Variants encoding a restricted carboxy-terminal domain of SLC12A2 cause hereditary hearing loss in humans
Variants encoding a restricted carboxy-terminal domain of SLC12A2 cause hereditary hearing loss in humans
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DOI：
10.1371/journal.pgen.1008643
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发表时间：
2020-04-01
期刊：
影响因子：
4.5
通讯作者：
Matsunaga, Tatsuo
中科院分区：
文献类型：
--
作者：
Mutai, Hideki;Wasano, Koichiro;Matsunaga, Tatsuo
声音是由听觉感觉细胞感知的，这是由于它们周围的组织，包括耳蜗外侧壁。耳蜗外侧壁的一部分，血管纹，对于产生和维持具有高钾浓度的内耳液，以及通过刺激边缘细胞分泌钾而在内耳中产生对声音感知重要的正电压是至关重要的。基因SLC 12 A2编码一种蛋白质，该蛋白质参与钠、钾和氯的转运，对血管纹中特定细胞的正常功能至关重要;然而，SLC 12 A2的人类变体以前与听力损失无关。通过对蛋白质编码序列的全面遗传分析，我们在四个先天性重度至极重度听力损失家族中确定了SLC 12 A2的四个候选变化。有趣的是，所有四种遗传变异体都在外显子21的3 '-剪接位点内或3'-剪接位点处，外显子21编码SLC 12 A2的羧基末端胞内结构域的一部分。在培养的细胞中的实验表明，跳跃或突变的外显子21显着减少由SLC 12 A2蛋白介导的氯离子内流。总之，我们的研究结果有力地表明，影响SLC 12 A2外显子21的突变代表了人类耳聋的一种新机制。遗传性听力损失是具有挑战性的诊断，因为致病基因的异质性。此外，一些与遗传性听力损失有关的基因尚未被确定。通过对三个先天性重度至极重度听力损失家系的全外显子组分析，我们在一个家系的五名受影响成员中发现了一个SLC 12 A2错义变异体，显示出显性遗传模式，沿着在两个散发病例中发现了SLC 12 A2的从头剪接位点和错义变异体，作为与听力损失相关的有希望的候选者。此外，我们发现另一个从头错义变异SLC 12 A2的散发病例。SLC 12 A2编码Na+，K+，2Cl（-）协同转运蛋白（NKCC）1，在富含K+的内淋巴的稳态中起关键作用。Slc 12 a2缺陷小鼠具有先天性的深度耳聋;然而，没有SLC 12 A2的人类变体被报道与听力损失相关。所有鉴定的SLC 12 A2变体均定位于外显子21或其3 '-剪接位点。体外分析表明，剪接位点变异体产生的外显子21跳过SLC 12 A2 mRNA转录本的表达水平远低于21号外显子的转录本，这表明21号外显子编码的区域在carboy末端结构域的组织特异性作用。体外功能分析表明，在所有研究的SLC 12 A2变体中，Cl-内流显著减少。免疫组织化学显示，SLC 12 A2位于耳蜗中几种类型细胞的质膜上，包括耳蜗边缘细胞，这对内淋巴稳态至关重要。总体而言，这项研究表明，影响SLC 12 A2转录本外显子21的变异是人类遗传性听力损失的原因。
Author summarySounds are perceived by auditory sensory cells, owing to tissues surrounding them, including the cochlear lateral wall. Part of the cochlear lateral wall, the stria vascularis, is critical for production and maintenance of inner-ear fluid with a high potassium concentration, and for generating the positive voltage in the inner ear, important for sound perception, by stimulating secretion of potassium from marginal cells. The gene SLC12A2 encodes a protein involved in sodium, potassium, and chloride transport essential for proper function of specific cells in the stria vascularis; however, human variants of SLC12A2 have not previously been associated with hearing loss. By comprehensive genetic analysis of protein-coding sequences, we identified four candidate changes in SLC12A2 in four families with congenital, severe-to-profound hearing loss. Intriguingly, all four genetic variants were either within or at the 3'-splice site of the exon 21 which encodes a part of the carboxy terminal intracellular domain of SLC12A2. Experiments in cultured cells showed that skipping or mutation of exon 21 significantly decreased chloride influx mediated by the SLC12A2 protein. Overall, our results strongly indicate that mutations influencing exon 21 of SLC12A2 represent a novel mechanism underlying deafness in humans.Hereditary hearing loss is challenging to diagnose because of the heterogeneity of the causative genes. Further, some genes involved in hereditary hearing loss have yet to be identified. Using whole-exome analysis of three families with congenital, severe-to-profound hearing loss, we identified a missense variant of SLC12A2 in five affected members of one family showing a dominant inheritance mode, along with de novo splice-site and missense variants of SLC12A2 in two sporadic cases, as promising candidates associated with hearing loss. Furthermore, we detected another de novo missense variant of SLC12A2 in a sporadic case. SLC12A2 encodes Na+, K+, 2Cl(-) cotransporter (NKCC) 1 and plays critical roles in the homeostasis of K+-enriched endolymph. Slc12a2-deficient mice have congenital, profound deafness; however, no human variant of SLC12A2 has been reported as associated with hearing loss. All identified SLC12A2 variants mapped to exon 21 or its 3'-splice site. In vitro analysis indicated that the splice-site variant generates an exon 21-skipped SLC12A2 mRNA transcript expressed at much lower levels than the exon 21-included transcript in the cochlea, suggesting a tissue-specific role for the exon 21-encoded region in the carboy-terminal domain. In vitro functional analysis demonstrated that Cl- influx was significantly decreased in all SLC12A2 variants studied. Immunohistochemistry revealed that SLC12A2 is located on the plasma membrane of several types of cells in the cochlea, including the strial marginal cells, which are critical for endolymph homeostasis. Overall, this study suggests that variants affecting exon 21 of the SLC12A2 transcript are responsible for hereditary hearing loss in humans.