交界型大疱性表皮松解症的LAMA3和LAMB3基因突变研究

发布时间：2018-05-19 15:31

  本文选题：交界型大疱性表皮松解症 + LAMA3基因　； 参考：《青岛大学》2017年硕士论文

【摘要】：目的:检测6例临床上确诊为喉-甲-皮肤综合征(LOCs)患者及1例致死性Herlitz型交界型大疱性表皮松解症(JEB-H)患者的LAMA3基因和LAMB3基因突变情况。方法:1.完善患者临床资料后,分别提取出6例喉-甲-皮肤综合征(LOCs)患者,1例致死性Herlitz型交界型大疱性表皮松解症(JEB-H)患者和其相关家属的外周血DNA;2.PCR方法扩增上述患者LAMA3和LAMB3基因所有外显子及其两侧相应的内含子序列,PCR扩增产物送公司测序检测其突变情况;3.以上测序结果结合文献中已经报道过的相关突变位点,综合分析;4.设计体外实验,通过Realtime-PCR方法和Western Blot方法检测文献中已经报道过的LAMA3基因的两个无义突变位点,及该突变导致的LAMA3截短体蛋白对伤口愈合的影响;5.体外设计minigene实验,预测JEB-H患者的LAMB3基因杂合剪切位点突变对m RNA剪接产生的影响。结果:1.基因突变检测结果:6例LOCs患者中的一例患者检测到LAMA3基因的as IVS39-1GA及p.R793*复合杂合突变;1例JEB-H患者存在LAMB3基因的as IVS11-22GA和p.C325*复合杂合突变。4个突变位点均为新发突变位点,且300例健康对照未发现此突变;2.收集到文献中已经报道过的LOCs患者存在的两个无义突变位点p.E292*与p.Q511*;3.Realtime-PCR和Western Blot结果:与正常对照(LAMA3全长蛋白)相比,两个LAMA3截短体蛋白在RNA水平和蛋白水平上均促进Ⅰ型胶原蛋白和Ⅲ型胶原蛋白的表达;4.RT-PCR结果:RT-PCR产物琼脂糖凝胶电泳后,将含目的条带的琼脂糖凝胶放置于凝胶成像仪上进行成像分析,分析结果为:空质粒显示248bp的单一条带,野生型质粒显示404bp的单一条带,突变型质粒则显示424bp的单一条带。结论:1.LAMA3截短体蛋白促进成纤维细胞分泌Ⅰ型胶原蛋白和Ⅲ型胶原蛋白,可能是影响患者伤口愈合的主要原因;2.该例JEB-H患者LAMB3基因的剪切位点突变as IVS11-22GA影响因m RNA剪接,该患者为LAMB3基因的无义突变合并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，

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