背景:Leigh综合征(LS)是一种常见的线粒体疾病,由线粒体和核基因突变引起。异亮氨酸tRNA合成酶2(IARS2)编码线粒体异亮氨酸tRNA合成酶,据报道,IARS2中的变体会导致LS。然而,IARS2变异的致病机制尚不清楚。
方法:两名无关患者,一个4岁的男孩和一个5岁的男孩被诊断为LS,被招募,并收集了详细的临床数据。从外周血中分离患者及其父母的DNA,用于使用下一代测序和Sanger测序鉴定致病变异。ClustalW计划,等位基因频率分析数据库(gnomAD和ExAc),和致病性预测数据库(Clinvar,突变Taster和PolyPhen2)用于预测变体的保守性和致病性。基因表达水平,耗氧率(OCR),呼吸链复合物活动,细胞三磷酸腺苷(ATP)生产,在患者来源的淋巴细胞和IARS2敲低HEK293T细胞中测量线粒体膜电位(MMP)和线粒体活性氧(ROS)水平,以评估变体的致病性.
结果:我们报道了2例表现为LS的无关中国患者,这些患者携带双等位基因IARS2变体(c.1_390del和c.2450G>来自一个4岁男孩,c.2090G>A和c.2122G>A来自一个5岁男孩),其中c.1_390del和c.2090G>A是新颖的。功能研究表明,携带c.1_390del和c.2450G>A变体的患者来源的淋巴细胞由于严重的线粒体复合物I和III缺陷而表现出线粒体功能受损,在IARS2敲低HEK293T细胞中也发现。体外细胞模型的补偿性实验证实了IARS2变体的致病性,因为重新表达野生型IARS2而不是突变体IARS2可以挽救复合物I和III的缺陷。耗氧量,和IARS2敲低细胞中的细胞ATP含量。
结论:我们的结果不仅扩大了LS的基因突变谱,而且还首次揭示了由于线粒体复合物I和III的联合缺乏而导致的IARS2变体的致病机制,有助于IARS2突变相关疾病的临床诊断。
BACKGROUND: Leigh syndrome (LS) is a common mitochondrial disease caused by mutations in both mitochondrial and nuclear genes. Isoleucyl-tRNA synthetase 2 (IARS2) encodes mitochondrial isoleucine-tRNA synthetase, and variants in IARS2 have been reported to cause LS. However, the pathogenic mechanism of IARS2 variants is still unclear.
METHODS: Two unrelated patients, a 4-year-old boy and a 5-year-old boy diagnosed with LS, were recruited, and detailed clinical data were collected. The DNA of the patients and their parents was isolated from the peripheral blood for the identification of pathogenic variants using next-generation sequencing and Sanger sequencing. The ClustalW program, allele frequency analysis databases (gnomAD and ExAc), and pathogenicity prediction databases (Clinvar, Mutation Taster and PolyPhen2) were used to predict the conservation and pathogenicity of the variants. The gene expression level, oxygen consumption rate (OCR), respiratory chain complex activity, cellular adenosine triphosphate (ATP) production, mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (ROS) levels were measured in patient-derived lymphocytes and IARS2-knockdown HEK293T cells to evaluate the pathogenicity of the variants.
RESULTS: We reported 2 unrelated Chinese patients manifested with LS who carried biallelic IARS2 variants (c.1_390del and c.2450G > A from a 4-year-old boy, and c.2090G > A and c.2122G > A from a 5-year-old boy), of which c.1_390del and c.2090G > A were novel. Functional studies revealed that the patient-derived lymphocytes carrying c.1_390del and c.2450G > A variants exhibited impaired mitochondrial function due to severe mitochondrial complexes I and III deficiencies, which was also found in IARS2-knockdown HEK293T cells. The compensatory experiments in vitro cell models confirmed the pathogenicity of IARS2 variants since re-expression of wild-type IARS2 rather than mutant IARS2 could rescue complexes I and III deficiency, oxygen consumption, and cellular ATP content in IARS2 knockdown cells.
CONCLUSIONS: Our results not only expand the gene mutation spectrum of LS, but also reveal for the first time the pathogenic mechanism of IARS2 variants due to a combined deficiency of mitochondrial complexes I and III, which is helpful for the clinical diagnosis of IARS2 mutation-related diseases.