HARS2基因突变致以耳聋为表现的Perrault综合征致病机制的初步研究
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西南医科大学附属医院

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泸州市科技计划项目(2021-SYF-30) ,项目负责人:李雷激


Preliminary study on the pathogenesis of Perrault syndrome with deafness due to mutation of HARS2 gene
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    摘要:

    目的 研究耳聋家系HARS2基因突变位点对HARS2蛋白的影响,为研究HARS2基因突变致Perrault综合征(Perrault syndrome,PRLTS)的发病机制奠定基础。方法 检测并验证该家系成员样本DNA的HARS2基因。使用软件对蛋白质建模,分析预测Asp117Asn和Leu303Pro突变对HARS2蛋白结构稳定性的影响。转染含野生型HARS2基因和HARS2基因Asp117Asn突变、Leu303Pro突变的慢病毒重组质粒以及空载体至HEK 293T细胞(human embryonic kidney cells,人胚肾细胞)中,分别获得WT细胞组、Asp117Asn细胞组、Leu303Pro细胞组、NC细胞组。Northern-blot检测线粒体tRNAHis氨酰化水平;Western-blot检测HARS2蛋白的表达;免疫荧光测定HARS2蛋白表达部位。结果 (1)先证者及其哥哥为耳聋患者,为HARS2 c.349G>A(p.Asp117Asn)和c.908T>C(p.Leu303Pro)复合杂合突变;其父亲为HARS2 c.908T>C(p.Leu303Pro)杂合突变;其外婆、母亲、舅舅为HARS2 349G>A(p.Asp117Asn)杂合突变。(2)突变可能导致HARS2蛋白稳定性下降。(3)WT细胞组线粒体tRNAHis氨酰化水平高于其余三组(P<0.05);Asp117Asn细胞组较Leu303Pro细胞组线粒体tRNAHis氨酰化水平高(P=0.016)。(4)HARS2蛋白均于线粒体表达。(5)HARS2蛋白在NC细胞组中无明显表达;WT细胞组HARS2蛋白表达与Asp117Asn细胞组无明显差异(P=0.356),高于Leu303Pro细胞组(p=0.000)。结论 1、明确了HARS2基因2个新突变位点c.349G>A和c.908T>C。2、该突变可能通过降低HARS2蛋白氨酰化能力和(或)表达,导致线粒体tRNAHis氨酰化能力降低,进而出现线粒体功能障碍,从而导致听力损失。

    Abstract:

    OObjective A basis for studying the pathogenesis of Perrault syndrome (PRLTS) caused by mutations in the HARS2 gene by studying of the effect of mutated loci of HARS2 gene on HARS2 protein in deaf families.Methods Detection and validation of the HARS2 gene in the DNA of samples from members of this family line.Analysis of protein modeling using software to predict the effect of Asp117Asn and Leu303Pro mutations on the structural stability of HARS2 protein.Lentiviral recombinant plasmids containing Leu303Pro mutation and Asp117Asn mutation of HARS2 gene ,wild-type HARS2 gene and empty vector were transfected into HEK 293T cells (human embryonic kidney cells) to obtain WT cell group, Asp117Asn cell group, Leu303Pro cell group and NC cell group.Northern-blot for mitochondrial tRNAHis amylation level;Western-blot for HARS2 protein expression;immunofluorescence for HARS2 protein expression site;Results (1)The preexisting patient and his brother were deaf and had a compound heterozygous mutation of HARS2 349G>A (p.Asp117Asn) and 908T>C (p.Leu303Pro);his father had a heterozygous mutation of HARS2 c.908T>C (p. Leu303Pro) heterozygous mutation; his grandmother, mother, and uncle had a heterozygous mutation of HARS2 349G>A (p.Asp117Asn);.(2)Mutations may affect HARS2 protein stability.(3)The mitochondrial tRNAHis amylation level in WT cell group was higher than the remaining three groups (P<0.05); Asp117Asn cell group had higher level of mitochondrial tRNA His amylation than Leu303Pro cell group (P=0.016).(4) HARS2 proteins are all expressed in mitochondria.(5)HARS2 protein was not significantly expressed in the NC cell group; HARS2 protein expression in WT cell group was not significantly different from Asp117Asn cell group (p=0.356) and higher than Leu303Pro cell group (p=0.000).Conclusion 1.Two new mutant loci c.349G>A and c.908T>C of the HARS2 gene were identified in this family.2.The mutation may lead to reduced mitochondrial tRNAHis ammonia-acylation capacity through reduced HARS2 protein ammonia-acylation capacity and/or expression, which in turn leads to mitochondrial dysfunction and thus hearing loss.

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  • 收稿日期:2022-06-01
  • 最后修改日期:2022-08-26
  • 录用日期:2022-08-30
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