應用長片段與短片段DNA序列分析以解決遺傳性聽損之診斷挑戰

Abstract

遺傳性聽力損傷（hereditary hearing impairment, HHI）是一種常見的遺傳疾病，具有高度的基因異質性與表現型變異性，因而診斷上充滿挑戰。雖然短片段的次世代定序（short-read next-generation sequencing, srNGS）已廣泛應用於臨床診斷，但針對遺傳性聽力損傷的診斷率僅約40%，顯示尚有改進診斷分析策略的需求。 本研究透過分析短片段與長片段定序資料，針對遺傳性聽力損傷未解的遺傳成因進行探討，與發展相關臨床檢測應用，主要包括三個方向： 1.GJB2相關的表現型變異性：重新分析短片段定序基因套組（short-read panel, srPanel）資料，發現除GJB2以外的聽力損傷相關基因致病變異，另有一可能影響表現型的修飾基因變異CRYL1 rs14236，可能與帶有GJB2 p.V37I同型合子的聽力損傷病人所觀察到的較嚴重聽損有關。 2.結構變異（structural variant, SV）的發現：建立一套整合長片段全基因體定序（long-read whole-genome sequencing, lrWGS）與短片段全基因體定序(short-read whole-genome sequencing, srWGS)資料的分析流程，成功從過去無法診斷家庭中找出新的可能致病非編碼區域缺失變異。 3.具成本效益的長片段定序套組 (long-read panel, lrPanel) 開發與應用：設計並驗證一個客製化長片段定序套組，針對短片段定序難以準確分析的STRC基因及其他容易出現結構變異的聽力相關基因進行分析，達成將長片段定序應用於臨床聽力損傷分子診斷的可能。 本研究利用短片段及長片段定序技術與發展分析策略，釐清未解的遺傳性聽力損傷遺傳病因，提供臨床參考依據，進而提升診斷率。

Hereditary hearing impairment (HHI) is a common genetic disorder characterized by high genetic heterogeneity and phenotypic variability, making diagnosis challenging. Although short-read next-generation sequencing (srNGS) is widely used in clinical diagnostics, the diagnostic yield for HHI remains around 40%, highlighting the need for improved strategies. This study investigates unresolved genetic causes of HHI and develops clinical testing approaches using both short- and long-read sequencing through three strategies: 1. GJB2-related phenotypic variability: Reanalysis of short-read panel (srPanel) data revealed additional pathogenic variants in other deafness genes and a potential modifier variant, CRYL1 rs14236, which may contribute to the more severe hearing loss observed in GJB2 p.V37I homozygotes. 2. Discovery of structural variants (SVs): A comprehensive analysis pipeline combining long-read whole-genome sequencing (lrWGS) with short-read WGS was developed, enabling the identification of novel non-coding deletions in previously undiagnosed families. 3. Cost-effective clinical application: A custom long-read panel targeting the STRC gene—difficult to analyze with srNGS—and other SV-prone deafness genes was developed and validated for clinical use. Overall, this study demonstrates how integrating multiple sequencing technologies and tailored analysis strategies can resolve complex genetic cases and improve the diagnostic outcomes of HHI.