Application Of Whole Exome Sequencing Research Articles

Application of Whole Exome Sequencing in Six Families with an Initial Diagnosis of Autosomal Dominant Retinitis Pigmentosa: Lessons Learned

This study aimed to identify the genetics underlying dominant forms of inherited retinal dystrophies using whole exome sequencing (WES) in six families extensively screened for known mutations or genes. Thirty-eight individuals were subjected to WES. Causative variants were searched among single nucleotide variants (SNVs) and insertion/deletion variants (indels) and whenever no potential candidate emerged, copy number variant (CNV) analysis was performed. Variants or regions harboring a candidate variant were prioritized and segregation of the variant with the disease was further assessed using Sanger sequencing in case of SNVs and indels, and quantitative PCR (qPCR) for CNVs. SNV and indel analysis led to the identification of a previously reported mutation in PRPH2. Two additional mutations linked to different forms of retinal dystrophies were identified in two families: a known frameshift deletion in RPGR, a gene responsible for X-linked retinitis pigmentosa and p.Ser163Arg in C1QTNF5 associated with Late-Onset Retinal Degeneration. A novel heterozygous deletion spanning the entire region of PRPF31 was also identified in the affected members of a fourth family, which was confirmed with qPCR. This study allowed the identification of the genetic cause of the retinal dystrophy and the establishment of a correct diagnosis in four families, including a large heterozygous deletion in PRPF31, typically considered one of the pitfalls of this method. Since all findings in this study are restricted to known genes, we propose that targeted sequencing using gene-panel is an optimal first approach for the genetic screening and that once known genetic causes are ruled out, WES might be used to uncover new genes involved in inherited retinal dystrophies.

Whole exome sequencing of suspected mitochondrial patients in clinical practice.

Mitochondrial disorders are characterized by a broad clinical spectrum. Identical clinical signs and symptoms can be caused by mutations in different mitochondrial or nuclear genes. Vice versa, the same mutation can lead to different phenotypes. Genetic syndromes and neuromuscular disorders mimicking mitochondrial disorders further complicate the diagnostic process. Whole exome sequencing (WES) is the state of the art next generation sequencing technique to identify genetic defects in mitochondrial disorders. Until recently it has mainly been used as a research tool. In this study, the use of WES in routine diagnostics is described. The WES data of 109 patients, referred under the suspicion of a mitochondrial disorder, were examined in two steps. First, the data were filtered using a virtual gene panel of genes known to be associated with mitochondrial disease. If negative, the entire exome was examined. A molecular diagnosis was achieved in 39 % of the heterogeneous cohort, and in 57 % of the subgroup of 42 patients with the highest suspicion for a mitochondrial disease. In addition to mutations in genes known to be associated with mitochondrial disorders (e.g. TUFM, MTFMT, FBXL4), in the subgroup of patients with the lowest suspicion for a mitochondrial disorder we found mutations in several genes associated with neuromuscular disorders (e.g. SEPN1, ACTA1) and genetic syndrome (e.g. SETBP1, ARID1B). Our results show that WES technology has been successfully implemented as a state-of-the-art, molecular diagnostic test for mitochondrial disorders as well as for the mimicking disorders in daily clinical practice. It also illustrates that clinical and biochemical phenotyping is essential for successful application of WES to diagnose individual patients.

Application of whole exome sequencing to a rare inherited metabolic disease with neurological and gastrointestinal manifestations: A congenital disorder of glycosylation mimicking glycogen storage disease

BackgroundRare inherited metabolic diseases with neurological and gastrointestinal manifestations can be misdiagnosed as other diseases or remain as disorders with indeterminate etiologies. This study aims to provide evidence to recommend the utility of whole exome sequencing in clinical diagnosis of a rare inherited metabolic disease. Methods and resultsA 4-month-old female baby visited an outpatient clinic due to poor weight gain, repeated seizure-like episodes, developmental delay, and unexplained hepatomegaly with abnormal liver function test results. Although liver biopsy revealed moderate fibrosis with a suggested diagnosis of glycogen storage disease (GSD), no mutations were identified either by single gene approach for GSD (G6PC and GAA) or by next generation sequencing panels for GSD (including 21 genes). Whole exome sequencing of the patient revealed compound heterozygous mutations of PMM2: c.580C>T (p.Arg194*) and c.713G>C (p.Arg238Pro) which mutations were associated with congenital disorder of glycosylation Ia (CDG-Ia: PMM2-CDG). ConclusionsWe successfully applied exome sequencing to diagnose the first reported Korean patient with CDG-Ia, which was misdiagnosed as GSD. Whole exome sequencing may prove to be the preferred strategy for analysis of clinical features that do not readily suggest a specific diagnosis, such as those observed in inherited metabolic diseases, including CDG.

Whole-Exome Sequencing as a Diagnostic Tool in a Family With Episodic Ataxia Type 1

Complex neurologic phenotypes are inherently difficult to diagnose. Whole-exome sequencing (WES) is a new tool in the neurologist’s diagnostic armamentarium. Whole-exome sequencing can be applied to investigate the “diagnostic odyssey” cases. These cases involve patients with rare diseases that likely have a genetic etiology but have failed to be diagnosed by clinical evaluation and targeted gene testing. We describe such a case, a 22-year-old man who had mild intellectual developmental disability and episodes of jerking ataxic movements that affected his whole body. He underwent numerous multidisciplinary and multicentric evaluations throughout his life that failed to establish a clear diagnosis. Following his visit to Mayo Clinic in Jacksonville, Florida, WES was applied for genetic determination of the unknown disorder in the proband and his biological parents and sister. Additional clinical evaluation, magnetic resonance neuroimaging, electromyography, and electroencephalography of the proband were performed to verify the phenotype after the WES results were available. To our knowledge, this is the first report of the application of WES to facilitate the diagnosis of episodic ataxia type 1. This case illustrates that WES supported by clinical data is a useful and time-saving tool in the evaluation of patients with rare and complex hereditary disorders.

Application of whole exome sequencing in the diagnosis of hereditary neurological diseases

Whole Exome Sequencing (WES) is a promising method in human genetics. Because the majority of pathogenic mutations that lead to the development of diseases are localized in exons and splice sites, WES could become a major tool for the diagnosis of diseases with a complex hereditary nature. This tool appears to be particularly useful for hereditary neurological diseases, such as autism spectrum disorders, Charcot-Marie-Tooth disease and others. In our review, we discuss the clinical application of WES, with special emphasis on the diagnosis of hereditary neurological diseases.

The Application of Whole-Exome Sequencing in Diagnosing Pediatric Rare Disease in Hong Kong

Whole-exome sequencing (WES) combines next generation sequencing (NGS) technology with capture methods to sequence all the coding regions of the genome. The application of WES has gained a lot of success worldwide in discovering new disease causing genes and in diagnosis. We reviewed some of the large collaborative efforts worldwide and summarized notable examples, observing an overall diagnostic yield of 16-30%. Locally in Hong Kong, there have been several applications of WES in research, as well as bioinformatics tools developed, and the field is continuing to grow. In our own department, we have applied this to pediatric rare diseases, by establishing our in-house research pipeline for WES, as well as utilizing core laboratory facilities in model animals and cell work for functional validation. We illustrate this approach using cases as examples. On the other hand, clinically, we are utilizing WES more as a diagnostic tool by analyzing selected pediatric cases via overseas laboratories. We see how this new tool is helping patients and families to obtain an answer for their condition, and subsequently helping them with their management and family planning. Finally, we discuss the challenges for WES in Hong Kong, and the future direction of the technology, with the potential to revolutionize clinical diagnosis and medical research. Key Words: gene, science, human resource, education, DNA INTRODUCTION Whole-exome sequencing (WES) combines next generation sequencing (NGS) technology with capture methods to sequence all the coding regions of the genome. The application of WES has gained a lot of success worldwide in discovering new disease causing genes and in diagnosis. We reviewed some of the large collaborative efforts worldwide and summarized notable examples, observing an overall diagnostic yield of 16-30%. Locally in Hong Kong, there have been several applications of WES in research, as well as bioinformatics tools developed, and the field is continuing to grow. In our own department, we have applied this to pediatric rare diseases, by establishing our in-house research pipeline for WES, as well as utilizing core laboratory facilities in model animals and cell work for functional validation. We illustrate this approach using cases as examples. On the other hand, clinically, we are utilizing WES more as a diagnostic tool by analyzing selected pediatric cases via overseas laboratories. We see how this new tool is helping patients and families to obtain an answer for their condition, and subsequently helping them with their management and family planning. Finally, we discuss the challenges for WES in Hong Kong, and the future direction of the technology, with the potential to revolutionize clinical diagnosis and medical research. Figure 1. We are good. Table 1. The data. dsffd dfgdg dfgdf gdfg dfgdfg dfgdf dfg gd fgdf dfgdf dfg gdfggfd gdfg dfg dfgd gdgdfg dfgfd dfg gdf Note: the test is published in a right format.

Molecular Genetic Diagnosis of a Bethlem Myopathy Family with an Autosomal-Dominant COL6A1 Mutation, as Evidenced by Exome Sequencing.

BackgroundWe describe herein the application of whole exome sequencing (WES) for the molecular genetic diagnosis of a large Korean family with dominantly inherited myopathy.Case ReportThe affected individuals presented with slowly progressive proximal weakness and ankle contracture. They were initially diagnosed with limb-girdle muscular dystrophy (LGMD) based on clinical and pathologic features. However, WES and subsequent capillary sequencing identified a pathogenic splicing-site mutation (c.1056+1G>A) in COL6A1, which was previously reported to be an underlying cause of Bethlem myopathy. After identification of the genetic cause of the disease, careful neurologic examination revealed subtle contracture of the interphalangeal joint in the affected members, which is a characteristic sign of Bethlem myopathy. Therefore, we revised the original diagnosis from LGMD to Bethlem myopathy.ConclusionsThis is the first report of identification of COL6A1-mediated Bethlem myopathy in Korea, and indicates the utility of WES for the diagnosis of muscular dystrophy.

Clinical application of whole exome sequencing reveals a novel compound heterozygous TK2-mutation in two brothers with rapidly progressive combined muscle-brain atrophy, axonal neuropathy, and status epilepticus

Mutations in several genes cause mtDNA depletion associated with encephalomyopathy. Due to phenotypic overlap, it is difficult to conclude from clinical phenotype to genetic defect. Here we report on two brothers who presented with rapid fatty muscle degeneration, axonal neuropathy, rapid loss of supratentorial white and gray matter, and status epilepticus. Whole exome sequencing coupled with ‘identity-by-state’ (IBS) analysis revealed a compound heterozygous missense mutation (p.M117V, p.A139V) in the thymidine kinase 2 (TK2) gene that segregated with the phenotype. Both mutations were located in the thymidine binding pouch of the enzyme. Residual mtDNA copy numbers in muscle were 8.5%, but normal in blood and fibroblasts. Our results broaden the clinical phenotype spectrum of TK2 mutations and promote WES as a useful method in the clinical setting for mutation detection, even in untypical cases. If two or more affected siblings from a non-consanguineous family can be investigated, IBS-analysis provides a powerful tool to narrow the number of disease candidates, similarly to autozygosity mapping in consanguineous families.

G.P.260: Clinical diversity in patients with nemaline myopathy

Nemaline myopathy (NM) is a clinically heterogeneous congenital myopathy characterized by the presence of nemaline rods in the skeletal muscle fibers. We investigated the clinical variation and pathological features in Korean patients with NM. Thirteen patients were diagnosed through muscle biopsy. Six patients had the typical congenital type, which showed the motor development delay and hypotonia at birth. Six patients showed the mild childhood type with the gait disturbance. One patient had the intermediate congenital type, who needed mechanical ventilation. Regarding the distribution of weakness, five patients presented distal dominant weakness across the clinical type. High arched palates and feet deformity were observed. Equino varus deformity was identified in two patients. But, we could not find the cardiac muscle involvement. Typical nemaline rods were recognized on light microscopy with muscle biopsy from 11 patients. However, we could identify the nemaline filamentous aggregation in electro microscope (EM) from 2 patients. One of them showed a mitochondrial abnormality in EM. Type 1 fiber predominance was in all patients. The causative mutation form 8 patients was investigated, we found a missense heterozygous mutation in exon 1 of <i>TPM3</i> gene (c.32T>A, p.Met11Lys). Seven patients showed negative results in <i>ACTA1</i> gene. The thirteen patients in this study did not share much common feature except the dysmorphic appearance. The clinical diversity was prominent with the distribution of muscle weakness and the severity of respiratory dysfunction. This study showed the phenotypic heterogeneity of NM is not only with clinical features but also with pathological findings in our patient pool. Wider application of whole exome sequencing may help overcome the phenotypic and genotypic diversity in the diagnosis of NM.

Recent advances in the genetics of dystonia.

Dystonia, a common and genetically heterogeneous neurological disorder, was recently defined as "a movement disorder characterized by sustained or intermittent muscle contractions causing abnormal, often repetitive, movements, postures, or both." Via the application of whole-exome sequencing, the genetic landscape of dystonia and closely related movement disorders is becoming exposed. In particular, several "novel" genetic causes have been causally associated with dystonia or dystonia-related disorders over the past 2 years. These genes include PRRT2 (DYT10), CIZ1 (DYT23), ANO3 (DYT24), GNAL (DYT25), and TUBB4A (DYT4). Despite these advances, major gaps remain in identifying the genetic origins for most cases of adult-onset isolated dystonia. Furthermore, model systems are needed to study the biology of PRRT2, CIZ1, ANO3, Gαolf, and TUBB4A in the context of dystonia. This review focuses on these recent additions to the family of dystonia genes, genotype-phenotype correlations, and possible cellular contributions of the encoded proteins to the development of dystonia.

An effort to examine the feasibility of whole exome and transcriptome sequencing in guiding personalized therapy for patients with non-small cell lung cancer.

e19136 Background: Usefulness of the next-generation sequencing in clinical practice for cancer treatment is in early development. We sought to evaluate the feasibility of clinical application of whole exome sequencing (WES) or whole exome plus transcriptome sequencing (WETS) to provide guidance for personalized therapy. Methods: Tumor samples and clinical information of 15 patients who were diagnosed with non-small cell lung cancer (NSCLC) were collected. Sequencing and bioinformatics analysis were then performed by BGI-Shenzhen to detect the genomic alterations and changes in RNA expressions. Interpretation was performed base on systematic review of publications that report the relationship between specific genomic alterations/gene expression level and the sensitivity/toxicity of the anticancer drugs. All agents involved in the interpretation were classified by FDA approval status and, subsequently, NCCN recommendations for NSCLC or other cancers. Results: All patients were analyzed for WES with 10 pati...

Application of whole exome sequencing in undiagnosed inherited polyneuropathies

BackgroundInherited polyneuropathies often go undiagnosed. We investigated whole exome sequencing (WES) in utility to identify the genetic causes of diverse forms of inherited polyneuropathies without genetic diagnosis.MethodsWES was applied to...

Puberté précocissime liée à un hamartome hypothalamique

Pituitary stalk interruption syndrome (PSIS) is a distinct developmental defect of the pituitary gland identified by magnetic resonance imaging and characterized by a thin, interrupted, attenuated or absent pituitary stalk, hypoplasia or aplasia of the adenohypophysis, and an ectopic posterior pituitary. The precise etiology of PSIS still remains elusive or incompletely confirmed in most cases. Adverse perinatal events, including breech delivery and hypoxia, were initially proposed as the underlying mechanism affecting the hypothalamic–pituitary axis. Nevertheless, recent findings have uncovered a wide variety of PSIS-associated molecular defects in genes involved in pituitary development, holoprosencephaly (HPE), neural development, and other important cellular processes such as cilia function. The application of whole exome sequencing (WES) in relatively large cohorts has identified an expanded pool of potential candidate genes, mostly related to the Wnt, Notch, and sonic hedgehog signaling pathways that regulate pituitary growth and development during embryogenesis. Importantly, WES has revealed coexisting pathogenic variants in a significant number of patients; therefore, pointing to a multigenic origin and inheritance pattern of PSIS. The disorder is characterized by inter- and intrafamilial variability and incomplete or variable penetrance. Overall, PSIS is currently viewed as a mild form of an expanded HPE spectrum. The wide and complex clinical manifestations include evolving pituitary hormone deficiencies (with variable timing of onset and progression) and extrapituitary malformations. Severe and life-threatening symptomatology is observed in a subset of patients with complete pituitary hormone deficiency during the neonatal period. Nevertheless, most patients are referred later in childhood for growth retardation. Prompt and appropriate hormone substitution therapy constitutes the cornerstone of treatment. Further studies are needed to uncover the etiopathogenesis of PSIS.

Exome sequencing reveals SCO2 mutations in a family presented with fatal infantile hyperthermia

We applied whole-exome sequencing (WES) for identification of an underlying genetic cause of a disease in a family presented with fatal infantile hyperthermia. Analysis of WES results revealed novel, deleterious compound missense mutations, Val160Ala and Pro233Thr, in the synthesis of cytochrome C oxidase 2 gene (SCO2) encoding a mitochondrial protein, Sco2, which is important for cytochrome C oxidase (COX) synthesis. Autosomal recessive mutations in SCO2 are known to be associated with COX deficiency recognized as fatal infantile cardio-encephalomyopathy (604272, OMIM). The Val160Ala and Pro233Thr mutations occurred in the conserved thioredoxin domain of Sco2 and predicted to disrupt protein folding and interaction of Sco2 with other proteins. Our results show applicability of WES in identification of disease-causing mutations and in establishing molecular diagnosis of severe, infantile onset disorder with a challenging diagnosis.

Clinical application of whole exome sequencing

aDepartment of Genetics bDepartment of Obstetrics and Gynecology cDepartment of Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama, USA Correspondence to Dr Nathaniel H. Robin, MD, Department of Genetics, University of Alabama at Birmingham, Kaul 210, 1530 3rd Avenue South, Birmingham, AL 35243, USA. Tel: +1 205 934 9528; fax: +1 205 975 6389; e-mail: [email protected]

Whole genomes, small children, big questions.

The child was first seen at the age of 15 months presenting with an unusual, severe and progressive Crohn’s disease-like illness. A definitive diagnosis could not be made, restricting effective clinical management. The boy underwent more than 100 surgical procedures over 3 years. A hematopoietic progenitor cell transplant, that might be a therapy of last resort, was considered too risky without a clear diagnosis of the underlying disorder. Established genetic tests for known congenital immune disorders presenting with inflammatory bowel disease-like illness were not informative. Ultimately, with institutional review board approval, research-stage, nonvalidated, WES was performed to make a clinical diagnosis and guide the treatment decision. The identification of the causative mutation enabled treatment of the underlying immune disorder and of the child’s bowel disease as well [3]. In a commentary article, alongside with the report describing the clinical and methodological details of the case, the Wisconsin group presents their ethical deliberations [4]. The authors raise some crucial questions – notwithstanding the impressive outcome and the indisputable huge benefit to the young patient – and suggest further discussion. This editorial aims to follow up on their suggestions. The first issue raised is about the elusive boundary between research and clinical care. As this case Whole-genome sequencing (WGS) is entering clinical practice. Expectations are high: a better understanding of disease, more accurate diagnosis and targeted therapies are hoped for; however, while some answers are being found, many new questions are arising. At the current stage, the dimensions of the -omics data sets in particular are overwhelming and physician–researchers are just starting to explore the ways in which they can be used. Apart from the bioinformatics challenges of genome data management there are the questions of meaningful interpretation of the emerging knowledge for health information users, both patients and professionals. At the individual and the societal level, questions are being asked about the extent and the usefulness of the newly available knowledge, for example: do whole-genome or -exome sequences tell us more than we want to know about ourselves, and doctors more than they need to know to treat their patients? In addition, do whole genomes tell parents more than they should know about their children? Since the early days of cytogenetic diagnosis, similar questions have been raised about the extent of the information yielded by testing, the communication of findings, and the ethical implications of testing [1]. Today, the availability of WGS and whole-exome sequencing (WES) for clinical diagnostics puts these old questions in a new context. Most recently, the feasibility of WGS was described for the application of noninvasive prenatal diagnosis [2].

Resolving the variable genome and epigenome in human disease

The individual human genome and epigenome are being defined at unprecedented resolution by current advances in sequencing technologies with important implications for human disease. This review uses examples relevant to clinical practice to illustrate the functional consequences of genetic and epigenetic variation. The insights gained from genome-wide association studies are described together with current efforts to understand the role of rare variants in common disease, set in the context of recent successes in Mendelian traits through the application of whole exome sequencing. The application of functional genomics to interrogate the genome and epigenome, build up an integrated picture of the regulatory genomic landscape and inform disease association studies is discussed, together with the role of expression quantitative trait mapping and analysis of allele-specific gene expression.

Application of Whole Exome Sequencing to Identify Disease-Causing Variants in Inherited Human Diseases

The recent advent of next-generation sequencing technologies has dramatically changed the nature of biomedical research. Human genetics is no exception-it has never been easier to interrogate human patient genomes at the nucleotide level to identify disease-associated variants. To further facilitate the efficiency of this approach, whole exome sequencing (WES) was first developed in 2009. Over the past three years, multiple groups have demonstrated the power of WES through robust disease-associated variant discoveries across a diverse spectrum of human diseases. Here, we review the application of WES to different types of inherited human diseases and discuss analytical challenges and possible solutions, with the aim of providing a practical guide for the effective use of this technology.

Making a definitive diagnosis: Successful clinical application of whole exome sequencing in a child with intractable inflammatory bowel disease

PurposeWe report a male child who presented at 15 months with perianal abscesses and proctitis, progressing to transmural pancolitis with colocutaneous fistulae, consistent with a Crohn disease-like illness. The age and severity of the presentation suggested an underlying immune defect; however, despite comprehensive clinical evaluation, we were unable to arrive at a definitive diagnosis, thereby restricting clinical management. MethodsWe sought to identify the causative mutation(s) through exome sequencing to provide the necessary additional information required for clinical management. ResultsAfter sequencing, we identified 16,124 variants. Subsequent analysis identified a novel, hemizygous missense mutation in the X-linked inhibitor of apoptosis gene, substituting a tyrosine for a highly conserved and functionally important cysteine. X-linked inhibitor of apoptosis was not previously associated with Crohn disease but has a central role in the proinflammatory response and bacterial sensing through the NOD signaling pathway. The mutation was confirmed by Sanger sequencing in a licensed clinical laboratory. Functional assays demonstrated an increased susceptibility to activation-induced cell death and defective responsiveness to NOD2 ligands, consistent with loss of normal X-linked inhibitor of apoptosis protein function in apoptosis and NOD2 signaling. ConclusionsBased on this medical history, genetic and functional data, the child was diagnosed as having an X-linked inhibitor of apoptosis deficiency. Based on this finding, an allogeneic hematopoietic progenitor cell transplant was performed to prevent the development of life-threatening hemophagocytic lymphohistiocytosis, in concordance with the recommended treatment for X-linked inhibitor of apoptosis deficiency. At >42 days posttransplant, the child was able to eat and drink, and there has been no recurrence of gastrointestinal disease, suggesting this mutation also drove the gastrointestinal disease. This report describes the identification of a novel cause of inflammatory bowel disease. Equally importantly, it demonstrates the power of exome sequencing to render a molecular diagnosis in an individual patient in the setting of a novel disease, after all standard diagnoses were exhausted, and illustrates how this technology can be used in a clinical setting. Genet Med 2011:13(3):255–262.

Top Advances in Functional Genomics and Translational Biology for 2010

In recent years, major advances have been made in transcriptomics, metabolomics, proteomics, and particularly population genetics. More than 850 genome-wide association studies (GWAS) were published by the end of 2010, now covering a vast number of diseases and phenotypes and often incorporating tens of thousands of subjects or, in some cases, more than 100 000 subjects, increasing statistical power to identify and replicate contributing loci. Sequencing throughput advances and decreasing costs per sample have allowed the application of genome-wide genotyping and sequencing technology to larger DNA samples as well as transcriptome and epigenome features. This year was marked not only by continuing progress with GWAS, sequencing, and copy number variation studies but also by follow-up of GWAS and other findings with functional studies that uncover new molecular pathways contributing to disease and pave the way for future clinical applications. The American Heart Association Functional Genomics and Translational Biology Interdisciplinary Council (http://www.americanheart.org/presenter.jhtml?identifier=3016539) provides a forum for a multidisciplinary group of volunteers committed to making a substantial contribution to reduce the burden of heart disease and stroke through genetics, genomics, proteomics, and metabolomics research and translational science. With input from the Early Career Committee of the Council on Functional Genomics and Translational Biology, we considered many papers and selected 10 outstanding manuscript groups (15 articles) published in 2010 that reflect these advances, and we briefly summarize each below in chronological order.