Sjogren-Larsson綜合征ALDH3A2基因突變篩查及致病機(jī)理研究
發(fā)布時(shí)間：2018-11-09 11:28
【摘要】：目的對(duì)一例sjogren-Larsson綜合征(Sjogren-Larsson syndrome,SLS)患者的ALDH3A2基因進(jìn)行突變檢測和功能學(xué)實(shí)驗(yàn)。研究SLS患者ALDH3A2基因突變的類型和特點(diǎn),為進(jìn)一步研究ALDH3A2基因突變導(dǎo)致SLS的致病機(jī)理奠定基礎(chǔ)。方法該實(shí)驗(yàn)對(duì)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?zhǔn)椒磻?yīng)(Polymerase Chain Reaction,PCR)擴(kuò)增ALDH3A2基因編碼區(qū)的全部外顯子并測序。本實(shí)驗(yàn)第二部分對(duì)在ALDH3A2基因篩查中發(fā)現(xiàn)的新突變(c.723CG)進(jìn)行功能學(xué)研究。從新鮮的肝組織中提取RNA并進(jìn)行逆轉(zhuǎn)錄,設(shè)計(jì)相應(yīng)引物擴(kuò)增ALDH3A2全編碼基因,構(gòu)建ALDH3A2野生型真核表達(dá)載體,利用定點(diǎn)誘變試劑盒構(gòu)建ALDH3A2突變體,對(duì)野生型表達(dá)載體和突變型表達(dá)載體進(jìn)行正反向測序驗(yàn)證。用野生型和突變型質(zhì)粒分別轉(zhuǎn)染人皮膚成纖維細(xì)胞,轉(zhuǎn)染48h后收集、裂解細(xì)胞,提取總蛋白,用多功能酶標(biāo)儀檢測突變對(duì)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(jié)果在受檢者ALDH3A2基因發(fā)現(xiàn)c.723CG(編碼區(qū)第723號(hào)核苷酸由C變?yōu)镚)、c.1157AG(編碼區(qū)第1157號(hào)核苷酸由A變?yōu)镚)的復(fù)合雜合核苷酸變異,上述變異分別導(dǎo)致第241號(hào)氨基酸由Cys變?yōu)門rp(p.Cys241Trp)、第386號(hào)氨基酸由Asn變?yōu)镾er(p.Asn386Ser),均為錯(cuò)義變異。受檢者其父在位點(diǎn)c.723發(fā)現(xiàn)CG的雜合變異,其母該位點(diǎn)未見異常;受檢者其母在位點(diǎn)c.1157發(fā)現(xiàn)AG的雜合變異,其父該位點(diǎn)未見異常。對(duì)野生型和突變型表達(dá)載體進(jìn)行酶活檢測,與野生型相比較,c.723CG造成FALDH活性的降低。結(jié)論在受檢者ALDH3A2基因所發(fā)現(xiàn)的復(fù)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(dǎo)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(xiàn)報(bào)道,與Sjoegren-Larsson綜合癥相關(guān)。變異c.723CG的致病性尚未見文獻(xiàn)報(bào)道,對(duì)其進(jìn)行功能學(xué)研究,證實(shí)了c.723CG是導(dǎo)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(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R596
本文編號(hào)：2320276
[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(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R596
【參考文獻(xiàn)】
相關(guān)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(xì)胞穩(wěn)定轉(zhuǎn)染的方法比較[J];四川大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2008年04期
2 陳昕,趙國華;Sjgren-Larsson綜合征研究進(jìn)展[J];中國當(dāng)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(bào)告[J];中國民間療法;1999年02期
，本文編號(hào)：2320276
本文鏈接：http://www.sikaile.net/kejilunwen/jiyingongcheng/2320276.html
最近更新
教材專著
