Lin Pei-Hsuan, Tsai Cheng-Yu, Chiang Yu-Ting, Ho Chang-Han, Lu Yue-Sheng, Hsu Jacob Shu-Jui, Cheng Yen-Fu, Tsai Shih-Feng, Hsu Chuan-Jen, Chen Pei-Lung, Wu Chen-Chi
Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, 100229, Taiwan.
Department of Otolaryngology, National Taiwan University Hospital, Taipei, 100225, Taiwan.
Mol Med. 2025 May 9;31(1):181. doi: 10.1186/s10020-025-01225-2.
Pathogenic variants in OTOF are a major cause of auditory synaptopathy. However, challenges remain in interpreting OTOF variants, including difficulties in confirming haplotype phasing using traditional short-read sequencing (SRS) due to the large gene size, the potential incomplete penetrance of certain variants, and difficulties in assessing variants at non-canonical splice sites. This study aims to revisit the genetic landscape of OTOF variants in a Taiwanese non-syndromic auditory neuropathy spectrum disorder (ANSD) cohort using a combination of sequencing technologies, predictive tools, and experimental validations.
We performed SRS to analyze OTOF variants in 65 unrelated Taiwanese patients diagnosed with non-syndromic ANSD, complemented by long-read sequencing (LRS) for haplotype phasing. A prediction-to-validation pipeline was implemented to assess the pathogenicity of cryptic variants using SpliceAI software and minigene assays.
Biallelic pathogenic OTOF variants were identified in 33 patients (50.8%), while monoallelic variants were found in five patients. Three novel variants, c.3864G > A (p.Ala1288 =), c.4501G > A (p.Ala1501Thr), and c.5813 + 2T > C, were detected. The pathogenicity of two non-canonical mis-splicing variants, c.3894 + 5G > C and c.3864G > A (p.Ala1288 =), was confirmed by minigene assays. LRS-based haplotype phasing revealed that the common missense variant c.5098G > C (p.Glu1700Gln) and the novel variant c.5975A > G (p.Lys1992Arg) are in cis and form a founder pathogenic allele in the Taiwanese population.
Our study highlights the genetic heterogeneity of DFNB9 and emphasizes the importance of population-specific variant interpretation. The integration of advanced sequencing technologies, predictive algorithms, and functional validation assays will improve the accuracy of molecular diagnosis and inform personalized treatment strategies for individuals with DFNB9.
OTOF基因的致病变异是听觉突触病变的主要原因。然而，在解释OTOF变异方面仍存在挑战，包括由于基因规模大，使用传统短读长测序（SRS）确认单倍型相位存在困难、某些变异可能存在不完全外显率，以及评估非经典剪接位点的变异存在困难。本研究旨在结合测序技术、预测工具和实验验证，重新审视台湾非综合征性听觉神经病谱系障碍（ANSD）队列中OTOF变异的遗传格局。
我们对65名诊断为非综合征性ANSD的无关台湾患者进行了SRS分析OTOF变异，并通过长读长测序（LRS）进行单倍型相位分析。实施了一个从预测到验证的流程，使用SpliceAI软件和小基因检测评估隐匿变异的致病性。
在33名患者（50.8%）中鉴定出双等位基因致病性OTOF变异，在5名患者中发现了单等位基因变异。检测到三个新变异，分别为c.3864G＞A（p.Ala1288=）、c.4501G＞A（p.Ala1501Thr）和c.5813+2T＞C。小基因检测证实了两个非经典错配剪接变异c.3894+5G＞C和c.3864G＞A（p.Ala1288=）的致病性。基于LRS的单倍型相位分析显示，常见的错义变异c.5098G＞C（p.Glu1700Gln）和新变异c.5975A＞G（p.Lys1992Arg）处于顺式状态，并在台湾人群中形成了一个始祖致病性等位基因。
我们的研究突出了DFNB9的遗传异质性，并强调了特定人群变异解释的重要性。先进测序技术、预测算法和功能验证检测的整合将提高分子诊断的准确性，并为DFNB9患者的个性化治疗策略提供依据。
