Enhanced identification of novel pathogenic variants in hereditary hearing loss through physical phasing with integrated short and long-read sequencing data.

Abstract

Haplotagged variant calling is essential for determining genetic etiologies in hereditary hearing loss (HHL) cases when familial testing is unavailable, and long-read whole-genome sequencing (lrWGS) enables this by outperforming in several key areas: enhanced detection of structural variations (SVs) and precise long-range haplotype phasing. In this study, we enrolled two HL cases from the China Deafness Genetics Consortium (CDGC) cohort, whose genetic tests were previously inconclusive due to a lack of pedigree segregation data. Small variants (including SNVs and InDels) profiles were generated by short-read whole-genome sequencing (srWGS), while SVs were identified and co-phased with small variants using a read-based approach. As a result, 87% and 83% of the chromosomal regions were successfully phased, and reached mean haplotype block lengths up to 661.9 kb and 309.9 kb, respectively. A total of 483 and 434 small variants, along with three and six heterozygous SVs in coding and splice regions of 201 HL-associated genes were phased. Pathogenic interpretations resolved compound heterozygosity in MARVELD2, identifying a pathogenic (P) variant NM_001038603.3:c.782G > A in trans with a novel pathogenic (P) deletion (NM_001038603.3:c.1183-1288_1503 + 195del). Additionally, we identified a known P variant NM_022124.6:c.5369-1G > A, which was oriented in trans with a P deletion NM_022124.6:c.-5-12_67 + 154del in the CDH23 gene. This study demonstrates the clinical utility of integrating srWGS and Nanopore lrWGS for comprehensive variant detection and haplotype determination in HL cases with limited family background details, providing a robust framework for resolving complex genetic etiologies and improving diagnostic precision.