Targeted panel sequencing in adult patients with left ventricular non-compaction reveals a large genetic heterogeneity

Left ventricular non-compaction (LVNC) is a cardiomyopathy that may be of genetic origin; however, few data are available about the yield of mutation, the spectrum of genes and allelic variations. The aim of this study was to better characterize the genetic spectrum of isolated LVNC in a prospective cohort of 95 unrelated adult patients through the molecular investigation of 107 genes involved in cardiomyopathies and arrhythmias. Fifty-two pathogenic or probably pathogenic variants were identified in 40 patients (42%) including 31 patients (32.5%) with single variant and 9 patients with complex genotypes (9.5%). Mutated patients tended to have younger age at diagnosis than patients with no identified mutation. The most prevalent genes were TTN, then HCN4, MYH7, and RYR2. The distribution includes 13 genes previously reported in LVNC and 10 additional candidate genes. Our results show that LVNC is basically a genetic disease and support genetic counseling and cardiac screening in relatives. There is a large genetic heterogeneity, with predominant TTN null mutations and frequent complex genotypes. The gene spectrum is close to the one observed in dilated cardiomyopathy but with specific genes such as HCN4. We also identified new candidate genes that could be involved in this sub-phenotype of cardiomyopathy.     

AudioGene: Predicting Hearing Loss Genotypes from Phenotypes to Guide Genetic Screening

Autosomal dominant nonsyndromic hearing loss (ADNSHL) is a common and often progressive sensory deficit. ADNSHL displays a high degree of genetic heterogeneity and varying rates of progression. Accurate, comprehensive, and cost‐effective genetic testing facilitates genetic counseling and provides valuable prognostic information to affected individuals. In this article, we describe the algorithm underlying AudioGene, a software system employing machine‐learning techniques that utilizes phenotypic information derived from audiograms to predict the genetic cause of hearing loss in persons segregating ADNSHL. Our data show that AudioGene has an accuracy of 68% in predicting the causative gene within its top three predictions, as compared with 44% for a majority classifier. We also show that AudioGene remains effective for audiograms with high levels of clinical measurement noise. We identify audiometric outliers for each genetic locus and hypothesize that outliers may reflect modifying genetic effects. As personalized genomic medicine becomes more common, AudioGene will be increasingly useful as a phenotypic filter to assess pathogenicity of variants identified by massively parallel sequencing.     

FLNC pathogenic variants in patients with cardiomyopathies: Prevalence and genotype-phenotype correlations

Pathogenic variants in FLNC encoding filamin C have been firstly reported to cause myopathies, and were recently linked to isolated cardiac phenotypes. Our aim was to estimate the prevalence of FLNC pathogenic variants in subtypes of cardiomyopathies and to study the relations between phenotype and genotype. DNAs from a cohort of 1150 unrelated index-patients with isolated cardiomyopathy (700 hypertrophic, 300 dilated, 50 restrictive cardiomyopathies, and 100 left ventricle non-compactions) have been sequenced on a custom panel of 51 cardiomyopathy disease-causing genes. An FLNC pathogenic variant was identified in 28 patients corresponding to a prevalence ranging from 1% to 8% depending on the cardiomyopathy subtype. Truncating variants were always identified in patients with dilated cardiomyopathy, while missense or in-frame indel variants were found in other phenotypes. A personal or family history of sudden cardiac death (SCD) was significantly higher in patients with truncating variants than in patients carrying missense variants (P = .01). This work reported the first observation of a left ventricular non-compaction associated with a unique probably causal variant in FLNC which highlights the role of FLNC in cardiomyopathies. A correlation between the nature of the variant and the cardiomyopathy subtype was observed as well as with SCD risk.     

Furthering the link between the sarcomere and primary cardiomyopathies: restrictive cardiomyopathy associated with multiple mutations in genes previously associated with hypertrophic or dilated cardiomyopathy

Mutations in genes that encode components of the sarcomere are well established as the cause of hypertrophic and dilated cardiomyopathies. Sarcomere genes, however, are increasingly being associated with other cardiomyopathies. One phenotype more recently recognized as a disease of the sarcomere is restrictive cardiomyopathy (RCM). We report on two patients with RCM associated with multiple mutations in sarcomere genes not previously associated with RCM. Patient 1 presented with NYHA Class III/IV heart failure at 22 years of age. She was diagnosed with RCM and advanced heart failure requiring heart transplantation. Sequencing of sarcomere genes revealed previously reported homozygous p.Glu143Lys mutations in MYL3, and a novel heterozygous p.Gly57Glu mutation in MYL2. The patient's mother is a double heterozygote for these mutations, with no evidence of cardiomyopathy. Patient 2 presented at 35 years of age with volume overload while hospitalized for oophorectomy. She was diagnosed with RCM and is being evaluated for heart transplantation. Sarcomere gene sequencing identified homozygous p.Asn279His mutations in TPM1. The patient's parents are consanguineous and confirmed heterozygotes. Her father was diagnosed with HCM at 42 years of age. This is the first report of mutations in TPM1, MYL3, and MYL2 associated with primary, non-hypertrophied RCM. The association of more sarcomere genes with RCM provides further evidence that mutations in the various sarcomere genes can cause different cardiomyopathy phenotypes. These cases also contribute to the growing body of evidence that multiple mutations have an additive effect on the severity of cardiomyopathies.     

Clinical and ECG variables to predict the outcome of genetic testing in hypertrophic cardiomyopathy

Knowledge on the influence of specific genotypes on the phenotypic expression of hypertrophic cardiomyopathy (HCM) is emerging. The objective of this study was to evaluate the genotype-phenotype relation in HCM patients and to construct a score to predict the genetic yield based to improve counseling. Unrelated HCM patients who underwent genetic testing were included in the analysis. Multivariate logistic regression was performed to identify variables that predict a positive genetic test. A weighted score was constructed based on the odds ratios. In total, 378 HCM patients were included of whom 141 carried a mutation (global yield 37%), 181 were mutation negative and 56 only carried a variant of unknown significance. We identified age at diagnosis <45 years, familial HCM, familial sudden death, arrhythmic syncope, maximal wall thickness ≥20 mm, asymmetrical hypertrophy and the absence of negative T waves in the lateral ECG leads as significant predictors of a positive genetic test. When we included these values in a risk score we found very high correlation between the score and the observed genetic yield (Pearson r = 0.98). MYBPC3 mutation carriers more frequently suffered sudden cardiac death compared to troponin complex mutations carriers (p = 0.01) and a similar trend was observed compared to MYH7 mutation carriers (p = 0.08) and mutation negative patients (p = 0.11). To conclude, a simple score system based on clinical variables can predict the genetic yield in HCM index patients, aiding in counseling HCM patients. MYBPC3 mutation carriers had a worse outcome regarding sudden cardiac death.     

Single,short in‐del,and copy number variations detection in monogenic dyslipidemia using a next‐generation sequencing strategy

Optimal molecular diagnosis of primary dyslipidemia is challenging to confirm the diagnosis, test and identify at risk relatives. The aim of this study was to test the application of a single targeted next‐generation sequencing (NGS) panel for hypercholesterolemia, hypocholesterolemia, and hypertriglyceridemia molecular diagnosis. NGS workflow based on a custom AmpliSeq panel was designed for sequencing the most prevalent dyslipidemia‐causing genes (ANGPTL3, APOA5, APOC2, APOB, GPIHBP1, LDLR, LMF1, LPL, PCSK9) on the Ion PGM Sequencer. One hundred and forty patients without molecular diagnosis were studied. In silico analyses were performed using the NextGENe software and homemade tools for detection of copy number variations (CNV). All mutations were confirmed using appropriate tools. Eighty seven variations and 4 CNV were identified, allowing a molecular diagnosis for 40/116 hypercholesterolemic patients, 5/13 hypocholesterolemic patients, and 2/11, hypertriglyceridemic patients respectively. This workflow allowed the detection of CNV contrary to our previous strategy. Some variations were found in previously unexplored regions providing an added value for genotype‐phenotype correlation and familial screening. In conclusion, this new NGS process is an effective mutation detection method and allows better understanding of phenotype. Consequently this assay meets the medical need for individualized diagnosis of dyslipidemia.     

The contribution of mosaicism to genetic diseases and de novo pathogenic variants

The contribution of mosaicism to diagnosed genetic disease and presumed de novo variants (DNV) is under investigated. We determined the contribution of mosaic genetic disease (MGD) and diagnosed parental mosaicism (PM) in parents of offspring with reported DNV (in the same variant) in the (1) Undiagnosed Diseases Network (UDN) (N = 1946) and (2) in 12,472 individuals electronic health records (EHR) who underwent genetic testing at an academic medical center. In the UDN, we found 4.51% of diagnosed probands had MGD, and 2.86% of parents of those with DNV exhibited PM. In the EHR, we found 6.03% and 2.99% and (of diagnosed probands) had MGD detected on chromosomal microarray and exome/genome sequencing, respectively. We found 2.34% (of those with a presumed pathogenic DNV) had a parent with PM for the variant. We detected mosaicism (regardless of pathogenicity) in 4.49% of genetic tests performed. We found a broad phenotypic spectrum of MGD with previously unknown phenotypic phenomena. MGD is highly heterogeneous and provides a significant contribution to genetic diseases. Further work is required to improve the diagnosis of MGD and investigate how PM contributes to DNV risk.     

Anti-calreticulin antibodies and calreticulin in sera of patients diagnosed with dilated or hypertrophic cardiomyopathy

Distinct cellular level of the Ca²⁺-binding chaperone calreticulin (CRT) is essential for correct embryonal cardiac development and postnatal function. However, CRT is also a potential autoantigen eliciting formation of antibodies (Ab), whose role is not yet clarified. Immunization with CRT leads to cardiac injury, while overexpression of CRT in cardiomyocytes induces dilated cardiomyopathy (DCM) in animals. Hence, we analysed levels of anti-CRT Ab and calreticulin in the sera of patients with idiopatic DCM and hypertrophic cardiomyopathy (HCM). ELISA and immunoblot using human recombinant CRT and Pepscan with synthetic, overlapping decapeptides of CRT were used to detect anti-CRT Ab. Serum CRT concentration was tested by ELISA. Significantly increased levels of anti-CRT Ab of isotypes IgA (p?p?p?相似文献   

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.     

Clinical and genetic characterization of Bardet–Biedl syndrome in Tunisia: defining a strategy for molecular diagnosis

Bardet–Biedl syndrome (BBS, OMIM 209900) is a rare genetic disorder characterized by obesity, retinitis pigmentosa, post axial polydactyly, cognitive impairment, renal anomalies and hypogonadism. The aim of this study is to provide a comprehensive clinical and molecular analysis of a cohort of 11 Tunisian BBS consanguineous families in order to give insight into clinical and genetic spectrum and the genotype–phenotype correlations. Molecular analysis using combined sequence capture and high‐throughput sequencing of 30 ciliopathies genes revealed 11 mutations in 11 studied families. Five mutations were novel and six were previously described. Novel mutations included c.1110G>A and c.39delA (p.G13fs*41) in BBS1, c.115+5G>A in BBS2, c.1272+1G>A in BBS6, c.1181_1182insGCATTTATACC in BBS10 (p.S396Lfs*6). Described mutations included c.436C>T (p.R146*) and c.1473+4A>G in BBS1, c.565C> (p.R189*) in BBS2, deletion of exons 4–6 in BBS4, c.149T>G (p.L50R) in BBS5, and c.459+1G>A in BBS8; most frequent mutations were described in BBS1 (4/11, 37%) and BBS2 (2/11, 18%) genes. No phenotype–genotype correlation was evidenced. This data expands the mutations profile of BBS genes in Tunisia and suggests a divergence of the genetic spectrum comparing Tunisian and other populations.     

Clinical efficacy of a next‐generation sequencing gene panel for primary immunodeficiency diagnostics

Primary immunodeficiencies (PIDs) are rare monogenic inborn errors of immunity that result in impairment of functions of the human immune system. PIDs have a broad phenotype with increased morbidity and mortality, and treatment choices are often complex. With increased accessibility of next‐generation sequencing (NGS), the rate of discovery of genetic causes for PID has increased exponentially. Identification of an underlying monogenic diagnosis provides important clinical benefits for patients with the potential to alter treatments, facilitate genetic counselling, and pre‐implantation diagnostics. We investigated a NGS PID panel of 242 genes within clinical care across a range of PID phenotypes. We also evaluated Phenomizer to predict causal genes from human phenotype ontology (HPO) terms. Twenty‐seven participants were recruited, and a total of 15 reportable variants were identified in 48% (13/27) of the participants. The panel results had implications for treatment in 37% (10/27) of participants. Phenomizer identified the genes harbouring variants from HPO terms in 33% (9/27) of participants. This study shows the clinical efficacy that genetic testing has in the care of PID. However, it also highlights some of the disadvantages of gene panels in the rapidly moving field of PID genomics and current challenges in HPO term assignment for PID.     

Economic evaluation of next-generation sequencing techniques in diagnosis of genetic disorders: A systematic review

In recent years, massively parallel sequencing or next generation sequencing (NGS) has considerably changed both the research and diagnostic fields, and rapid developments have led to the combination of NGS techniques in clinical practice, ease of analysis, and detection of genetic mutations. This article aimed at reviewing the economic evaluation studies of the NGS techniques in the diagnosis of genetic diseases. In this systematic review, scientific databases (PubMed, EMBASE, Web of Science, Cochrane, Scopus, and CEA registry) were searched from 2005 to 2022 to identify the related literature on the economic evaluation of NGS techniques in the diagnosis of genetic diseases. Full-text reviews and data extraction were all performed by two independent researchers. The quality of all the articles included in this study was evaluated using the Checklist of Quality of Health Economic Studies (QHES). Out of 20 521 screened abstracts, 36 studies met the inclusion criteria. The mean score of the QHES checklist for the studies was 0.78 (high quality). Seventeen studies were conducted based on modeling. Cost-effectiveness analysis, cost-utility analysis, and cost-minimization analysis were done in 26 studies, 13 studies, and 1 study, respectively. Based on the available evidence and findings, exome sequencing, which is one of the NGS techniques, could have the potential to be used as a cost-effective genomic test to diagnose children with suspected genetic diseases. The results of the present study support the cost-effectiveness of exome sequencing in diagnosing suspected genetic disorders. However, the use of exome sequencing as a first- or second-line diagnostic test is still controversial. Most studies have been conducted in high-income countries, and research on the cost-effectiveness of NGS methods is recommended in low- and middle-income countries.     

Feedback of individual genetic results to research participants: in favor of a qualified disclosure policy

This article discusses whether and when researchers have a moral obligation to feedback individual genetic research results. This unsettled debate has rapidly gained in urgency in view of the emergence of biobanks and the advances in next-generation sequencing technology, which has the potential to generate unequalled amounts of genetic data. This implies that the generation of many known and unknown genetic variants in individual participants of genetics/genomics research as intentionally or collaterally obtained byproducts is unavoidable. As we conclude that valid reasons exist to adopt a duty to return genetic research results, a qualified disclosure policy is proposed. This policy contains a standard default package, possibly supplemented with (one or more of) three additional packages. Whereas the default package, containing life-saving information of immediate clinical utility, should be offered routinely and mandatory to all research participants, offering (one of) the three additional packages is context-specific. Such a qualified disclosure policy in our opinion best balances the potential benefits of disclosure with the potential risks for research participants and the harms of unduly hindering biomedical research. We appeal to the genetics community to make a joint effort to further refine the packages and set thresholds for result selection.     

A characterization of postzygotic mutations identified in monozygotic twins

Postzygotic mutations are DNA changes acquired from the zygote stage onwards throughout the lifespan. These changes lead to differences in DNA sequence among cells of an individual, potentially contributing to the etiology of complex disorders. Here we compared whole genome DNA sequence data of two monozygotic twin pairs, 40 and 100 years old, to detect somatic mosaicism. DNA samples were sequenced twice on two Illumina platforms (13X and 40X read depth) for increased specificity. Using differences in allelic ratios resulted in sets of 1,720 and 1,739 putative postzygotic mutations in the 40‐year‐old twin pair and 100‐year‐old twin pair, respectively, for subsequent enrichment analysis. This set of putative mutations was strongly (p < 4.37e–91) enriched in both twin pairs for regulatory elements. The corresponding genes were significantly enriched for genes that are alternatively spliced, and for genes involved in GTPase activity. This research shows that somatic mosaicism can be detected in monozygotic twin pairs by using allelic ratios calculated from DNA sequence data and that the mutations which are found by this approach are not randomly distributed throughout the genome.     

Prevalence of pathogenic and likely pathogenic variants in the RASopathy genes in patients who have had panel testing for cardiomyopathy

RASopathies are a group of developmental disorders caused by pathogenic variants in the RAS‐MAPK pathway. Cardiomyopathy is a major feature of this group of disorders, specifically hypertrophic cardiomyopathy (HCM). HCM can be the first presenting feature in individuals with RASopathies. We conducted a retrospective study of all individuals who have had a cardiomyopathy gene panel ordered through our institution to determine the prevalence of pathogenic or likely pathogenic variants in RAS pathway genes in individuals with cardiomyopathy. We evaluated variants in the following genes: BRAF, CBL, HRAS, KRAS, MAP2K1, MAP2K2, NF1, NRAS, PTPN11, RAF1, SHOC2, and SOS1. We reviewed 74 cases with cardiomyopathy, including 32 with HCM, 24 with dilated cardiomyopathy (DCM), nine with both left ventricular noncompaction (LVNC) and DCM, four with LVNC only, two with arrhythmogenic right ventricular cardiomyopathy (ARVC) and three with unspecified cardiomyopathy. We identified four patients (5.41%) with pathogenic or likely pathogenic variants in HRAS, PTPN11 and RAF1 (two individuals). Indication for testing for all four individuals was HCM. The prevalence of pathogenic or likely pathogenic variants in RASopathy genes in our HCM patient cohort is 12.5% (4/32). We conclude that the RASopathy genes should be included on multi‐gene panels for cardiomyopathy to increase diagnostic yield for individuals with HCM.     

Clinical and genetic study of Friedreich ataxia in an Australian population

Friedreich ataxia is an autosomal recessive disorder caused by mutations in the FRDA gene that encodes a 210-amino acid protein called frataxin. An expansion of a GAA trinucleotide repeat in intron 1 of the gene is present in more than 95% of mutant alleles. Of the 83 people we studied who have mutations in FRDA, 78 are homozygous for an expanded GAA repeat; the other five patients have an expansion in one allele and a point mutation in the other. Here we present a detailed clinical and genetic study of a subset of 51 patients homozygous for an expansion of the GAA repeat. We found a correlation between the size of the smaller of the two expanded alleles and age at onset, age into wheelchair, scoliosis, impaired vibration sense, and the presence of foot deformity. There was no significant correlation between the size of the smaller allele and cardiomyopathy, diabetes mellitus, loss of proprioception, or bladder symptoms. The larger allele size correlated with bladder symptoms and the presence of foot deformity. The duration of disease is correlated with wheelchair use and the presence of diabetes, scoliosis, bladder symptoms and impaired proprioception, and vibration sense but no other complications studied. Am. J. Med. Genet. 87:168–174, 1999. © 1999 Wiley-Liss, Inc.     

The role of tandem duplicator phenotype in tumour evolution in high-grade serous ovarian cancer

High-grade serous ovarian carcinoma (HGSOC) is characterized by genomic instability, ubiquitous TP53 loss, and frequent development of platinum resistance. Loss of homologous recombination (HR) is a mutator phenotype present in 50% of HGSOCs and confers hypersensitivity to platinum treatment. We asked which other mutator phenotypes are present in HGSOC and how they drive the emergence of platinum resistance. We performed whole-genome paired-end sequencing on a model of two HGSOC cases, each consisting of a pair of cell lines established before and after clinical resistance emerged, to describe their structural variants (SVs) and to infer their ancestral genomes as the SVs present within each pair. The first case (PEO1/PEO4), with HR deficiency, acquired translocations and small deletions through its early evolution, but a revertant BRCA2 mutation restoring HR function in the resistant lineage re-stabilized its genome and reduced platinum sensitivity. The second case (PEO14/PEO23) had 216 tandem duplications and did not show evidence of HR or mismatch repair deficiency. By comparing the cell lines to the tissues from which they originated, we showed that the tandem duplicator mutator phenotype arose early in progression in vivo and persisted throughout evolution in vivo and in vitro, which may have enabled continual evolution. From the analysis of SNP array data from 454 HGSOC cases in The Cancer Genome Atlas series, we estimate that 12.8% of cases show patterns of aberrations similar to the tandem duplicator, and this phenotype is mutually exclusive with BRCA1/2 carrier mutations.