Exome Sequencing as a Diagnostic Tool for Pediatric‐Onset Ataxia

Ataxia demonstrates substantial phenotypic and genetic heterogeneity. We set out to determine the diagnostic yield of exome sequencing in pediatric patients with ataxia without a molecular diagnosis after standard‐of‐care assessment in Canada. FORGE (Finding Of Rare disease GEnes) Canada is a nation‐wide project focused on identifying novel disease genes for rare pediatric diseases using whole‐exome sequencing. We retrospectively selected all FORGE Canada projects that included cerebellar ataxia as a feature. We identified 28 such families and a molecular diagnosis was made in 13; a success rate of 46%. In 11 families, we identified mutations in genes associated with known neurological syndromes and in two we identified novel disease genes. Exome analysis of sib pairs and/or patients born to consanguineous parents was more likely to be successful (9/13) than simplex cases (4/15). Our data suggest that exome sequencing is an effective first line test for pediatric patients with ataxia where a specific single gene is not immediately suspected to be causative.     

Diagnostic Exome Sequencing to Elucidate the Genetic Basis of Likely Recessive Disorders in Consanguineous Families

Rare, atypical, and undiagnosed autosomal‐recessive disorders frequently occur in the offspring of consanguineous couples. Current routine diagnostic genetic tests fail to establish a diagnosis in many cases. We employed exome sequencing to identify the underlying molecular defects in patients with unresolved but putatively autosomal‐recessive disorders in consanguineous families and postulated that the pathogenic variants would reside within homozygous regions. Fifty consanguineous families participated in the study, with a wide spectrum of clinical phenotypes suggestive of autosomal‐recessive inheritance, but with no definitive molecular diagnosis. DNA samples from the patient(s), unaffected sibling(s), and the parents were genotyped with a 720K SNP array. Exome sequencing and array CGH (comparative genomic hybridization) were then performed on one affected individual per family. High‐confidence pathogenic variants were found in homozygosity in known disease‐causing genes in 18 families (36%) (one by array CGH and 17 by exome sequencing), accounting for the clinical phenotype in whole or in part. In the remainder of the families, no causative variant in a known pathogenic gene was identified. Our study shows that exome sequencing, in addition to being a powerful diagnostic tool, promises to rapidly expand our knowledge of rare genetic Mendelian disorders and can be used to establish more detailed causative links between mutant genotypes and clinical phenotypes.     

Hidden Mutations in Cornelia de Lange Syndrome Limitations of Sanger Sequencing in Molecular Diagnostics

Cornelia de Lange syndrome (CdLS) is a well‐characterized developmental disorder. The genetic cause of CdLS is a mutation in one of five associated genes (NIPBL, SMC1A, SMC3, RAD21, and HDAC8) accounting for about 70% of cases. To improve our current molecular diagnostic and to analyze some of CdLS candidate genes, we developed and established a gene panel approach. Because recent data indicate a high frequency of mosaic NIPBL mutations that were not detected by conventional sequencing approaches of blood DNA, we started to collect buccal mucosa (BM) samples of our patients that were negative for mutations in the known CdLS genes. Here, we report the identification of three mosaic NIPBL mutations by our high‐coverage gene panel sequencing approach that were undetected by classical Sanger sequencing analysis of BM DNA. All mutations were confirmed by the use of highly sensitive SNaPshot fragment analysis using DNA from BM, urine, and fibroblast samples. In blood samples, we could not detect the respective mutation. Finally, in fibroblast samples from all three patients, Sanger sequencing could identify all the mutations. Thus, our study highlights the need for highly sensitive technologies in molecular diagnostic of CdLS to improve genetic diagnosis and counseling of patients and their families.     

Exome sequencing is a useful diagnostic tool for complicated forms of hereditary spastic paraplegia

Hereditary spastic paraplegias constitute a heterogeneous group of neurodegenerative diseases encompassing pure and complicated forms, for which at least 52 loci and 31 causative genes have been identified. Although mutations in the SPAST gene explain approximately 40% of the pure autosomal dominant forms, molecular diagnosis can be challenging for the sporadic and recessive forms, which are often complicated and clinically overlap with a broad number of movement disorders. The validity of exome sequencing as a routine diagnostic approach in the movement disorder clinic needs to be assessed. The main goal of this study was to explore the usefulness of an exome analysis for the diagnosis of a complicated form of spastic paraplegia. Whole‐exome sequencing was performed in two Spanish siblings with a neurodegenerative syndrome including upper and lower motor neuron, ocular and cerebellar signs. Exome sequencing revealed that both patients carry a novel homozygous nonsense mutation in exon 15 of the SPG11 gene (c.2678G>A; p.W893X), which was not found in 584 Spanish control chromosomes. After many years of follow‐up and multiple time‐consuming genetic testing, we were able to diagnose these patients by making use of whole‐exome sequencing, showing that this is a cost‐efficient diagnostic tool for the movement disorder specialist.     

Consecutive medical exome analysis at a tertiary center: Diagnostic and health‐economic outcomes

The utility of whole exome analysis has been extensively demonstrated in research settings, but its clinical utility as a first‐tier genetic test has not been well documented from diagnostic and health economic standpoints in real‐life clinical settings. We performed medical exome analyses focusing on a clinically interpretable portion of the genome (4,813 genes) as a first‐tier genetic test for 360 consecutive patients visiting a genetics clinic at a tertiary children's hospital in Japan, over a 3‐year period. Bioinformatics analyses were conducted using standard software. A molecular diagnosis was made in 171 patients involving a total of 107 causative genes. Among these 107 causative genes, 57 genes were classified as genes with potential organ‐specific interventions and management strategies. Clinically relevant results were obtained in 26% of the total cohort and 54% of the patients with a definitive molecular diagnosis. Performing the medical exome analysis at the time of the initial visit to the tertiary center, rather than after visits to pertinent specialists, brain MRI examination, and G‐banded chromosome testing, would have reduced the financial cost by 197 euros according to retrospective calculation under multiple assumption. The present study demonstrated a high diagnostic yield (47.5%) for singleton medical exome analysis as a first‐tier test in a real‐life setting. Medical exome analysis yielded clinically relevant information in a quarter of the total patient cohort. The application of genomic testing during the initial visit to a tertiary medical center could be a rational approach to the diagnosis of patients with suspected genetic disorders.     

Targeted Next‐Generation Sequencing can Replace Sanger Sequencing in Clinical Diagnostics

Mutation detection through exome sequencing allows simultaneous analysis of all coding sequences of genes. However, it cannot yet replace Sanger sequencing (SS) in diagnostics because of incomplete representation and coverage of exons leading to missing clinically relevant mutations. Targeted next‐generation sequencing (NGS), in which a selected fraction of genes is sequenced, may circumvent these shortcomings. We aimed to determine whether the sensitivity and specificity of targeted NGS is equal to those of SS. We constructed a targeted enrichment kit that includes 48 genes associated with hereditary cardiomyopathies. In total, 84 individuals with cardiomyopathies were sequenced using 151 bp paired‐end reads on an Illumina MiSeq sequencer. The reproducibility was tested by repeating the entire procedure for five patients. The coverage of ≥30 reads per nucleotide, our major quality criterion, was 99% and in total ～21,000 variants were identified. Confirmation with SS was performed for 168 variants (155 substitutions, 13 indels). All were confirmed, including a deletion of 18 bp and an insertion of 6 bp. The reproducibility was nearly 100%. We demonstrate that targeted NGS of a disease‐specific subset of genes is equal to the quality of SS and it can therefore be reliably implemented as a stand‐alone diagnostic test.     

Novel mosaic variants in two patients with Cornelia de Lange syndrome

Cornelia de Lange syndrome (CdLS) is a dominantly inherited developmental disorder caused by mutations in genes that encode for either structural (SMC1A, SMC3, RAD21) or regulatory (NIPBL, HDAC8) subunits of the cohesin complex. NIPBL represents the major gene of the syndrome and heterozygous mutations can be identified in more than 65% of patients. Interestingly, large portions of these variants were described as somatic mosaicism and often escape standard molecular diagnostics using lymphocyte DNA.Here we discuss the role of somatic mosaicism in CdLS and describe two additional patients with NIPBL mosaicism detected by targeted gene panel or exome sequencing. In order to verify the next generation sequencing data, Sanger sequencing or pyrosequencing on DNA extracted from different tissues were applied. None of the pathogenic variants was originally detected by Sanger sequencing on blood DNA.Patient 1 displays an unusual combination of clinical features: he is cognitively only mildly affected, but shows severe limb reduction defects. Patient 2 presents with a moderate phenotype. Interestingly, Sanger sequencing analysis on fibroblast DNA of this patient did not detect the disease-causing variant previously observed on the same DNA sample by exome sequencing. Subsequent analyses could confirm the variants by Sanger sequencing on buccal mucosa DNA. Notably, this is the first report of a higher mutational load in buccal mucosa than in fibroblast cells of a CdLS patient.Detection of low-level mosaicism is of utmost importance for an accurate molecular diagnosis and a proper genetic counseling of patients with a clinical diagnosis of CdLS. Next-generation sequencing technologies greatly facilitate the detection of low-level mosaicism, which might otherwise remain undetected by conventional sequencing approaches.     

基于靶基因文库的高通量测序平台建立肥厚型心肌病患者致病基因突变检测方法

目的 基于靶基因文库,应用下一代半导体高通量测序平台,建立快速、 准确的肥厚型心肌病(hypertrophic cardiomyopathy,HCM)常见致病基因突变检测方法 ,有利于HCM患者的早期预防及临床分子诊断.方法 选择国内外公认的与HCM致病相关的常见基因(MYH7、MYBPC3、TNNT2、TNNI3、A...     

Performant Mutation Identification Using Targeted Next‐Generation Sequencing of 14 Thoracic Aortic Aneurysm Genes

At least 14 causative genes have been identified for both syndromic and nonsyndromic forms of thoracic aortic aneurysm/dissection (TAA), an important cause of death in the industrialized world. Molecular confirmation of the diagnosis is increasingly important for gene‐tailored patient management but consecutive, conventional molecular TAA gene screening is expensive and labor‐intensive. To circumvent these problems, we developed a TAA gene panel for next‐generation sequencing of 14 TAA genes. After validation, we applied the assay to 100 Marfan patients. We identified 90 FBN1 mutations, 44 of which were novel. In addition, Multiplex ligation‐dependent probe amplification identified large deletions in six of the remaining samples, whereas false‐negative results were excluded by Sanger sequencing of FBN1, TGFBR1, and TGFBR2 in the last four samples. Subsequently, we screened 55 syndromic and nonsyndromic TAA patients. We identified causal mutations in 15 patients (27%), one in each of the six following genes: ACTA2, COL3A1, TGFBR1, MYLK, SMAD3, SLC2A10 (homozygous), two in NOTCH1, and seven in FBN1. We conclude that our approach for TAA genetic testing overcomes the intrinsic hurdles of consecutive Sanger sequencing of all candidate genes and provides a powerful tool for the elaboration of clinical phenotypes assigned to different genes.     

Effective Immunological Guidance of Genetic Analyses Including Exome Sequencing in Patients Evaluated for Hemophagocytic Lymphohistiocytosis

We report our experience in using flow cytometry-based immunological screening prospectively as a decision tool for the use of genetic studies in the diagnostic approach to patients with hemophagocytic lymphohistiocytosis (HLH). We restricted genetic analysis largely to patients with abnormal immunological screening, but included whole exome sequencing (WES) for those with normal findings upon Sanger sequencing. Among 290 children with suspected HLH analyzed between 2010 and 2014 (including 17 affected, but asymptomatic siblings), 87/162 patients with “full” HLH and 79/111 patients with “incomplete/atypical” HLH had normal immunological screening results. In 10 patients, degranulation could not be tested. Among the 166 patients with normal screening, genetic analysis was not performed in 107 (all with uneventful follow-up), while 154 single gene tests by Sanger sequencing in the remaining 59 patients only identified a single atypical CHS patient. Flow cytometry correctly predicted all 29 patients with FHL-2, XLP1 or 2. Among 85 patients with defective NK degranulation (including 13 asymptomatic siblings), 70 were Sanger sequenced resulting in a genetic diagnosis in 55 (79%). Eight patients underwent WES, revealing mutations in two known and one unknown cytotoxicity genes and one metabolic disease. FHL3 was the most frequent genetic diagnosis. Immunological screening provided an excellent decision tool for the need and depth of genetic analysis of HLH patients and provided functionally relevant information for rapid patient classification, contributing to a significant reduction in the time from diagnosis to transplantation in recent years.     

Diagnostic exome sequencing in early‐onset Parkinson's disease confirms VPS13C as a rare cause of autosomal‐recessive Parkinson's disease

Parkinson's disease (PD) is a genetically heterogeneous disorder and new putative disease genes are discovered constantly. Therefore, whole‐exome sequencing could be an efficient approach to genetic testing in PD. To evaluate its performance in early‐onset sporadic PD, we performed diagnostic exome sequencing in 80 individuals with manifestation of PD symptoms at age 40 or earlier and a negative family history of PD. Variants in validated and candidate disease genes and risk factors for PD and atypical Parkinson syndromes were annotated, followed by further analysis for selected variants. We detected pathogenic variants in Mendelian genes in 6.25% of cases and high‐impact risk factor variants in GBA in 5% of cases, resulting in overall maximum diagnostic yield of 11.25%. One individual was compound heterozygous for variants affecting canonical splice sites in VPS13C, confirming the causal role of protein‐truncating variants in this gene linked to autosomal‐recessive early‐onset PD. Despite the low diagnostic yield of exome sequencing in sporadic early‐onset PD, the confirmation of the recently discovered VPS13C gene highlights its advantage over using predefined gene panels.     

Contribution of high‐throughput DNA sequencing to the study of primary immunodeficiencies

Primary immunodeficiencies (PIDs) are inborn errors of the immune system. PIDs have been characterized immunologically for the last 60 years and genetically, principally by Sanger DNA sequencing, over the last 30 years. The advent of next‐generation sequencing (NGS) in 2011, with the development of whole‐exome sequencing in particular, has facilitated the identification of previously unknown genetic lesions. NGS is rapidly generating a stream of candidate variants for an increasing number of genetically undefined PIDs. The use of NGS technology is ushering in a new era, by facilitating the discovery and characterization of new PIDs in patients with infections and other phenotypes, thereby helping to improve diagnostic accuracy. This review provides a historical overview of the identification of PIDs before NGS, and the advances and limitations of the use of NGS for the diagnosis and characterization of PIDs.     

Enhanced utility of family-centered diagnostic exome sequencing with inheritance model–based analysis: results from 500 unselected families with undiagnosed genetic conditions

PurposeDiagnostic exome sequencing was immediately successful in diagnosing patients in whom traditional technologies were uninformative. Herein, we provide the results from the first 500 probands referred to a clinical laboratory for diagnostic exome sequencing.MethodsFamily-based exome sequencing included whole-exome sequencing followed by family inheritance−based model filtering, comprehensive medical review, familial cosegregation analysis, and analysis of novel genes.ResultsA positive or likely positive result in a characterized gene was identified in 30% of patients (152/500). A novel gene finding was identified in 7.5% of patients (31/416). The highest diagnostic rates were observed among patients with ataxia, multiple congenital anomalies, and epilepsy (44, 36, and 35%, respectively). Twenty-three percent of positive findings were within genes characterized within the past 2 years. The diagnostic rate was significantly higher among families undergoing a trio (37%) as compared with a singleton (21%) whole-exome testing strategy.ConclusionOverall, we present results from the largest clinical cohort of diagnostic exome sequencing cases to date. These data demonstrate the utility of family-based exome sequencing and analysis to obtain the highest reported detection rate in an unselected clinical cohort, illustrating the utility of diagnostic exome sequencing as a transformative technology for the molecular diagnosis of genetic disease.Genet Med 17 7, 578–586.     

Highly recurrent H3F3A mutations with additional epigenetic regulator alterations in giant cell tumor of bone

Recurrent H3F3A and IDH2 mutations have been reported in giant cell tumor of bone (GCTB). However, the reported incidences have varied, and other molecular genetic alterations have not been identified due to the small number of cases analyzed with comprehensive methods. Moreover, the relative sensitivities of Sanger sequencing and next‐generation sequencing (NGS) for the detection of H3F3A mutations in DNA extracted from archival formalin‐fixed paraffin‐embedded (FFPE) samples for clinical diagnosis have not been assessed. To address these issues, we conducted whole‐exome sequencing of 7 GCTBs and integrated the previously published genomic sequencing data of 6 GCTBs. We subsequently performed targeted sequencing of an additional 39 GCTBs, including 2 atypical cases and an extremely rare case of primary malignant transformation of GCTB. We also evaluated the sensitivity of Sanger sequencing for detecting H3F3A mutations in FFPE samples that are usually used for clinical diagnosis. H3F3A glycine hotspot mutations were the most frequently detected mutations (96%) in the 52 GCTBs by NGS. Of the 50 hotspot mutations, p.G34W was observed in 48 cases and p.G34L/G34R was detected in one. One of two atypical GCTB cases with wild‐type H3F3A had a H3F3B mutation (p.G34V). Other mutated genes were not recurrent. Sanger sequencing did not detect H3F3A mutations in 10 of 15 H3F3A NGS mutation‐positive FFPE samples. In conclusion, we confirmed that H3F3A is the most frequently mutated GCTB driver gene, and that H3F3A mutations are not present in atypical GCTBs. Sanger sequencing was much less sensitive than targeted NGS for detecting H3F3A mutations in FFPE samples.     

Network‐Informed Gene Ranking Tackles Genetic Heterogeneity in Exome‐Sequencing Studies of Monogenic Disease

Genetic heterogeneity presents a significant challenge for the identification of monogenic disease genes. Whole‐exome sequencing generates a large number of candidate disease‐causing variants and typical analyses rely on deleterious variants being observed in the same gene across several unrelated affected individuals. This is less likely to occur for genetically heterogeneous diseases, making more advanced analysis methods necessary. To address this need, we present HetRank, a flexible gene‐ranking method that incorporates interaction network data. We first show that different genes underlying the same monogenic disease are frequently connected in protein interaction networks. This motivates the central premise of HetRank: those genes carrying potentially pathogenic variants and whose network neighbors do so in other affected individuals are strong candidates for follow‐up study. By simulating 1,000 exome sequencing studies (20,000 exomes in total), we model varying degrees of genetic heterogeneity and show that HetRank consistently prioritizes more disease‐causing genes than existing analysis methods. We also demonstrate a proof‐of‐principle application of the method to prioritize genes causing Adams‐Oliver syndrome, a genetically heterogeneous rare disease. An implementation of HetRank in R is available via the Website http://sourceforge.net/p/hetrank/ .     

Classification of Genes: Standardized Clinical Validity Assessment of Gene–Disease Associations Aids Diagnostic Exome Analysis and Reclassifications

Ascertaining a diagnosis through exome sequencing can provide potential benefits to patients, insurance companies, and the healthcare system. Yet, as diagnostic sequencing is increasingly employed, vast amounts of human genetic data are produced that need careful curation. We discuss methods for accurately assessing the clinical validity of gene–disease relationships to interpret new research findings in a clinical context and increase the diagnostic rate. The specifics of a gene–disease scoring system adapted for use in a clinical laboratory are described. In turn, clinical validity scoring of gene–disease relationships can inform exome reporting for the identification of new or the upgrade of previous, clinically relevant gene findings. Our retrospective analysis of all reclassification reports from the first 4 years of diagnostic exome sequencing showed that 78% were due to new gene–disease discoveries published in the literature. Among all exome positive/likely positive findings in characterized genes, 32% were in genetic etiologies that were discovered after 2010. Our data underscore the importance and benefits of active and up‐to‐date curation of a gene–disease database combined with critical clinical validity scoring and proactive reanalysis in the clinical genomics era.     

Nonoptical Massive Parallel DNA Sequencing of BRCA1 and BRCA2 Genes in a Diagnostic Setting

The introduction of the benchtop massive parallel sequencers made it possible for the majority of clinical diagnostic laboratories to gain access to this fast evolving technology. In this study, using the Ion Torrent Personal Genome Machine, we present a strategy for the molecular diagnosis of hereditary breast and ovarian cancer and respective analytical validation. The methodology relies on a multiplex PCR amplification of the BRCA1 and BRCA2 genes combined with a variant prioritization pipeline, designed to minimize the number of false‐positive calls without the introduction of false‐negative results. A training set of samples was used to optimize the entire process, and a second set was used to validate and independently evaluate the performance of the workflow. Performing the study in a blind manner relative to the variants in the samples and using conventional Sanger sequencing as standard, the workflow resulted in a strategy with a maximum analytical sensitivity ≥98.6% with a confidence of 95% and a specificity of 96.9%. Importantly, no true variant was missed. This study presents a comprehensive massive parallel sequencing–Sanger sequencing based strategy, which results in a high analytical sensitivity assay that provides a time‐ and cost‐effective strategy for the identification of mutations in the BRCA1 and BRCA2 genes.