Exome Sequencing In Patients Research Articles

Compound heterozygous variants in NBAS as a cause of atypical osteogenesis imperfecta

BackgroundOsteogenesis imperfecta (OI), the commonest inherited bone fragility disorder, affects 1 in 15,000 live births resulting in frequent fractures and reduced mobility, with significant impact on quality of life. Early diagnosis is important, as therapeutic advances can lead to improved clinical outcome and patient benefit. ReportWhole exome sequencing in patients with OI identified, in two patients with a multi-system phenotype, compound heterozygous variants in NBAS (neuroblastoma amplified sequence). Patient 1: NBAS c.5741G>A p.(Arg1914His); c.3010C>T p.(Arg1004*) in a 10-year old boy with significant short stature, bone fragility requiring treatment with bisphosphonates, developmental delay and immunodeficiency. Patient 2: NBAS c.5741G>A p.(Arg1914His); c.2032C>T p.(Gln678*) in a 5-year old boy with similar presenting features, bone fragility, mild developmental delay, abnormal liver function tests and immunodeficiency. DiscussionHomozygous missense NBAS variants cause SOPH syndrome (short stature; optic atrophy; Pelger-Huet anomaly), the same missense variant was found in our patients on one allele and a nonsense variant in the other allele. Recent literature suggests a multi-system phenotype. In this study, patient fibroblasts have shown reduced collagen expression, compared to control cells and RNAseq studies, in bone cells show that NBAS is expressed in osteoblasts and osteocytes of rodents and primates. These findings provide proof-of-concept that NBAS mutations have mechanistic effects in bone, and that NBAS variants are a novel cause of bone fragility, which is distinguishable from ‘Classical’ OI. ConclusionsHere we report on variants in NBAS, as a cause of bone fragility in humans, and expand the phenotypic spectrum associated with NBAS. We explore the mechanism underlying NBAS and the striking skeletal phenotype in our patients.

ASCIA‐P74: AUSTRALIAN AUTOINFLAMMATORY DISEASES REGISTRY (AADRY): A NATIONAL APPROACH TO THE GENETIC AND IMMUNOLOGICAL EVALUATION OF PATIENTS WITH SUSPECTED AUTOINFLAMMATORY DISEASE

Traditionally defined as periodic fever syndromes lacking markers of autoimmunity such as high titre self-reactive T cells and auto-antibodies, the spectrum of monogenic autoinflammatory diseases has broadened substantially in recent years. The clinical heterogeneity of newly described conditions such as STING associated vasculopathy with onset in infancy (SAVI) and Deficiency in ADA2 (DADA2) has forced reconsideration of traditional designations, with the common link between these disorders being inappropriate inflammation and cytokine dysregulation. These new diagnoses can be attributed to the introduction of whole exome sequencing (WES) into the diagnostic algorithm of patients with suspected, but currently genetically undefined, autoinflammatory diseases. We believe it's important to take a national approach to the diagnosis of autoinflammatory disorders given their rarity and introduce the establishment of AADRY, an Australian Autoinflammatory Diseases RegistrY. AADRY proposes to collect epidemiological and clinical data on patients with suspected and confirmed autoinflammatory diseases, and will perform whole exome sequencing in patients who are yet to be genetically characterised. We also aim to perform in vitro testing and harness CRISPR-Cas9 gene editing technology to determine the functional significance of novel variants and their potential contribution to clinical phenotype. We describe bioinformatic analyses and other results from the first 12 AADRY cases analysed, including one trio and one family of five.

Improved diagnostic yield of neuromuscular disorders applying clinical exome sequencing in patients arising from a consanguineous population

Neuromuscular diseases (NMDs) include a broad range of disorders affecting muscles, nerves and neuromuscular junctions. Their overlapping phenotypes and heterogeneous genetic nature have created challenges in diagnosis which calls for the implementation of massive parallel sequencing as a candidate strategy to increase the diagnostic yield. In this study, total of 45 patients, mostly offspring of consanguineous marriages were examined using whole exome sequencing. Data analysis was performed to identify the most probable pathogenic rare variants in known NMD genes which led to identification of causal variants for 33 out of 45 patients (73.3%) in the following known genes: CAPN3, Col6A1, Col6A3, DMD, DYSF, FHL1, GJB1, ISPD, LAMA2, LMNA, PLEC1, RYR1, SGCA, SGCB, SYNE1, TNNT1 and 22 novel pathogenic variants were detected. Today, the advantage of whole exome sequencing in clinical diagnostic strategies of heterogeneous disorders is clear. In this cohort, a diagnostic yield of 73.3% was achieved which is quite high compared to the overall reported diagnostic yield of 25% to 50%. This could be explained by the consanguineous background of these patients and is another strong advantage of offering clinical exome sequencing in diagnostic laboratories, especially in populations with high rate of consanguinity.

Whole exome sequencing in patients with white matter abnormalities.

Here we report whole exome sequencing (WES) on a cohort of 71 patients with persistently unresolved white matter abnormalities with a suspected diagnosis of leukodystrophy or genetic leukoencephalopathy. WES analyses were performed on trio, or greater, family groups. Diagnostic pathogenic variants were identified in 35% (25 of 71) of patients. Potentially pathogenic variants were identified in clinically relevant genes in a further 7% (5 of 71) of cases, giving a total yield of clinical diagnoses in 42% of individuals. These findings provide evidence that WES can substantially decrease the number of unresolved white matter cases. Ann Neurol 2016;79:1031-1037.

Determining the frequency of pathogenic germline variants from exome sequencing in patients with castrate-resistant prostate cancer

ObjectivesTo determine the frequency of pathogenic inherited mutations in 157 select genes from patients with metastatic castrate-resistant prostate cancer (mCRPC).DesignObservational.SettingMultisite US-based cohort.ParticipantsSeventy-one adult male patients with histological confirmation of prostate...

Whole Exome Sequencing in Patients with the Cuticular Drusen Subtype of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in elderly people worldwide. Cuticular drusen (CD) is a clinical subtype of AMD, which typically displays an earlier age at onset, and has a strong genetic component. Genetic studies support a role for rare sequence variants in CD susceptibility, and rare sequence variants in the CFH gene have been identified in 8.8% of CD cases. To further explore the role of rare variants in CD, we performed whole exome sequencing (WES) in 14 affected members of six families and 12 sporadic cases with CD. We detected rare sequence variants in CFH and FBLN5, which previously were shown to harbor rare variants in patients with CD. In addition, we detected heterozygous rare sequence variants in several genes encoding components of the extracellular matrix (ECM), including FBLN1, FBLN3/EFEMP1, FBLN5, FBLN6/HMCN1, FBN2, and COL15A1. Two rare pathogenic variants were identified in the COL15A1 gene: one in a sporadic case and another was found to segregate in a family with six affected individuals with CD. In addition, two rare pathogenic variants were identified in the FGL1 gene in three unrelated CD cases. These findings suggest that alterations in the ECM and in the coagulation pathway may play a role in the pathogenesis of CD. The identified candidate genes require further analyses in larger cohorts to confirm their role in the CD subtype of AMD. No evidence was found of rare sequence variants in a single gene that segregate with CD in the six families, suggesting that the disease is genetically heterogeneous.

The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease.

Despite recent progress in the genetic characterization of congenital muscle diseases, the genes responsible for a significant proportion of cases remain unknown. We analysed two branches of a large consanguineous family in which four patients presented with a severe new phenotype, clinically marked by neonatal-onset muscle weakness predominantly involving axial muscles, life-threatening respiratory failure, skin abnormalities and joint hyperlaxity without contractures. Muscle biopsies showed the unreported association of multi-minicores, caps and dystrophic lesions. Genome-wide linkage analysis followed by gene and exome sequencing in patients identified a homozygous nonsense mutation in TRIP4 encoding Activating Signal Cointegrator-1 (ASC-1), a poorly characterized transcription coactivator never associated with muscle or with human inherited disease. This mutation resulted in TRIP4 mRNA decay to around 10% of control levels and absence of detectable protein in patient cells. ASC-1 levels were higher in axial than in limb muscles in mouse, and increased during differentiation in C2C12 myogenic cells. Depletion of ASC-1 in cultured muscle cells from a patient and in Trip4 knocked-down C2C12 led to a significant reduction in myotube diameter ex vivo and in vitro, without changes in fusion index or markers of initial myogenic differentiation. This work reports the first TRIP4 mutation and defines a novel form of congenital muscle disease, expanding their histological, clinical and molecular spectrum. We establish the importance of ASC-1 in human skeletal muscle, identify transcriptional co-regulation as novel pathophysiological pathway, define ASC-1 as a regulator of late myogenic differentiation and suggest defects in myotube growth as a novel myopathic mechanism.

Editorial: Recent Advances in Voltage-Gated Sodium Channels, their Pharmacology and Related Diseases.

Editorial: Recent Advances in Voltage-Gated Sodium Channels, their Pharmacology and Related Diseases.

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.

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.

Abstract 16484: The Transcriptional Repressor NR1D2 is Associated With Congenital Heart Disease and Plays an Evolutionarily Conserved Role in Cardiac Development

Title: The Transcriptional Repressor NR1D2 is Associated with Congenital Heart Disease and Plays an Evolutionarily Conserved Role in Cardiac Development. Introduction: Congenital heart disease (CHD) is a leading cause of morbidity and mortality in young children and the underlying genetic etiology is complex and poorly defined. Recent genetic studies of mixed cardiac phenotypes focusing on de novo events have identified novel disease loci in up to 10% of participants and uncovered new functional categories of genes related to cardiac development. Hypothesis: We hypothesize that exome sequencing of patients with CHD will uncover novel genes related to cardiac disease and development. Methods: To identify disease-associated mutations, we analyzed exome sequencing data in a multi-institutional discovery cohort of 59 trios with a novel analytical pipeline. We phenotyped knockout animals for orthologous genes across multiple genetic phyla (mouse and fruit fly) for aspects of cardiac development. Additionally we performed Chromatin Immuno-Precipitation Sequencing (ChIP-seq) in a HL-1 mouse atrial cell line. Results: Exome sequencing uncovered a de novo missense variant R175W in the highly conserved DNA binding domain of NR1D2 a key component of the molecular clock. Knockout mice for Nr1d2tm1-Dgen display atrioventricular septal defects and related malformations, while knockdowns of the drosophila homolog Eip75B displays abnormal heart tube fusion during late embryonic development. ChIP-seq data for Nr1d2 suggests transcriptional repression at key loci controlling differentiation into both valve development (Twist1, Twist2, Bmpr1b) and atrial left-right patterning (Pitx2, Cited2). Conclusions: A patient with a deleterious de novo mutation in NR1D2 and knockout in multiple model organisms show cardiac defects, while genomic analysis of ChIP-seq data supports a modulatory role for this transcriptional repressor in the complex 3-dimensional process of cardiac morphogenesis. In conclusion, these data suggest that NR1D2 plays an evolutionarily conserved role in cardiac development and congenital heart disease. Further experiments are needed to understand the mechanism by which NR1D2 might link the molecular clock to cardiac development.

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 .

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.

Exome sequencing of patients with histiocytoid cardiomyopathy reveals a de novo NDUFB11 mutation that plays a role in the pathogenesis of histiocytoid cardiomyopathy.

Histiocytoid cardiomyopathy (Histiocytoid CM) is a rare form of cardiomyopathy observed predominantly in newborn females that is fatal unless treated early in life. We have performed whole exome sequencing on five parent-proband trios and identified nuclear-encoded mitochondrial protein mutations in three cases. The molecular genetic basis of Histiocytoid CM remains unknown despite several hypotheses in medical literature. The findings presented in this manuscript may represent components of genetic etiologies for this heterogeneous disease. Two probands had de novo non-sense mutations in the second exon of the X-linked nuclear gene NDUFB11. A third proband was doubly heterozygous for inherited rare variants in additional components of complex I, NDUFAF2 and NDUFB9, confirming that Histiocytoid CM is genetically heterogeneous. In a fourth case, the proband with Histiocytoid CM inherited a mitochondrial mutation from her heteroplasmic mother, as did her brother who presented with cardiac arrhythmia. Strong candidate recessive or compound heterozygous variants were not found for this individual or for the fifth case. Although NDUFB11 has not been implicated before in cardiac pathology, morpholino-mediated knockdown of ndufb11 in zebrafish embryos generated defective cardiac tissue with cardiomegaly, looping defects, and arrhythmia which suggests the role of NDUFB11 in the pathogenesis of this abnormal cardiac pathology. Taken together, the unbiased whole exome sequencing approach confirms the suspected genetic heterogeneity of Histiocytoid CM. Therefore, the novel NDUFB11 mutation may cause a complex 1 deficiency in synergy with additional unknown mtDNA variants.

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.

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.

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.