Multiplex targeted high‐throughput sequencing for Mendelian cardiac disorders

Abstract

Mendelian cardiomyopathies and arrhythmias are characterized by an important genetic heterogeneity, rendering Sanger sequencing very laborious and expensive. As a proof of concept, we explored multiplex targeted high-throughput sequencing (HTS) as a fast and cost-efficient diagnostic method for individuals suffering from Mendelian cardiac disorders. We designed a DNA capture assay including all exons from 130 genes involved in cardiovascular Mendelian disorders and analysed simultaneously four samples by multiplexing. Two patients had familial hypertrophic cardiomyopathy (HCM) and two patients suffered from long QT syndrome (LQTS). In patient 1 with HCM, we identified two known pathogenic missense variants in the two most frequently mutated sarcomeric genes MYH7 and MYBPC. In patient 2 with HCM, a known acceptor splice site variant in MYBPC3 was found. In patient 3 with LQTS, two missense variants in the genes SCN5A and KCNQ were identified. Finally, in patient 4 with LQTS a known missense variant was found in MYBPC3, which is usually mutated in patients with cardiomyopathy. Our results showed that multiplex targeted HTS works as an efficient and cost-effective tool for molecular diagnosis of heterogeneous disorders in clinical practice and offers new insights in the pathogenesis of these complex diseases.

Unbiased targeted next-generation sequencing molecular approach for primary immunodeficiency diseases

“…Next-generation sequencing (NGS) has been applied to comprehensively study several Mendelian monogenic disorders, as well as complex Abbreviations used CID: Combined immunodeficiency CNV: Copy number variation ePCR: Emulsion PCR HGMD: Human Gene Mutation Database indel: Insertion/deletion NGS: Next-generation sequencing PID: Primary immunodeficiency disease SCID: Severe combined immunodeficiency disease SNP: Single nucleotide polymorphism SNV: Single nucleotide variant WES: Whole-exome sequencing WGS: Whole-genome sequencing diseases. [9][10][11] Whole-exome sequencing (WES) with NGS can provide a rapid diagnosis and has now been adopted by several laboratories as the method of choice for the diagnosis of various PIDs. [12][13][14][15][16] This is because of its speed and comprehensiveness, with the coding regions of the whole set of genes in the genome (the exome) able to be screened in 1 run.…”

Genetics of hypertrophic cardiomyopathy: advances and pitfalls in molecular diagnosis and therapy

“… 58 Sanger sequencing is nevertheless important for cosegregation studies of the supposed causative mutation within HCM families and also as a gold standard validation technique in parallel with novel approaches. 6 , 46 , 56 , 59 – 61 The wide genetic heterogeneity of HCM renders Sanger sequencing tedious and too expensive for routine clinical practice, 62 prompting DNA microarrays as an alternative technique. 63 – 65 …”

“…Mutation detection using the iPLEX MassARRAY matrix-assisted laser desorption/ionization time-of-flight system (Sequenom Inc., San Diego, CA, USA) has been used recently for diagnosis of HCM. 56 , 60 , 62 , 65 , 75 In contrast with automated Sanger sequencing, high-throughput techniques such as iPLEX ® MassARRAY allow rapid and cost-effective testing for a large number of mutations simultaneously. iPLEX MassARRAY involves multiplex primary PCR using outer primers that flank HCM mutation sites followed by a homogeneous mass extend reaction with multiple, single, inner primers that together generate fragments of different mass specific for each genotype (iPLEX).…”

“… 76 In the last 5 years, several of these NGS enrichment methods for genotyping HCM-related mutations have been tested, and were generally observed to provide faster, accurate, and efficient clinical genetic analysis. 53 , 60 , 62 , 81 – 88 …”

Application of Next Generation Sequencing for personalized medicine for sudden cardiac death

“…In recent years, NGS has been applied to comprehensively study several Mendelian monogenic disorders as well as complex diseases including cancer ( Navin and Hicks, 2011 ) and cardiac diseases ( Fokstuen et al, 2014 ). For these multifactorial diseases, a very important advantage of NGS is the possibility of testing many genes in a relatively short time and with low cost.…”