofibromatosis-Noonan syndrome (NFNS). They can also be denoted as neuro-cardio-facio-cutaneous syndromes (NCFCs) or RAS-MAPK-syndromes. The two most common syndromes within the RASopathies are NS and NF1, with and incidence of 1/1000-2500 and 1/2500-3000 respec-tively, whereas the remaining syndromes are much less frequent. [48, 49]

Mutations associated with the RASopathies have been identified in 14 different genes, all regulating the RAS-MAPK signalling pathway; hence, the name RASopathies (Figure 4). [41, 50] This pathway is often affected in various types of cancers; however, most mutations identified in the RASopa-thies do not overlap with the cancer mutations. In general, it is believed that both types of mutations lead to dysregulation of the pathway, but somatic oncogenic mutations cause a stronger activation than germline mutations, which might explain the absence of overlapping mutations. This common pathogenic mechanism, dysregulation of the RAS-MAPK pathway, explains the clinical similarities within the RASopathies, where reduced growth, typi-cal facial features, cardiac defects, ectodermal abnormalities, variable cogni-tive deficits and susceptibilities to certain malignancies are all identified characteristics. Despite this clinical overlap between the different syn-dromes, an extensive clinical variability is seen within each syndrome. [31]

Both the clinical overlap and the clinical variability can cause a difficulty in diagnosing patients with RASopathies correctly. Since different syn-dromes have different prognoses, for example mental retardation is more common in CFCS than NS and the increased risk of developing malignan-cies differs with each syndrome, setting the correct diagnosis is of great im-portance for future follow-up. By combining clinical characteristics with genetic defects, diagnosing is greatly improved. Genetics can also be of help to better understand clinical variability between patients. [49, 51] In the present thesis, the focus will be on Noonan spectrum disorders, in-cluding NS, NS-like syndromes and NFNS, with detailed clinical and genet-ic descriptions of each of them. In addition, NF1 will also be discussed in further detail, due to its overlap with NFNS.

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Figure 4. The RAS-MAPK pathway and the different RASopathies together with their associated genes. (Updated and adapted from Ekvall et al. [26])

Noonan and Noonan-like syndromes Clinical description Jacqueline Noonan was one of the first to publish a comprehensive descrip-tion of this group of patients in 1963. In 1985, it was therefore suggested to change the name from male Turner syndrome to Noonan syndrome.

NS (OMIM 163950) is one of the most common monogenic disorders in humans with an incidence of one in 1000-2500 births. The inheritance pat-tern is autosomal dominant and both familial and sporadic cases exist. Clini-cally, NS is a very variable disorder, both within families and between unre-lated patients harbouring the same mutation. [52, 53]

The main characteristics of NS are congenital heart defects, short stature, typical facial features and unusual pectus deformity. Pulmonic stenosis is the most common heart defect (50-65%), followed by hypertrophic cardiomyo-pathy (~20%). Other types of heart defects include atrioventricular canal defects and atrial and ventricular septal defects. [54] The adult height of females (without growth hormone treatment) is 148.4±5.6cm and of males

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(without growth hormone treatment) 157.4±8.0cm, but around 30% of pa-tients with NS have an adult height in the normal range. [54, 55] Typical facial features include a broad forehead, hypertelorism, ptosis, downslanting palpebral fissures, low-set posteriorly rotated ears with thick helices, deep philtrum, high arched palate, low posterior hairline and broad webbed neck. The facial features usually become less prominent with age. Besides the main characteristics, a number of associated features exist, such as neonatal feeding difficulties, developmental and motor delay, learning disabilities, bleeding abnormalities (e.g. coagulation deficits or thrombocytopenia), skin manifestations (e.g. café-au-lait spots, pigmented naevi, lentigines or kerato-sis), cryptorchidism, ocular problems (e.g. strabismus or refractive errors) and skeletal defects (e.g. scoliosis). [54, 56]

NS-like disorder with loose anagen hair (NS/LAH, OMIM 607721) is a syndrome that greatly resembles NS. This syndrome was first presented in 2003, and patients show characteristics such as more severe growth and cog-nitive deficits, distinctive hyperactive behaviour, diffuse skin pigmentation, hoarse/hypernasal voice, easily pluckable, sparse, thin and slowly growing hair and cardiac defects, with a significant overrepresentation of mitral valve dysplasia and septal defects compared to the general NS population. [57, 58]

Another NS-like condition is NS-like disorder with or without JMML (OMIM 613563), which was reported in 2010. These patients have a rela-tively variable phenotype, although clearly overlapping with NS, with short stature, developmental delay, cryptorchidism and predisposition to JMML. [40, 44, 50, 59]

A predisposition to develop cancer exists in patients with NS, but the risk is relatively low considering the mutations in the RAS-MAPK pathway, which is often implicated in cancer pathogenesis. JMML, acute lympho-blastic leukaemia, rhabdosarcoma and neuroblastoma are types of cancer observed in NS patients [53, 60]. Furthermore, tumour-like lesions such as giant cell lesions affecting the jawbones or joints have also been observed in patients with NS. [50]

Genetic description Of the 14 genes associated with RASopathies, patients with NS or NS-like conditions have been found to harbour mutations in ten of these genes. How-ever, these ten NS-associated genes are not only associated with NS, but the majority of them are associated with other RASopathies as well, such as LS or CFCS (Figure 4). This further explains the clinical similarities within RASopathies. Despite association to these ten genes, the genetic aetiology in ~25% of patients with NS is still unknown.

In 2001, PTPN11 on chromosome 12q24.13 was the first gene to be associ-ated with NS [61]. The gene consists of 16 exons and encodes a tyrosine

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phosphatase, termed SHP2, ubiquitously expressed in the cytoplasm. Two tandemly arranged N-terminal src-homology 2 domains (N-SH2 and C-SH2), a catalytic protein tyrosine phosphatase (PTP) domain and a C-terminal tail, containing two tyrosol phosphorylation sites and a proline-rich stretch, build up SHP2. The two SH2 domains bind to phosphotyrosol resi-dues on other target proteins, which promote localization of SHP2 to e.g. cell surface receptors or scaffolding proteins. SHP2 alternates between an active and inactive form by the release or binding of the N-SH2 domain to the PTP domain. [56]

Approximately 50% of patients with NS have mutations in PTPN11, mak-ing it the major gene associated with NS. The mutations identified are main-ly missense mutations, whereas deletions, insertions/duplications and indels are rare. ([62] and www.hgmd.org) The pathogenic mechanism is suggested to be destabilization of the inactive form of SHP2, i.e. a gain-of-function mechanism, and most mutations are located in residues involved in, or in close proximity to, the interaction between the N-SH2 and the PTP domain.

Mutations in PTPN11 have also been identified in patients with LS, who are sometimes diagnosed as NS in very young ages. [51, 63, 64].

The next gene to be associated with NS was KRAS on chromosome 12p12.1 [65]. KRAS consists of six exons and encodes a GTPase with two splice var-iants, KRASA and KRASB, where KRASB is predominant and often denot-ed as KRAS. The expression of KRAS is ubiquitous. Like all RAS proteins, KRAS consists of a conserved G domain, required for signalling, and a less conserved C-terminal tail, denoted as the hypervariable region, which medi-ates post-translational processing and plasma membrane anchoring. The protein cycles between inactive GDP-bound state and active GTP-bound state.

Less than 2% of NS patients harbour mutations in KRAS and, so far, only missense mutations have been identified. The outcome of all KRAS muta-tions is a gain-of-function, generated by different mechanisms, such as im-paired intrinsic GTPase hydrolysis or increased GDP/GTP dissociation rate. [66]

Patients with CFCS have large clinical overlap with NS, and CFCS has previously even been suggested to be a variant of NS. In 2006, mutations in KRAS were also identified in patients with CFCS. [67]

Mutations in SOS1 on chromosome 2p22.1 and RAF1 on chromosome 3p25.2 have also been identified in patients with NS (~13% and 3-17% re-spectively). [68-71] SOS1 is comprised of 23 exons and encodes SOS1, which is a ubiquitously expressed guanine-nucleotide-exchange factor of RAS, catalysing conversion of inactive RAS-GDP to active RAS-GTP. SOS1 is build up by five different domains: a histone domain (HD), a Dbl homology (DH) domain, a pleckstrin homology (PH) domain, a RAS ex-

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changer motif (Rem) domain and a Cdc25 domain. Furthermore, the C-terminal contains recognition sites, which together with the PH domain and the HD domain promote interaction with certain adaptor proteins, allowing localization to the plasma membrane upon stimulation. SOS1 is autoinhibit-ed by interaction between the DH and the Rem domain, which blocks bind-ing site for RAS. The majority of mutations in SOS1 are missense mutations, located in regions predicted to be involved in maintaining the catalytically inactive conformation. These mutations then destabilize this inactive con-formation, resulting in a gain-of-function. [56] Rarely, a mutation in SOS1 has been identified in patients with CFCS. [16, 72]

RAF1 is comprised of 17 exons and its protein RAF1, a serine threonine kinase, is also ubiquitously expressed. Three functional domains reside in RAF1, conserved regions 1 to 3 (CR1-3). CR1 is involved in RAS-GTP binding and promotes localization to the membrane, CR2 also regulates translocation to the membrane, but is also responsible for the catalytic activi-ty, and CR3 mediates phosphorylation. RAF1 has one inactive and one ac-tive conformation, where the N-terminal part of the protein interacts with the kinase domain in CR3 and inactivates it. Missense mutations are the main type of mutation in RAF1 as well, and they are clustered mainly in CR2, but also in CR3 or just C-terminal of CR3. The majority of these mutations cause a gain-of-function. [56] A few patients with LS have also been found to harbour mutations in RAF1. [68]

In 2009, two additional genes, SHOC2 and NRAS, were found to harbour mutations in patients with NS or NS-like conditions [57, 73]. SHOC2 on chromosome 10q25.2 is a nine-exon gene and encodes a widely expressed protein, SHOC2, mainly composed of leucine-rich repeats. SHOC2 has been found to have two functions, either it can act as a scaffold and guide RAS to downstream targets or it is part of PP1C and promotes PP1C’s translocation to the plasma membrane. Once at the plasma membrane, PP1C mediates RAF1 dephosphorylation at Ser259, which is a requirement for stable trans-location of RAF1 to the plasma membrane and catalytic activation. [56]

Mutations in SHOC2 are found in patients with the NS-like condition NS/LAH, which corresponds to less than 5% of the entire NS population. Hitherto, only one single missense mutation has been identified in SHOC2-positive patients. This mutation changes serine to glycine in residue two of the protein, which introduces an N-myristolation site. N-myristolation is a process where a 14-carbon saturated fatty acid is attached to an N-terminal glycine residue with a satisfactory consensus sequence surrounding it, which promotes anchoring to the plasma membrane. Mutated SHOC2 fulfils the consensus requirements and becomes N-myristolated, resulting in constitu-tive membrane translocation of SHOC2. In turn, this constitutive transloca-tion promotes prolonged dephosphorylation of RAF1 at Ser259 mediated by PP1C, thereby sustaining RAF1-stimulated activation of the pathway.

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Hence, this single missense mutation of SHOC2 is a gain-of-function muta-tion. [56, 57]

The other gene, NRAS on chromosome 1p13.2, is constituted by six exons and as KRAS, it encodes a small GTPase, cycling between an active GTP-bound state and an inactive GDP-bound state. Only a few patients with NS have been identified with mutations in NRAS and the observed mutation type is only missense, where the mutation in each case results in enhanced phos-phorylation of MEK and ERK, i.e. a gain-of-function. [73-75]

Furthermore, a few reports have been published on NS patients with mis-sense mutations in BRAF and MEK1 [16, 69, 76, 77]. BRAF, located on chromosome 7q34, consists of 18 exons and as RAF1, it encodes a serine threonine kinase expressed in various tissues and contains the three con-served regions (CR1-3), but BRAF has been shown to have higher MEK kinase activity compared to RAF1 and ARAF. Mutations in BRAF are the major cause in CFCS; however, most mutations identified in NS patients do not overlap with mutations associated with CFCS. Two patients with LS have also been found to harbour mutations in BRAF. [77, 78]

MEK1 on chromosome 15q22.31 consists of eleven exons and encodes mitogen-activated protein kinase kinase 1, a kinase downstream of RAF. MEK1 consists of one negative regulatory domain at the N-terminal and a single kinase domain. Mutations in MEK1, and also the functionally related MEK2, are mainly associated with CFCS. [56]

The last two genes to be associated with NS or NS-like conditions were CBL and MYST4/KAT6B. CBL is a 16-exon gene located on chromosome 11q23.3. It encodes a ubiquitously expressed RING finger E3 ubiquitin lig-ase, one of the enzymes required for targeting substrates for degradation by the proteasome. Four domains comprise CBL: one N-terminal tyrosine ki-nase-binding domain, involved in protein-protein interaction, one zinc-finger RING-finger domain, mediating E3 ubiquitin ligase activity, one proline-rich region and one ubiquitin-associated domain, promoting ubiquitin binding and overlapping with a leucine zipper motif, involved in protein dimeriza-tion. Mutations in CBL have been shown to impair ubiquitylation of recep-tors, causing increased pathway signalling, and patients with mutations in CBL have features more or less reminiscent of NS. [40, 44, 59] Somatic mu-tations in this gene are found in patients with different myeloid malignan-cies, and in fact, the spectrum of somatic mutations overlaps with the germline mutations. [40]

MYST4/KAT6B is located on chromosome 10q22.2 and consists of 16 ex-ons. It encodes a histone acetyltransferase, which regulates the RAS-MAPK pathway via H3 acetylation. This gene was found to be associated with an NS-like phenotype in a patient with a balanced chromosomal translocation, where one of the breakpoints was located within the MYST4/KAT6B gene.

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Quantitative RT-PCR in this patient revealed a 50% decrease in mRNA ex-pression levels of MYST4/KAT6B. This haploinsufficiency resulted in in-creased phosphorylation of MEK1/2 and ERK1/2, and also enhanced AKT phosphorylation. However, no further mutations in this gene could be identi-fied in a cohort of 131 subjects with suggestive NS features, who were nega-tive for mutations in previously associated NS-genes. [41]

Genotype-phenotype correlations Although operating within the same pathway, and in the majority of cases causing increased signalling, mutations in different genes or residues are associated with different clinical features (Table 1). However, NS patients with mutations in the same gene or even the same nucleotide position still can display clinical variability.

A significant relationship between NS patients harbouring a PTPN11 muta-tion and pulmonic stenosis has been revealed, whereas the frequency of hy-pertrophic cardiomyopathy was significantly lower in patients with a PTPN11 mutation. [46, 79] Familial cases also have a significantly higher occurrence of mutations in PTPN11 compared to sporadic cases. In addition, PTPN11-positive patients more often have easy bruising, thorax deformities, short stature and cryptorchidism compared to other genotypes. [71, 80] Fur-thermore, a specific mutation in PTPN11, p.T73I, has been identified as a JMML risk genotype. [50]

Patients with SOS1 mutations have a similar spectrum of heart defects as PTPN11-positive patients, but are less likely to have short stature and to need special education. However, they have significantly more often thorax deformities and ectodermal manifestations, such as curly hair, sparse eye-brows or keratosis pilaris. [71, 80, 81]

Hypertrophic cardiomyopathy and hyperpigmented cutaneous lesions occur at a significantly higher frequency (75-95% and 33% respectively) in pa-tients with a RAF1 mutation compared to NS patients in general. [48, 50, 68]

KRAS-positive patients more often have cognitive impairment and a more severe phenotype than the general NS population; otherwise the phenotype is quite variable. [48, 50]

Patients with mutations in SHOC2 represent a separate entity, NS/LAH; hence, they have several features associated with the genotype. Loose anagen hair is one feature, which is only observed in this condition. Other associated features include hoarse/hypernasal voice, reduced growth caused

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Table 1. Genotype-phenotype correlations in NS and NS-like syndromes

Mutated gene Clinical feature

PTPN11

+ Pulmonic stenosis + Thorax deformities + Easy bruising + Short stature + Cryptorchidism (in males) + Familial cases + JMML (p.T73I)

- Hypertrophic cardiomyopathy

SOS1

+ Pulmonic stenosis + Thorax deformities + Ectodermal manifestations

- Hypertrophic cardiomyopathy - Short stature - Special education

RAF1 + Hypertrophic cardiomyopathy + Hyperpigmented cutaneous lesions

KRAS + Cognitive impairment + Severe phenotype in general

SHOC2

+ Loose anagen hair + Hoarse/hypernasal voice + Reduced growth (GH deficiency) + Cognitive impairment + Distinctive hyperactive behaviour + Darkly pigmented skin with eczema or ichtyosis + Mitral valve dysplasia + Septal defects

CBL

+ JMML + Short stature + Developmental delay + Café-au-lait spots + Cryptorchidism (in males)

BRAF

+ Neonatal growth failure (due to feeding difficulties) + Mild-to-moderate cognitive deficits + Hypotonia + Multiple naevi or dark colored lentigines + More severe phenotype in adulthood

MEK1, NRAS, MYST4/KAT6B No clear correlations

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by GH deficiency, cognitive deficits, distinctive hyperactive behaviour and darkly pigmented skin with eczema or ichtyosis. These patients also have a significant overrepresentation of the heart defects mitral valve dysplasia and septal defects compared to the general NS population. [57]

Likewise, patients with mutations in CBL are denoted as a separate disorder. The main associated feature is JMML, although CBL-positive patients with-out hematologic abnormalities exist. Short stature, developmental delay, café-au-lait spots and cryptorchidism are additional associated features. [40, 44, 59]

BRAF-positive NS patients are associated with neonatal growth failure due to feeding difficulties, mild-to-moderate cognitive deficits, hypotonia and multiple naevi or dark colored lentigines. They also present with a more severe phenotype in adulthood compared to patients with mutations in PTPN11 or SOS1. [48]

For MEK1, NRAS and MYST4/KAT6B, no clear genotype-phenotype correla-tions have been identified.

Neurofibromatosis type 1 Clinical description The first description of neurofibromatosis type 1 (NF1, OMIM 162200), made by von Recklinghausen, dates back to 1882. [82] NF1 is one of the most common disorders with an autosomal dominant inheritance pattern, and the incidence is approximately one in 2500-3000 births. About half of the cases are inherited and the other half are caused by de novo mutations. As in NS, there is a clinical variability in NF1 both within families and between unrelated patients harbouring the same mutation. [49]

To establish a clinical diagnosis of NF1, the National Institutes of Health (NIH) consensus statement is used, which is a statement of diagnostic crite-ria for NF1 proposed by the NIH Consensus Development Conference in 1987 and later reviewed in 1997. [83] The statement requires at least two of the following seven clinical criteria to be present in order to set the diagnosis to NF1:

Six or more café-au-lait spots with a greatest diameter

>5mm in children >15mm in adults

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Two or more neurofibromas of any type or one plexiform neurofi-broma

Axillary or inguinal freckling

Two or more Lisch nodules

Optic pathway glioma (OPG)

Skeletal abnormalities, e.g. scoliosis, pseudoarthrosis, sphenoid dys-plasia or thinning of long bone cortex

A first-degree relative with NF1

The presence of café-au-lait spots in NF1 patients is high, >99% of patients have these spots. The benign tumours neurofibromas are, as the name sug-gests, one of the main hallmarks of NF1. Cutaneous neurofibromas are the most common type, present in >99% of patients with NF1, whereas plexi-form neurofibromas are less common (30-50%). Both café-au-lait spots and neurofibromas are less common in infants, but develop later in childhood. Axillary or inguinal freckling is also a quite common symptom, present in approximately 85% of NF1 patients. Like neurofibromas, Lisch nodules are also a type of benign tumour, affecting the iris. These tumours are very common (90-95%) in patients with NF1 and is one of the best markers for NF1 in older children and adults. A third type of benign tumour found in NF1 patients, but at a much lower frequency (15%) than neurofibromas or Lisch nodules, are OPGs, which affect the central nervous system. Skeletal abnormalities are found in ~60% of patients with NF1. [82, 84, 85]

Besides these seven clinical criteria, NF1 patients display several addi-tional features, for instance learning disabilities (>50%), epilepsy (6-7%), congenital heart defects (~3%), hypertension, gastrointestinal problems, delayed puberty, mildly short stature or macrocephaly [82, 85-88].

As noted, benign tumours occur in NF1 patients with high frequency. Never-theless, the presence of malignant tumours is relatively low (10-20%), alt-hough with an increased frequency compared to the general population. The most common reason for premature death in NF1 patients is presence of malignant peripheral nerve sheath tumours (MPNSTs), which are more prone to develop in plexiform neurofibromas. Other malignant tumours pre-sent in NF1 patients are central nervous system tumours (particularly associ-ated with OPGs), rhabdomyosarcoma and leukaemias (especially JMML). [83, 85, 87]

Genetic description As opposed to NS, NF1 has only been associated with mutations in one gene, NF1 on chromosome 17q11.2, and the first mutations were identified

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in 1990. [45] (Figure 1) More than 90% of patients with a clinical diagnosis of NF1 harbour mutations in NF1. [48]

NF1 is a large gene consisting of 61 exons and encoding several different transcripts, where the most common transcript is a 2818 amino acid long protein, named neurofibromin. Neurofibromin is built up of four domains: a cysteine/serine-rich domain (CSRD), a GAP-related domain (GRD), a SEC14 domain and a pleckstrin homology (PH)-like domain (Figure 5). [89] The GRD is the most clearly defined functional domain of neurofibromin and it is responsible for accelerated intrinsic hydrolysis of RAS-GTP to RAS-GDP. The CSRD harbours three potential cAMP-dependent PKA recognition sites, which can be phosphorylated by PKA and in turn, affect the cAMP pathway. [90, 91] A link between cAMP and the RAS-MAPK pathway exist, in which cAMP inhibits the signal from RAS to RAF1. [92] The SEC14 domain is found in secretory proteins and lipid-regulated pro-teins, and it mediates interactions between proteins and phospholipids. [38, 93] Binding of a ligand by the SEC14 domain is suggested to be regulated by the neighbouring PH-like domain. [94]

In addition, at least twelve NF1 pseudogenes are present in the human ge-nome. These pseudogenes are located on a number of chromosomes and some chromosomes harbour more than one NF1 pseudogene. [95] The com-plexity of NF1 is further demonstrated by the presence of three active genes, OMGP, EVI2B and EVI2A, in intron 27b of NF1, but in opposite orientation of NF1. [96]

Figure 5. An overview of neurofibromin, the protein encoded by NF1. The four different domains are indicated by colored boxes.

Many different types of mutations in NF1 exist, such as missense/nonsense mutations, splicing mutations, small deletions, small insertions, small indels, gross deletions, gross insertions/duplications, complex rearrangements and repeat variations, where the first three make up the most common types (~70%, 968/1354; www.hgmd.org). There are no clear clustered regions for the mutations in NF1; instead they are relatively widespread over the entire gene (see Fig.2 in Paper IV). However, some mutations are more recurrent than others and certain exons also seem to be minor hot spots, e.g. exon 7 and exon 37. [90, 97-99] These recurrences and minor hot spots might in part be due to either methylation-mediated deamination of 5-methylcytosine, i.e. a C to T transition at CpG dinucleotides, or some structural elements, such as the quasi-symmetric element present in exon 37. [90, 100-102]

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Genomic microdeletions encompassing the entire NF1 and a number of flanking genes are the genetic cause of approximately 5-10% of patients with NF1. These microdeletions are divided into four types: type-1, type-2, type-3 and atypical deletions. [103] The type-1 microdeletion is ~1.4Mb long, in-cluding 14 protein-coding genes, five pseudogenes and five microRNAs, and is by far the most common microdeletion in NF1 patients. The cause of this deletion is non-allelic homologous recombination between two paralogous sequences of low copy repeats (LCRs) proximal and distal of NF1. [104]

The main outcome of all mutations in NF1 is, or is predicted to be, a trun-cated protein (>80%), resulting in haploinsufficiency of neurofibromin [90, 97, 99, 105, 106]. Only ~10% of NF1 mutations are missense mutations, not affecting splicing. [90] Thus, the NF1 mutations are loss-of-function muta-tions in contrast to the mutations in NS-associated genes, which were gain-of-function mutations. However, since neurofibromin works as a negative regulator of the pathway and the genes involved in NS are positive regula-tors, the outcome will still be the same, i.e. increased signalling.

Mutations in NF1 have also been identified in patients with Watson syn-drome (OMIM 193520) and NFNS (described later).

Genotype-phenotype correlations Several genotype-phenotype studies have been performed for patients with NF1, but only a few have rendered convincing results.

Comparing NF1 patients with intragenic mutations to NF1 patients with genomic microdeletions, a more severe phenotype is often seen in the latter, including facial dysmorphism, learning disabilities, cardiovascular malfor-mations, childhood overgrowth, excessive number of and earlier onset of benign neurofibroma, and developmental delay. A higher incidence of MPNSTs and other malignancies are also seen in NF1 patients with micro-deletions. [103, 107-109]

Another clear correlation is lack of cutaneous neurofibromas in NF1 pa-tients with an in-frame deletion of three base pairs in exon 17, c.2970-2972delAAT. [98, 110]

For NF1 patients with OPGs, a significant clustering of mutations in the first third of NF1 has been identified. [97, 111] Furthermore, most of these mutations seem to cause a truncated neurofibromin. [97, 105, 111]

Comparison of NF1 patients with missense mutations to NF1 patients with nonsense or frame-shift mutations, a lower risk of developing Lisch nodules has been detected in patients with missense mutations, although with borderline statistical significance [112]. Furthermore, NF1 patients with in-frame defects have a clear association with the development of pulmonic stenosis. [113]

Lastly, Ars et al. identified a large number of mutations in exon 10b in NF1 patients with scoliosis and a mutation in NF1 (9/22). Interestingly,

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among all 22 patients, none of them harboured a mutation located within the GRD domain. [97, 105]

Neurofibromatosis-Noonan syndrome Clinical description An overlap between NF1 and NS exist, for example pulmonic stenosis, café-au-lait spots, skeletal abnormalities, mild mental retardation and short stature are symptoms reported in both NF1 and NS, whereas Lisch nodules and neurofibromas are specific to NF1 and facial dysmorphism is specific to NS. [114, 115]

In 1985, Allanson et al. described a group of patients displaying features of both NF1 and NS and suggested the name neurofibromatosis-Noonan syndrome (NFNS, OMIM 601321). [116] Furthermore, studies on previously reported patients with NF1 have revealed the presence of NS features in 4-10% of them. [87, 117]

For patients with NFNS, a low incidence of plexiform neurofibromas, skeletal anomalies and internal tumours are found, but café-au-lait spots, hypertelorism, ptosis, low-set posteriorly rotated ears and cardiac defects are often present. [118]

Genetic description Ever since NFNS was first presented in 1985, it has been debated whether NFNS is a separate syndrome distinct from NF1 and NS, a phenotypic varia-bility of either NF1 or NS, or a coincidence of NF1 and NS. [119]

In the majority of cases, the gene involved in NFNS is NF1 (Figure 4), suggesting NFNS to be a variant of NF1. [114, 115, 118, 120-122] However, a few reports have identified a mutation in PTPN11, in addition to an NF1 mutation, indicating that NFNS is not solely a variant of NF1, but also a coincidence of NF1 and NS, where an additive effect of the two mutations causes the NFNS phenotype. [123, 124] NFNS patients with a mutation in PTPN11 alone or any other NS-associated genes have not been identified, suggesting NFNS to be genetically distinct from NS.

The mutations in NF1 associated with an NFNS phenotype are in some cases only identified in patients with NFNS, and in other cases found both in pa-tients with NFNS and in classical NF1 patients without any features sugges-tive of NS.

However, a significantly higher prevalence of missense mutations and in-frame deletions in NFNS patients compared to NF1 patients has been identi-fied. Furthermore, these in-frame defects in NFNS patients are clustered in

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the GRD to a higher extent than in-frame defects in patients with NF1. [90, 118]

Genotype-phenotype correlations To date, the number of NFNS patients reported in the literature is relatively low. Therefore, the only genotype-phenotype correlation identified within this group of patients is an increased risk of pulmonic stenosis in NFNS pa-tients with an in-frame defect compared to patients with a truncating muta-tion. [113, 118]

Animal models and future treatments Several mouse models as well as models of zebra fish and fruit fly exist with mutations in e.g. Ptpn11, Sos1, Raf1 or Nras. The mutated mice display fea-tures of RASopathies, such as short stature, typical facial features, congenital heart defects, learning disabilities, motor deficits and blood cell anomalies. The majority of RASopathy-associated genes are lethal during embryogene-sis in homozygous knock-out mice, indicating that these genes are important for embryonic development. [125-130]

Since increased signalling of the RAS-MAPK pathway is also seen in differ-ent cancers, this pathway has been a hot target for development of drugs for cancer treatment. During the years, different inhibitor drugs have been de-veloped and several of these are now being tested in RASopathy-mouse models with promising results. Wu et al. generated knock-in mice with an NS-associated mutation in Raf1. These mice displayed short stature, facial dysmorphism and cardiac defects, but when treated postnatal with a MEK inhibitor, these features were normalized. [131] Similarly promising results, although not with complete reduction of the heart defects, were demonstrat-ed in Sos1-mutated mice treated with a MEK inhibitor as well. [130]

Furthermore, treatments with an already approved immunosuppressant drug, called rapamycin, in mice with an LS-associated mutation in Ptnp11 could completely normalize and reverse the cardiac defects present in these mice. [132] However, the same results were not obtained by rapamycin treatment in the Raf1-mutated mice. (Personal communication) This is due to the fact that rapamycin is an inhibitor of the interacting AKT-mTOR pathway and the previous Ptpn11 mutation actually results in an enhanced activation of this pathway and not the RAS-MAPK pathway, whereas the latter Raf1 mutation demonstrated enhanced RAS-MAPK activation only. [131, 132] Supporting results were demonstrated in the Sos1-mutated mice. These mice showed increased activation of both Ras and Rac, a protein transducing signals in the cardiovascular system, and treatment with a MEK

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inhibitor did reduce the penetrance of cardiac defects, but some cardiac de-fects still existed. This is possibly because of increased activation of Rac, which is not affected by MEK inhibitors. [130]

Together, these differences in response to certain drugs show the im-portance of knowing the genetic mechanisms behind the RASopathies to be able to treat patients correctly in the future.

Another approved drug is lovastatin, which is used for hypercholesterolemia. Treatment with lovastatin in Nf1-mutated mice has been shown to improve cognitive function in these mice. [133] Actually, a Phase-1 trial with lovas-tatin in children with NF1 has been performed, which suggests that this drug might be effective against cognitive deficits in NF1 patients. [134] However, the use of a similar drug, simvastatin, did not present similar results. [135]

These studies of animal models with RASopathies and clinical trials present very promising results; however, further studies are needed before we will be able to fully treat patients with RASopathies.

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Present investigations

Background The first clinical description of a RASopathy, NF1, dates back as far as 1882, whereas the first genetic association of a RASopathy, the NF1 gene in NF1, was not identified until 1990. Then a further decade passed before in-volvement of a second gene was discovered, which was PTPN11 in NS. At that time, the term RASopathies (or any other previously known names for this group of syndromes), or even grouping of the syndromes, did not exist. With further investigations, both clinical and genetic, this group of syn-dromes started to evolve. First, the group was named after the clinical find-ings, neuro-cardio-facio-cutaneous syndromes (NCFCs). But with identifica-tion of defects within genes involved in the RAS-MAPK pathway, the name changed to RAS-MAPK syndromes or RAS-MAPK pathway disorders and later RASopathies, which is the main term used today.

Despite the clinical overlap between RASopathies, an extensive clinical var-iability is seen within each syndrome and further within families and be-tween unrelated individuals harbouring the same mutation. Both clinical overlap and clinical variability can cause difficulties in diagnosing patients with RASopathies correctly, which is of great importance for future follow-up, due to the fact that different syndromes have different prognoses con-cerning certain features.

Clinical variability, specifically within Noonan spectrum disorders, will be genetically investigated in the present thesis to gain knowledge of possible genetic mechanisms that could explain this clinical variability. In addition, the four hypotheses of NFNS, debated since 1985, will also be investigated. Furthermore, evidences of the difficulties in determining correct diagnosis, but where the diagnosing was greatly improved by use of genetic analysis, will also be presented.

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Aims Specific aims of the present investigations were:

Paper I and II

To investigate clinical variability within NS, by determining genetic defects in two patients with severe/complex NS or NS-like phenotype.

Paper III and IV

To investigate clinical variability within families displaying features of both NF1 and NFNS, by determining genetic de-fects in four families with NF1 and NFNS.

To investigate the hypotheses of NFNS being a separate syn-drome distinct from NF1 and NS, a phenotypic variant of ei-ther of the syndromes or a coincidence of both disorders, by using the same analyses as above.

Paper I Paper I presents a two-generation family, where the index patient suffered from an atypical severe form of NS with additional symptoms, including Arnold-Chiari I malformation, hypoplasia of the corpus callosum, syringo-myelia, hydronephrosis, malrotation of the bowel and premature menopause. Furthermore, the index patient as well as four additional family members presented with multiple café-au-lait (CAL) spots segregating in an autosomal dominant manner in the family; this suggested two different genetic origins for NS in the index patient and the multiple CAL spots in the index patient and four other family members.

Since mutations in PTPN11 are the major cause of NS, the index patient was screened for mutations in this gene. The analysis revealed a previously re-ported heterozygous missense mutation in exon 7, c.853T>C; p.F285L. This mutation was not identified in any of the other family members, which sup-ported our hypothesis of two different genetic origins for NS in the index patient and the multiple CAL spots present in the family.

Although PTPN11 p.F285L as well as other mutations affecting the same codon have been detected in several NS patients previously, and also in pa-tients affected by Noonan-like/multiple giant cell lesion syndrome and cherubism, this was the first report on a patient harbouring a mutation in p.F285 and affected by additional severe manifestations. [136-143] It was

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also the first reported case of an NS patient with Arnold-Chiari I malfor-mation where a mutation has been identified. [140, 144-146]

CAL spots are one of the characteristics of NF1 and NFNS, which both are associated with mutations in NF1, but a clinical investigation, including a slit lamp examination, of the index patient, her father and her sister, excluded NF1 as well as NFNS as a diagnosis in the family. However, CAL spots alone (OMIM 114030) have been linked to NF1 in one previous study. [147]

Thus, to investigate the cause of CAL spots in this family, NF1 was first screened for mutations, both by traditional Sanger sequencing of exons and exon-intron boundaries and by MLPA to investigate for presence of dele-tions or duplications, which are found in 5-10% of NF1 patients. The MLPA analysis detected no deletions or duplications, but the sequencing analysis revealed an alteration in exon 29, c.5425C>T; p.R1809C, in the index pa-tient, her sister and their father; all presenting with multiple CAL spots. However, the same alteration was not found in the index patient’s uncle or his daughter, who both presented with multiple CAL spots as well, indicat-ing that this alteration was not the genetic cause of CAL spots in this family. Nevertheless, p.R1809C has been identified in a few NF1 patients and one NFNS family (see Paper IV), but also in unaffected relatives. [97, 148]

Another gene associated with CAL spots is SPRED1, whose protein forms dimers with SPRED2, encoded by SPRED2. Mutations in SPRED1 have been associated with the RASopathy Legius syndrome, which besides CAL spots also display multiple lipomas as a feature. [149, 150] Due to presence of CAL spots in this family and also multiple lipomas in the father of the index patient, both SPRED1 and SPRED2 were investigated by link-age analysis. However, the result from this analysis showed no linkage to any of the SPRED genes. Thus, genetic aetiology of the CAL spots still re-mained unknown.

To summarize the investigation of the CAL spots, we did identify an altera-tion in NF1, p.R1809C, but it did not segregate with the CAL spots in the family and has been reported previously in unaffected relatives in one fami-ly. To explain identification of this variant in unaffected individuals, one can speculate that this variant might have reduced penetrance or cause a very mild phenotype. Indeed, the cohort where this variant was identified in unaf-fected individuals was very large, and these individuals may not have under-gone a detailed clinical investigation and might in fact display a very mild phenotype, e.g. CAL spots only. [97]

It is also tempting to believe that the CAL spots in the present family might even be of two different genetic origins, suggesting that p.R19809C identified here is in fact the cause of CAL spots in the index patient, her father and her sister. Occurrence of independent mutations in NF1 in fami-lies with NF1 has been described in two previous studies. [151, 152] Of

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note, p.R1809C was not identified in 85 healthy unrelated individuals from the Swedish population in this study (nor in an additional 190 healthy unre-lated individuals from the Swedish population in Paper IV) and neither in NCBI’s dbSNP135 nor the 1000 Genomes database (brows-er.1000genomes.org). Furthermore, p.R1809 is a highly conserved amino acid across 13 species and computational prediction programs predicted p.R1809C to be probably damaging. All this indicates that the alteration might still affect the protein, but perhaps to a lesser degree than other vari-ants.

Since both NF1 and PTPN11 function in the RAS-MAPK pathway and sev-eral studies have reported on presence of co-occurring mutations in patients with RASopathies, one might speculate that p.R1809C acts as a modifying locus and together with PTPN11 p.F285L causes the severe symptoms in the index patient. [123, 124, 153-157] (See also Paper II)

Other modifying loci that together with PTPN11 p.F285L might contrib-ute to the severe symptoms in the index patient are for instance, the not yet identified cause of CAL spots in the family or perhaps some additional vari-ants in regulatory regions of the genes screened here or in other genes in-volved in the RAS-MAPK pathway or interacting pathways.

In conclusion, the findings in Paper I demonstrate that presence of two dif-ferent genetic defects, resulting in an additive effect, could be an explanation for the clinical variability within NS.

Paper II In Paper II, a patient with NS/LAH and several additional features, including osteoporosis, gingival hyperplasia, spinal neuroblastoma, liver hemangioma and intrathoracic extramedullary hematopoiesis (EMH), was investigated.

The index patient was screened for mutations in eight NS-associated genes as well as MEK2, a gene associated with the RASopathy CFCS. The muta-tion screening revealed two independent heterozygous missense mutations, one in exon 11 of PTPN11, c.1226G>C; p.G409A, and one in exon 2 of SHOC2, c.4A>G; p.S2G. These mutations have previously been reported separately in NS patients [57, 158], but to our knowledge, this was the first report of a co-occurrence with PTPN11 and SHOC2 mutations.

To investigate the inheritance pattern, both parents of the index patient were screened for these mutations, revealing that SHOC2 p.S2G represented a de novo mutation, whereas PTPN11 p.G409A was maternally inherited. The PTPN11 mutation was also detected in the brother of the index patient.

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Both the mother and the brother displayed features of NS, although very mild phenotypes.

PTPN11 p.G409A is located in a highly conserved region in the PTP domain of SHP2, the protein encoded by PTPN11. Furthermore, it was not present in 218 healthy unrelated individuals from the Swedish population and has not been reported in the 1000 Genomes’ deep catalogue of human genetic varia-tion database (browser.1000genomes.org) nor NCBI’s dbSNP137. However, only one family with NS has previously been reported to harbour this muta-tion. [158] The phenotype of that family was very mild and variable, where some carriers of the mutation had no evident NS phenotype; thus, the muta-tion was suggested to be associated with a mild, partial NS phenotype. This is further supported by the findings in Paper II, since the mother and the brother of the index patient harboured the same mutation and presented with very mild phenotypes as well.

SHOC2 p.S2G has been found in >30 NS/LAH patients, where the majority (25 patients) were reported in one single study by Cordeddu et al. [57, 159-162] Hitherto, it is the only mutation identified in SHOC2. Functional stud-ies of this specific mutation have demonstrated an introduction of an N-myristoylation site, which causes constitutive membrane targeting of SHOC2, resulting in enhanced RAS-MAPK activity. Patients with this spe-cific mutation often present with some additional features not characteristic of classical NS patients, and several of these features, such as macrocephaly, loose anagen hair and hoarse/nasal voice, were also present in the index pa-tient in this paper. [57]

In addition to features associated with SHOC2 p.S2G and PTPN11p.G409A, the index patient here suffered from several other symptoms, rarely associat-ed with NS. To our knowledge, intrathoracic EMH has not been previously reported in patients with RASopathies; however, a few of the other features have been associated with two other RASopathies, NF1 and CS.

As in Paper I, the two mutations identified in Paper II are located in genes both functioning in the RAS-MAPK pathway. Thereby, one explanation for the additional symptoms in the index patient might be an additive effect of the two mutations present. Although, PTPN11 p.G409A was suggested to cause a mild, partial NS phenotype, it can in this case act as a modifier and together with SHOC2 p.S2G result in an even more highly increased activa-tion of the pathway compared to a mutation in only SHOC2. This increased activation could then lead to development of additional features displayed in the index patient. The suggestion of an additive effect of two mutations, where one of them operates as a modifier to cause a more severe NS pheno-type, has been proposed in other studies (see also Paper I). [123, 124, 155-157]

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Prada et al. reported on a patient with co-occurring mutations in NF1 and PTPN11, who was much more severely affected than would be expected with either syndrome alone and suggested that hyperactivation of the RAS-MAPK pathway was the most likely cause of disease for this patient. [156]

Furthermore, Longoni et al. reported on a patient with mutations in SOS1 and RAF1. The RAF1 mutation had previously been found to increase ERK activity and was probably the main reason for the NS phenotype presented in the patient, whereas the SOS1 mutation was novel. SOS1 can function as a GEF for both RAS and another protein named RAC1, which is involved in the RAC1-JNK pathway. Interestingly, functional studies demonstrated that this novel SOS1 mutation did not increase ERK activity compared to the wild-type, but showed an increased JNK activity instead, which could be responsible for cutaneous anomalies displayed in the patient. [157]

However, co-occurrence of mutations does not always cause a more se-vere phenotype, which has been demonstrated in two studies by Brasil et al. [153, 154] Although, in one of the studies, one of the mutations was predict-ed to be benign and might not contribute to the outcome at all. [154]

As mentioned, the index patient in Paper II suffered from osteoporosis and spinal cord compression. Due to these complications, she was treated with bisphosphonate. Bisphosphonate binds and blocks the enzyme farnesyl di-phosphate synthetase (FPPS), which leads to decreased prenylation of pro-teins. Interestingly, one of the proteins that undergo prenylation is RAS. Prenylation is necessary for RAS to acquire full biologic activity and several inhibitors of prenylation have been developed as anticancer agents. [163] Thus, by treating with bisphosphonate, prenylation will be decreased; hence, possibly resulting in decreased levels of RAS with full biological activity and reduced RAS-MAPK signalling.

Since RASopathies in general are caused by an increased signalling of the RAS-MAPK pathway, this causes one to speculate if bisphosphonate treat-ment would have an effect on overall RAS signalling throughout the body, thereby possibly affecting the general health condition, and not only osteo-porosis and spinal cord compression, of these patients in a positive manner.

In conclusion, Paper II is in concordance with previous findings in Paper I and further demonstrates that clinical variability seen within NS and NS-like conditions could be explained by the co-occurrence of mutations, resulting in an additive effect, where one of the mutations can act as modifier.

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Paper III Paper III presents a five-generation family with clinical diagnosis NFNS. Seven members of the family were described in 1995 as having NS with CAL spots [164]; here, the family was clinically re-examined and additional family members were added.

Upon clinical examination/re-examination of the family, the initial clinical diagnosis was changed to NFNS. This was due to fulfilment of the NIH-consensus statement for diagnosing NF1 in all but one affected family mem-ber, together with presence of features of NS in several affected family members. Furthermore, the affection status of two individuals was changed from unaffected to affected after re-examination. The reason to why they were stated as unaffected in the previous study is not known, but one could speculate that it was due to absence of a congenital heart defect, which in earlier days was almost a requirement for the diagnosis NS.

Although this family supported the findings described earlier for patients with NFNS, it still stood out among all NFNS cases reported in terms of complete absence of dermal or visible plexiform neurofibromas in all adults, which might also be an explanation for why the diagnosis of NFNS or NF1 was not considered in the previous study.

Since 1985, when NFNS was first presented, a debate has been ongoing as to whether NFNS is a separate syndrome from NF1 and NS, a phenotypic vari-ability of either NF1 or NS, or a coincidence of NF1 and NS. [119] The ma-jority of cases support the hypothesis of NFNS being a phenotypic variant of NF1, but a few studies have demonstrated a coincidence of NF1 and NS, due to presence of mutations in both NF1 and PTPN11. [123, 124] Hitherto, the two hypotheses of NFNS being a separate syndrome or a variant of NS have not been reported. To investigate which of these hypotheses the NFNS fami-ly in Paper III supported, NF1 was first screened for mutations.

The mutation screening of NF1 revealed a novel heterozygous missense mutation in exon 24, c.4168C>T; p.L1390F, which segregated with the dis-order in the family and was not identified in 108 healthy unrelated individu-als from the Swedish population. Furthermore, the mutated residue, p.L1390, showed strong evolutionary conservation in several species and is located in the GRD, which is the domain interacting with RAS. In fact, p.L1390 is even part of the most highly conserved sequence motif for RasGAPs and func-tional studies in a neurofibromin homologue, p120-GAP, demonstrated an immense decrease in GTPase activity when the corresponding p.L1390 resi-due was mutated compared to the wild-type. [165, 166] Together, this sup-ported p.L1390F to be the disease-causing mutation in this family.

Since quite a high number of mutations in NF1, including missense muta-tions, have been reported to affect mRNA splicing, this was also investigated

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for the novel p.L1390F mutation identified here. [105] However, p.L1390F did not affect splicing, based on cDNA analysis. This suggested an impair-ment of the catalysis of neurofibromin as the functional impact, which was supported by previous studies. [167]

In order to determine the possibility of a coincidence of both NF1 and NS, an additional mutation screening of eight RASopathy-associated genes was performed. Notably, in previous studies, only PTPN11 has been investi-gated for additional mutations. At the time of this study, association of SHOC2, NRAS, CBL and MYST4/KAT6B with RASopathies had not been discovered. However, this additional screening revealed no further mutations in the family. Thus, this family supported previous studies of NFNS being a variant of NF1 [118]. Notably, despite harbouring the same mutation, the clinical expressivity varies within the family, where some individuals dis-play more features of NF1 and others more NS features. This might be due to changes in modifier genes not yet identified or changes within regulatory regions of the screened RASopathy-associated genes.

A higher prevalence of in-frame defects in NFNS compared to NF1 has pre-viously been reported by De Luca et al. [118] Paper III reviewed all previ-ously reported NFNS patients and confirmed a higher prevalence of in-frame defects in NFNS. De Luca et al. also detected a clustering of in-frame de-fects to GRD in neurofibromin, which was further supported by the findings in Paper III. Since NF1 is a very large gene, a suggestion to first screen the GRD for mutations in NFNS patients was proposed in Paper III.

This study evidenced the difficulty in diagnosing NFNS correctly in patients presenting with suggestive NS features and CAL spots, but no visible signs of neurofibromas. As mentioned, the diagnosis of the family was changed from NS to NFNS and the status of two family members were changed from unaffected to affected after re-examination, which identified Lisch nodules in the two previously unaffected individuals as well as one of the affected individual. This stresses the importance of performing a slit lamp examina-tion to detect Lisch nodules, and we suggested to include this in the evalua-tion of patients with NS and skin manifestations, such as CAL spots or freckling, to identify NFNS.

In conclusion, the findings in Paper III support the hypothesis of NFNS be-ing a variant of NF1 and genetically distinct from NS. In addition, it presents an evidence of clinical variability within families harbouring the same muta-tion and also demonstrates the difficulty of determining correct diagnosis, but where the diagnosing was greatly improved by genetic analysis.

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Paper IV In Paper IV, three unrelated families displaying features of NF1 and NFNS were investigated. The study included eight affected individuals in total. All affected individuals fulfilled the NIH diagnostic criteria for NF1. In addition, one member of each family had features suggestive or typical of NS; hence, the diagnosis NFNS.

To investigate the clinical variability demonstrated within these families, we performed a comprehensive genetic analysis of twelve RASopathy-associated genes: NF1, PTPN11, SOS1, KRAS, NRAS, BRAF, RAF1, SHOC2, SPRED1, MAP2K1, MAP2K2 and CBL. This analysis revealed three different heterozygous mutations in NF1, all segregating with the dis-order in each of the three families. The identified mutations have been previ-ously reported in patients with NF1, however, not in patients with NFNS. No additional mutation was detected in any of the remaining eleven genes.

The first family, NFNS-1, was a three-generation family, where the index patient as well as his paternal grandmother were diagnosed as NF1, whereas the father presented with NFNS. Here, a missense mutation, p.R1809C, was identified in exon 29, which is the same mutation identified in Paper I. This mutation has previously been identified in two unrelated NF1 patients, but also in unaffected relatives of one of them. [97, 148] However, as discussed previously, this mutation could have reduced penetrance, which would ex-plain why some individuals do not present with a phenotype. Another expla-nation could be that these carriers display a very mild phenotype, which was not detected in the previous study, speculatively due to lack of detailed clini-cal investigations. Notably, affected members of NFNS-1 do display rela-tively mild symptoms with no congenital heart defects or neurofibroma. In addition, the individuals harbouring only p.R1809C in Paper I also present a very mild phenotype, with multiple lipomas and/or CAL spots only.

Together with the findings in Paper I, this suggests that p.R1809C is in-deed the disease-causing mutation, although probably mild, in this family.

The second family, NFNS-2, was a two-generation family, where the index patient was diagnosed as NFNS, whereas the father and the brother were diagnosed as NF1. This family harboured a 4bp-deletion in exon 37, c.6789_6792delTTAC, which causes a frameshift in the ORF, resulting in a stop codon six positions downstream. This mutation does not affect splicing, as substitutions located within the same four base pairs have been shown to do. [168] Eight NF1 patients have been reported previously with this dele-tion, but no data concerning presence of coinciding NS features have been presented. [90, 98-100, 102, 169] Exon 37 of NF1 has been found to be a small mutation hot spot, especially within these four base pairs. This is prob-

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ably due to presence of a so-called quasi-symmetric element, which forms a loop-like structure, but where six bases, including these four bases, are left unpaired and therefore might be prone to generate deletions. [100-102, 170]

The last family, NFNS-3, was also a two-generation family, in which the index patient presented with NFNS, whereas her father only displayed signs of NF1 and received his diagnosis after the daughter was diagnosed. In this family, a splice site mutation in intron 17, c.2991-1G>A, was detected. Sub-stitution of G to A in this position disrupts the splice acceptor site, resulting in skipping of exon 18 and a 41 amino acid shorter protein. Several NF1 patients harbouring the same mutation as well as other mutations also result-ing in skipping of exon 18 have been reported previously, supporting this to be the disease-causing mutation in the NFNS-3 family as well. [90, 97, 171-173]

Since we did not detect a mutation in any of the other eleven genes screened, the clinical variability within these families cannot be explained by presence of two genetic defects in coding regions of RASopathy-associated genes as in Paper I and II. However, pathogenic variants or modifying loci in other genes not investigated, or in regulatory regions of the genes screened, might still exist and contribute to phenotypic variability.

The number of reported NFNS cases is relatively low, where one explana-tion might be incorrect diagnosing (as in Paper III initially), due to the clini-cal overlap with NF1 and NS. Therefore, we proposed that screening of NF1 should also be considered for NS patients negative for mutations in NS-associated genes, especially when CAL spots are present.

Paper IV also summarized all mutations in NF1 reported in HGMD, to-gether with some additional mutations reported in NFNS patients. Mutations reported in NF1 patients are relatively widespread over the entire gene, whereas mutations associated with NFNS seem to cluster mainly to the GRD, and also the CSRD, which supports previous studies, including Paper III. [118] Furthermore, a tendency for small deletions being clustered to the CSRD and missense/nonsense mutations to the GRD is also demonstrated.

In conclusion, the results in Paper IV support that NFNS is a variant of NF1 and genetically distinct from NS. Like Paper III, it presents an evidence of clinical variability within families, which cannot be explained by co-occurrence of mutations in coding regions of RASopathy-associated genes as in Paper I and II. However, with further investigations and increased knowledge, one could speculate that an explanation in resemblance to the previous one, but perhaps more complicated will be presented in future.

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Concluding remarks and future perspectives

A general issue for Noonan spectrum disorders, as well as RASopathies, is the difficulty of determining correct diagnosis, which is due to both clinical overlap between the syndromes and clinical variability within each syn-drome or even within families. Setting correct diagnosis is of great im-portance, since different syndromes are associated with different risks of developing, for example, mental retardation or cancer. To be able to improve the diagnostics, we need to better understand and increase our knowledge about mechanisms that might be responsible for this clinical variability.

Solving the puzzle about clinical variability and correct diagnosis is even more important for future treatments of patients with RASopathies, since some drugs have been demonstrated to be mutation-specific in studies with mice. That is, mice with a mutation in one RASopathy-associated gene re-sponded well to certain drugs, whereas mice with mutations in another RASopathy-associated gene did not respond at all to the same drug. This was found to be due to mutation-specific activation of different signalling path-ways. (Personal communication and [131-133])

Evidences of clinical variability within families were seen in all four papers in the present thesis. In Paper I and II, a possible explanation for this varia-bility was demonstrated by the co-occurrence of mutations within RASopa-thy-associated genes, which could cause an additive effect. However, pres-ence of two mutations within RASopathy-associated genes does not always cause a more severe phenotype or additive effect. [153, 154] Further studies are needed to be able to understand these differences.

In Paper III and IV, although nine/twelve RASopathy-genes were screened, the clinical variability could not be explained as in Paper I and II. Of note, Paper III and IV were the first and third to investigate involvement of additional RASopathy-associated genes, besides NF1 and PTPN11, in NFNS patients. However, since we only screened the coding regions of these genes, a variant affecting the clinical outcome might be present within some regulatory region of these genes or possibly in another gene affecting the RAS-MAPK pathway or interacting pathways. Another mechanism that could also be involved in the clinical variability of the families in Paper III and IV is skewed allele-specific expression of NF1. Jentarra et al. investigat-ed allele-specific expression in normal subjects and found that ~30% had a significant variation in the expression of their two NF1 alleles. [174] If this

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may have been present in the families in Paper III and IV, one could specu-late that individuals displaying more features have lower expression of the normal allele compared to individuals with less features.

In addition, evidences of the difficulties in setting the diagnosis were seen particularly in Paper III, but also in Paper IV. In Paper III, the health status of two of the family members was changed from unaffected to affected upon re-examination and also the diagnosis of the family was changed from NS to NFNS. We then stressed the importance of performing a slit lamp examina-tion in individuals with NS and skin manifestations, such as CAL spots or freckling, in order to more easily identify NFNS as the diagnosis. In Paper IV, we discussed the idea of “hidden” NFNS cases, i.e. incorrectly diagnosed cases, and suggested to include NF1 in the mutation screening of NS patients negative for mutations in the known NS-associated genes, especially when CAL spots are present.

Indeed, such a study has been initiated. A cohort of patients with a pheno-type fitting or suggestive of NS and negative for known mutations in most of the NS-associated genes has been collected. Patients from this cohort are then screened for mutations in the GRD of NF1, since previous studies of NFNS patients have demonstrated a clustering to this region, including Paper III. [118] To date, two variants have been identified. However, both variants were inherited from an apparently healthy parent, and probably represent rare polymorphisms, since neither of them were present in 190 healthy unre-lated individuals from the Swedish population. Notably, the patients includ-ed in this cohort did not undergo a detailed clinical examination, which negatively affects the outcome of the study. Furthermore, although a cluster-ing of mutations to the GRD has been demonstrated previously, several NFNS-associated mutations are located outside the GRD as seen in Paper IV, which suggest that the entire NF1 gene should be screened. NF1 is a very large gene; thus, to continue screening remaining exons will probably require more thorough clinical data from each patient included in the cohort or the development of a faster and more efficient method for screening, such as the method in development described below.

Since the clinical variability within RASopathies is quite extensive and pa-tients are misdiagnosed clinically, one strategy would be to screen patients with features suggestive of a RASopathy for all RASopathy-associated genes. However, to do this with traditional Sanger sequencing, which is the molecular diagnostic technique mainly used today, is very time-consuming and hence, costly. A way to solve this is to introduce the relatively new techniques of next-generation sequencing into the clinics. Currently, we are developing and evaluating a new type of diagnostics for RASopathy patients, where these techniques are used to screen 13 RASopathy-associated genes

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(all but MYST4/KAT6B) in a targeted re-sequencing manner. (Manuscript in preparation)

Although some explanations for clinical variability as well as genotype-phenotype correlations exist, much remains to be elucidated and more stud-ies are required in future. It would be interesting to further molecularly com-pare patients with different phenotypes, but with the same genetic cause, both families and unrelated individuals. Also, molecular comparisons of patients with very similar phenotypes, but different mutated genes, would be of interest, as well as patients with co-occurring mutations in comparison to patients with only one of the mutations. These molecular studies could in-clude several different focuses, such as expression levels of single genes, expression profiles of all genes within a certain network, expression profiles of miRNA or activation levels of the RAS-MAPK pathway by measuring for example phosphorylation levels of ERK, to determine if there are differences or similarities that could further explain a certain outcome.

An example of such a study was reported by Ferrero et al., in which glob-al mRNA expression profiles in patients with a PTPN11, SOS1 or SHOC2 mutation were resolved. They demonstrated that robust transcriptional signa-tures could not only specifically discriminate each of the three mutation groups from controls, but also from each other, although with some partial overlap. [175]

Another issue is the unknown genetic aetiology of RASopathy patients nega-tive for mutations in known RASopathy-associated genes, which is especial-ly true for patients with a Noonan spectrum disorder. To date, 14 genes have been associated with RASopathies. Ten of these 14 genes have been reported in the pathogenesis of NS or NS-like syndromes; still the genetic aetiology for ~25% of NS patients is unknown. [16, 40, 41, 57, 69, 73, 76, 77, 176]

With the introduction of techniques of next-generation sequencing, new possibilities have opened up, which allow us to identify the remaining genet-ic defects. A well-suited technique for RASopathy patients is the whole-exome approach, where all exons in a human genome are sequenced. [177] Such approaches within the RASopathies are ongoing; however, no studies have been published yet. (Personal communication) Due to RASopathies being autosomal dominant disorders and the assumption that genes with future association to RASopathies will only be responsible for a small num-ber of cases, investigating trios on whole-exome sequencing will facilitate following analyses and the determination of disease-causing mutations. An-other clue to which variants are of importance is the involvement in the RAS-MAPK pathway for all RASopathy-associated genes so far. In this context, the expansion of computer programs dealing with interaction net-works of genes will be of great use. [178]

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To further understand the RASopathies, studies of different animal models are of great importance, especially for development of future treatments. Several studies in mice have shown very promising results for treating fea-tures of RASopathies, and it will be extremely exciting to see whether they have the same effect in humans with RASopathies or not. Although several animal models already exist, further studies are needed as we gain more and more knowledge about additional genes and pathways involved in RAS-opathies. As mentioned previously, a number of studies have shown that some mutations associated with RASopathies, not only affect the RAS-MAPK pathway, but also several interacting pathways. These results are highly significant for future treatments and express the importance of deter-mining the genetic defects in patients with RASopathies.

In summary, the findings in the present thesis demonstrate an extensive clin-ical variability within families with Noonan spectrum disorders, indicating the clinical and genetic complexity of this subgroup of syndromes, as well as the RASopathies. As new techniques develop, the possibilities of generating genetic information will increase and the secrets of the RASopathies, as well as the human genome in general will eventually be revealed.

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Populärvetenskaplig svensk sammanfattning

Vår kropp är uppbyggd av ca.100 000 miljarder celler. Cellerna kan se olika ut och ha olika uppgifter i kroppen. I alla celler finns dock en cellkärna och i den finns vår arvsmassa, även kallad DNA. Arvsmassan har ett eget alfabet, som består av fyra bokstäver: A, C, T och G. I arvmassan finns våra arvsan-lag, eller gener som de också kallas. Generna fungerar som recept för de proteiner som cellerna tillverkar, och som vi behöver för att kroppen ska byggas upp och fungera. Generellt finns alla gener i två uppsättningar; en uppsättning som vi fått från mamma och en från pappa.

För att kroppen ska fungera som den ska, behöver cellerna kunna föröka sig och det gör de genom att dela på sig. När celler ska dela på sig, måste DNAt göra en kopia av sig självt för att kunna sprida informationen vidare till båda cellerna. Vid denna kopiering uppstår ibland fel, som kan leda till att receptet för ett visst protein ändras, vilket i sin tur kan innebära att protei-net inte fungerar som det ska. Det finns många olika fel, eller mutationer som det också kallas, som kan uppstå, t.ex. kan en bokstav bytas ut mot en annan eller så kan en bokstav försvinna eller läggas till. Ibland kan flera tusen eller t.o.m. miljoner bokstäver bytas ut, försvinna eller läggas till. Det är mutationer som kan ge upphov till olika sjukdomar.

I den här avhandlingen har en grupp av sjukdomar som kallas för RASopa-tier studerats. RASopatierna orsakas av medfödda mutationer i arvsmassan, där den vanligaste mutationen är att en bokstav bytts ut mot en annan bok-stav. Sju stycken olika sjukdomar ingår i RASopatierna: Noonans syndrom, neurofibromatos typ 1, neurofibromatos-Noonans syndrom, kardio-facio-kutant syndrom, LEOPARDs syndrom, Costellos syndrom och Legius syndrom. Att ha ett syndrom innebär att man har en kombination av flera olika symptom. Noonans syndrom och neurofibromatos typ 1 är de två van-ligaste syndromen, där ett barn per ca.1000-3000 drabbas, medan de andra syndromen är mycket mer sällan förekommande.

Alla sju syndromen kännetecknas generellt av hjärtfel, kortvuxenhet, ty-piska ansiktsdrag, hudproblem, påverkan på den mentala och motoriska ut-vecklingen och en ökad risk för vissa typer av tumörer. Det finns dock vik-tiga skillnader mellan de olika syndromen när det gäller t.ex. risken för tu-mörer, graden av utvecklingsstörning eller typen av hjärtfel. En mängd andra symptom förekommer också i varierande grad bland dessa syndrom, vilket tillsammans med de karaktäristiska dragen gör att det är stor s.k. klinisk va-

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riabilitet inom de olika syndromen, d.v.s. patienter med ett visst syndrom har inte exakt samma symptom, och patienter med olika syndrom kan ha lik-nande symptom. Allt detta har lett till att det i många fall är svårt att bedöma vilket av de här sju syndromen en patient har. En korrekt diagnos är oerhört viktig för att kunna erbjuda uppföljande kontroller som tar hänsyn till de olika riskerna med de olika syndromen. Det är även viktigt för framtida be-handling av dessa patienter.

Celler kommunicerar med varandra och med omvärlden genom att känna av olika ämnen runt omkring sig. När en cell upptäcker ett visst ämne, startar en slags kedja av signaler från cellens yttre in till cellkärnan, som då kan akti-vera eller inaktivera rätt sorts proteiner och utvecklas. Det finns många olika slags signalkedjor eller signalvägar i cellen.

Anledningen till att RASopatierna kallas som de gör är för att de mutat-ioner i arvsmassan som orsakar dem finns i gener som alla är involverade i en och samma signalkedja, och den signalkedjan kallas RAS-MAPK. Totalt har man hittat mutationer i 14 olika gener för RASopatierna. Vissa av dem finns bara i ett enskilt syndrom, medan andra är gemensamma för olika syndrom. I och med att man hittat orsakerna till RASopatierna, har bedöm-ningen av patienter underlättats genom att kombinera den kliniska diagno-sen, d.v.s. symptombilden hos en patient, med en genetisk analys av patien-tens DNA, för att se vilken gen som innehåller en mutation.

Den här avhandlingen är baserad på fyra artiklar. I arbete I och II studerades orsaker till den kliniska variabiliteten hos patienter med Noonans syndrom. Här har vi undersökt två patienter som visar upp en mycket mer allvarlig symptombild än vad som generellt sett är vanligt. Vi fann att båda patienter-na hade mutationer i två olika gener, istället för en som är det vanligaste. Dessa två gener verkar båda inom RAS-MAPK-signalkedjan och slutsatsen är att två mutationer i en och samma patient ger en förstärkt effekt, vilket leder till svårare symptom.

I arbete III och IV studerade vi den kliniska variabiliteten i fyra familjer, där vissa familjemedlemmar bara visade symptom av neurofibromatos typ 1, medan andra inom samma familj hade diagnosen neurofibromatos-Noonans syndrom, som är som namnet låter en blandning av neurofibromatos typ 1 och Noonans syndrom. Här, till skillnad från arbete I och II, hittade vi endast en mutation i vardera familj och mutationerna låg alla i genen som orsakar neurofibromatos typ 1. Detta bekräftade dock tidigare resultat att majoriteten av patienter med neurofibromatos-Noonans syndrom endast har mutation i genen som orsakar neurofibromatos typ 1.

Sammanfattningsvis har resultaten från de här studierna gett ökad kunskap om de bakomliggande orsakerna till RASopatierna, vilket på lång sikt kan ge förbättrad diagnostik, prognos, riskbedömning och behandling av patienter.

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Acknowledgements

The work presented in this thesis was carried out at the Department of Im-munology, Genetics and Pathology at Uppsala University. Financial support was provided by the Foundation of Sävstaholm, the Swedish Research Council, Borgström’s foundation, the Medical faculty of Uppsala University and Uppsala University Hospital.

I would like to express my sincere gratitude to everyone who has supported and helped me during these years. In particular, I would like to thank the following persons:

The patients and their families for participating in these studies.

My supervisor, Marie-Louise Bondeson, for giving me the opportunity to do research in your group. I don’t think one can have a better supervisor than you!!! Thank you for always being so supportive and encouraging!!

My co-supervisor, Göran Annerén, for all the great support and for al-ways being such a positive and enthusiastic person. Thank you for bringing me with you on the road sometimes! It’s nice to actually see that our re-search makes a difference in real life!

My former labmate, Maja Molin (f.d. Nyström), for introducing me to clin-ical genetics and creating such a nice working environment. The days at Rudbeck are still not the same without you! Thank you for all the nice chats both about work and life in general!

All present and former members of the Dahl-group for “adopting” me into your group and all your activities, such as journal clubs and group meetings, but most importantly BBQs and julbord. It’s nice to feel part of something!

Lena and Patricia, for believing in me and putting in a good word for me. Without you, I would probably not have ended up where I am today! Marcella, thanks for all the nice back-to-back conversations.

The present and former members of the Wadelius-group, for creating such a nice working atmosphere in the office. Special thanks to Helena and Marco for all the fun chats during lunch (and working time..).

Present and former “members” of the round table in the lunch room, for enjoying conversations during lunch time.

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All the people in the DNA-lab, Lotta, Berri and everyone else at the Clini-cal genetics department, for making me feel so welcomed every time I’m there. I would especially like to thank those in the DNA-lab who have helped me with all kinds of problems and numerous of DNA-preps! You have saved me a lot of time and energy!

My other co-authors, Judith Allanson, Gerd Holmström, Kerstin Sjörs, Anders Jonzon, Lars Hagenäs, Bo Strömberg, Christina Edeby and Ma-lin Elinder, for good collaborations and valuable inputs.

My former students, Teresia, Fatemeh and Malin, for all the great work you’ve helped me with. You as well have saved me a lot of time and energy!

My former colleagues, Carina and Linshu, for believing in me and mak-ing me feel self-confident about what I do and what I know.

Everyone at the Administration, for all the help and guidance you have given me during these years.

All other Rudbeckian friends, for making Rudbeck a really nice place to work at. Special thanks to the former Rudbeckian, Fiona, for proofreading this thesis!

All my non-Rudbeckian friends, especially: Gänget med stort G, for all the fun happenings we’ve had through the

years. Thanks for keeping my mind off science sometimes and for all the love and support you’ve given me! Special thanks to Sofié, for all the hard work we did as undergraduates at Ångström, Pollax, Café Linné, Storken,.. You made calculating integrals and reading tons of pages funny!

The frisbee people, for “forcing” me to do some great workout with not only my brain. It has helped me a lot when biking up the hill to Rudbeck every morning!

All my old friends from Örebro, for all the good times we’ve had and for contributing to the person I am today. I miss you here in Uppsala! In particular, I would like to thank Sandra for funny studying techniques. Re-member studying physics while roller blading?!

The last part of the acknowledgements goes to my family: My “family-in-law”, the Klippels, for always supporting and helping me.

I can’t ask for a better family-in-law! The Ekvall-family, my mother, my father, my sister and Gustav, for all

the endless love and support you have given me. Thank you for always be-lieving in me and helping me with all kinds of things! I love you all!

Last, but most of all, I would like to thank my own little Ekvall/Klippel-family. Marcus, words cannot describe how happy and grateful I am to have you in my life! Thank you for putting up with me all these years! You are truly the best! Alexander, my sweetest little guy, you have brought so much joy and happiness into my life. Jag älskar er av hela mitt hjärta! ♥

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