Application of Long-Read Nanopore Sequencing to the Search for Mutations in Hypertrophic Cardiomyopathy

Abstract

Increasing evidence suggests that both coding and non-coding regions of sarcomeric protein genes can contribute to hypertrophic cardiomyopathy (HCM). Here, we introduce an experimental workflow (tested on four patients) for complete sequencing of the most common HCM genes (MYBPC3, MYH7, TPM1, TNNT2, and TNNI3) via long-range PCR, Oxford Nanopore Technology (ONT) sequencing, and bioinformatic analysis. We applied Illumina and Sanger sequencing to validate the results, FastQC, Qualimap, and MultiQC for quality evaluations, MiniMap2 to align data, Clair3 to call and phase variants, and Annovar’s tools and CADD to assess pathogenicity of variants. We could not amplify the region encompassing exons 6–12 of MYBPC3. A higher sequencing error rate was observed with ONT (6.86–6.92%) than with Illumina technology (1.14–1.35%), mostly for small indels. Pathogenic variant p.Gln1233Ter and benign polymorphism p.Arg326Gln in MYBPC3 in a heterozygous state were found in one patient. We demonstrated the ability of ONT to phase single-nucleotide variants, enabling direct haplotype determination for genes TNNT2 and TPM1. These findings highlight the importance of long-range PCR efficiency, as well as lower accuracy of variant calling by ONT than by Illumina technology; these differences should be clarified prior to clinical application of the ONT method.

Monogenic diseases associated with cardiomyopathy genes and their phenotypic manifestations

“…Фенотип аритмогенной КМП может встречаться при других генетически обусловленных кардиомиопатиях [24]. Однако аритмии не всегда проявляются у пациентов с моногенными КМП даже при наличии одного и того же патогенного варианта, что наблюдалось, в частности, для пациентов с ГКМП, вызванной заменой p.Gln1233Ter в гене MYBPC3 [25].…”

“…), биохимических показателей и клеточного состава крови [27]. На наш взгляд, с учетом сложности генетической детерминации (от моногенного до полигенного компонента), неполной пенетрантности и экспрессивности генетических вариантов, возможного модифицирующего влияния различных генетических факторов на клиническую картину КМП [25,28,29] этот аспект в дальнейшем требует более детального рассмотрения.…”

Special Issue: “Genes and Human Diseases”

“…Additionally, despite the fairly good knowledge of the genes leading to rare/monogenic hereditary diseases, the emergence and development of new genetic technologies for studying genomes (new generation sequencing [29,30], etc.) allow us to obtain new data on the genetic factors (mutations) that cause these diseases, which is of great practical importance [31,32]. So, the above arguments clearly indicate the need to continue active research on the genetic nature of human diseases, which is undoubtedly important not only for understanding the causes/mechanisms of development of these diseases but will also create prerequisites for their introduction into practical medicine.…”

Targeted Phasing of 2-200 Kilobase DNA Fragments with a Short-Read Sequencer and a Single-Tube Linked-Read Library Method

“…On average, humans carry a heterozygosity every 1-2 kilobases (kb), depending on genetic ancestry and admixture [4][5][6][7][8] ; thus, sequencing platforms such as Pacific Biosciences Ò (PacBio) and Oxford Nanopore Technology Ò (ONT) that process 20-100 kb long reads have drawn much attention 9 . However, long-read sequencers remain less cost-effective and can have lower genotyping fidelity and throughput than short-read sequencers, especially for reads longer than 20 kb 10 . Moreover, newer short-read technologies are evolving and further pushing down sequencing costs (e.g., Ultima Genomics Ò , Singular Genomics Ò , and Element Biosciences Ò ).…”