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目的 检测分析X连锁型Alport综合征(AS)患者α5(Ⅳ)链mRNA及其序列并探讨其基因型与表型之间的关系。方法 提取21例X连锁型AS患者的培养的皮肤成纤维细胞总RNA,应用逆转录-聚合酶链反应(RT-PCR)扩增覆盖全部α5(Ⅳ)链编码区的mRNA,经直接测序的方法分析。同时根据cDNA序列异常位置,应用PCR和直接测序的方法从基因组DNA水平分析COL4A5基因。结果 21例患者的mRNA分析均发现α5(Ⅳ)链序列异常,其中17个突变为新发现的突变。15例患者在mRNA水平检测到的结果与在基因组DNA检测到的结果一致;6例患者因发生剪接位点突变,在mRNA水平和基因组DNA水平检测到的COL4A5基因突变不一致。16/21例患者属于青少年型AS,2/21例属于成年型AS。结论 各种类型的COL4A5基因突变均可导致严重的临床表型,而且从mRNA水平分析AS基因型,可以发现并证实一些在基因组DNA水平无法发现和证实的异常。
Objective To detect and analyze the mRNA and sequence of α5 (Ⅳ) chain of patients with X-linked Alport syndrome (AS) and explore the relationship between the genotypes and phenotypes. Methods The total RNA was extracted from cultured skin fibroblasts of 21 patients with X-linked AS. The mRNA covering the entire coding region of α5 (Ⅳ) chain was amplified by reverse transcription-polymerase chain reaction (RT-PCR) Methodological analysis. At the same time, COL4A5 gene was analyzed from genomic DNA level by PCR and direct sequencing according to the abnormal position of cDNA sequence. Results All the 21 patients had abnormal α5 (Ⅳ) chain, of which 17 were found to be newly discovered mutations. Fifteen patients had the same result at the mRNA level as the one detected in the genomic DNA. Six of the patients had mutations in the COL4A5 gene at mRNA and genomic levels due to mutations in the splicing site. 16/21 patients belonged to adolescent AS and 2 of 21 belonged to adult AS. CONCLUSIONS: Various types of COL4A5 mutations lead to severe clinical phenotypes. Furthermore, analysis of AS genotypes at mRNA level may reveal and confirm some abnormalities that could not be detected and confirmed at genomic DNA level.