Abstract Background Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy mainly caused by desmosomal gene variants. Previously, we reported that DSG2 variants were the most common in 153 Japanese ARVC probands (DSG2 multiple, n=62 and single, n=4; PKP2, n=28; others, n=7). Among the multiple DSG2 variant carriers, 22 probands were compound heterozygous carriers, and 25 were homozygous, which were confirmed by genetic testing for family members (Figure A). On the other hand, 88% of their family members with single DSG2 variant carriers were not diagnosed with ARVC, which meant that recessive DSG2 variants were the major cause of ARVC in Japan. However, zygosities in remaining 15 probands with multiple DSG2 variants were unknown due to the lack of genome data from their parents. Purpose This study aimed to clarify the zygosities of multiple DSG2 variants using amplicon-based long-read sequencing. Methods Genomic DNA was extracted from whole blood using standard protocols. The range containing multiple variants was amplified by long-range PCR. Sequencing libraries were prepared using ONT Ligation Sequencing Kit. Each library was loaded onto a flow cell for sequencing on ONT GridION Mk1 running MinKNOW control software. FASTQ files were aligned to hg38 using minimap2. Variants were called using Clair3 and phased using WhatsHap. Integrative Genomics Viewer (IGV) was used for visualization of the phased variants. Results We successfully phased all the multiple DSG2 variants found in the 15 proband. A typical result of amplicon-based LRS was shown in Figure B. It was IGV screen shot of reads at DSG2 containing c.874C>T (p.R292C) and c.1481A>C (p.D494A). The reads colored in red belonged to one haplotype, while the ones in blue belonged to the other. Each of the two variants was on different color reads, indicating they were compound heterozygous variants. Using LRS, we confirmed all the 15 probands had compound heterozygous variants in DSG2. Overall, the 62 probands (40.5%) with DSG2 variants were recessive variant carriers (Figure A). Conclusions Japanese ARVC was mainly caused by autosomal recessive variants in DSG2. Clarifying the zygosity of causative variants is crucial for the diagnosis in recessive diseases including Japanese ARVC. Amplicon-based LRS was helpful for phasing the multiple DSG2 variants directly without genetic testing of the parents.Figure AFigure B
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