Whole Exome Sequencing In Patients Research Articles

Whole-exome tumor sequencing study in biliary cancer patients with a response to MEK inhibitors.

We previously conducted a phase-II study with selumetinib (AZD6244), a small molecule inhibitor of MEK1/2, in advanced biliary tract cancers (BTC), where the primary endpoint was response rate. Several patients experienced objective response. These findings were confirmed with MEK162 in a similar patient population. To assess for tumor-specific genetic variants that mediate sensitivity to MEK inhibition in BTC, we performed whole-exome sequencing in patients with an objective response to selumetinib. Normal and tumor DNA from FFPE tissue from two patients who experienced an objective response underwent whole-exome sequencing. Raw sequence reads were processed with GATK workflow and tumor specific variants were identified using MuTect and VarScan2. Ensemble Variant Effect Predictor was used to determine functional consequences of these variants. Copy number changes and potential gene fusion events were also screened. Findings were compared to assess for any commonality between the two tumor samples, and whether the identified variants were intrinsic to the MAPK pathway. 1169 and 628 tumor-specific variants were identified in the two samples. Further analysis demonstrated 60 and 53 functional and novel variants, respectively. Of the identified tumor-specific variants, fusion events or copy number changes, no commonality was seen. Several variants in genes associated with ERK signaling were present in each tumor sample. Although there were no common tumor-specific variants in the two patients who exhibited an objective response to selumetinib, several genes associated with ERK signaling were identified. Confirmatory studies investigating the role of the identified genes and other potential tumor independent factors need further investigation.

A Human Bone Marrow Failure Syndrome Caused By a Homozygous Mutation in MYSM1

Inherited bone marrow failure syndromes comprise a heterogeneous group of genetic disorders characterized by dysfunction of hematopoietic stem or progenitor cells. We present a consanguineous pedigree with two siblings with early-onset, progressive bone marrow failure (neutropenia, anemia, thrombocytopenia) associated with trigonocephaply, hyperplastic gingiva, cataract, accessory mammilla, dental changes and mild neurocognitive developmental delay. Pancytopenia at birth as well as non-compaction cardiomyopathy was observed in one of the patients.Bone marrow histology showed marked hypocellularity with decrease of all hematopoietic cell lines but without cellular atypia, monocytosis, blast excess or fibrosis. Fanconi anemia was excluded by mitomycin C induced chromosomal breakage studies.Immunophenotyping of peripheral blood cells showed a reduction of mature B cells, reduced switched memory B cells, whereas the level of activated B-cells and plasma blasts were increased. T cell proliferation upon PHA and specific antigens was normal.Using whole exome sequencing in patients and both parents we could identify a single potentially disease causing homozygous stop codon mutation (NM_001085487: c.1168G>T: p.E390*) in MYSM1 (Myb-Like, SWIRM And MPN Domain-Containing Protein 1). A family with two affected patients carrying the same mutation has been reported previously (Alsultan A et al, Blood 122:3844, 2013). MYSM1 is a Histone H2A deubiquitinase that has previously been implicated in controlling hematopoietic stem cells, progenitor B cells, and NK cells in mice (Nijnik A et al, Blood 119:1370, 2012).MYSM1 protein was absent in patients' EBV-transformed B cell lines (EBV-LCLs). MYSM1-deficient EBV-LCLs had elevated γ-H2AX levels, indicative of increased genomic instability. Upon exposure to UV light, we observed a sustained expression of p53 and phospho-p53 as well as p38 MAPK and phospho-p38 MAPK in patients' fibroblasts in comparison to fibroblasts from healthy individuals.In view of the progressive pancytopenia, both siblings underwent allogeneic hematopoietic stem cell transplantation from 10/10 HLA-matched family donors after reduced intensity conditioning with alemtuzumab, fludarabine and treosulfan at 2 and 4 years of age, respectively. The procedure was well tolerated, and both siblings exhibit full donor hematopoietic reconstitution at 21 and 26 months after transplant.In summary, we here identify a novel human bone marrow failure syndrome caused by MYSM1-deficiency that can be cured by allogeneic hematopoietic stem cell transplantation. DisclosuresNo relevant conflicts of interest to declare.

Whole Exome Sequencing in Evaluation of Thrombophilia: A Novel 33-Gene Panel

Introduction:Venous thromboembolism (VTE) occurs with an incidence of 1-2 per 1000 individuals per year. Approximately 10-20% of patients with VTE have a heritable thrombophilia involving one of five known genes: Factor V, prothrombin (PT), antithrombin (AT), protein C (PC), and protein S (PS). Significant variability of laboratory functional assays as well as fluctuating plasma levels of AT, PC and PS lead not only to delay in initial thrombophilia screening but also to multiple rounds of costly testing. Whole exome sequencing (WES) is a potentially useful diagnostic tool for inherited thrombophilias as it avoids dependence on laboratory and situational variation in protein levels. We compiled a panel of 33 genes involved in thrombosis with a goal of investigating the diagnostic yield and cost of WES in comparison to traditional thrombophilia testing.Methods:Since January of 2014, we have been performing WES in patients with a personal and family history of VTE seen at Yale New Haven Hospital. Thus far, 18 such patients have had a complete WES analysis. Data regarding patient demographics, number and type of VTE events, family/surgical history, medical co-morbidities, and traditional laboratory testing for inherited thrombophilias was recorded. Costs of each test were determined based on the amount billed to insurance. WES focusing on 33 genes involved in thrombosis was performed and analyzed by the DNA Diagnostic Lab at the Yale School of Medicine. Positive WES testing was defined as identification of a pathogenic variant in a gene known to be associated with thrombophilia, found at a frequency consistent with frequency of the disease, and with evidence that the variant predisposes to thrombosis. Positive laboratory testing was defined as any test that led to an unequivocal diagnosis of Factor V Leiden, PT mutation, homozygous MTHFR mutation with hyperhomocysteinemia, or a deficiency in AT, PC, or PS.Results:All 18 patients (7 male, 11 female) were included in the final analysis. Median age at first VTE was 35.5 years (range, 14-78 years); median number of independent VTE events was 2 (range, 1-9). WES with our 33-gene thrombophilia panel was positive in 11 of 18 (61.1%) patients, while traditional laboratory testing was positive in only 2 of 16 (12.5%) cases (Table 1). There were no statistically significant differences in clinical characteristics between those patients with positive findings on WES versus those without. Identified variants included those in genes with well known roles in thrombosis (SERPINC1, PROS1, F5), and in genes with emerging data regarding thrombosis (HABP2, SERPINA10, SERPIND1). Two patients identified on WES as having PS deficiency, one with AT deficiency, and one with a non-Leiden Factor V mutation had laboratory testing that was either normal or uninterpretable. The total cost of WES at our institution was $1935.00; by comparison, among 16 patients who underwent laboratory testing, median cost of laboratory testing was $2892.70 (range, $406.90-$11419.80), and the cost of laboratory testing exceeded WES in 13 patients.Conclusions:WES using our 33-gene thrombophilia panel has higher diagnostic yield and is more cost effective than traditional thrombophilia testing. With increasing availability and declining cost, this thrombophilia gene panel has the potential to truly transform thrombophilia testing. Further investigation of the diagnostic power and phenotypic correlation of identified mutations is in progress.Table 1Mutations detected by the 33 gene thrombophilia panelGenderAge (yrs)Lab testing resultWES resultM40NegativeSERPINA10 (Q384R) heterozygousM67NegativeSERPINC1 (S426W) heterozygousM30NegativePROS1 (Y234C) heterozygousF34NegativeHABP2 (G508E) heterozygousF56APC resistanceF5 (R506Q) heterozygousF27F2 20210 G>A heterozygousF2 20210G>A heterozygous; vWF P2063S heterozygousF37NegativeSERPIND1 (R468C) heterozygousF48NegativeF5 (T915S) heterozygousM20NegativePROS1 (P76L) heterozygousM55NegativeSERPINA10 (21_23delCCT ) heterozygousF78NegativeHABP2 (G534E) heterozygousF49NegativeNegativeF41NegativeNegativeM32NegativeNegativeF64NegativeNegativeM42NegativeNegativeF51NegativeNegativeF28NegativeNegative DisclosuresNo relevant conflicts of interest to declare.

Abstract A13: Establishment and characterization of patient-derived xenograft models of gastrointestinal stromal tumor resistant to standard tyrosine kinase inhibitors

Abstract BACKGROUND: The prognosis of gastrointestinal stromal tumor (GIST) has been dramatically improved after introduction of imatinib which can inhibit the driver mutated oncoproteins,KIT or PDGFRa. However, most patients eventually develop resistance to the tyrosine kinase inhibitors (TKIs) targeting these oncoproteins including imatinib, sunitinib, or regorafenib. The paucity of TKIs-resistant commercially available GIST cell lines hampers development of effective therapy for drug-resistant GIST. Therefore, we established patient-derived xenograft (PDX) models that faithfully recapitulate the genetic and phenotypic features of drug-resistant GIST. METHODS: PDXs have been established in NOD-SCID mice with tumor fragments of patients with metastatic and/or unresectable GIST after failure of at least imatinib and/or sunitinib. The histological and genomic similarities between all xenografts and the parental tumors have been confirmed using H&E and KIT staining, and short tandem repeat (STR) analysis, respectively. Mutation was detected by whole exome sequencing of patients' tumors on a HiSeq2000 and validated by Sanger sequencing of patients tumors and PDXs. After sequential passaging to BALB/c nude mouse, drug sensitivity assay (imatininb, sunitinib, and regorafenib) was conducted in established PDX models along with Western blotting regarding to KIT and FGFR signaling pathway. As an imatinib-sensitive control model, a xenograft established with GIST-T1 cell line was used. RESULTS: Three GIST PDX models were established from an imatinib/sunitinib/sorafenib-resistant GIST harboring KIT exon 11 (p.Y570-L576del) and 17 (p.D816E) mutations (AMC-GX1), an imatinib-resistant GIST harboring KIT exon 11 (p.K550_K558del) and 14 (p.T670I) mutations (AMC-GX2), and an imatinib/sunitinib-resistant GIST harboring KIT exon 11 (p.565-577GNNYVVIDPTQLP>Q) and 17 (p.D820Y) mutations (AMC-GX3). Histologic and genetic similarities were confirmed between primary patients' tumors and PDXs. The mutation status of GIST PDXs was consistent with primary tumors. The GIST PDX models showed TKI sensitivity profiles comparable to clinical responses in patients. At day 21 after treatmemt with imatinib, tumor growth was inhibited by 17.8, 53.9, and 12.6% in AMC-GX1, GX2, and GX3, respectively, while by 80.0% in GIST-T1 xenografts. Western blotting analysis showed that KIT phosphorylation was inhibited by imatinib treatment in AMC-GX1 similar to GIST-T1, but not in AMC-GX2 and AMC-GX3. In contrast to GIST-T1, PI3K downstream of KIT was not inhibited with imatinib in AMC-GX1, GX2, and GX3. In addition, TKIs-resistant PDX models showed FGFR1 activation at baseline which was not evident in GIST-T1 xenografts. CONCLUSION: We have established 3 TKI-resistant GIST PDX models harboring variable KIT mutations which show different KIT and FGFR signaling features from imatinib-sensitive models. The established GIST PDX models will play a role for further studies on mechanisms of resistance to TKI and evaluation of novel targeted therapies in GIST. Citation Format: Young-Soon Na, Min-Hee Ryu, Sook Ryun Park, Ju-Kyung Lee, Hanui Kim, Chae-Won Lee, Sun Young Lee, Young-Kyoung Shin, Ja-Lok Ku, Sung-Min Ahn, Yoon-Koo Kang. Establishment and characterization of patient-derived xenograft models of gastrointestinal stromal tumor resistant to standard tyrosine kinase inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A13.

MG-117 Clinical features of prc2 complex-related overgrowth due to mutations in eed

Our lab was the first to publish that constitutional mutations in the epigenetic regulator EZH2 cause Weaver Syndrome (WS). WS is characterised by overgrowth, increased height, large head, intellectual disability and susceptibility to various cancers. We found pathogenic mutations in EZH2 among 7 out of 45 individuals with Weaver-like features, of which two had developed malignancies prior to referral. Our efforts focus on determining characteristics that will help us predict the likelihood of WS patients developing cancer, through phenotype/genotype correlations and functional studies. As of yet, it is not clear whether screening for haematological malignancies in patients with Weaver syndrome would increase the patients’ chances of survival. Given that EZH2 is a histone-modifying enzyme known to be mutated in various somatic cancers, we hypothesised that constitutional mutations in other epigenetic regulators could explain the overgrowth features seen in our undiagnosed patients. To investigate this, we carried out whole exome sequencing in patients with paediatric syndromes similar to WS who did not have coding mutations in EZH2 . We have identified novel de novo mutations in EZH2’s partner protein EED in two patients to date. Both proteins are members of the Polycomb Repressive Complex 2 that maintains gene silencing. Based on the similarities of the patients’ phenotypes to WS, and on other lines of evidence including mouse Eed hypomorphs and an epigenetic signature that clustered with Weaver syndrome patients, we characterised these mutations as pathogenic. Here we describe additional features of overgrowth associated with constitutional mutations in human EED , which is also mutated somatically in various cancers. Samples from additional families are required to make a definitive link between mutations in these genes, Weaver-like syndromes, and cancer development. We continue to perform exome sequencing in patients with Weaver-like syndromes who have normal microarray studies and no mutations in EZH2 or NSD1 .

Separation of seizures from comorbidities in the Fitful mouse model of DNM1epileptic encephalopathy

The epileptic encephalopathies (EE) are a family of severe brain disorders manifesting early in life and characterized by spasms and/or intractable seizures. The relentless seizure activity impacts both cognitive and behavioral development in the patient . Many patients do not respond to typical anti-epileptic therapies and can experience early death. Of late, several novel mutations in newly revealed genes have been associated with the EEs. Through the efforts of such groups as the Epi4K and EuroEPINOMIC-RES consortiums, the Epilepsy Phenome/Genome Project and the Deciphering Developmental Disorders Study, de novo variants have surfaced in such genes as GABRB3, ALG13, GRIN1, NEDD4L, DNM1 and others . Whole-exome sequencing of patients has revealed 9 DNM1 variants . This research highlight describes our study of the mouse Dnm1 EE model to illustrate how certain classes of de novo variants may give rise to EEs and emphasize the contribution of specific neuronal populations to the comorbid phenotypes. Dynamin-1, the product of the DNM1 gene, is a large GTPase required for synaptic vesicle recycling in neurons . The highly brain specific Dynamin-1 is expressed during synaptogenesis through adulthood and undergoes alternative splicing resulting in the regulated expression of several splice variants.

Whole exome sequencing in patients with congenital cataracts

SummaryNext‐generation sequencing (NGS) represents one of the most significant technological advances in the biological sciences of the past decade. NGS is now being introduced by many laboratories for routine diagnostic use. For genetically heterogeneous disorders approaches include custom‐designed target enrichment permitting analysis of disease‐associated gene panels and whole exome sequencing (WES). The sensitivity, speed and cost per sample makes WES a valuable tool and it is increasingly being used to identify the molecular genetic causes of inherited cataracts. The detection rate of disease‐causing mutations is high and the results enhance clinical diagnosis and genetic counselling of the affected families.

Mutations in SLC25A46, encoding a UGO1-like protein, cause an optic atrophy spectrum disorder.

Dominant optic atrophy (DOA)1,2 and axonal peripheral neuropathy (Charcot-Marie-Tooth Type 2 or CMT2)3 are hereditary neurodegenerative disorders most commonly caused by mutations in the canonical mitochondrial fusion genes OPA1 and MFN2, respectively4. In yeast, homologs of OPA1(Mgm1) and MFN2(Fzo1) work in concert with Ugo15,6, which has no human equivalent to date7. By whole exome sequencing patients with optic atrophy and CMT2, we identified four families with recessive mutations in SLC25A46. We demonstrate that SLC25A46, like Ugo1, is a modified carrier protein that has been recruited to the outer mitochondrial membrane and interacts with the inner membrane remodeling protein, mitofilin(Fcj1). Loss-of-function in cultured cells and in zebrafish unexpectedly leads to increased mitochondrial connectivity, while severely affecting the development and maintenance of neurons in the fish. The discovery of SLC25A46 strengthens the genetic overlap between optic atrophy and CMT2, while exemplifying a novel class of modified solute transporters linked to mitochondrial dynamics.

Novel genes in LDL metabolism--a comprehensive overview.

To summarize recent findings from genome-wide association studies (GWAS), whole-exome sequencing of patients with familial hypercholesterolemia and 'exome chip' studies pointing to novel genes in LDL metabolism. The genetic loci for ATP-binding cassette transporters G5 and G8, Niemann-Pick C1-Like protein 1, sortilin-1, ABO blood-group glycosyltransferases, myosin regulatory light chain-interacting protein and cholesterol 7α-hydroxylase have all consistently been associated with LDL cholesterol levels and/or coronary artery disease in GWAS. Whole-exome sequencing and 'exome chip' studies have additionally suggested several novel genes in LDL metabolism including insulin-induced gene 2, signal transducing adaptor family member 1, lysosomal acid lipase A, patatin-like phospholipase domain-containing protein 5 and transmembrane 6 superfamily member 2. Most of these findings still require independent replications and/or functional studies to confirm the exact role in LDL metabolism and the clinical implications for human health. GWAS, exome sequencing studies, and recently 'exome chip' studies have suggested several novel genes with effects on LDL cholesterol. Novel genes in LDL metabolism will improve our understanding of mechanisms in LDL metabolism, and may lead to the identification of new drug targets to reduce LDL cholesterol levels.

High Diagnostic Yield of Whole Exome Sequencing in Participants With Retinal Dystrophies in a Clinical Ophthalmology Setting

To assess the diagnostic yield and the practicality of implementing whole exome sequencing within a clinical ophthalmology setting. Evaluation of a diagnostic protocol. setting: Patient participants were enrolled during clinical appointments in a university-based ophthalmic genetics clinic. Twenty-six patients with a variety of presumed hereditary retinal dystrophies. Participants were offered whole exome sequencing in addition to clinically available sequencing gene panels between July 2012 and January 2013 to determine the molecular etiology of their retinal dystrophy. Diagnostic yield and acceptability of whole exome sequencing in patients with retinal disorders. Twenty-six of 29 eligible patients (∼90%) who were approached opted to undergo molecular testing. Each participant chose whole exome sequencing in addition to, or in lieu of, clinically available sequencing gene panels. Time to obtain informed consent was manageable in the clinical context. Whole exome sequencing successfully identified known pathogenic mutations or suspected deleterious variants in 57.7% of participants. Additionally, 1 participant had 2 autosomal dominant medically actionable incidental findings (unrelated to retinopathy) that were reported to enable the participant to take preventive action and reduce risk for future disease. In this study, we identified the molecular etiology for more than half of all participants. Additionally, we found that participants were widely accepting of whole exome sequencing and the possibility of being informed about medically actionable incidental findings.

Whole-exome sequencing in patients with ichthyosis reveals modifiers associated with increased IgE levels and allergic sensitizations

Whole-exome sequencing in patients with ichthyosis reveals modifiers associated with increased IgE levels and allergic sensitizations

KCNT1 gain-of-function mutations linked to human epilepsy are modulated by quinidine

Epilepsy of infancy with migrating focal seizures (EIMFS) and a severe form of autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) are strikingly different epilepsy syndromes, which have very recently been associated with mutations in the same gene. EIMFS is a rare early infantile epileptic encephalopathy characterised by heterogeneous migrating focal seizures, and associated with arrest or regression of development resulting in profound disability. In contrast, ADNFLE begins in mid-childhood and is characterized by clusters of motor seizures arising from sleep and is associated with major comorbidities of intellectual disability and psychiatric features. Whole exome sequencing of patients with EIMFS and severe ADNFLE has revealed a number of KCNT1 mutations with 100% penetrance, many of which are de novo . KCNT1 , widely expressed in the mammalian central nervous system, encodes a potassium channel that is activated by large elevations in intracellular sodium, which occur during normal physiological signalling in the nervous system. While KCNT1 channels are thought to play important roles in regulating neuronal excitability, their precise contribution to electrical activity differs in different neuronal cell types. KCNT1 disorders are resistant to standard anti-epileptic drugs and have a severe prognosis, creating an urgent need for novel therapies. Several studies have demonstrated that antiarrhythmic compounds, such as quinidine, bepridil and clofilium are effective blockers of KCNT1 channels and despite their known adverse effect profile our recent study suggests that they may hold promise as effective therapies in EIMFS. This research highlight describes this study, evaluating the electrophysiological and pharmacological gain-of-function phenotype of KCNT1 mutations in vitro and examining the neurodevelopmental time frame for the potential contribution of this channel to neuronal excitability in vivo .

G.P.261: Whole exome sequencing in patients with congenital myopathies

Congenital myopathies (CM) are a group of disorders presenting at birth or early infancy, characterised by muscle weakness and specific changes in the muscle biopsy. During the recent decade a number of genes have been discovered, however, additional novel genes are yet to be identified as genetic diagnosis cannot be currently established in many CM patients. With the aim to identify the genetic defect in 33 CM patients, in whom mutations in suspected genes have been previously excluded, we carried out whole exome sequencing (WES). From the 33 patients, 19 have been resolved and 14 patients are still being investigated as the initial WES data analysis failed to identify possible candidate genes. Among the resolved cases, two patients with muscle biopsy suggestive of core myopathy and one with centronuclear myopathy, carried heterozygous truncating <i>TTN</i> mutations supporting the emerging data that <i>TTN</i> mutations should be investigated as causative in patients with unresolved centronuclear and core myopathy. We also identified a homozygous missense mutation in <i>STAC3</i> gene in a patient with King-Denborough syndrome and core-like changes on muscle biopsy, indicating a causative role of <i>STAC3</i> mutations in King–Denborough syndrome. Recently, a homozygous missense mutation in <i>STAC3</i> gene was identified in patients with Native American myopathy. A patient with a severe phenotype and muscle biopsy changes suggestive of nemaline myopathy, carried a homozygous missense mutation in <i>KLHL40</i>. Mutations in <i>KLHL40</i> have been very recently identified as a frequent cause of severe autosomal-recessive nemaline myopathy. Ten of the resolved patients carried mutations in potentially causative genes which are currently under investigation.

Whole exome sequencing identifies new causative mutations in Tunisian families with non-syndromic deafness.

Identification of the causative mutations in patients affected by autosomal recessive non syndromic deafness (DFNB forms), is demanding due to genetic heterogeneity. After the exclusion of GJB2 mutations and other mutations previously reported in Tunisian deaf patients, we performed whole exome sequencing in patients affected with severe to profound deafness, from four unrelated consanguineous Tunisian families. Four biallelic non previously reported mutations were identified in three different genes: a nonsense mutation, c.208C>T (p.R70X), in LRTOMT, a missense mutation, c.5417T>C (p.L1806P), in MYO15A and two splice site mutations, c.7395+3G>A, and c.2260+2T>A, in MYO15A and TMC1 respectively. We thereby provide evidence that whole exome sequencing is a powerful, cost-effective screening tool to identify mutations causing recessive deafness in consanguineous families.

A community-based resource for automatic exome variant-calling and annotation in Mendelian disorders.

BackgroundMendelian disorders are mostly caused by single mutations in the DNA sequence of a gene, leading to a phenotype with pathologic consequences. Whole Exome Sequencing of patients can be a cost-effective alternative to standard genetic screenings to find causative mutations of genetic diseases, especially when the number of cases is limited. Analyzing exome sequencing data requires specific expertise, high computational resources and a reference variant database to identify pathogenic variants.ResultsWe developed a database of variations collected from patients with Mendelian disorders, which is automatically populated thanks to an associated exome-sequencing pipeline. The pipeline is able to automatically identify, annotate and store insertions, deletions and mutations in the database. The resource is freely available online http://exome.tigem.it. The exome sequencing pipeline automates the analysis workflow (quality control and read trimming, mapping on reference genome, post-alignment processing, variation calling and annotation) using state-of-the-art software tools. The exome-sequencing pipeline has been designed to run on a computing cluster in order to analyse several samples simultaneously. The detected variants are annotated by the pipeline not only with the standard variant annotations (e.g. allele frequency in the general population, the predicted effect on gene product activity, etc.) but, more importantly, with allele frequencies across samples progressively collected in the database itself, stratified by Mendelian disorder.ConclusionsWe aim at providing a resource for the genetic disease community to automatically analyse whole exome-sequencing samples with a standard and uniform analysis pipeline, thus collecting variant allele frequencies by disorder. This resource may become a valuable tool to help dissecting the genotype underlying the disease phenotype through an improved selection of putative patient-specific causative or phenotype-associated variations.

Whole-exome sequencing in patients with inherited neuropathies: outcome and challenges

Inherited peripheral neuropathies (IPN) are one of the most frequent inherited causes of neurological disability characterized by considerable phenotypic and genetic heterogeneity. Based on clinical and electrophysiological properties, they can be subdivided into three main groups: HMSN, dHMN, and HSN. At present, more than 50 IPN genes have been identified. Still, many patients and families with IPN have not yet received a molecular genetic diagnosis because clinical genetic testing usually only covers a subset of IPN genes. Moreover, a considerable proportion of IPN genes has to be identified. Here we present results of WES in 27 IPN patients excluded for mutations in many known IPN genes. Eight of the patients received a definite diagnosis. While six of these patients carried bona fide pathogenic mutations in known IPN genes, two patients had mutations in genes known to be involved in other types of neuromuscular disorders. A further group of eight patients carried sequence variations in IPN genes that could not unequivocally be classified as pathogenic. In addition, combining data of WES and linkage analysis identified SH3BP4, ITPR3, and KLHL13 as novel IPN candidate genes. Moreover, there was evidence that particular mutations in PEX12, a gene known to cause Zellweger syndrome, could also lead to an IPN phenotype. We show that WES is a useful tool for diagnosing IPN and we suggest an expanded phenotypic spectrum of some genes involved in other neuromuscular and neurodegenerative disorders. Nevertheless, interpretation of variants in known and potential novel disease genes has remained challenging.

P17 Whole exome sequencing in patients with congenital myopathies

Congenital myopathies (CM) are a group of disorders presenting at birth or early infancy, characterised by muscle weakness and specific changes in the muscle biopsy. During the recent decade a number of genes have been discovered, however, additional novel genes are yet to be identified as genetic diagnosis cannot be currently established in many CM patients. With the aim to identify the genetic defect in 33 CM patients, in whom mutations in suspected genes have been previously excluded, we carried out whole exome sequencing (WES). From the 33 patients, 19 have been resolved and 14 patients are still being investigated as the initial WES data analysis failed to identify possible candidate genes. Among the resolved cases, two patients with muscle biopsy suggestive of core myopathy and one with centronuclear myopathy, carried heterozygous truncating TTN mutations supporting the emerging data that TTN mutations should be investigated as causative in patients with unresolved centronuclear and core myopathy. We also identified a homozygous missense mutation in STAC3 gene in a patient with King-Denborough syndrome and core-like changes on muscle biopsy, indicating a causative role of STAC3 mutations in King–Denborough syndrome. Recently, a homozygous missense mutation in STAC3 gene was identified in patients with Native American myopathy. A patient with a severe phenotype and muscle biopsy changes suggestive of nemaline myopathy, carried a homozygous missense mutation in KLHL40. Mutations in KLHL40 have been very recently identified as a frequent cause of severe autosomal-recessive nemaline myopathy. Ten of the resolved patients carried mutations in potentially causative genes which are currently under investigation.

P56 Whole-exome sequencing in patients with sensory and motor inherited neuropathies

P56 Whole-exome sequencing in patients with sensory and motor inherited neuropathies

Whole Exome Analysis Of Germline Alterations Associated With Myelodysplastic Syndrome

While in the past germline (GL) events have been investigated in myeloid disorders, these studies focused on targeted genotyping of empirically selected candidate genes, and a comprehensive characterization of GL-encoded susceptibility has not been undertaken in these diseases. Whole exome sequencing (WES) data sets obtained in the molecular discovery projects, allow for a targeted or unbiased analysis of inherited alterations to identify those with a pathogenic importance. To that end, initial WES studies, including published analyses from TCGA leukemia cohort, have focused mainly on somatic mutations.MDS is a disease of the elderly, typically presenting with complex phenotypes and clinical histories. Inherited predisposition, if present, would be expected to follow complex, non-mendelian genetic traits, have low penetrance and interact with various extrinsic factors such as exposures or coexisting conditions, and family histories may not be informative. Therefore, in this disorder predisposing GL alterations may be difficult to discern.Here, we have applied WES in patients with MDS and other myeloid neoplasms (N=117). We also investigated AML data set available through TCGA (N=201). In addition to defining somatic mutations in paired (tumor, GL DNA), we also performed an unbiased search for GL SNPs and mutations.Our strategy was comprehensive; firstly it focused on selection of non-synonymous and, possibly deleterious SNPs. Subsequently, putative candidates were further prioritized based on their genotypic frequency in the general population, and finally, according to perceived importance for the leukemogenesis. For the purpose of this study, we used a maximum minor allele frequency threshold of .05 in matched general population for alterations to be prioritized.As a result, we identified a large variety of new and, as expected, previously reported GL polymorphisms and mutations. Known pathogenic as well as novel GL alterations (42 non-synonymous sites, reported (N=33) and novel (N=9)) were found in telomerase genes (TERT, DKC1, GAR1, POT1, SMG6, NOP10, TINF2, NHP2, WRAP53) and other bone marrow failure genes (ELANE, GFI1, HAX1, GATA2, CSF3R, WAS) or many others.Subsequently, we hypothesized that there may also be GL variants of pathogenic significance in genes that are also frequently affected by somatic events. Known examples of such genes include SETBP1, NF1, CBL and many others. Such GL events may be directly disease-prone or indirectly predispose to somatic lesions within the same gene (as recently shown for JAK2 V617F mutation). Since the complexity of the entire analysis exceeds the boundaries of this abstract we focus here on exemplary results of genes most frequently affected by somatic mutations, including e.g., TET2 (8%), ASXL1 (15%), DNMT3A (26%), CEBPA (6%), TP53 (8%) and others. In this illustrative subset alone, 647 non-synonymous variants across 74 sites, in 14/16 genes of interest were found (FLT3, TET2 and TP53 were most frequently affected). Following, bio-analytic filtering we identified 30 rare variants, of which 11/30 were present at a significantly higher frequency compared to control population. Of these, 5/11 were located within such gene as ASXL1: S737N (p=.002), P1213R (p=.005), G543S (p=.023), L1286V (p=.026), L1216F (p=.036), 3/11 in FLT3: D324N (p<.001), A912V (p=.002) ,F298L (p=.003), the remaining were in DNMT3A: R693H (p=.042), KIT: N293S (p=.019), and KRAS: C180X (p=.002). Of note is the FLT3 polymorphism, D324N, which has been previously linked to AML susceptibility. Excluding reported polymorphic sites, 33% of non-synonymous sites were not reported in population databases.Our data identified several GL variants that appear to associate with specific phenotypic features. For instance, a significantly decreased overall survival was seen in patients who had a RUNX1/RUNX1-IT (p=.03) GL mutation. Furthermore, patients diagnosed with MDS/MPN were found to have a greater odds of presenting with concurrent GL and somatic variants in TET2 gene compared to non-MDS/MPN patients [OR 7.4; 95% CI 2.5-21.8).In conclusion, our data suggests that both GL and somatic events in myeloid disorders are important for the pathogenesis of myeloid malignancies. They may interact with each other and with cytogenetic abnormalities, which can lead to deletion of protective alleles or amplification of disease-prone alleles. Disclosures:Makishima:AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

Diagnostic utility of whole exome sequencing in patients showing cerebellar and/or vermis atrophy in childhood

Cerebellar and/or vermis atrophy is recognized in various types of childhood disorders with clinical and genetic heterogeneity. Although careful evaluation of clinical features and neuroimaging can lead to correct diagnosis of disorders, their diagnosis is sometimes difficult because clinical features can overlap with each other. In this study, we performed family-based whole exome sequencing of 23 families including 25 patients with cerebellar and/or vermis atrophy in childhood, who were unable to be diagnosed solely by clinical examination. Pathological mutations of seven genes were found in ten patients from nine families (9/23, 39.1%): compound heterozygous mutations in FOLR1, C5orf42, POLG, TPP1, PEX16, and de novo mutations in CACNA1A, and ITPR1. Patient 1A with FOLR1 mutations showed extremely low concentration of 5-methyltetrahydrofolate in the cerebrospinal fluid and serum, and Patient 6 with TPP1 mutations demonstrated markedly lowered tripeptidyl peptidase 1 activity in leukocytes. Furthermore, Patient 8 with PEX16 mutations presented a mild increase of very long chain fatty acids in the serum as supportive data for genetic diagnosis. The main clinical features of these ten patients were nonspecific and mixed, and included developmental delay, intellectual disability, ataxia, hypotonia, and epilepsy. Brain MRI revealed both cerebellar and vermis atrophy in eight patients (8/10, 80%), vermis atrophy/hypoplasia in two patients (2/10, 20%), and brainstem atrophy in one patient (1/10, 10%). Our data clearly demonstrate the utility of whole exome sequencing for genetic diagnosis of childhood cerebellar and/or vermis atrophy.