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.     

Feasibility of whole-exome sequencing in fine-needle aspiration specimens of papillary thyroid microcarcinoma for the identification of novel gene mutations

Genetic profiling is important for assisting the management of papillary thyroid microcarcinoma (PTMC). Although whole-exome sequencing (WES) of surgically resected PTMC tissue has been performed and revealed potential prognostic biomarkers, its application in PTMC fine-needle aspiration (FNA) specimens has not been explored. This study aimed to evaluate the feasibility of WES using FNA specimens of PTMC. Five PTMC patients were enrolled with clinical characteristics gathered. Fine aspiration cytology needle (23 gauges) was used to collect FNA biopsy with ultrasound guidance. WES analysis of FNA specimens from five PTMC patients and matched blood samples was performed. The WES of FNA samples yielded an average sequencing depth of 281× and average coverage of 99.5%. We identified 534 somatic single-nucleotide variants and 13 indels in total, and per sample, we found a mean of 24 exonic mutations, which affected a total of 120 genes. In the PTMC FNA samples, the most frequently mutated genes were BRAF and ANKRD18B, and the four driver genes were BRAF, AFF3, SRCAP, and EGFR. We also identified several germline cancer predisposing gene mutations. The results suggest that WES of FNA specimens is feasible for PTMC and can identify novel genetic mutations.     

Whole exome and targeted gene sequencing to detect pathogenic recessive variants in early onset cerebellar ataxia

Over 100 genetically distinct causal known loci for hereditary ataxia phenotype poses a challenge for diagnostic work-up for ataxia patients in a clinically relevant time and precision. In the present study using next-generation sequencing, we have investigated pathogenic variants in early-onset cerebellar ataxia cases using whole exome sequencing in singleton/family-designed and targeted gene-panel sequencing. A total of 98 index patients were clinically and genetically (whole exome sequencing (WES) in 16 patients and targeted gene panel of 41 ataxia causing genes in 82 patients) evaluated. Four families underwent WES in family based design. Overall, we have identified 24 variants comprising 20 pathogenic and four likely-pathogenic both rare/novel, variations in 21 early onset cerebellar ataxia patients. Among the identified variations, SACS (n = 7) and SETX (n = 6) were frequent, while ATM (n = 2), TTPA (n = 2) and other rare loci were observed. We have prioritized novel pathogenic variants in RARS2 and FA2H loci through family based design in two out of four families.     

Using whole-exome sequencing to identify variants inherited from mosaic parents

Whole-exome sequencing (WES) has allowed the discovery of genes and variants causing rare human disease. This is often achieved by comparing nonsynonymous variants between unrelated patients, and particularly for sporadic or recessive disease, often identifies a single or few candidate genes for further consideration. However, despite the potential for this approach to elucidate the genetic cause of rare human disease, a majority of patients fail to realize a genetic diagnosis using standard exome analysis methods. Although genetic heterogeneity contributes to the difficulty of exome sequence analysis between patients, it remains plausible that rare human disease is not caused by de novo or recessive variants. Multiple human disorders have been described for which the variant was inherited from a phenotypically normal mosaic parent. Here we highlight the potential for exome sequencing to identify a reasonable number of candidate genes when dominant disease variants are inherited from a mosaic parent. We show the power of WES to identify a limited number of candidate genes using this disease model and how sequence coverage affects identification of mosaic variants by WES. We propose this analysis as an alternative to discover genetic causes of rare human disorders for which typical WES approaches fail to identify likely pathogenic variants.     

Detection of genomic deletions of PKP2 in arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited myocardial disease that predominantly affects the right ventricle and is associated with ventricular arrhythmias that may lead to sudden cardiac death. Mutations within at least seven separate genes have been identified to cause ARVC, however a genetic culprit remains elusive in approximately 50% of cases. Although negative genetic testing may be secondary to pathogenic mutations within undiscovered genes, an alternative explanation may be the presence of large deletions or duplications involving known genes. These large copy number variants may not be detected with standard clinical genetic testing which is presently limited to direct DNA sequencing. We describe two cases of ARVC possessing large deletions involving plakophilin‐2 (PKP2) identified with microarray analysis and/or multiplex ligation‐dependent probe amplification (MLPA) that would have been classified as genotype negative with standard clinical genetic testing. A deletion of the entire coding region of PKP2 excluding exon 1 was identified in patient 1 and his son. In patient 2, MLPA analysis of PKP2 revealed deletion of the entire gene with subsequent microarray analysis demonstrating a de novo 7.9 Mb deletion of chromosome 12p12.1p11.1. These findings support screening for large copy number variants in clinically suspected ARVC cases without clear disease causing mutations following initial sequencing analysis.     

An exome-wide exploration of cases of primary ovarian insufficiency uncovers novel sequence variants and candidate genes

Primary ovarian insufficiency (POI) implies the cessation of menstruation for several months in women before the age of 40 years and is a major cause of infertility. The study of the contribution of genetic factors to POI has been fueled by the use of whole exome sequencing (WES). Here, to uncover novel causative pathogenic variants and risk alleles, WES has been performed in 12 patients with familial POI (eight unrelated index cases and two pairs of sisters) and six women with early menopause and family history of POI (four index cases and one pair of sisters). Likely causative variants in NR5A1 and MCM9 genes were identified as well as a variant in INHA that requires further investigation. Moreover, we have identified more than one candidate variant in 3 out of 15 familial cases. Taken together, our results highlight the genetic heterogeneity of POI and early menopause and support the hypothesis of an oligogenic inheritance of such conditions, in addition to monogenic inheritance.     

Molecular diagnosis of putative stargardt disease probands by exome sequencing

ABSTRACT: BACKGROUND: The commonest genetic form of juvenile or early adult onset macular degeneration is Stargardt Disease (STGD) caused by recessive mutations in the gene ABCA4. However, high phenotypic and allelic heterogeneity and a small but non-trivial amount of locus heterogeneity currently impede conclusive molecular diagnosis in a significant proportion of cases. METHODS: We performed whole exome sequencing (WES) of nine putative Stargardt Disease probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Follow-up dideoxy sequencing was performed for confirmation and to screen for mutations in an additional set of affected individuals lacking a definitive molecular diagnosis. RESULTS: Whole exome sequencing revealed seven likely disease-causing variants across four genes, providing a confident genetic diagnosis in six previously uncharacterized participants. We identified four previously missed mutations in ABCA4 across three individuals. Likely disease-causing mutations in RDS/PRPH2, ELOVL, and CRB1 were also identified. CONCLUSIONS: Our findings highlight the enormous potential of whole exome sequencing in Stargardt Disease molecular diagnosis and research. WES adequately assayed all coding sequences and canonical splice sites of ABCA4 in this study. Additionally, WES enables the identification of disease-related alleles in other genes. This work highlights the importance of collecting parental genetic material for WES testing as the current knowledge of human genome variation limits the determination of causality between identified variants and disease. While larger sample sizes are required to establish the precision and accuracy of this type of testing, this study supports WES for inherited early onset macular degeneration disorders as an alternative to standard mutation screening techniques.     

Whole exome sequencing identifies multiple diagnoses in congenital glaucoma with systemic anomalies

The genetic basis of congenital glaucoma with systemic anomalies is largely unknown. Whole exome sequencing (WES) in 10 probands with congenital glaucoma and variable systemic anomalies identified pathogenic or likely pathogenic variants in three probands; in two of these, a combination of two Mendelian disorders was found to completely explain the patients' features whereas in the third case only the ocular findings could be explained by the genetic diagnosis. The molecular diagnosis for glaucoma included two cases with compound heterozygous or homozygous pathogenic alleles in CYP1B1 and one family with a dominant pathogenic variant in FOXC1; the second genetic diagnosis for the additional systemic features included compound heterozygous mutations in NPHS1 in one family and a heterozygous 18q23 deletion in another pedigree. These findings show the power of WES in the analysis of complex conditions and emphasize the importance of CYP1B1 screening in patients with congenital glaucoma regardless of the presence/absence of other systemic anomalies.     

Comprehensive analysis via exome sequencing uncovers genetic etiology in autosomal recessive nonsyndromic deafness in a large multiethnic cohort

PurposeAutosomal recessive nonsyndromic deafness (ARNSD) is characterized by a high degree of genetic heterogeneity, with reported mutations in 58 different genes. This study was designed to detect deafness-causing variants in a multiethnic cohort with ARNSD by using whole-exome sequencing (WES).MethodsAfter excluding mutations in the most common gene, GJB2, we performed WES in 160 multiplex families with ARNSD from Turkey, Iran, Mexico, Ecuador, and Puerto Rico to screen for mutations in all known ARNSD genes.ResultsWe detected ARNSD-causing variants in 90 (56%) families, 54% of which had not been previously reported. Identified mutations were located in 31 known ARNSD genes. The most common genes with mutations were MYO15A (13%), MYO7A (11%), SLC26A4 (10%), TMPRSS3 (9%), TMC1 (8%), ILDR1 (6%), and CDH23 (4%). Nine mutations were detected in multiple families with shared haplotypes, suggesting founder effects.ConclusionWe report on a large multiethnic cohort with ARNSD in which comprehensive analysis of all known ARNSD genes identifies causative DNA variants in 56% of the families. In the remaining families, WES allows us to search for causative variants in novel genes, thus improving our ability to explain the underlying etiology in more families.     

Using medical exome sequencing to identify the causes of neurodevelopmental disorders: Experience of 2 clinical units and 216 patients

Although whole‐exome sequencing (WES) is the gold standard for the diagnosis of neurodevelopmental disorders (NDDs), it remains expensive for some genetic centers. Commercialized panels comprising all OMIM‐referenced genes called “medical exome” (ME) constitute an alternative strategy to WES, but its efficiency is poorly known. In this study, we report the experience of 2 clinical genetic centers using ME for diagnosis of NDDs. We recruited 216 consecutive index patients with NDDs in 2 French genetic centers, corresponded to the daily practice of the units and included non‐syndromic intellectual disability (NSID, n = 33), syndromic ID (NSID = 122), pediatric neurodegenerative disorders (n = 7) and autism spectrum disorder (ASD, n = 54). We sequenced samples from probands and their parents (when available) with the Illumina TruSight One sequencing kit. We found pathogenic or likely pathogenic variants in 56 index patients, for a global diagnostic yield of 25.9%. The diagnosis yield was higher in patients with ID as the main diagnosis (32%) than in patients with ASD (3.7%). Our results suggest that the use of ME is a valuable strategy for patients with ID when WES cannot be used as a routine diagnosis tool.     

Genetic spectrum and clinical profiles in a southeast Chinese cohort of Charcot-Marie-Tooth disease

Charcot-Marie-Tooth (CMT) disease is a heterogeneous group of inherited sensorimotor neuropathies. To clarify the genetic spectrum and clinical profiles in Chinese CMT patients, we enrolled 150 unrelated CMT patients from southeast China. We performed multiplex ligation-dependent probe amplification (MLPA) testing in all patients and next-generation sequencing (NGS) among those patients without PMP22 rearrangements. We identified PMP22 duplications in 40 patients and deletions in 12 patients. In addition, we found 19 novel variants and 36 known mutations in 57 patients. Among these 55 variants, 45 pathogenic or likely pathogenic variants were identified in 48 cases, and 10 variants with uncertain significance were identified in 9 cases. Therefore, we obtained a genetic diagnosis in 63.8% (88/138) of CMT patients and 66.7% (100/150) of all included patients. PMP22, GJB1, and MFN2 are the most common causative genes in CMT1 (demyelinated form), intermediate CMT, and CMT2 (axonal form), respectively. In this study, we identified a higher proportion of intermediate CMT, a relatively high frequency of NDRG1 mutations and clinical features of later onset age in CMT1A patients. Our results broaden the genetic and clinical spectrum of CMT patients, which can help optimize the genetic and clinical diagnosis.     

Utilization of amplicon-based targeted sequencing panel for the massively parallel sequencing of sporadic hearing impairment patients from Saudi Arabia

Background

Hearing Impairment (HI) can have genetic or environmental causes and in some cases, an interplay of both. Genetic causes are difficult to determine as mutations in more than 90 genes have been shown recently to be responsible for HI. Providing a genetic diagnostic test for HI is therefore a challenge especially for ethnic groups where GJB2 mutations are shown to be rare.

Results

Here we show the design and implementation of an amplicon-based targeted sequencing panel that allows the simultaneous sequencing of 87 HI genes. Mutations identified included known pathogenic mutations and novel variants with unknown significance. The diagnostic rate of this panel is 28 % when only pathogenic variants were reported. However, an additional 28 % harbored recurrent combinations of novel or rare single nucleotide variants in the OTOF or PCDH15 genes. Such combinations were not identified in healthy individuals.

Conclusions

Targeted sequencing approach is a very useful strategy for the identification of mutations affecting the HI genes because of its relatively fast turn-around time and cost effectiveness compared to whole-exome sequencing. Further novel or rare variants could be identified by implementing a large-scale screening of HI using our panel which will eventual lead to a higher diagnostic rate.

     

Novel recessive PDZD7 biallelic mutations in two Chinese families with non‐syndromic hearing loss

Autosomal recessive non‐syndromic hearing loss (ARNSHL) is a highly heterogeneous genetic condition. PDZD7 has emerged as a new genetic etiology of ARNSHL. Biallelic mutations in the PDZD7 gene have been reported in two German families, four Iranian families, and a Pakistani family with ARNSHL. The effect of PDZD7 on ARNSHL in other population has yet to be elucidated. Two Chinese ARNSHL families, each of which had two affected siblings, were included in this study. The families underwent target region capture and high‐throughput sequencing to analyze the exonic, splice‐site, and intronic sequences of 128 genes. Furthermore, 1751 normal Chinese individuals served as controls, and 122 Chinese families segregating with apparent ARNSHL, who had been previously excluded for variants in the common deafness genes GJB2 and SLC26A4, were subjected to screening for candidate mutations. We identified a novel homozygous missense mutation (p.Arg66Leu) and novel compound heterozygous frameshift mutations (p.Arg56fsTer24 and p.His403fsTer36) in Chinese families with ARNSHL. This is the first report to identify PDZD7 as an ARNSHL‐associated gene in the Chinese population. Our finding could expand the pathogenic spectrum and strengthens the clinical diagnostic role of the PDZD7 gene in ARNSHL patients.     

Differential diagnosis of vacuolar myopathies in the NGS era

Altered autophagy accompanied by abnormal autophagic (rimmed) vacuoles detectable by light and electron microscopy is a common denominator of many familial and sporadic non‐inflammatory muscle diseases. Even in the era of next generation sequencing (NGS), late‐onset vacuolar myopathies remain a diagnostic challenge. We identified 32 adult vacuolar myopathy patients from 30 unrelated families, studied their clinical, histopathological and ultrastructural characteristics and performed genetic testing in index patients and relatives using Sanger sequencing and NGS including whole exome sequencing (WES). We established a molecular genetic diagnosis in 17 patients. Pathogenic mutations were found in genes typically linked to vacuolar myopathy (GNE, LDB3/ZASP, MYOT, DES and GAA), but also in genes not regularly associated with severely altered autophagy (FKRP, DYSF, CAV3, COL6A2, GYG1 and TRIM32) and in the digenic facioscapulohumeral muscular dystrophy 2. Characteristic histopathological features including distinct patterns of myofibrillar disarray and evidence of exocytosis proved to be helpful to distinguish causes of vacuolar myopathies. Biopsy validated the pathogenicity of the novel mutations p.(Phe55*) and p.(Arg216*) in GYG1 and of the p.(Leu156Pro) TRIM32 mutation combined with compound heterozygous deletion of exon 2 of TRIM32 and expanded the phenotype of Ala93Thr‐caveolinopathy and of limb‐girdle muscular dystrophy 2i caused by FKRP mutation. In 15 patients no causal variants were detected by Sanger sequencing and NGS panel analysis. In 12 of these cases, WES was performed, but did not yield any definite mutation or likely candidate gene. In one of these patients with a family history of muscle weakness, the vacuolar myopathy was eventually linked to chloroquine therapy. Our study illustrates the wide phenotypic and genotypic heterogeneity of vacuolar myopathies and validates the role of histopathology in assessing the pathogenicity of novel mutations detected by NGS. In a sizable portion of vacuolar myopathy cases, it remains to be shown whether the cause is hereditary or degenerative.     

Truncating mutations on myofibrillar myopathies causing genes as prevalent molecular explanations on patients with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure with high morbidity and mortality. More than 40 genes have been reported to cause DCM. To provide new insights into the pathophysiology of dilated cardiomyopathy, a next‐generation sequencing (NGS) workflow based on a panel of 48 cardiomyopathies‐causing genes was used to analyze a cohort of 222 DCM patients. Truncating variants were detected on 63 unrelated DCM cases (28.4%). Most of them were identified, as expected, on TTN (29 DCM probands), but truncating variants were also identified on myofibrillar myopathies causing genes in 17 DCM patients (7.7% of the DCM cohort): 10 variations on FLNC and 7 variations on BAG3 . This study confirms that truncating variants on myofibrillar myopathies causing genes are frequently associated with dilated cardiomyopathies and also suggest that FLNC mutations could be considered as a common cause of dilated cardiomyopathy. Molecular approaches that would allow to detect systematically truncating variants in FLNC and BAG3 into genetic testing should significantly increase test sensitivity, thereby allowing earlier diagnosis and therapeutic intervention for many patients with dilated cardiomyopathy.     

Whole exome sequencing is an efficient,sensitive and specific method for determining the genetic cause of short‐rib thoracic dystrophies

Short‐rib thoracic dystrophies (SRTDs) are congenital disorders due to defects in primary cilium function. SRTDs are recessively inherited with mutations identified in 14 genes to date (comprising 398 exons). Conventional mutation detection (usually by iterative Sanger sequencing) is inefficient and expensive, and often not undertaken. Whole exome massive parallel sequencing has been used to identify new genes for SRTD (WDR34, WDR60 and IFT172); however, the clinical utility of whole exome sequencing (WES) has not been established. WES was performed in 11 individuals with SRTDs. Compound heterozygous or homozygous mutations were identified in six confirmed SRTD genes in 10 individuals (IFT172, DYNC2H1, TTC21B, WDR60, WDR34 and NEK1), giving overall sensitivity of 90.9%. WES data from 993 unaffected individuals sequenced using similar technology showed two individuals with rare (minor allele frequency <0.005) compound heterozygous variants of unknown significance in SRTD genes (specificity >99%). Costs for consumables, laboratory processing and bioinformatic analysis were <AU$850 per sample. WES is sensitive, specific, efficient and cost‐effective for mutation screening as well as gene discovery in SRTDs and can be considered a first‐line methodology for mutation identification in affected individuals.     

Expanding the genotype–phenotype correlation of de novo heterozygous missense variants in YWHAG as a cause of developmental and epileptic encephalopathy

Developmental and Epileptic encephalopathies (DEE) describe heterogeneous epilepsy syndromes, characterized by early‐onset, refractory seizures and developmental delay (DD). Several DEE associated genes have been reported. With increased access to whole exome sequencing (WES), new candidate genes are being identified although there are fewer large cohort papers describing the clinical phenotype in such patients. We describe 6 unreported individuals and provide updated information on an additional previously reported individual with heterozygous de novo missense variants in YWHAG. We describe a syndromal phenotype, report 5 novel, and a recurrent p.Arg132Cys YWHAG variant and compare developmental trajectory and treatment strategies in this cohort. We provide further evidence of causality in YWHAG variants. WES was performed in five patients via Deciphering Developmental Disorders Study and the remaining two were identified via Genematcher and AnnEX databases. De novo variants identified from exome data were validated using Sanger sequencing. Seven out of seven patients in the cohort have de novo, heterozygous missense variants in YWHAG including 2/7 patients with a recurrent c.394C > T, p.Arg132Cys variant; 1/7 has a second, pathogenic variant in STAG1. Characteristic features included: early‐onset seizures, predominantly generalized tonic–clonic and absence type (7/7) with good response to standard anti‐epileptic medications; moderate DD; Intellectual Disability (ID) (5/7) and Autism Spectrum Disorder (3/7). De novo YWHAG missense variants cause EE, characterized by early‐onset epilepsy, ID and DD, supporting the hypothesis that YWHAG loss‐of‐function causes a neurological phenotype. Although the exact mechanism of disease resulting from alterations in YWHAG is not fully known, it is possible that haploinsufficiency of YWHAG in developing cerebral cortex may lead to abnormal neuronal migration resulting in DEE.     

Genetic variants in DGAT1 cause diverse clinical presentations of malnutrition through a specific molecular mechanism

BackgroundDGAT1, a gene encoding a protein involved in lipid metabolism, has been recently implicated in causing a rare nutritional and digestive disease presenting as Congenital Diarrheal Disorder (CDD). Genetic causes of malnutrition can be classified as metabolic disorders, caused by loss of a specific enzyme's function. However, disease driven by genetic variants in lipid metabolism genes is not well understood, and additional information is needed to better understand these effects.MethodsWe gathered a multi-institutional cohort of undiagnosed patients with a constellation of phenotypes presenting as malnutrition and metal ion dysregulation. Clinical Whole Exome Sequencing (WES) was performed on four patients and their unaffected parents. We prioritized genetic variants based on multiple criteria including population allele frequency and presumed inheritance pattern, and identified a candidate gene. Computational modeling was used to investigate if the altered amino acids are likely to result in a dysfunctional enzyme.ResultsWe identified a multi-institutional cohort of patients presenting with malnutrition-like symptoms and likely pathogenic genomic variants within DGAT1. Multiple approaches were used to profile the effect these variants have on protein structure and function. Laboratory and nutritional intervention studies showed rapid and robust patient responses.ConclusionsThis report adds on to the database for existing mutations known within DGAT1, a gene recently implicated with CDD, and also expands its clinical spectrum. Identification of these DGAT1 mutations by WES has allowed for changes in the patients’ nutritional rehabilitation, reversed growth failure and enabled them to be weaned off of total parenteral nutrition (TPN).     

High diagnostic yield and novel variants in very late-onset spasticity

Hereditary spastic paraplegias (HSPs) are a diverse group of genetic conditions with variable severity and onset age. From a neurogenetic clinic, we identified 14 patients with very late-onset HSP, with symptoms starting after the age of 35. In this cohort, sequencing of known genetic causes was performed using clinically available HSP sequencing panels. We identified 4 patients with mutations in SPG7 and 3 patients with SPAST mutations, representing 50% of the cohort and indicating a very high diagnostic yield. In the SPG7 group, we identified novel variants in two patients. We have also identified two novel mutations in the SPAST group. We present sequencing data from cDNA and RT-qPCR to support the pathogenicity of these variants, and provide observations regarding the poor genotype-phenotype correlation in these conditions that should be the subject of future study.