本文選題：交界型大皰性表皮松解癥 + LAMA3基因�。� 參考：《青島大學》2017年碩士論文

【摘要】：目的:檢測6例臨床上確診為喉-甲-皮膚綜合征(LOCs)患者及1例致死性Herlitz型交界型大皰性表皮松解癥(JEB-H)患者的LAMA3基因和LAMB3基因突變情況。方法:1.完善患者臨床資料后,分別提取出6例喉-甲-皮膚綜合征(LOCs)患者,1例致死性Herlitz型交界型大皰性表皮松解癥(JEB-H)患者和其相關家屬的外周血DNA;2.PCR方法擴增上述患者LAMA3和LAMB3基因所有外顯子及其兩側相應的內含子序列,PCR擴增產物送公司測序檢測其突變情況;3.以上測序結果結合文獻中已經報道過的相關突變位點,綜合分析;4.設計體外實驗,通過Realtime-PCR方法和Western Blot方法檢測文獻中已經報道過的LAMA3基因的兩個無義突變位點,及該突變導致的LAMA3截短體蛋白對傷口愈合的影響;5.體外設計minigene實驗,預測JEB-H患者的LAMB3基因雜合剪切位點突變對m RNA剪接產生的影響。結果:1.基因突變檢測結果:6例LOCs患者中的一例患者檢測到LAMA3基因的as IVS39-1GA及p.R793*復合雜合突變;1例JEB-H患者存在LAMB3基因的as IVS11-22GA和p.C325*復合雜合突變。4個突變位點均為新發(fā)突變位點,且300例健康對照未發(fā)現此突變;2.收集到文獻中已經報道過的LOCs患者存在的兩個無義突變位點p.E292*與p.Q511*;3.Realtime-PCR和Western Blot結果:與正常對照(LAMA3全長蛋白)相比,兩個LAMA3截短體蛋白在RNA水平和蛋白水平上均促進Ⅰ型膠原蛋白和Ⅲ型膠原蛋白的表達;4.RT-PCR結果:RT-PCR產物瓊脂糖凝膠電泳后,將含目的條帶的瓊脂糖凝膠放置于凝膠成像儀上進行成像分析,分析結果為:空質粒顯示248bp的單一條帶,野生型質粒顯示404bp的單一條帶,突變型質粒則顯示424bp的單一條帶。結論:1.LAMA3截短體蛋白促進成纖維細胞分泌Ⅰ型膠原蛋白和Ⅲ型膠原蛋白,可能是影響患者傷口愈合的主要原因;2.該例JEB-H患者LAMB3基因的剪切位點突變as IVS11-22GA影響因m RNA剪接,該患者為LAMB3基因的無義突變合并as IVS10-22GA而致病。
[Abstract]:Objective: to detect the mutation of LAMA3 gene and LAMB3 gene in 6 patients with laryngo-A-skin syndrome and 1 patient with fatal Herlitz type epidermolysis bullosa. Method 1: 1. After improving the patient's clinical data, All exons of LAMA3 and LAMB3 gene were extracted from 6 patients with laryngo-A-skin syndrome and 1 patient with fatal Herlitz type epidermolysis bullous epidermolysis (JEB-H) and their relatives. All exons of LAMA3 and LAMB3 gene were amplified by PCR; and The corresponding intron sequences on both sides of the PCR products were sent to the company for sequencing to detect the mutation. The above sequencing results were combined with the related mutation sites reported in the literature. In vitro experiments were designed to detect the two nonsense mutation sites of LAMA3 gene and the effect of LAMA3 truncated body protein (LAMA3 truncated body protein) on wound healing, which had been reported in the literature by Realtime-PCR and Western Blot methods. Minigene assay was designed in vitro to predict the effect of LAMB3 gene heterozygous shear site mutation on m RNA splicing in JEB-H patients. The result is 1: 1. The results of gene mutation analysis showed that one of 6 patients with LOCs detected LAMA3 gene as IVS39-1GA and p. R793* heterozygosity mutation. One patient with JEB-H had LAMB3 gene as IVS11-22GA and p. C325 * heterozygosity mutation. All the 4 mutation sites were new mutations. This mutation was not found in 300 healthy controls. Two nonsense mutation sites, p.E292* and p.Q5113Realtime-PCR and Western Blot results were collected in LOCs patients reported in the literature: compared with the full-length protein of Lama 3 in normal controls. Two LAMA3 truncated body proteins promoted the expression of collagen type 鈪，

本文編號：1910644