PDF(Pubmed)
Abstract：
Developmental anomalies of the hearing organ, the cochlea, are diagnosed in approximately one-fourth of individuals with congenital deafness. Most patients with cochlear malformations remain etiologically undiagnosed due to insufficient knowledge about underlying genes or the inability to make conclusive interpretations of identified genetic variants. We used exome sequencing for genetic evaluation of hearing loss associated with cochlear malformations in three probands from unrelated families. We subsequently generated monoclonal induced pluripotent stem cell (iPSC) lines, bearing patient-specific knockins and knockouts using CRISPR/Cas9 to assess pathogenicity of candidate variants. We detected FGF3 (p.Arg165Gly) and GREB1L (p.Cys186Arg), variants of uncertain significance in two recognized genes for deafness, and PBXIP1(p.Trp574*) in a candidate gene. Upon differentiation of iPSCs towards inner ear organoids, we observed significant developmental aberrations in knockout lines compared to their isogenic controls. Patient-specific single nucleotide variants (SNVs) showed similar abnormalities as the knockout lines, functionally supporting their causality in the observed phenotype. Therefore, we present human inner ear organoids as a tool to rapidly validate the pathogenicity of DNA variants associated with cochlear malformations.
摘要：
听觉器官发育异常,耳蜗,大约四分之一的先天性耳聋患者被诊断出。由于对潜在基因的知识不足或无法对已确定的遗传变异做出结论性解释，大多数耳蜗畸形患者在病因上仍未被诊断。我们使用外显子组测序技术对来自无关家庭的三个先证者中与耳蜗畸形相关的听力损失进行了遗传评估。我们随后产生了单克隆诱导多能干细胞（iPSC）系，使用CRISPR/Cas9进行患者特异性敲入和敲除以评估候选变体的致病性。我们检测到FGF3（p。Arg165Gly）和GREB1L（p。Cys186Arg），在两个公认的耳聋基因中具有不确定意义的变异，和PBXIP1（p。Trp574*)中的一个候选基因。iPSCs向内耳类器官分化后，与同基因对照相比，我们在敲除品系中观察到显着的发育异常。患者特异性单核苷酸变体（SNV）显示出与敲除系相似的异常，在功能上支持它们在观察到的表型中的因果关系。因此,我们提出了人类内耳类器官作为快速验证与耳蜗畸形相关的DNA变异的致病性的工具。
