Genetic and Clinical Investigation of Noonan Spectrum Disorders

Transcription

1 Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 830 Genetic and Clinical Investigation of Noonan Spectrum Disorders SARA EKVALL ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2012 ISSN ISBN urn:nbn:se:uu:diva

2 Dissertation presented at Uppsala University to be publicly examined in Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, Friday, December 7, 2012 at 09:15 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Abstract Ekvall, S Genetic and Clinical Investigation of Noonan Spectrum Disorders. Acta Universitatis Upsaliensis. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine pp. Uppsala. ISBN Noonan spectrum disorders belong to the RASopathies, a group of clinically related developmental disorders caused by dysregulation of the RAS-MAPK pathway. This thesis describes genetic and clinical investigations of six families with Noonan spectrum disorders. In the first family, the index patient presented with severe Noonan syndrome (NS) and multiple café-au-lait (CAL) spots, while four additional family members displayed multiple CAL spots only. Genetic analysis of four RAS-MAPK genes revealed a de novo PTPN11 mutation and a paternally inherited NF1 mutation, which could explain the atypically severe NS, but not the CAL spots trait in the family. The co-occurrence of two mutations was also present in another patient with a severe/complex NS-like phenotype. Genetic analysis of nine RASopathy-associated genes identified a de novo SHOC2 mutation and a maternally inherited PTPN11 mutation. The latter was also identified in her brother. Both the mother and the brother displayed mild phenotypes of NS. The results from these studies suggest that an additive effect of co-occurring mutations contributes to severe/complex NS phenotypes. The inherent difficulty in diagnosing Noonan spectrum disorders is evident in families with neurofibromatosis-noonan syndrome (NFNS). An analysis of nine RASopathy-associated genes in a five-generation family with NFNS revealed a novel NF1 mutation in all affected family members. Notably, this family was initially diagnosed with NS and CAL spots. The clinical overlap between NS and NFNS was further demonstrated in three additional NFNS families. An analysis of twelve RASopathy-associated genes revealed three different NF1 mutations, all segregating with the disorder in each family. These mutations have been reported in 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 clinical overlap between NS and NFNS, we propose screening for NF1 mutations in NS patients negative for 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 the RAS-MAPK pathway contributes to the clinical variability observed within Noonan spectrum disorders 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 Uppsala, Sweden. Sara Ekvall 2012 ISSN ISBN urn:nbn:se:uu:diva (

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5 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

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7 List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I II III IV 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 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): 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): 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.

8 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-faciocutaneous syndromes: two clinically and genetically overlapping disorders. J Med Genet, 45(8): 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 amplification analysis. Eur J Med Genet, 53(3): Wittström E., Ekvall S., Schatz P., Bondeson M.L., Ponjavic V., Andréasson S. (2011) Morphological and functional changes in multifocal vitelliform retinopathy and biallelic mutations in BEST1. Ophthalmic Genet, 32(2):83-96

9 Contents Introduction The human genome Human genetic variation Disease-causing variants Human genetic disorders Monogenic disorders Methods in disease-gene identification Linkage analysis Sanger sequencing Restriction fragment length polymorphism (RFLP) Multiplex ligation-dependent probe amplification (MLPA) SNP arrays Next-generation sequencing The RAS-MAPK pathway Activation of the RAS-MAPK pathway Regulation of the RAS-MAPK pathway Phosphorylation and dephosphorylation Scaffolding proteins, phosphatases and inhibitors Internalization and degradation of receptors Histone modifications Post-transcriptional regulation Determination of signal specificity of the RAS-MAPK pathway Signal strength and duration Cross-talk with other pathways Subcellular localization of components of the pathway Cancer and the RAS-MAPK pathway Drug development RASopathies Noonan and Noonan-like syndromes Clinical description Genetic description Genotype-phenotype correlations Neurofibromatosis type Clinical description... 37

10 Genetic description Genotype-phenotype correlations Neurofibromatosis-Noonan syndrome Clinical description Genetic description Genotype-phenotype correlations Animal models and future treatments Present investigations Background Aims Paper I Paper II Paper III Paper IV Concluding remarks and future perspectives Populärvetenskaplig svensk sammanfattning Acknowledgements References... 62

11 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

12 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

13 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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15 Introduction The human genome Almost 60 years have passed since Watson and Crick discovered the structure of deoxyribonucleic acid, DNA, in [1] They put forward the principle 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 released a number of publications, where regions of transcription, transcription factor association, chromatin structure and histone modification were systematically mapped. Together, these researchers could assign biochemical functions for 80% of the genome, providing new insights into the organization of our genome and the mechanisms of gene regulation. This demonstrates that much more than just the protein-coding genes are of great importance 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 segments. A genetic variant present in more than 1% of the population is considered to be a polymorphism. [6] Two large projects, the International HapMap project and the 1000 Genomes project, have been initiated to identify genetic similarities and differences 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, 15

16 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, microsatellites and mononucleic tracts, depending on the length of the total repeat tract (more than 10 5 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 involving segments of DNA larger than 1kb and can be divided into five subcategories: 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 Variants 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 duplications 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 interchromosomal. 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. 16

17 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-offunction mutation. A point mutation is a substitution, a deletion or an insertion of a single nucleotide and it can be located in either coding regions or non-coding regions. In the coding region, point mutations can be classified as missense, nonsense, frameshift, splice site or silent mutations, all depending on the outcome 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 disorders, 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; the Online Mendelian Inheritance in Man (OMIM; ncbi.nlm.nih.gov/omim) and the Leiden Open Variation Database (LOVD; 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 involvement of the environment. The categories are monogenic (also called Mendelian) disorders, complex (also called multifactorial) disorders, chromosomal disorders and mitochondrial disorders. The present thesis will focus 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 17

18 present, i.e. when different mutations in the same gene cause the same disorder. In some monogenic disorders, mutations at different loci cause the same phenotype, called locus heterogeneity. A third type of heterogeneity is clinical 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 transmitted by either sex. Autosomal recessive, where affected individuals are homozygous or compound heterozygous for the mutated allele, located on one of the autosomes. 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 disorders, since they carry only one X-chromosome. The mother of an affected 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 express 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. 18

19 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 suitable 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 recombination is less likely to occur between closely positioned loci. By using linkage analysis, genomic regions associated with the disease can be identified 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 genetic 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 significant 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 certain 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 sequencing can be performed directly on that particular gene or those particular genes of interest. 19

20 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 nucleotide terminator will exist in one reaction. These fragments are then separated 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 performed by Sanger sequencing, but another method of choice is RFLP. This method is based on the fact that restriction enzymes recognize specific 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 compared 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 genome. 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 amplification products are then separated according to size by capillary gel electrophoresis and the unique length of each ligated probe pair makes it possible 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] 20

21 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, requiring 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 enzyme and ligation of adaptors to the digested fragments. The fragments are then amplified simultaneously by PCR, using primers recognizing the adaptor sequences. This is followed by fragmentation of the amplified fragments, labelling and finally hybridization to the SNP array. The resulting hybridization 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. ( 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 technology (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 research and patient care in the future. [21] Already, they have been used successfully 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 compared to using regular Sanger sequencing. [23] Sequencing of the exons of the X-chromosome in patients with mental retardation and control individuals 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] 21

22 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 surroundings. 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 cascade 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 stimulus and the desired outcome in different cellular processes, such as differentiation, 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 proliferation, 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 proteincoupled receptors and ion channels, can initiate the activation of the RAS- MAPK pathway. Upon stimulation of the extracellular domain of these receptors, kinase activity in the cytoplasmic domain of the receptors is induced. 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 22

23 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 phosphorylate 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, approximately 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 23

24 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 similar 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, dephosphorylation of ERK by ERK-dependent DUSPs also inhibits the signalling. 24

25 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 phosphatase 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 degradation 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 subjected 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 RNAbinding 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 25

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 components of the RAS-MAPK pathway. A) PUM2 inhibits translation of ERK directly, but can also indirectly function as a positive regulator of ERK signalling. B) HuR promotes MEK1 translation directly, but can also negatively regulate the pathway indirectly by promoting MKP-1 translation. C) mirna let-7 can directly repress 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 specificity 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 extracellular 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 proliferation of the cells, whereas in NGF-stimulated cells, ERK activation was sustained and cells were differentiating. An explanation for this difference was the effect of immediate early genes, which induce different cellular processes 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- 26

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 operating 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 primarily 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 cytosol and can translocate to the nucleus or other organelles in the cell to perform further interactions. However, a portion of the components stay attached to their anchors upon stimulation, to be directed to other specific targets in the cytoplasm. Together, these specific localizations in the cell influence 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 proliferation, motility and survival; all processes where the RAS-MAPK pathway is of great importance, making this pathway a hot target for many human cancers. [35] In fact, several components within the pathway have been associated with different types of cancers. Mutations in RAS genes have been identified 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- 27

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 myelomonocytic leukaemia (JMML) and mutations in NF1, encoding neurofibromin, 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 development 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 signalling have been more successful. However, these drugs have no effect in patients 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 switching 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] 28

29 RASopathies The RASopathies are a group of clinically and genetically related developmental disorders, including Noonan syndrome (NS) and NS-like syndromes, cardio-facio-cutaneous syndrome (CFCS), LEOPARD syndrome (LS), Costello 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-MAPKsyndromes. The two most common syndromes within the RASopathies are NS and NF1, with and incidence of 1/ and 1/ respectively, 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 RASopathies 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, typical facial features, cardiac defects, ectodermal abnormalities, variable cognitive deficits and susceptibilities to certain malignancies are all identified characteristics. Despite this clinical overlap between the different syndromes, 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 syndromes have different prognoses, for example mental retardation is more common in CFCS than NS and the increased risk of developing malignancies differs with each syndrome, setting the correct diagnosis is of great importance 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, including NS, NS-like syndromes and NFNS, with detailed clinical and genetic descriptions of each of them. In addition, NF1 will also be discussed in further detail, due to its overlap with NFNS. 29

30 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 description of this group of patients in In 1985, it was therefore suggested to change the name from male Turner syndrome to Noonan syndrome. NS (OMIM ) is one of the most common monogenic disorders in humans with an incidence of one in births. The inheritance pattern is autosomal dominant and both familial and sporadic cases exist. Clinically, NS is a very variable disorder, both within families and between unrelated 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 cardiomyopathy (~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 30

31 (without growth hormone treatment) 157.4±8.0cm, but around 30% of patients 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 keratosis), 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 ) is a syndrome that greatly resembles NS. This syndrome was first presented in 2003, and patients show characteristics such as more severe growth and cognitive 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 ), which was reported in These patients have a relatively 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 lymphoblastic 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. However, 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 associated with NS [61]. The gene consists of 16 exons and encodes a tyrosine 31

32 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 residues 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, making it the major gene associated with NS. The mutations identified are mainly missense mutations, whereas deletions, insertions/duplications and indels are rare. ([62] and 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 variants, KRASA and KRASB, where KRASB is predominant and often denoted 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 mediates 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 mutations is a gain-of-function, generated by different mechanisms, such as impaired 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% respectively). [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- 32

33 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 autoinhibited by interaction between the DH and the Rem domain, which blocks binding 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 conformation, 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 activity, and CR3 mediates phosphorylation. RAF1 has one inactive and one active 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 translocation 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 constitutive membrane translocation of SHOC2. In turn, this constitutive translocation promotes prolonged dephosphorylation of RAF1 at Ser259 mediated by PP1C, thereby sustaining RAF1-stimulated activation of the pathway. 33

34 Hence, this single missense mutation of SHOC2 is a gain-of-function mutation. [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 GTPbound 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 phosphorylation of MEK and ERK, i.e. a gain-of-function. [73-75] Furthermore, a few reports have been published on NS patients with missense 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 conserved 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 ligase, one of the enzymes required for targeting substrates for degradation by the proteasome. Four domains comprise CBL: one N-terminal tyrosine kinase-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 dimerization. Mutations in CBL have been shown to impair ubiquitylation of receptors, causing increased pathway signalling, and patients with mutations in CBL have features more or less reminiscent of NS. [40, 44, 59] Somatic mutations in this gene are found in patients with different myeloid malignancies, 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 exons. 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. 34

35 Quantitative RT-PCR in this patient revealed a 50% decrease in mrna expression levels of MYST4/KAT6B. This haploinsufficiency resulted in increased phosphorylation of MEK1/2 and ERK1/2, and also enhanced AKT phosphorylation. However, no further mutations in this gene could be identified in a cohort of 131 subjects with suggestive NS features, who were negative 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 mutation and pulmonic stenosis has been revealed, whereas the frequency of hypertrophic 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] Furthermore, 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 eyebrows or keratosis pilaris. [71, 80, 81] Hypertrophic cardiomyopathy and hyperpigmented cutaneous lesions occur at a significantly higher frequency (75-95% and 33% respectively) in patients 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 35

36 Table 1. Genotype-phenotype correlations in NS and NS-like syndromes Mutated gene PTPN11 SOS1 RAF1 KRAS SHOC2 CBL BRAF MEK1, NRAS, MYST4/KAT6B Clinical feature + Pulmonic stenosis + Thorax deformities + Easy bruising + Short stature + Cryptorchidism (in males) + Familial cases + JMML (p.t73i) - Hypertrophic cardiomyopathy + Pulmonic stenosis + Thorax deformities + Ectodermal manifestations - Hypertrophic cardiomyopathy - Short stature - Special education + Hypertrophic cardiomyopathy + Hyperpigmented cutaneous lesions + Cognitive impairment + Severe phenotype in general + 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 + JMML + Short stature + Developmental delay + Café-au-lait spots + Cryptorchidism (in males) + Neonatal growth failure (due to feeding difficulties) + Mild-to-moderate cognitive deficits + Hypotonia + Multiple naevi or dark colored lentigines + More severe phenotype in adulthood No clear correlations 36

37 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 without 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 correlations have been identified. Neurofibromatosis type 1 Clinical description The first description of neurofibromatosis type 1 (NF1, OMIM ), made by von Recklinghausen, dates back to [82] NF1 is one of the most common disorders with an autosomal dominant inheritance pattern, and the incidence is approximately one in 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 criteria for NF1 proposed by the NIH Consensus Development Conference in 1987 and later reviewed in [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 37

38 Two or more neurofibromas of any type or one plexiform neurofibroma Axillary or inguinal freckling Two or more Lisch nodules Optic pathway glioma (OPG) Skeletal abnormalities, e.g. scoliosis, pseudoarthrosis, sphenoid dysplasia 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 suggests, one of the main hallmarks of NF1. Cutaneous neurofibromas are the most common type, present in >99% of patients with NF1, whereas plexiform 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 additional 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. Nevertheless, the presence of malignant tumours is relatively low (10-20%), although 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 present in NF1 patients are central nervous system tumours (particularly associated 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 38

39 in [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 proteins, 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 genome. These pseudogenes are located on a number of chromosomes and some chromosomes harbour more than one NF1 pseudogene. [95] The complexity 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; 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, ] 39