【摘要】：目的對一例sjogren-Larsson綜合征(Sjogren-Larsson syndrome,SLS)患者的ALDH3A2基因進行突變檢測和功能學實驗。研究SLS患者ALDH3A2基因突變的類型和特點,為進一步研究ALDH3A2基因突變導致SLS的致病機理奠定基礎。方法該實驗對象為山東省青島市1例SLS患兒及其父母。采集患兒及其父母外周靜脈血并提取DNA,采用聚合酶鏈式反應(Polymerase Chain Reaction,PCR)擴增ALDH3A2基因編碼區(qū)的全部外顯子并測序。本實驗第二部分對在ALDH3A2基因篩查中發(fā)現(xiàn)的新突變(c.723CG)進行功能學研究。從新鮮的肝組織中提取RNA并進行逆轉錄,設計相應引物擴增ALDH3A2全編碼基因,構建ALDH3A2野生型真核表達載體,利用定點誘變試劑盒構建ALDH3A2突變體,對野生型表達載體和突變型表達載體進行正反向測序驗證。用野生型和突變型質(zhì)粒分別轉染人皮膚成纖維細胞,轉染48h后收集、裂解細胞,提取總蛋白,用多功能酶標儀檢測突變對脂肪醛脫氫酶活性的影響,與野生型比較分析得出突變體活性的改變。結果在受檢者ALDH3A2基因發(fā)現(xiàn)c.723CG(編碼區(qū)第723號核苷酸由C變?yōu)镚)、c.1157AG(編碼區(qū)第1157號核苷酸由A變?yōu)镚)的復合雜合核苷酸變異,上述變異分別導致第241號氨基酸由Cys變?yōu)門rp(p.Cys241Trp)、第386號氨基酸由Asn變?yōu)镾er(p.Asn386Ser),均為錯義變異。受檢者其父在位點c.723發(fā)現(xiàn)CG的雜合變異,其母該位點未見異常;受檢者其母在位點c.1157發(fā)現(xiàn)AG的雜合變異,其父該位點未見異常。對野生型和突變型表達載體進行酶活檢測,與野生型相比較,c.723CG造成FALDH活性的降低。結論在受檢者ALDH3A2基因所發(fā)現(xiàn)的復合雜合變異分別遺傳自受檢者其父母,父母均為雜合子,該變異是導致受檢者發(fā)病的致病性變異。變異c.1157AG的致病性已經(jīng)文獻報道,與Sjoegren-Larsson綜合癥相關。變異c.723CG的致病性尚未見文獻報道,對其進行功能學研究,證實了c.723CG是導致患者發(fā)病的致病性突變。
[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.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R596

【參考文獻】

相關期刊論文 前3條

1 蘆小燕;解慧琪;李莉;智偉;陳曉禾;鄧力;;人成纖維細胞穩(wěn)定轉染的方法比較[J];四川大學學報(醫(yī)學版);2008年04期

2 陳昕,趙國華;Sjgren-Larsson綜合征研究進展[J];中國當代兒科雜志;2002年06期

3 曲春華;痙攣性癱瘓 智力發(fā)育不全魚鱗癬綜合征1例報告[J];中國民間療法;1999年02期

，

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