发根DNA检测Alport综合征COL4A5基因突变

Detection of mutation in COL4A5 gene of Alport's syndrome by extracting DNA from hair root

目的:通过发根DNA提取分析Alport综合征(Alportsyndrome,AS)一家系中COL4A5基因突变,试图通过该方法简化AS家系的收集工作和推广COL4A5基因检测。方法:通过PCR和直接测序的方法,对先证者外周血DNACOL4A5基因所有51个外显子及其相邻内含子的DNA序列进行检测。发现突变位点后,提取6份该家系成员发根部DNA进行该突变位点检测。结果:①成功地从发根部提取DNA,进行了PCR和直接测序。②在COL4A5基因第50号外显子4944位点发现一错义突变,碱基G被T取代,导致其编码1648位氨基酸由色氨酸变为半胱氨酸(4944G>TW1648C),男性患者为纯合突变,女性患者为杂合突变,正常家系成员和92例对照中均未发现此位点异常,说明W1648C为引起该家系临床病变的突变位点,并非多态性位点。在性连锁Alport综合征中,COL4A5基因此突变位点为首次报道。结论:通过毛发收集、DNA提取,可使家系收集工作更为简便易行,可帮助未开展基因检测的地区进行基因诊断。本研究通过发根DNA提取检测Alport综合征一家系中新的COL4A5基因突变,遗传方式符合性连锁显性遗传。

Objective： By collecting and sequencing the DNA of hair root in a Chinese family with X-linked Alport＇s syndrome （XLAS）, to establish a method that can simplify the investigation of family members and generalize genetic diagnosis of AS. Methodology： We extracted genomic DNA from peripheral blood cells of the proband and from hair root of six family members. By means of PCR and direct sequencing, all 51 exons and their neighboring intronic sequences of the COL4A5 gene in the proband were analyzed at first. Then this mutation position was detected using the DNA extracted from 6 family members＇hair root. Results： ①The DNA from root region of hair was extracted and amplified successfully. ②At those position 4944 in exon 50, a missense mutation was found（4944G〉T）, which changed the 1648 th amino acid from Tryptophan to Cysteine（W1648C）. The male patient had homozygous mutation; all the female patients had heterozygous mutation, whereas no mutation was found in normal family members and 92 control healthy individuals, h was suggested that mutation was the underlying cause of XLAS in this family, and not a polymorphism. ③This mutation of COL4A5 gene is first reported in XLAS. Conclusion： Collection and extraction of DNA from root region of hair can simplify the investigation of family members and help to genetic diagnosis in area without gene sequencing. We detected a new mutation in a Chinese family with XLAS by means of amplifying DNA of hair root.