Myocardial hypertrophy characterizes hypertrophic cardiomyopathy (HCM) and has an autosomal dominant pattern of inheritance. Mutations in various genes are responsible for the phenotype of the disease. However, genetic screening for detecting the underlying mutations causing HCM with conventional sequencing techniques still is challenging and time consuming. Nanopore based amplicon sequencing is a third-generation sequencing technology that enables the analysis of different candidate genes as well as different patients in a single sequencing run. Here, we describe Nanopore sequencing of eight HCM candidate genes (myosin-binding protein c (mybpc3), β-myosin heavy chain (myh7), troponin T (tnnt2), troponin I (tnni3, α-tropomyosin (tpm1), cardiac actin (actc1), and regulatory and essential myosin light chains (myl2 and myl3)) in four members of a  family with a known myh7 mutation and one patient with suspected HCM.

Methods: We developed a workflow for genetic testing of the entire candidate genes from four members of a family with the known p.Arg840His mutation in the myh7 gene and one patient with suspected HCM in a single sequencing run. For this purpose, long amplicons (up to 9000 bp) were generated using a longAmp PCR reaction. The long amplicons were then used to construct a sequencing library with the Oxford Nanopore Ligation Kit (LSK109). Additionally, each patient was assigned with its own barcode for later differentiation. Sequencing was performed using a flow cell with a Minion MK1C (Oxford Nanopore Technologies) and stopped after generation of  approximately 500k sequencing reads. Bioinformatic analysis was performed using the Nanopipe platform (Institute of Bioinformatics Münster). The fastq files generated were assembled separately for each barcode, converted to fasta files, uploaded to Nanopipe and analysed against the human DNA sequence (hg38).

Results: Our study detected the known heterozygous p.Arg840His mutation and additional homozygous and heterozygous polymorphisms in the DNA of the HCM family. In addition our study revealed that a total of 15K sequencing reads per gene is sufficient to generate enough coverage to provide an accurate genetic diagnosis of HCM. Candidate gene analysis of the patient with suspected HCM revealed a known amino acid change in mybpc3 (p.Val896Met), but described as benign or likely benign, in addition to various polymorphisms in introns, up- and downstream regions, and silent mutations.

Our study shows that it is feasible to use Nanopore sequencing for rapid and cost effective genetic testing of HCM. The workflow is scalable and enables cost-effective and timely analysis making it an attractive technology for any clinical diagnostic laboratory. The workflow can be applied to any candidate gene and is therefore very suitable for complex genetic diseases such as cardiomyopathies.