后极性表型先天性白内障的疾病相关候选基因定位

发布时间：2018-02-27 23:15

  本文关键词： 先天性白内障 CRYBB2基因 基因突变 聚合酶链反应 限制性片段长度多态性　出处：《浙江大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的： 先天性白内障是儿童期视力缺损的首要病因之一。遗传突变是最常见的致病因素。迄今为止,至少已发现与遗传性的先天性白内障相关的24个疾病相关基因。本实验对一个后极性表型的先天性白内障家系进行疾病相关候选基因定位。 方法： 本实验拟研究一四代的先天性白内障大家系,共27人,患者9名。所有家系成员均接受病史调查和眼科检查,包括视力、裂隙灯、散瞳眼底检查等。抽取外周血样本,提取基因组DNA。聚合酶链反应(PCR)扩增相关的候选基因的编码区及内含子、外显子交界区。扩增产物双向测序,并进行序列分析。限制性片段长度多态性(RFLP)进一步验证突变。SIFT, PolyPhen, Align-GVGD三种计算法和疏水性分析预测突变对蛋白质结构、功能的影响。 结果： 裂隙灯下检查该家系表型为后极性。分析家系图,遗传方式为常染色体显性遗传。测序结果发现CRYBB2基因编码区一条等位基因的第5位碱基有C—T改变,导致了第2位氨基酸由高保守的丙氨酸变为缬氨酸(CRYBB2-A2V).该碱基改变于9名患者中共分离,而不存在于27名健康家系成员及100名健康者对照。RFLP证实该突变致使患者丢失一个HaeⅢ酶切位点,而健康的家庭成员和100个健康者对照中未有改变。SIFT, PolyPhen, Align-GVGD分数均提示A2V为可影响蛋白质结构和功能的有意义的突变。疏水性分析结果显示A2V突变蛋白在突变区域的疏水性高于野生型β-晶状体蛋白。 结论： 本研究首次证实了CRYBB2基因第2外显子中第5位碱基发生C—T改变是与后极性先天性白内障表型共分离的错义突变,CRYBB2基因为后极性先天性白内障的疾病相关候选基因。进一步证实了先天性白内障的遗传异质性,以及白内障表型与基因型的相对相关性,支持了βB2-晶状体蛋白在人晶状体发育及白内障形成过程中具有重要作用的理论。
[Abstract]:Objective:. Congenital cataract is one of the leading causes of childhood visual impairment. Genetic mutation is the most common cause. At least 24 disease-related genes associated with inherited congenital cataract have been identified. Methods:. In this study, we studied 14 and 4 generations of congenital cataract, including 27 patients and 9 patients. All the family members underwent medical history investigation and ophthalmologic examination, including visual acuity, slit lamp, mydriasis fundus examination, etc. Peripheral blood samples were taken. Genomic DNA.Polymerase chain reaction (PCR) was used to amplify the coding region, intron and exon junctions of the candidate genes. Restriction fragment length polymorphism (RFLP) further verified the effect of mutation SIFT, PolyPhen, Align-GVGD and hydrophobic analysis on protein structure and function. Results:. The phenotype of the pedigree was examined to be post polarity under slit lamp. The genetic pattern was autosomal dominant inheritance. The results of sequencing showed that the fifth base of an allele in the coding region of CRYBB2 gene had C-T changes. The result was that the second amino acid changed from a highly conserved alanine to a valine, CRYBB2-A2V, which was isolated from nine patients. RFLP did not exist in 27 healthy family members and 100 healthy controls. RFLP confirmed that the mutation resulted in the loss of a Hae 鈪，

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