Department of Otolaryngology-Head and Neck Surgery, Molecular Otolaryngology & Renal Research Labs, University of Iowa Hospitals and Clinics, Iowa City, Iowa, USA.
J Med Genet. 2013 Sep;50(9):627-34. doi: 10.1136/jmedgenet-2013-101749. Epub 2013 Jun 26.
Non-syndromic hearing loss (NSHL) is the most common sensory impairment in humans. Until recently its extreme genetic heterogeneity precluded comprehensive genetic testing. Using a platform that couples targeted genomic enrichment (TGE) and massively parallel sequencing (MPS) to sequence all exons of all genes implicated in NSHL, we tested 100 persons with presumed genetic NSHL and in so doing established sequencing requirements for maximum sensitivity and defined MPS quality score metrics that obviate Sanger validation of variants.
We examined DNA from 100 sequentially collected probands with presumed genetic NSHL without exclusions due to inheritance, previous genetic testing, or type of hearing loss. We performed TGE using post-capture multiplexing in variable pool sizes followed by Illumina sequencing. We developed a local Galaxy installation on a high performance computing cluster for bioinformatics analysis.
To obtain maximum variant sensitivity with this platform 3.2-6.3 million total mapped sequencing reads per sample were required. Quality score analysis showed that Sanger validation was not required for 95% of variants. Our overall diagnostic rate was 42%, but this varied by clinical features from 0% for persons with asymmetric hearing loss to 56% for persons with bilateral autosomal recessive NSHL.
These findings will direct the use of TGE and MPS strategies for genetic diagnosis for NSHL. Our diagnostic rate highlights the need for further research on genetic deafness focused on novel gene identification and an improved understanding of the role of non-exonic mutations. The unsolved families we have identified provide a valuable resource to address these areas.
非综合征型听力损失（NSHL）是人类最常见的感觉障碍。直到最近，其极端的遗传异质性使得全面的遗传检测变得不可能。我们使用一种将靶向基因组富集（TGE）和大规模平行测序（MPS）相结合的平台来对所有与 NSHL 相关的基因的外显子进行测序，对 100 名疑似遗传性 NSHL 患者进行了测试，从而确定了最大灵敏度的测序要求，并定义了 MPS 质量评分指标，可以避免对变异进行 Sanger 验证。
我们检查了 100 名连续收集的推定遗传性 NSHL 患者的 DNA，没有因遗传、先前的遗传测试或听力损失类型而排除在外。我们使用基于捕获的多重化进行 TGE，然后进行 Illumina 测序。我们在高性能计算集群上开发了一个本地 Galaxy 安装程序，用于生物信息学分析。
为了在该平台上获得最大的变异灵敏度，每个样本需要 320-630 万条总映射测序reads。质量评分分析表明，95%的变异不需要进行 Sanger 验证。我们的总体诊断率为 42%，但因临床特征而异，从不对称听力损失患者的 0%到双侧常染色体隐性 NSHL 患者的 56%不等。
这些发现将指导 TGE 和 MPS 策略在 NSHL 的遗传诊断中的应用。我们的诊断率突出了需要进一步研究遗传耳聋，重点是新基因的鉴定和更好地理解非外显子突变的作用。我们鉴定的未解决的家族为解决这些问题提供了宝贵的资源。
