Sjogren-Larsson综合征ALDH3A2基因突变筛查及致病机理研究

发布时间：2018-11-09 11:28

【摘要】：目的对一例sjogren-Larsson综合征(Sjogren-Larsson syndrome,SLS)患者的ALDH3A2基因进行突变检测和功能学实验。研究SLS患者ALDH3A2基因突变的类型和特点,为进一步研究ALDH3A2基因突变导致SLS的致病机理奠定基础。方法该实验对象为山东省青岛市1例SLS患儿及其父母。采集患儿及其父母外周静脉血并提取DNA,采用聚合酶链式反应(Polymerase Chain Reaction,PCR)扩增ALDH3A2基因编码区的全部外显子并测序。本实验第二部分对在ALDH3A2基因筛查中发现的新突变(c.723CG)进行功能学研究。从新鲜的肝组织中提取RNA并进行逆转录,设计相应引物扩增ALDH3A2全编码基因,构建ALDH3A2野生型真核表达载体,利用定点诱变试剂盒构建ALDH3A2突变体,对野生型表达载体和突变型表达载体进行正反向测序验证。用野生型和突变型质粒分别转染人皮肤成纤维细胞,转染48h后收集、裂解细胞,提取总蛋白,用多功能酶标仪检测突变对脂肪醛脱氢酶活性的影响,与野生型比较分析得出突变体活性的改变。结果在受检者ALDH3A2基因发现c.723CG(编码区第723号核苷酸由C变为G)、c.1157AG(编码区第1157号核苷酸由A变为G)的复合杂合核苷酸变异,上述变异分别导致第241号氨基酸由Cys变为Trp(p.Cys241Trp)、第386号氨基酸由Asn变为Ser(p.Asn386Ser),均为错义变异。受检者其父在位点c.723发现CG的杂合变异,其母该位点未见异常;受检者其母在位点c.1157发现AG的杂合变异,其父该位点未见异常。对野生型和突变型表达载体进行酶活检测,与野生型相比较,c.723CG造成FALDH活性的降低。结论在受检者ALDH3A2基因所发现的复合杂合变异分别遗传自受检者其父母,父母均为杂合子,该变异是导致受检者发病的致病性变异。变异c.1157AG的致病性已经文献报道,与Sjoegren-Larsson综合症相关。变异c.723CG的致病性尚未见文献报道,对其进行功能学研究,证实了c.723CG是导致患者发病的致病性突变。
[Abstract]:Objective to detect the mutation and function of ALDH3A2 gene in a patient with sjogren-Larsson syndrome (Sjogren-Larsson syndrome,SLS). To study the types and characteristics of ALDH3A2 gene mutation in patients with SLS lay a foundation for further study on the pathogenesis of SLS caused by ALDH3A2 gene mutation. Methods one child with SLS and his parents in Qingdao, Shandong Province, were studied. Peripheral venous blood and DNA, were extracted from peripheral venous blood of children and their parents. All exons of ALDH3A2 gene coding region were amplified by polymerase chain reaction (Polymerase Chain Reaction,PCR) and sequenced. In the second part of this experiment, the new mutation (c.723CG) found in ALDH3A2 gene screening was studied. RNA was extracted from fresh liver tissue and reverse transcription was carried out. Corresponding primers were designed to amplify the full coding gene of ALDH3A2 and construct ALDH3A2 wild-type eukaryotic expression vector. ALDH3A2 mutants were constructed by site-directed mutagenesis kit. The wild type expression vector and mutant type expression vector were confirmed by forward and reverse sequencing. Human skin fibroblasts were transfected with wild-type and mutant plasmids respectively. After 48 hours of transfection, the cells were collected, lysed, total proteins were extracted, and the effects of mutation on the activity of aliphatic aldehyde dehydrogenase were detected by multifunctional enzyme marker. Compared with wild type, the change of activity of mutants was obtained. Results the complex heterozygote mutations of c.723CG (coding region 723 nucleotides changed from C to G), c.1157AG (coding region 1157 nucleotides from A to G) were found in the ALDH3A2 gene. The results showed that the 241 amino acid changed from Cys to Trp (p.Cys241Trp) and the 386-amino acid from Asn to Ser (p.Asn386Ser). The heterozygosity of CG was found at locus c. 723, but not abnormal at locus c. 1157. The heterozygosity of AG was found at locus c. 1157, but not abnormal at locus c. 1157. The enzyme activity of wild type and mutant type expression vector was detected. Compared with wild type, c.723CG resulted in the decrease of FALDH activity. Conclusion the complex heterozygosity found in the ALDH3A2 gene of the tested subjects was inherited from the parents and parents of the tested subjects, respectively. The variation is the pathogenicity variation leading to the onset of the disease in the subjects. The pathogenicity of mutated c.1157AG has been reported to be associated with Sjoegren-Larsson syndrome. The pathogenicity of mutated c.723CG has not been reported in the literature. The functional study has confirmed that c.723CG is a pathogenicity mutation leading to the pathogenesis of c.723CG in patients.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R596

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