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Gene Panel – Noonan Syndrome (Reference – 2014.01.004)

Notice of Assessment

June 2014

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1 GENERAL INFORMATION

1.1 Requester: Centre hospitalier universitaire de Sherbrooke (CHUS)

1.2 Application for Review Submitted to MSSS: January 26, 2014

1.3 Application Received by INESSS: March 1, 2014

1.4 Notice Issued: June 30, 2014

Note:

This notice is based on the scientific and commercial information submitted by the requester and on a complementary review of the literature according to the data available at the time that this test was assessed by INESSS.

2 TECHNOLOGY, COMPANY, AND LICENCE(S)

2.1 Name of the Technology

High-throughput gene panel sequencing (next generation (NGS) or massively parallel sequencing).

2.2 Brief Description of the Technology, and Clinical and Technical Specifications

Next generation sequencing provides rapid and less expensive simultaneous sequencing of several genes; exomes and whole genomes can also be sequenced. The requester plans to use this approach to sequence exons and sequences adjacent to splice sites of 14 genes associated with Noonan syndrome and certain related syndromes. The BRAF, CBL, HRAS, KRAS, MAP2K1, MAP2K2, NF1, NRAS, PTPN11, RAF1, RIT1, SHOC2, SOS1 and SPRED1 genes were selected following a review of the scientific evidence on their clinical relevance.

Sequencing involves three steps: sample preparation/genebank production (library), sequencing, and results analysis. In short, after DNA fragmentation and the addition of oligonucleotide adapters that allow for sequencing, areas of interest are enriched by hybrid capture followed by PCR using a custom SeqCap Ez kit from Nimblegen. The oligonucleotides in the kit are chosen and synthesized in accordance with the requester’s specifications (Appendix A). DNA fragments are then sequenced using a basic principle similar to the Sanger approach: nucleotide sequence is determined during cyclic synthesis of a complementary strand. This reaction uses fluorescent nucleotides for detection and is performed with Illumina’s MiSeq platform. Unlike traditional sequencing, NGS generates and deciphers millions of reactions simultaneously. Lastly, the sequences obtained are assembled and aligned to a reference sequence. Variations are detected and annotated automatically and manually. Automated bioinformatic analysis is performed using software based on the Burrows-Wheeler Aligner (BWA) program and the Genome Analysis Toolkit (GATK) (Broad Institute) and their recommendations [Van der Auwera et al., 2013; McKenna et al., 2010]. Variants are then confirmed by Sanger sequencing (analysis is performed on the Genetic Analyzer 3500, Life Technologies) as recommended by the American College of Medical Genetics and Genomics (ACMG). The minimum quality score accepted for sequencing is Q40, with a minimum of 20 sequences per base. Variants are assessed using ClinVar, HGMD, LOVD, dbSNP and Exome Server Project databases, and reported in accordance with ACMG recommendations [Rehm et al., 2013].

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2.3 Company or Developer

Illumina designed the sequencing platform, and Nimblegen developed the kit for enrichment of areas of interest.

2.4 Licence(s) (Health Canada, FDA)

The products and technologies used have not been licenced by Health Canada or the FDA. The FDA approved Illumina’s MiSeqDx platform for clinical use at the end of 2013.

2.5 Weighted Value: 858.81

3 CLINICAL INDICATIONS, PRACTICE SETTINGS, AND TESTING PROCEDURES

3.1 Targeted Patient Group

The test targets individuals with clinical signs compatible with Noonan syndrome and other related syndromes such as cardiofaciocutaneous syndrome, Costello syndrome, LEOPARD syndrome, Watson syndrome and Legius syndrome.

3.2 Targeted Disease

Noonan syndrome (NS) is a congenital genetic disorder with a prevalence of 1 per 1,000 to 2,500 births [Sharland et al., 1992; Mendez and Opitz, 1985; Noonan and Ehmke, 1963]. Approximately 60% of cases are sporadic [Rohrer, 2009]. Affected individuals have distinctive facial features, short stature despite normal birth size, and congenital heart defects in up to 90% of patients [Kaski and Limongelli, 2014]. Other characteristics include various coagulation defects, lymphatic dysplasias, ocular abnormalities, feeding problems in early childhood, typical deformity of the spinal column and shield chest deformity of the sternum combining pectus carinatum superiorly and pectus excavatum inferiorly, and developmental delays of varying severity. Up to one third of patients have a mild intellectual deficit. The most commonly associated cardiovascular abnormalities are pulmonary valve dysplasia combined with pulmonary stenosis (50% to 60% of cases) and hypertrophic cardiomyopathy (20% of cases) which can be present at birth or develop during childhood. Approximately 9% of patients die from these abnormalities between the ages of a few months and 61 years [Shaw et al., 2007]. Myeloproliferative neoplasias and juvenile myelomonocytic leukemia (JMML) have been reported in these patients [Aoki et al., 2008; Kratz et al., 2005; Tartaglia and Gelb, 2005].

NS is part of a group of disorders recently referred to as “RASopathies”. Like cardiofaciocutaneous syndrome (CFC), Costello syndrome (CS), neurofibromatosis type 1 (NF1) and LEOPARD syndrome (LS), this syndrome is caused by mutations in various genes involved in the RAS/MAPK signalling pathway (Appendix B). This pathway, which has been studied extensively in oncogenesis, plays a role in transduction of extracellular signals (like growth factors and cytokines) into biochemical and transcriptional responses regulating cell proliferation, differentiation and senescence [Ferrero et al., 2012; Matozaki et al., 2009; Schubbert et al., 2007]. Its central role in multiple cellular processes results in the diversity of phenotypic characteristics observed in NS and related syndromes. It also contributes to the overlap observed among the various syndromes (Table 1) [Tartaglia and Gelb, 2010].

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Table 1: Noonan syndrome and related syndromes (“RASopathies”)

DISORDER OMIM* PREVALENCE†

PHENOTYPIC CHARACTERISTICSǁ

Noonan syndrome 163950 609942 610733 611553 613224 613706

1/1,000 – 1/2,500 Distinctive dysmorphic craniofacial features

Congenital heart defects Small stature Ophthalmic abnormalities Coagulation defects Normal or slightly reduced

neurocognitive function Cryptorchidism Predisposition to cancer

Noonan-like syndrome with loose anagen hair

607721 1/1,000,000 Variant of NS Similar phenotype to NS Brittle hair

Costello syndrome 218040 ˂ 250 reported cases

Similar craniofacial features to NS but may be more pronounced

Congenital heart defects Very severe feeding problems Small stature Ophthalmic abnormalities Cutaneous lesions Reduced neurocognitive function Hypotonia Predisposition to cancer

LEOPARD syndrome 151100 ˂ 200 reported cases

Similar phenotype to NS Appearance of multiple lentigines Predisposition to cancer is unknown

Capillary malformation - arteriovenous malformation

608354 Unknown Capillary malformations that may be associated with arteriovenous malformations

Predisposition to cancer is uncertain

Cardiofaciocutaneous syndrome

115150 More than 100 reported cases

Similar craniofacial features to NS but may be more pronounced

Congenital heart defects Severe feeding problems Small stature Ophthalmic abnormalities Cutaneous lesions Feeding problems when young Mild to severe reduction in

neurocognitive function Hypotonia Predisposition to cancer is uncertain

Legius syndrome 611431 ˂ 200 reported cases

Café-au-lait macules Macroencephaly Normal or slightly reduced

neurocognitive function

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DISORDER OMIM* PREVALENCE†

PHENOTYPIC CHARACTERISTICSǁ

Intertriginous freckling No predisposition to cancer

Watson syndrome 193520 Unknown Pulmonary stenosis Café-au-lait macules Reduced neurocognitive function Small stature Macroencephaly Lisch nodules

Abbreviation: OMIM: Online Mendelian Inheritance in Man. *www.omim.org †www.orphanet.net, genetics home reference (http:// ghr.nlm.nih.gov) ǁRauen, 2013.

In NS as in most other RASopathies, causal mutations are heterozygous and result in activation of the RAS/MAPK signalling pathway, but to a lesser extent than somatic mutations associated with oncogenesis. Familial recurrence is consistent and follows autosomal dominant inheritance. However de novo mutations are more common (approximately 60% of cases) [Hafner and Groesser, 2013; Shaw et al., 2007].

Genetic etiology can be seen in 70% to 90% of affected individuals [Bhambhani and Muenke, 2014; Lepri et al., 2014]. Approximately 50% of NS cases have a missense mutation in the PTPN11 gene, 10% to 15% in the SOS1 gene, and 3% to 17% in the RAF1 gene. The KRAS gene contains mutations in less than 2% to 5% of affected individuals. Other genes associated with NS contain mutations in less than 1% of cases (NRAS, BRAF and MAP2K1).

The differential diagnosis includes syndromes characterized by facial dysmorphology, small stature and heart defects, like Williams syndrome and Aarskog syndrome, as well as Turner syndrome, Watson syndrome, in utero exposure to alcohol or primidone, and other RASopathies such as Costello syndrome, cardiofaciocutaneous syndrome, Noonan-like syndrome with loose anagen hair and LEOPARD syndrome [Van der Burgt, 2007].

3.3 Number of Patients Targeted

120 patients are targeted per year.

The test will be performed on children and adults. It could be available as prenatal screening based on volumes and results of other NGS test panels.

3.4 Medical Specialties and Other Professions Involved

Pediatrics, genetics and genetic counselling.

3.5 Testing Procedure

Only medical geneticists order this type of test. Requests come from large centres like CHU Sainte-Justine, Montreal Children’s Hospital, CHUS and Centre hospitalier universitaire de Québec (CHUQ).

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4 TECHNOLOGY BACKGROUND

4.1 Nature of the Diagnostic Technology: Single test

4.2 Brief Description of the Current Technological Context

The recommended diagnostic approach is sequential Sanger sequencing of candidate genes selected based on observed phenotype and the prevalence of observed mutations. Therefore this approach requires accurate clinical assessment to limit delays and associated costs [Romano et al., 2010].

Following the development of NGS sequencing technologies, several suppliers outside Quebec offer NGS gene panel sequencing. The panels generally consist of genes associated with NS and related syndromes. For example, GeneDx offers sequencing of the BRAF, KRAS, HRAS, NRAS, MAP2K1, MAP2K2, PTPN11, RAF1, CBL, SOS1, and SHOC2 genes, and Blueprint Genetics, Harvard Medical School’s Partners HealthCare Center for Personalized Genetic Medicine (PCPGM), Children's Hospital of Philadelphia and Emory Genetics Laboratory offer a panel with SPRED1 in addition to the previous genes. The requester currently sends its test requisitions to these suppliers.

At present, the Quebec Index of Biomedical Tests does not include any sequencing of genes associated with NS, either singly or as a panel. In 2012–2013, 116 test requisitions were sent to the United States at a total cost of $187,000.

4.3 Brief Description of the Advantages Cited for the New Technology

NS has a heterogeneous genetic etiology. Additionally, when mutated, the genes associated with NS result in phenotypic heterogeneity, complicating diagnosis.

The requester’s approach allows simultaneous sequencing of all genes associated with NS. NGS gene panel sequencing eliminates the need for successive sequencing, resulting in more rapid and less costly diagnosis and care. The genetic cause can be confirmed in 75% to 90% of cases.

NS testing is the 5th most expensive test performed outside Quebec, according to data from the MSSS and information presented at the Réseaux universitaires intégrés de santé (RUIS, integrated university health networks) sectoral table on genetics (data provided by the requester). The requester assesses the weighted value of the test at 858.81. The annual production cost for the anticipated 120 tests would be $103,057.

4.4 Cost of Technology and Options: Not assessed.

5 EVIDENCE

5.1 Clinical Relevance

5.1.1 Other Tests Replaced

Does not replace any test in the Quebec Index of Biomedical Tests. It would replace those currently sent outside Quebec.

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5.1.2 Diagnostic or Prognostic Value

Diagnosis of NS is based on the patient’s clinical characteristics. If NS is suspected, genetic testing is recommended to confirm the diagnosis and genetic etiology, and to establish the differential diagnosis. The information obtained determines the prognosis, ensures appropriate care for the patient, and provides reproduction options to couples, particularly individuals with Noonan syndrome [Roberts et al., 2013; Romano et al., 2010].

Groups of experts, including Romano et al. [2010], have issued recommendations for follow-up. Significant differences in the risk of complications have been reported based on the syndrome diagnosed, and follow-up/treatment varies based on the syndrome and each patient’s particular clinical characteristics. Care may include follow-up for eating problems in early childhood, assessment of cardiac function, and monitoring of growth and motor development, among others. A complete eye exam and hearing test should be performed during the first years at school, and coagulation studies should be performed before any surgery. The risk of several cancers is also slightly higher with some syndromes than in the normal population [Aoki and Matsubara, 2013; Cizmarova et al., 2013; Van der Burgt, 2007].

Several phenotypic-genetic concordance studies have associated certain genes or mutations with a predisposition to certain traits or risks, despite significant phenotypic heterogeneity. For example, follow-up for increased risk of cancerous lesions could be indicated for patients with mutations in the HRAS gene. Development of various tumours, benign and malignant, has been reported in 15% of these patients [Gripp et al., 2006]. Hypertrophic cardiomyopathies are over-represented in NS cases with mutations in the RAF1 gene (75% to 90% compared with 20% in the general cohort of NS patients) [Tartaglia and Gelb, 2010; Verloes and Cavé, 2010]. Therefore, closer follow-up could also be advisable for these patients.

When they receive appropriate care, most children with NS can go on to lead a normal adult life. Signs and symptoms lessen with age, and most affected adults do not require any special medical treatment [Van der Burgt, 2007; Tartaglia and Gelb, 2005; Van der Burgt et al., 1994; Noonan, 1963]. However, adults with NS may have a higher risk of sudden death [Binder et al., 2012]. Additional prospective studies are required to assess the risks associated with NS in adults.

5.1.3 Therapeutic Value

There is currently no curative treatment for patients with NS or any of the related syndromes. With respect to cancer treatment, several molecular inhibitors of various RAS/MAPK pathway effector proteins have been developed. Some have been approved by the FDA and Health Canada while others are in development and undergoing clinical trials [Burgess, 2013; Sebolt-Leopold, 2008]. Rauen et al. [2011] and Chen et al. [2010] have suggested that RAS pathway inhibitors such as farnesyltransferase and BRAF and MEK inhibitors could represent possible treatments for RASopathies.

Two clinical trials have been published to date on the use of RAS/MAPK pathway inhibitors in RASopathies. The first study examined the effect of 12 weeks of treatment with simvastatin, an RAS activity inhibitor, on cognitive function in 62 children aged 8 to 16 years with neurofibromatosis. The authors concluded that there was no improvement, based on their criteria [Krab et al., 2008].

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The second study examined the effect of 3 months of treatment with lovastatin in a cohort of 24 children aged 10 to 17 years with neurofibromatosis. The study’s primary objective was to assess safety and toxicity. Secondary results revealed significant improvement in verbal and non-verbal memory, attention and visual efficiency [Acosta et al., 2011].

A phase II clinical trial by Novartis is currently recruiting. The purpose of the study is to determine whether MEK162 (an MEK inhibitor) is able to antagonize MEK activation in individuals with NS with hypertrophic cardiomyopathy (study NCT01556568).1 Hypertrophy regression will be assessed after six months of treatment.

5.2 Clinical Validity

Semi-automated Sanger sequencing has been used for many years and is considered the gold standard. However, NGS now allows simultaneous analysis of several genes, the exome or even the entire genome using a high-throughput, low-cost approach. As a result, NGS is poised to being adopted in clinical settings. Against this backdrop, the FDA approved Illumina’s MiSeqDx platform for clinical use at the end of 2013.

Use of the NGS approach in multi-gene disorders has identified several variations described as pathogenic and subsequently confirmed by traditional sequencing [Lepri et al., 2014; Umbarger et al., 2014; Li et al., 2013; Sikkema-Raddatz et al., 2013]. Simultaneous sequencing of several genes as performed with NGS would be difficult, if not impossible, for diagnosis using the traditional Sanger approach.

Sanger sequencing is required to investigate regions with insufficient read depth and to confirm observed variants. However, NGS should be introduced in clinical settings, as the benefits of the technique outweigh the associated risks (FDA memorandum on the introduction of MiSeqDx, ACMG [Rehm et al., 2013; Sikkema-Raddatz et al., 2013]).

Association of NF1 with neurofibromatosis type 1, identification of PTPN11 as the first NS gene, and overlap in the phenotypic characteristics of NS with several other related syndromes, led to the search for other genes that code for proteins in the RAS/MAPK pathway in NS and other RASopathies. These studies and, more recently, a few rare NGS exome sequencing studies revealed a limited number of genes responsible for NS and related syndromes.

Clinical Validity of Panel Genes

PTPN11

The role of the PTPN11 gene, located on chromosome 12q24, in the etiology of NS was discovered through the work of Tartaglia et al. [2001] in a study on positional candidate genes. The gene, with 15 exons spread over 85 kb, codes for SHP2, a tyrosine phosphatase protein with 593 amino acids containing two tandem Src homology 2 (SH2) domains in the N-terminal side of a PTPase catalytic domain. The protein positively regulates the RAS pathway.

Subsequent studies of cohorts of unrelated subjects and families revealed that approximately 50% of NS patients have a mutation in this gene. The mode of transmission is autosomal dominant. The more than 47 documented mutations are almost exclusively missense mutations that affect SH2 function. Affected residues reside in the N-terminal

1 ClinicalTrials.gov. Safety, tolerability, pharmacokinetics and pharmacodynamics of MEK162 in Noonan syndrome hypertrophic cardiomyopathy [Web site]. Available at: http://clinicaltrials.gov/show/NCT01556568.

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domain or PTP domain, which includes the SH2 interface. The most common mutation is N308D. Structural studies have suggested that some mutations could cause gain of function resulting in excessive SHP2 activity [Yoon et al., 2013; Zenker et al., 2004; Musante et al., 2003; Digilio et al., 2002; Kosaki et al., 2002; Legius et al., 2002; Tartaglia et al., 2002; Maheshwari et al., 2002; Tartaglia et al., 2001].

Approximately ten cases of duplication of region 12q24 encompassing PTPN11 have been reported in NS patients [Chen et al., 2014; Graham et al., 2009].

Eleven mutations, different from those associated with NS, have been related to LEOPARD syndrome in approximately 90% of cases, including familial and sporadic cases [Martinez-Quintana and Rodriguez-González, 2012]. The p.Tyr259Cys and p.Thr468Met mutations were the most common [Yoshida et al., 2004; Zenker et al., 2004; Sarkozy et al., 2003; Digilio et al., 2002; Legius et al., 2002]. The mechanism by which the mutations cause the disorder is not clear, but LS mutations typically affect PTP domain residues, which result, in transient in vitro transfection assays, in decreased catalytic activity and lower RAS/ERK activation [Qiu et al., 2014; Verloes and Cavé, 2010].

Mutations in the PTPN11 gene have also been associated with several types of cancers, including juvenile myelomonocytic leukemia, acute myeloid leukemia and retinoblastomas [Cheng et al., 2013; Blackford et al., 2009; Silva et al., 2009; Jongmans, 2005; Kratz et al., 2005; Bentires-Alj et al., 2004].

SOS1

The SOS1 gene codes for a GTP (guanosine triphosphate) exchange factor, or GEF, with 1,333 amino acids (150 kDa). This multi-domain protein activates RAS by allowing GDP-GTP exchange. Its gene is located on chromosome 2p22.1 and consists of 23 coding exons spread over 135 kb.

Roberts [2007], Tartaglia [2007], Zenker [2007] et al. reported an association between the SOS1 gene and NS using patient cohorts with Noonan syndrome. Subsequent studies confirmed that the vast majority of mutations detected are missense mutations spread over several domains. Most of the regions involved contribute structurally to maintaining an active form of the protein that stimulates RAS and ERK activation. The prevalence of SOS1 mutations has been assessed at approximately 20% to 28% of the population with NS.

From a phenotypic standpoint, NS patients with SOS1 mutations present frequent and distinct ectodermal characteristics or abnormalities, including keratosis pilaris and curly hair. In addition to the association with NS, mutations in this gene are also associated with gingival fibrosis.

RAF1

The RAF1 gene, with 16 coding exons over 34 kb, is located on chromosome 3p25, and codes for a protein that is part of the serine/threonine kinase family. The protein acts as an RAS effector by phosphorylating MEK1 and MEK2 kinases.

Pandit et al. [2007] and Razzaque et al. [2007] used a candidate gene approach to sequence RAF1 in 237 and 30 patients, respectively, with NS or LS in whom sequencing for PTPN11, SOS1, KRAS and HRAS was unable to establish genetic etiology. These studies associated activating missense mutations in the RAF1 gene with NS with a prevalence of approximately 5% to 15%. Mutations in RAF1 have also been reported in 33% of LS patients.

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Three regions of the protein are affected by mutations: 70% of mutations are located in the 14-3-3 recognition sequence within the N-terminal (or conserved region 2) containing Ser 259, 15% of mutations affect residues in the kinase domain (Asp486 and Thr491) and 15% affect 2 adjacent residues (Ser612 and Leu613) in the C-terminal. Mutations affecting phosphorylation of serine in position 259 cause gain of function, while those affecting the kinase activation loop cause a loss.

Mutation of residues in the CR2 region has been related to increased prevalence of hypertrophic cardiomyopathy (95% versus 18% in the total population affected by NS) [Kobayashi et al., 2010; Pandit et al., 2007; Razzaque et al., 2007]. Mutations in the RAF1 gene have also been associated with pulmonary adenocarcinoma, colorectal cancer, melanoma and non-small cell lung cancer.

KRAS

The KRAS gene is located on chromosome 12p12 and is 45 kb in length. It consists of 4 exons that code for a protein of the GTPase family. Two isoforms are produced by alternative splicing. The protein is activated by GEFs, including SOS1, and transduces the signal downstream toward various effectors including MAPK and PI3PK/AKT.

Several groups have found more than ten different de novo heterozygous missense mutations in patients with NS, CFC and CS [Nava et al., 2007; Zenker et al., 2007; Carta et al., 2006; Niihori et al., 2006; Schubbert et al., 2006]. However these mutations affect a small percentage of patients with NS and CFC (less than 2% to 5%). Initial studies suggested that NS patients with a KRAS mutation are more severely affected [Carta et al., 2006]. In two case reports, Brasil et al. [2012] and Stark et al. [2012] indicated rare familial cases of CFC with KRAS mutations.

The KRAS gene has an activating mutation in an estimated 17% to 25% of human tumours [Kranenburg, 2005]. Cancers associated with this gene include pulmonary adenocarcinomas and colorectal and pancreatic carcinomas [Rajagopalan et al., 2002; Lee et al., 1995; Sidransky et al., 1992; Almoguera et al., 1988; Smit et al., 1988; Rodenhuis et al., 1987].

NRAS

The NRAS gene, located on chromosome 1p13 and consisting of 4 exons, was associated with NS in a candidate gene study by Cirstea et al. [2010]. A cohort of 917 subjects with an NS-related phenotype and no genetic etiology underwent investigation for NRAS mutations. Two heterozygous missense mutations were reported in four unrelated subjects (c.149C˃T and c.179G˃A, resulting in p.Thr50Ile and p.Gly60Glu, respectively). Three cases were sporadic, while the fourth carried an inherited mutation that segregated with the phenotype in the family. Work by Kraoua et al. [2012] revealed a mutation (c.179G>A) in two patients in a cohort of 125 with an NS-related phenotype in whom no causal mutation in the PTPN11, KRAS, SOS1, MEK1, MEK2, RAF1, BRAF and SHOC2 genes was found. These studies confirmed the low prevalence of NRAS mutations in NS.

Somatic mutations in the NRAS gene are also associated with follicular thyroid cancer, some cases of acute myeloid leukemia, colorectal cancer and autoimmune lymphoproliferative syndrome, among others [Cancer Genome Atlas Research Network, 2013; Bezieau et al., 2001].

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BRAF

The BRAF gene consists of 18 coding exons spread over 190 kb of chromosome 7q34. The protein produced is a member of the serine/threonine kinase family that phosphorylates and activates MEK1 and MEK2 when activated by RAS [Martinez-Quintana and Rodriguez-González, 2012].

The gene’s association with NS, LS and CFC was observed in candidate gene studies of the syndromes’ genetic etiology. Niihori et al. [2006] sequenced BRAF in 40 unrelated patients with CFC, and they observed 8 different missense mutations in 16 patients. In a group of CFC patients, Rodriguez-Viciana et al. [2006] found 11 sporadic mutations in 18 of 23 CFC patients (78%), while Schulz et al. [2008] reported 12 different mutations in a cohort of 24 CFC patients (47%).

More rarely (in less than 2% of cases), BRAF mutations have been reported in patients presenting diagnostic criteria for NS or LS [Sarkozy et al., 2009; Nystrom et al., 2008; Razzaque et al., 2007]. More than 40 BRAF mutations have been described, all missense; they are located in 2 regions: the cysteine-rich domain and the protein kinase domain (exons 11, 12, 14 and 15). Somatic BRAF mutations have also been associated with colorectal cancer, melanoma, pulmonary adenocarcinoma, and non-small cell lung cancer [Namba et al., 2003; Brose et al., 2002; Davies et al., 2002; Rajagopalan et al., 2002].

SHOC2

SHOC2 is a gene located on chromosome 10q25 that consists of 5 coding exons spread over 7 kb. The protein encoded by SHOC2 consists essentially of a protein interaction domain (leucine repeats). It seems to function as a scaffold allowing RAS to bind to downstream effector proteins, including RAF1.

Cordeddu et al. [2009] associated the SHOC2 gene with Noonan-like syndrome with loose anagen hair. Sequencing of candidate genes identified following examination of the human interactome2 revealed a missense mutation at position c.4A˃G of the gene (p.Ser2Gly) in 96 individuals with a NS phenotype. Sequencing of 410 additional subjects revealed the mutation in 21 additional cases.

The p.Ser2Gly mutation is the only mutation described in this gene. It causes aberrant N-terminal myristoylation,3 resulting in constitutive cell membrane targeting of the protein, leading to prolonged RAF1-stimulated MAPK activation.

CBL

CBL is a gene with 16 coding exons spread over 93 KB on chromosome 11q23. The protein produced by this gene is an E3 ubiquitin ligase that negatively regulates signalling downstream of receptor tyrosine kinases by targeting substrates for degradation by the proteasome [Martinelli et al., 2010].

Two independent studies conducted with an unrelated cohort and with familial cases, respectively, initially reported an association between a NS-related phenotype and heterozygous missense mutations in the CBL gene [Martinelli et al., 2010; Niemeyer et al.,

2 Interactome: network of all proteins that interact directly or otherwise in this signalling pathway. 3 Myristoylation: part of the post-translational modifications that some proteins undergo. It is involved in certain protein-protein interactions, protein stability and certain protein-membrane interactions.

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2010]. Martinelli et al. reported 2 sporadic cases and 2 familial cases in a cohort of 365 subjects presenting NS-associated traits. They observed phenotypic heterogeneity, that is, some individuals presented NS phenotype concordance while others presented only certain traits.

Missense mutations found in CBL are concentrated in two domains: the RING (Really Interesting New Gene) finger domain and the linker connecting this domain to the N-terminal tyrosine kinase-binding domain. The mutations reported cause loss of function and prolong RTK lifespan [Verloes and Cavé, 2010].

Homozygous somatic CBL mutations are also reported with myeloid leukemias, especially JMML. These mutations are primarily small in-frame deletions, modifications to the splice site and missenses [Niemeyer et al., 2010; Pérez, 2010].

HRAS

The HRAS gene consists of 4 coding exons located on chromosome 11p15. The gene is part of the RAS oncogene family. It codes for a protein with GTPase activity that is involved in membrane receptor signal transduction to intracellular effectors.

A candidate gene approach allowed Aoki et al. [2005] to associate HRAS gene mutations with Costello syndrome. They detected 4 different heterozygous mutations in 12 of the 13 subjects in their cohort presenting Costello syndrome. Some of these mutations had been found previously in various tumours [Bos, 1989].

Several other subsequent studies reported that approximately 87% of CS cases have mutations in this gene. Mutations have also been described in CFC cases [Gremer et al., 2010; Lo et al., 2008; Gripp et al., 2007; Zampino et al., 2007; Gripp et al., 2006; Kerr et al., 2006; Rauen et al., 2006; Sol-Church et al., 2006;]. Several heterozygous missense mutations were described. However more than 90% affect glycine residues at position 12 or 13 and result in gain of function [Gripp and Lin, 2012; Estep et al., 2006]. These are generally de novo mutations, although a few cases of inherited mutations have been reported [Gripp and Lin, 2012].

HRAS mutations have been reported in a variety of cancers, including bladder cancer, follicular thyroid cancer and oral squamous cell carcinoma [Groesser et al., 2012; Di Micco et al., 2006; Vasko et al., 2003].

MAP2K1 and MAP2K2 (MEK1 and MEK2)

MAP2K1 and MAP2K2 are located on chromosomes 15q22 and 19p13, respectively. MAP2K1 consists of 11 exons spread over 103 kb, compared with 7 exons over 11 kb for MAP2K2. The proteins encoded by these genes, MEK1 and MEK2, are RAF-MAP kinase effectors.

The association between these genes and CFC was first reported by Rodriguez-Viciana et al. [2006] in a cohort of 23 patients diagnosed with CFC, and by Niihori et al. [2006] in a cohort of 43 subjects with a CFC phenotype. Their studies and other later ones assessed the prevalence of activating missense mutations at approximately 20% to 25% in the CFC patient population. MAP2K1 has been associated with NS on rare occasions. Mutations are clustered primarily in two regions of the gene: the negative regulatory region (NRR) and the kinase domain [Armour and Allanson, 2008; Nystrom et al., 2008; Schulz et al., 2008; Gripp et al., 2007; Narumi et al., 2007]. Most of the mutations studied seem to result in increased MEK activity [review by Bromberg-White et al., 2012].

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CFC is considered sporadic. However, Rauen et al. [2010] and Linden and Price [2011] independently reported two families with autosomal dominant inheritance of heterozygous mutations of the MAP2K2 gene.

Mutations in this gene have been reported in various human cancers such as melanoma and non-small cell lung cancer [Pao and Girard, 2011 and a review by Bromberg-White et al., 2012].

NF1

NF1 is a 127 kb gene located on chromosome 17q11. It has 15 coding exons and produces neurofibromin, a protein that regulates several signalling pathways and catalyzes inactivation of RAS proteins. It was the first gene to reveal an association between RAS/MAPK pathway mutations and a developmental syndrome (neurofibromatosis type 1). Inherited and de novo mutations have been reported in many studies [reviewed by Pasmant et al., 2012].

NF1 mutations have also been associated with Watson syndrome, which has autosomal dominant transmission. Tassabehji et al. [1993] reported a deletion of 42 base pairs (bp) in exon 28 in a family with 3 affected members. The mutation results in loss of function of the protein.

Mutations in the gene are also associated with several types of cancer, including rhabdomyosarcoma, carcinoid tumours, certain types of gliomas, breast cancer and JMML [reviewed by Patil and Chamberlain, 2012].

RIT1

RIT1 is a 6-exon gene located on chromosome 1q22 that codes for a protein of the GTPase family (RAS) [Hynds et al., 2003].

A single study reported mutations in the gene in individuals presenting a NS-related phenotype. In the study, Aoki and Matsubara [2013] used exome sequencing to find mutations that could be causal in 14 subjects with NS. Other mutations were then detected in 166 additional subjects through RIT1 targeted sequencing. Of these, 17 (9%) had 9 missense mutations in RIT1.

Mutations in the gene have also been associated, in low numbers, with pulmonary adenocarcinoma and myeloid neoplasms [Berger et al., 2014; Gómez-Segui et al., 2013].

SPRED1

The SPRED1 gene has 7 coding exons spread over more than 98 kb on chromosome 15q14, and codes for a protein that negatively regulates the MAP kinase cascade by dephosphorylating RAF [Nonami et al., 2004].

Brems et al. [2007] described the association between this gene and Legius syndrome in members of five families and 86 other unrelated subjects with no detected mutations in NF1. The authors report 11 heterozygous mutations. Their studies indicate that the mutations lead to a loss of function that prevents SPRED1 from inhibiting RAF/MEK signalling.

The association between this gene and Legius syndrome was subsequently confirmed in other studies of sporadic and familial cases [Denayer et al., 2011, Laycock-van Spyk et al., 2011; Spencer et al., 2011; Pasmant et al., 2009, among others].

13

To date, more than 100 different mutations have been identified in the Leiden Open Variation Database and the University of Utah’s pathology department database.4 The mutations, all associated with Legius syndrome, vary: missense, frameshift, nonsense, copy number variations, splicing mutations , deletions and start codon mutations.

Summary of Prevalence Studies of NS-Associated Mutations

Several publications have reported the role of each of the genes presented above in NS. In 2010, Romano et al. compiled the results of various studies that involved more than 20 patients and examined NS-associated mutations (Tables 3, 4 and 5).

Table 3: Summary of mutation studies of NS patients prior to 2010

GENE NUMBER OF

PATIENTS

NUMBER OF

STUDIES

NUMBER OF PATIENTS WITH

MUTATIONS (% OF COHORT)

% OF ALL CASES

COMMENTS

PTPN11 877 13 359 (40.9) 40.9

KRAS 616 6 15 (2.4) 1.4

SOS1 311 5 60 (19.3) 11.1 PTPN11 and KRAS negative

RAF1 304 4 31 (10.2) 4.7 PTPN11, KRAS and SOS1 negative

BRAF 334 3 6 (1.8) 0.8 PTPN11, KRAS, SOS1 and RAF1 negative

SHOC2 96 1 4 (4.2) 1.7 PTPN11, KRAS, SOS1, RAF1 and BRAF negative

NRAS 733 1 4 (0.5) 0.2 PTPN11, KRAS, SOS1, RAF1 and BRAF negative

Adapted from Romano et al., 2010.

Following the publication by Romano et al., a few other studies reproduced their results in additional cohorts, testing additional genes.

4 Mendelian genes [Web site], available at: https://grenada.lumc.nl/LOVD2/mendelian_genes/home and Mutation databases [Web site], available at: http://www.arup.utah.edu/database/ (viewed April 24, 2014).

14

Table 4: Summary of mutation studies of NS patients published after 2010

STUDY NO. OF PATIENTS

% OF PATIENTS WITH MUTATIONS COMMENTS

PTPN11 KRAS SOS1 RAF1 BRAF NRAS SHOC2

Ezquieta 2012 643 26.7 0 2.2 1.4 0.8 NA NA 182 family members

Smpokou 2012 35 37 NA 23 NA 3 NA NA

Lee 2011 59 39 5.1 20 6.8 1.7 No cases

No cases

Denayer 2011 115 NA NA NA NA NA 2.6 0 PTPN11, SOS1, RAF1 and KRAS negative cohort

Abbreviations: NA: not assessed.

Clinical Validity of NGS Gene Panel Sequencing

Only one study examining the clinical validity of NGS gene panel sequencing was found. Lepri et al. [2014] recently performed mass sequencing of 80 patients for the PTPN11, SOS1, RAF1, BRAF, HRAS, KRAS, NRAS, SHOC2, MAP2K1, MAP2K2 and CBL genes. Mutations were identified in 6 genes, and 38 mutations were reported: PTPN11 (22/38 = 58%), SOS1 (9/38 = 23%), BRAF (2/38 = 5%), MAP2K2 (2/38 = 5%), RAF1 (2/38 = 5%), CBL (1/38 = 3%). The relative frequency of the mutations observed met expectations based on the literature, and Sanger sequencing of all variants observed confirmed all mutations.

15

Table 5: Genes associated with NS and other related syndromes

DISORDER OMIM ASSOCIATED GENES

Noonan syndrome 163950 609942 610733 611553 613224 613706

PTPN11 KRAS SOS1 RAF1 NRAS BRAF

MAP2K2 RIT1

Noonan-like syndrome with loose anagen hair

607721 SHOC2

CBL syndrome 613563 CBL

Costello syndrome 218040 HRAS

LEOPARD syndrome 151100 PTPN11 RAF

BRAF

Capillary malformation – arteriovenous malformation

608354 RASA1

Cardiofaciocutaneous syndrome 115150 KRAS BRAF

MAP2K1/2

Legius syndrome 611431 SPRED1

Neurofibromatosis type 1 166200 NF1

Watson syndrome 193520 NF1

Abbreviations: CBL = Noonan syndrome-like disorder with juvenile myelomonocytic leukemia; OMIM = Online Mendelian Inheritance in Man.

TERM PRESENCE ABSENCE NOT APPLICABLE

Sensitivity X

Specificity X

Positive predictive value (PPV) X

Negative predictive value (NPV) X

Likelihood ratio (LR) X

ROC Curve X

Accuracy X

1

5.3 Analytical (or Technical) Validity

In recent years, numerous publications have reported on the usefulness and validity of each NGS platform for different disorders. In summary, compared with the Sanger method, the sensitivity, specificity and reproducibility of the MiSeq platform is almost 100% in regions with sufficient coverage. Table 7 presents a few recent validation studies.

Table 7: Summary of a few recent comparative studies of NGS platforms

TYPE (NUMBER) OF

SAMPLE

TYPE OF TESTING CRITERIA EVALUATED COMMENTS

Tan, 2014 Human genomic DNA (25)

Inherited polycystic kidney disease panel

Sensitivity 95% CI, 99.2% 96.8% -99.9% CI Specificity 95% CI, 99.9% 100% -99.7% CI

The panel consisted of two genes. Cost and turnaround time were reduced by 70% compared with the Sanger method.

Li, 2013 Human genomic DNA (15)

Inherited heart disease panel

Sensitivity and Specificity 100% (MiSeq) 99.1% (PGM) Positive Predictive Value 95.9% (MiSeq) 95.5% (PGM) Coverage 97.9% (MiSeq) 96.8% (PGM)

Insufficient coverage of regions with high GC content.

Koshimizu, 2013

Human genomic DNA (38)

Autism panel Sensitivity (7 samples) 100% (MiSeq) 85.7% (PGM) 10X Coverage 93.7% (PGM) 96.8% (MiSeq) 20X Coverage 85.9% (PGM) 93.2% (MiSeq) Quality Control Q30 (MiSeq) Q20 to Q25 (PGM)

Higher false-positive rate with PGM

Sikkema-Raddatz, 2013

Human genomic DNA (84)

Hereditary cardiomyopathy panel

Sensitivity 100% (MiSeq) Reproducibility 100% (MiSeq) 30X Coverage 99% (MiSeq)

Quail, 2012 Four microbial genomes (GC content

Whole genome Error rate: ˂ 0.4% (MiSeq) 1.8% (PGM)

PGM detects more variants. MiSeq has a lower false-positive rate than the

2

TYPE (NUMBER) OF

SAMPLE

TYPE OF TESTING CRITERIA EVALUATED COMMENTS

from 19.3 to 67.7%)

13% (Pacific Biosciences)

others. AT-rich sequences have better coverage with PGM.

Abbreviations: AT = adenine-thymine; CI = confidence interval; DNA = deoxyribonucleic acid; GC = guanine-cytosine; PGM = Ion Torrent Personal Genome Machine.

A review of the literature on the use of NGS gene panel sequencing for diagnosing NS revealed only one study by Lepri et al., published in 2014.

Lepri et al. [2014] assessed the analytical validity of the NGS approach. Their study is divided into two steps:

1) Validation, during which a limited number of samples for which genotype was previously determined by Sanger sequencing (6 positive and 4 negative) undergo NGS sequencing;

2) Genetic characterization of 80 patients diagnosed with NS by NGS.

The TruSeq Custom Amplicon kit (Illumina, San Diego, CA) was used to prepare the gene library and amplify targeted sequences (244 amplicons). Testing was performed on the MiSeq platform (Illumina, San Diego, CA). MiSeq Reporter software and the Genome Analysis Toolkit (GATK) were used for bioinformatic analysis. When a variant was observed, it was sequenced using the Sanger approach.

Concordance

The authors report 100% concordance.

Sensitivity and Specificity

The authors report 100% specificity and sensitivity with a minimum read depth of 30 and a minimum Q score of 30. Coverage of targeted regions was 98%.

Repeatability

Analytical repeatability was 100%, assessed over 120 exons from 80 patients (3 independent repetitions).

CHUS Validation

The technologies and chemistries used with panel sequencing include capturing sequences of interest using Nimblegen’s SeqCap EZ system, sequencing with Illumina’s MiSeq platform, and bioinformatic analysis with automated and manual steps using tools based on the Burrows-Wheeler Aligner (BWA) and Genome Analysis Toolkit (GATK). This technical approach was validated using a CEPH5 reference material (NA12878) from the National Institute of Standards and Technology (NIST). Exons and junction points were sequenced for 96 genes in two independent tests. Variations obtained were compared with those previously reported by NIST.6 For bioinformatic analysis of the sequences, minimum parameters for variations had a quality score greater than Q30 (1 error in 1,000) and coverage was greater than or equal to 20X.

5 CEPH: Centre d’étude du polymorphisme humain. 6 National Institute of Standards and Technology (NIST). Want to better understand the accuracy of your human genome sequencing? [web site]. Available at: http://www.nist.gov/mml/bbd/ppgenomeinabottle2.cfm.

3

Coverage

Mean sequencing coverage for the 96 genes was 90X, with less than 0.3% of sequences showing less than 20X coverage. Noonan panel genes were sequenced for 20 subjects, which allowed the requester to determine that 3 exons (exons 1 of BRAF, NF1 and PTPN11) had to be sequenced using the Sanger approach, as NGS provided insufficient coverage of those exons.

Sensitivity

The approach adopted by the requester detected the 373 variants expected in specimen NA12878, including 14 indels. Therefore, the requester reports 100% sensitivity (95% CI: 99%–100%).

Testing of 197 variations (including 18 indels) of the GAA gene found in 20 subjects, 19 of whom had undergone previous NGS sequencing (by the Laboratory for Molecular Medicine-Partners Healthcare and the Connective Tissue Gene Test Lab), also revealed 100% sensitivity (95% CI: 98%–100%).

Specificity

Specificity was assessed using sample NA12878. Of the 208 variations observed, 4 were false-positives, representing 98% specificity (95% CI: 95%–99%). Of the false-positives, 1 variant in 4 was an indel.

External Quality Control

There is no external quality control test at this time for NGS. However, the requester is implementing a sample exchange program with Emory Genetics Laboratory until a test is developed by the College of American Pathologists (CAP).

The requester is taking part in CAP sequencing and interpretation tests for Sanger sequencing.

Table 8: Summary

PARAMETER PRESENCE ABSENCE NOT APPLICABLE

Repeatability X

Reproducibility X

Analytical sensitivity X

Analytical specificity X

Matrix effect X

Concordance X

Correlation between test and comparator X

5.4 Recommendations from Other Organizations: None identified.

4

6 ANTICIPATED OUTCOMES OF INTRODUCING THE TEST

6.1 Impact on Material and Human Resources

The complexity of both the technology and the interpretation of results requires specialized personnel with the necessary initial and continuing professional training. Additionally, interpreting the variants found involves significant human and information technology resources. This includes frequent updating of working databases and software, given the rapid growth of scientific discoveries and knowledge.

6.2 Economic Consequences of Introducing the Test Into Quebec's Health Care and Social Services System

NGS gene panel sequencing is a less expensive option than the current practice of sending tests outside Quebec. However, depending on test volumes and the breakdown of the various tests of this kind, there may always be some, such as those pertaining to prenatal screening, that have to be sent outside Quebec to meet the necessary turnaround time.

6.3 Main Organizational, Ethical, and Other (Social, Legal, Political) Issues

Next generation sequencing is still a new technology, and, like the field of knowledge surrounding it, it is developing rapidly. Results can affect personal and clinical decisions. However, interpretation of results is sometimes variable or uncertain [Marshall, 2013; Evans and Rothschild, 2012; Makrythanasis and Antonarakis, 2012].

7 IN BRIEF

7.1 Clinical Relevance

Noonan syndrome is phenotypically and etiologically heterogeneous. Detecting specific mutations in a patient ensures the correct diagnosis and appropriate care. Reproduction options can also be considered when familial mutations are found.

7.2 Clinical Validity

The genetic cause of Noonan syndrome can be found in approximately 70% to 90% of patients. Several associated genes have been validated fairly extensively in several cohorts of sporadic and familial cases.

7.3 Analytical Validity

NGS sequencing is an interesting alternative to Sanger sequencing because of its high throughput and low cost. Sensitivity and specificity are almost 100%. However, the ACMG recommends confirmation of variants using another technical approach.

7.4 Recommendations from Other Organizations

For now, no recommendations have been proposed on the use of this approach to investigate cases of Noonan syndrome. However, a certain number of experts and national and international bodies (ACMG, CAP, CCMG, CDC, CMGS, etc.) have made some recommendations on methodology and considerations for implementing next generation sequencing in a clinical setting, quality assurance related to use of the technology and establishment of interpretation criteria.

5

8 INESSS NOTICE IN BRIEF

Gene Panel - Noonan Syndrome

Status of the Diagnostic Technology

Established

Innovative

Experimental (for research purposes only)

Replacement for technology: , which becomes obsolete

INESSS Recommendation

Keep test in the Index

Remove test from the Index

Reassess test when local validation data are available

Additional Recommendation

Draw connection with listing of drugs, if companion test

Produce an optimal use manual

Identify indicators, when monitoring is required

Notes

This is a relevant test.

There is interest in and a need to conduct the test in Quebec instead of sending it outside the province.

6

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APPENDIX A Enrichment of Genomic DNA Regions Using Nimblegen’s SeqCAP EZ (adapted from www.nimblegen.com)

1. Oligonucleotides are directed against genome target regions.

2. A gene library is generated from genomic DNA using the shotgun approach.

3. The gene bank is hybridized with oligonucleotides.

4. Magnetic beads capture DNA fragments bound to oligonucleotides.

5. Unbound fragments are eluted.

6. PCR targeted fragments are amplified.

7. Enrichment quality control is performed using PCR.

8. Sequencing is performed.

8. Sequencing

6. mplification

7. Quality Control

5. ashing

4. Bead Capture

3. ybridiation

2. Library Preparation

Capture jln;ljvfg;kfjgkfjsdf;kgjragipjergprjdg;kbeads

Probees

NGS Adaptors

1. Genomic DNA

3. Hybridization

Capture Beads

5. Washing

6. Amplification

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APPENDIX B RAS-MAPK Signalling Pathway (adapted from Rauen, 2013)

This signalling pathway is involved in cellular differentiation, motility, apoptosis and senescence. RASopathies are associated with mutations that affect the genes of various effectors in the pathway. The genes involved in various RASopathies are indicated by dashed lines.

Legend:

NS: Noonan syndrome

CFC: Cardiofaciocutaneous syndrome

NF-1: Neurofibromatosis type 1

CM-AVM: Capillary malformation-arteriovenous malformation syndrome

CS: Costello syndrome

LS: LEOPARD syndrome

LS

LS

Cystolic and nuclear effectors