Identification of Variants of Uncertain Significance in the Genes Associated with Thoracic Aortic Disease in Russian Patients with Nonsyndromic Sporadic Subtypes of the Disorder.

Nonsyndromic sporadic thoracic aortic aneurysm (nssTAA) is characterized by diverse genetic variants that may vary in different populations. Our aim was to identify clinically relevant variants in genes implicated in hereditary aneurysms in Russian patients with nssTAA. Forty-one patients with nssTAA without dissection were analyzed. Using massive parallel sequencing, we searched for variants in exons of 53 known disease-causing genes. Patients were found to have no (likely) pathogenic variants in the genes of hereditary TAA. Six variants of uncertain significance (VUSs) were identified in four (9.8%) patients. Three VUSs [FBN1 c.7841C>T (p.Ala2614Val), COL3A1 c.2498A>T (p.Lys833Ile), and MYH11 c.4993C>T (p.Arg1665Cys)] are located in genes with "definitive" disease association (ClinGen). The remaining variants are in "potentially diagnostic" genes or genes with experimental evidence of disease association [NOTCH1 c.964G>A (p.Val322Met), COL4A5 c.953C>G (p.Pro318Arg), and PLOD3 c.833G>A (p.Gly278Asp)]. Russian patients with nssTAA without dissection examined in this study have ≥1 VUSs in six known genes of hereditary TAA (FBN1, COL3A1, MYH11, NOTCH1, COL4A5, or PLOD3). Experimental studies expanded genetic testing, and clinical examination of patients and first/second-degree relatives may shift VUSs to the pathogenic (benign) category or to a new class of rare "predisposing" low-penetrance variants causing the pathology if combined with other risk factors.

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

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.