Practical considerations in the clinical application of whole‐exome sequencing

Despite the exciting advent of whole‐exome sequencing (WES) in medical genetics practices, the optimal interpretation of results requires further actions such as reconsidering clinical information and obtaining further laboratory testing. There are no published data to guide clinicians in this process. In a retrospective study on 93 patients who underwent clinical WES, we set out to assess and resolve these practical challenges. With the laboratories reporting a molecular diagnostic rate of 25.8%, the medical geneticists and the laboratories were 90% concordant in their interpretation of the WES results. Divergence occurred when the medical geneticist reconsidered clinical information and/or additional information regarding pathogenicity of a variant. Variants of uncertain significance were reported in 86% of patients, with 53.7% needing follow‐up, such as additional laboratory tests and genotyping of family members. By layering clinical data (e.g. mode of inheritance and phenotypic fit) on to the laboratory results, we developed clinical categories for the WES results. These categories of definite diagnosis (14/93), likely diagnosis (8/93), possible diagnosis (13/93) and no diagnosis (58/93) could be used to convey results to patients uniformly. Our framework for a clinically informed interpretation of the results enhances the utility of WES within medical genetics practices.     

Diagnostic odyssey in severe neurodevelopmental disorders: toward clinical whole‐exome sequencing as a first‐line diagnostic test

The current standard of care for diagnosis of severe intellectual disability (ID) and epileptic encephalopathy (EE) results in a diagnostic yield of ～50%. Affected individuals nonetheless undergo multiple clinical evaluations and low‐yield laboratory tests often referred to as a ‘diagnostic odyssey’. This study was aimed at assessing the utility of clinical whole‐exome sequencing (WES) in individuals with undiagnosed and severe forms of ID and EE, and the feasibility of its implementation in routine practice by a small regional genetic center. We performed WES in a cohort of 43 unrelated individuals with undiagnosed ID and/or EE. All individuals had undergone multiple clinical evaluations and diagnostic tests over the years, with no definitive diagnosis. Sequencing data analysis and interpretation were carried out at the local molecular genetics laboratory. The diagnostic rate of WES reached 32.5% (14 out of 43 individuals). Genetic diagnosis had a direct impact on clinical management in four families, including a prenatal diagnostic test in one family. Our data emphasize the clinical utility and feasibility of WES in individuals with undiagnosed forms of ID and EE and highlight the necessity of close collaborations between ordering physicians, molecular geneticists, bioinformaticians and researchers for accurate data interpretation.     

Somatic copy number alterations by whole‐exome sequencing implicates YWHAZ and PTK2 in castration‐resistant prostate cancer

Castration‐resistant prostate cancer (CRPC) is the most aggressive form of prostate cancer (PCa) and remains a significant therapeutic challenge. The key to the development of novel therapeutic targets for CRPC is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to identify therapeutic targets for CRPC by assessing somatic copy number alterations (SCNAs) by whole‐exome sequencing on five CRPC/normal paired formalin‐fixed paraffin‐embedded (FFPE) samples, using the SOLiD4 next‐generation sequencing (NGS) platform. Data were validated using fluorescence in situ hybridization (FISH) on a PCa progression cohort. PTK2 and YWHAZ amplification, mRNA and protein expression were determined in selected PCa cell lines. Effects of PTK2 inhibition using TAE226 inhibitor and YWHAZ knock‐down on cell proliferation and migration were tested in PC3 cells in vitro. In a larger validation cohort, the amplification frequency of YWHAZ was 3% in localized PCa and 48% in CRPC, whereas PTK2 was amplified in 1% of localized PCa and 35% in CRPC. YWHAZ knock‐down and PTK2 inhibition significantly affected cell proliferation and migration in the PC3 cells. Our findings suggest that inhibition of YWHAZ and PTK2 could delay the progression of the disease in CRPC patients harbouring amplification of the latter genes. Furthermore, our validated whole‐exome sequencing data show that FFPE tissue could be a promising alternative for SCNA screening using next‐generation sequencing technologies. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Utility of whole‐exome sequencing for those near the end of the diagnostic odyssey: time to address gaps in care

An accurate diagnosis is an integral component of patient care for children with rare genetic disease. Recent advances in sequencing, in particular whole‐exome sequencing (WES), are identifying the genetic basis of disease for 25–40% of patients. The diagnostic rate is probably influenced by when in the diagnostic process WES is used. The Finding Of Rare Disease GEnes (FORGE) Canada project was a nation‐wide effort to identify mutations for childhood‐onset disorders using WES. Most children enrolled in the FORGE project were toward the end of the diagnostic odyssey. The two primary outcomes of FORGE were novel gene discovery and the identification of mutations in genes known to cause disease. In the latter instance, WES identified mutations in known disease genes for 105 of 362 families studied (29%), thereby informing the impact of WES in the setting of the diagnostic odyssey. Our analysis of this dataset showed that these known disease genes were not identified prior to WES enrollment for two key reasons: genetic heterogeneity associated with a clinical diagnosis and atypical presentation of known, clinically recognized diseases. What is becoming increasingly clear is that WES will be paradigm altering for patients and families with rare genetic diseases.     

Trio‐whole‐exome sequencing and preimplantation genetic diagnosis for unexplained recurrent fetal malformations

Whole‐exome sequencing (WES) is widely used to detect genetic mutations that cause Mendelian diseases, and has been successfully applied in combination with preimplantation genetic diagnosis (PGD) to avoid the transmission of genetic defects. We investigated 40 nonconsanguineous families with unexplained, recurrent fetal malformations (two or more malformed fetuses) from May 2016 to December 2018. Using Trio‐WES, we identified 32 disease‐associated variants in 40 families (80% positive rate), which were subsequently verified. Known Mendelian diseases were identified in 12 families (30%), highly suspected Mendelian diseases in 12 families (30%), variants with uncertain significance in 8 families (20%), and no noticeable variants for 8 families (20%). Further analysis showed variants in 22 genes may cause fetal malformations. Four gene variants were detected in fetuses for the first time, which expanded the spectrum of the disease phenotype. Two novel candidate genes may be related to fetal malformations. Of 26 couples receiving PGD on disease‐associated genes, 3 healthy newborns were delivered, and 4 couples are undergoing pregnancies. We reported the fetal data and developed an optimized genetic testing strategy. Our finding strongly suggests the presence of single gene Mendelian disorders in 60% of those families, and PGD services for couples to have healthy babies.     

Genetics professionals' perspectives on reporting incidental findings from clinical genome‐wide sequencing

Whole exome or whole genome analysis using massively parallel sequencing technologies will undoubtedly solve diagnostic dilemmas; however, incidental findings (IF) that may have medical and social implications will also be discovered. While there is consensus in the literature that analytically valid and medically actionable IF should be returned to patients if requested, there is debate regarding the return of other IF. There are currently no guidelines established for managing IF in the clinical context. We therefore distributed an online questionnaire to 496 geneticists and genetic counselors in Canada to explore this unresolved issue, and 210 professionals participated (response rate = 42%). The proportion of respondents who indicated that they would return IF to patients depended on the nature of the finding, ranging from 95% for information pertaining to a serious and treatable condition to 12% for information with only social implications (e.g., non‐paternity). There was a lack of consensus around the disclosure of certain IF such as genetic carrier status, especially for pediatric patients. The most important considerations identified as impacting IF disclosure included condition‐specific factors such as treatment availability, test accuracy, and evidence indicating pathogenicity. This is the first study to document the views of geneticists and genetic counselors in Canada towards the disclosure of IF, and represents a step towards evidence‐based guidelines for clinical genome‐wide sequencing investigations. © 2013 Wiley Periodicals, Inc.     

Personal genome sequencing: current approaches and challenges

The revolution in DNA sequencing technologies has now made it feasible to determine the genome sequences of many individuals; i.e., “personal genomes.” Genome sequences of cells and tissues from both normal and disease states have been determined. Using current approaches, whole human genome sequences are not typically assembled and determined de novo, but, instead, variations relative to a reference sequence are identified. We discuss the current state of personal genome sequencing, the main steps involved in determining a genome sequence (i.e., identifying single-nucleotide polymorphisms [SNPs] and structural variations [SVs], assembling new sequences, and phasing haplotypes), and the challenges and performance metrics for evaluating the accuracy of the reconstruction. Finally, we consider the possible individual and societal benefits of personal genome sequences.     

A novel heterozygous variant p.(Trp538Arg) of SYNM is identified by whole‐exome sequencing in a Chinese family with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a relatively frequent myocardial disease that may lead to heart failure, syncope, and sudden cardiac death. Genetic factors play important roles in the etiology of the disease. To date, at least 50 genes have been identified in patients with DCM, among them, only three mutations have been reported in Synemin (SYNM) gene. In this study, we investigate a Chinese family of three generations with four patients with DCM. Employing whole‐exome sequencing (WES) and bioinformatics strategies, a novel heterozygous missense mutation p.(Trp538Arg) of SYNM was identified and cosegregated with the affected family members. The missense mutation locates in the C‐terminal domain of SYNM and leads to a substitution of tryptophan by arginine and may cause the structure change of synemin protein. In conclusion, we employed WES to detect the mutations of DCM patients and identified a novel likely pathogenic mutation in SYNM gene. Our study not only expands the spectrum of SYNM mutations, it further confirms that mutations in SYMN may underlie nonfamilial DCM, and offers genetic testing information to additional DCM patients.     

Detection of mosaic variants using genome sequencing in a large pediatric cohort

The increased use of next-generation sequencing has expanded our understanding of the involvement and prevalence of mosaicism in genetic disorders. We describe a total of eleven cases: nine in which mosaic variants detected by genome sequencing (GS) and/or targeted gene panels (TGPs) were considered to be causative for the proband's phenotype, and two of apparent parental mosaicism. Variants were identified in the following genes: PHACTR1, SCN8A, KCNT1, CDKL5, NEXMIF, CUX1, TSC2, GABRB2, and SMARCB1. In addition, we identified one large duplication including three genes, UBE3A, GABRB3, and MAGEL2, and one large deletion including deletion of ARFGAP1, EEF1A2, CHRNA4, and KCNQ2. All patients were enrolled in the NYCKidSeq study, a research program studying the communication of genomic information in clinical care, as well as the clinical utility and diagnostic yield of GS for children with suspected genetic disorders in diverse populations in New York City. We observed variability in the correlation between reported variant allele fraction and the severity of the patient's phenotype, although we were not able to determine the mosaicism percentage in clinically relevant tissue(s). Although our study was not sufficiently powered to assess differences in mosaicism detection between the two testing modalities, we saw a trend toward better detection by GS as compared with TGP testing. This case series supports the importance of mosaicism in childhood-onset genetic conditions and informs guidelines for laboratory and clinical interpretation of mosaic variants detected by GS.     

Using whole‐exome sequencing to investigate the genetic bases of lysosomal storage diseases of unknown etiology

Lysosomes are membrane‐bound, acidic eukaryotic cellular organelles that play important roles in the degradation of macromolecules. Mutations that cause the loss of lysosomal protein function can lead to a group of disorders categorized as the lysosomal storage diseases (LSDs). Suspicion of LSD is frequently based on clinical and pathologic findings, but in some cases, the underlying genetic and biochemical defects remain unknown. Here, we performed whole‐exome sequencing (WES) on 14 suspected LSD cases to evaluate the feasibility of using WES for identifying causal mutations. By examining 2,157 candidate genes potentially associated with lysosomal function, we identified eight variants in five genes as candidate disease‐causing variants in four individuals. These included both known and novel mutations. Variants were corroborated by targeted sequencing and, when possible, functional assays. In addition, we identified nonsense mutations in two individuals in genes that are not known to have lysosomal function. However, mutations in these genes could have resulted in phenotypes that were diagnosed as LSDs. This study demonstrates that WES can be used to identify causal mutations in suspected LSD cases. We also demonstrate cases where a confounding clinical phenotype may potentially reflect more than one lysosomal protein defect.     

A familial study of twins with severe asthenozoospermia identified a homozygous SPAG17 mutation by whole‐exome sequencing

Asthenozoospermia (AZS) is a common cause of male infertility, characterized by abnormal reduction in the motility of ejaculated spermatozoa. Here, in a patient from a consanguineous family, we identified a homozygous mutation (c.G4343A, p.R1448Q) in SPAG17 by whole‐exome sequencing. The encoded protein, SPAG17, localizes to the axonemal central apparatus and is considered essential for flagellar waveform. In silico analysis revealed that R1448Q is a potential pathogenic mutation. Immunostaining and western blot assays showed that the R1448Q mutation may exert a negative effect on the steady‐state of the SPAG17 protein. Therefore, SPAG17 may be a new pathogenic gene causing AZS.     

Choices of incidental findings of individuals undergoing genome wide sequencing,a single center's experience

Genome wide sequencing is an emerging clinical tool that may provide information on genetic variants that are not directly related to the patient's primary disorder. These incidental findings (IFs) may include information about conditions that can be treated and may also indicate conditions for which treatments are not currently available. Data is currently limited regarding what IFs an individual would want to disclose. This study reports on 305 individual choices for return of IFs that were completed at the Medical College of Wisconsin's clinical sequencing laboratory. Individuals were given access to five categories of IFs to select from: no incidental findings, untreatable childhood disorders, treatable adulthood disorders, untreatable adulthood disorders, and carrier of a disorder. Retrospective chart review was conducted and individual choices were recorded and analyzed. The majority of individuals (76.1%) selected every IF to be reported, 14.4% wanted a subset of the options, and 9.5% did not want any IFs reported. This study contributes to the limited data that demonstrates what an individual would actually choose when undergoing genetic sequencing. Furthermore, this data supports the opinion that individuals want and utilize the ability to choose the findings reported.     

Whole exome and whole genome sequencing with dried blood spot DNA without whole genome amplification

Newborn screening (NBS) for rare conditions is performed in all 50 states in the USA. We have partnered with the California Department of Public Health Genetic Disease Laboratory to determine whether sufficient DNA can be extracted from archived dried blood spots (DBS) for next‐generation sequencing in the hopes that next‐generation sequencing can play a role in NBS. We optimized the DNA extraction and sequencing library preparation protocols for residual infant DBS archived over 20 years ago and successfully obtained acceptable whole exome and whole genome sequencing data. This sequencing study using DBS DNA without whole genome amplification prior to sequencing library preparation provides evidence that properly stored residual newborn DBS are a satisfactory source of DNA for genetic studies.     

Critical points for an accurate human genome analysis

Next‐generation sequencing is radically changing how DNA diagnostic laboratories operate. What started as a single‐gene profession is now developing into gene panel sequencing and whole‐exome and whole‐genome sequencing (WES/WGS) analyses. With further advances in sequencing technology and concomitant price reductions, WGS will soon become the standard and be routinely offered. Here, we focus on the critical steps involved in performing WGS, with a particular emphasis on points where WGS differs from WES, the important variables that should be taken into account, and the quality control measures that can be taken to monitor the process. The points discussed here, combined with recent publications on guidelines for reporting variants, will facilitate the routine implementation of WGS into a diagnostic setting.