IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy.
Experimental Therapeutics Program, IFOM ETS -The AIRC Institute of Molecular Oncology, Milan, Italy.
PLoS One. 2023 Jan 23;18(1):e0273586. doi: 10.1371/journal.pone.0273586. eCollection 2023.
Diaphanous related formins are regulatory cytoskeletal protein involved in actin elongation and microtubule stabilization. In humans, defects in two of the three diaphanous genes (DIAPH1 and DIAPH3) have been associated with different types of hearing loss. Here, we investigate the role of the third member of the family, DIAPH2, in nonsyndromic hearing loss, prompted by the identification, by exome sequencing, of a predicted pathogenic missense variant in DIAPH2. This variant occurs at a conserved site and segregated with nonsyndromic X-linked hearing loss in an Italian family. Our immunohistochemical studies indicated that the mouse ortholog protein Diaph2 is expressed during development in the cochlea, specifically in the actin-rich stereocilia of the sensory outer hair cells. In-vitro studies showed a functional impairment of the mutant DIAPH2 protein upon RhoA-dependent activation. Finally, Diaph2 knock-out and knock-in mice were generated by CRISPR/Cas9 technology and auditory brainstem response measurements performed at 4, 8 and 14 weeks. However, no hearing impairment was detected. Our findings indicate that DIAPH2 may play a role in the inner ear; further studies are however needed to clarify the contribution of DIAPH2 to deafness.
细丝相关formin 是一种调节细胞骨架的蛋白质，参与肌动蛋白伸长和微管稳定。在人类中，三种细丝相关formin 基因（DIAPH1 和 DIAPH3）中的两种缺陷与不同类型的听力损失有关。在这里，我们通过外显子组测序鉴定出 DIAPH2 中的一个预测致病性错义变异，从而研究了该家族的第三个成员 DIAPH2 在非综合征性听力损失中的作用。该变体发生在保守位点，与意大利一个家族的非综合征性 X 连锁听力损失共分离。我们的免疫组织化学研究表明，小鼠同源蛋白 Diaph2 在耳蜗发育过程中表达，特别是在感觉外毛细胞的富含肌动蛋白的静纤毛中表达。体外研究表明，突变的 DIAPH2 蛋白在 RhoA 依赖性激活时表现出功能障碍。最后，通过 CRISPR/Cas9 技术生成了 Diaph2 敲除和敲入小鼠，并在 4、8 和 14 周时进行了听觉脑干反应测量。然而，未检测到听力损伤。我们的发现表明 DIAPH2 可能在内耳中发挥作用；然而，需要进一步的研究来阐明 DIAPH2 对耳聋的贡献。
