X连锁显性遗传Alport综合征的产前基因诊断

Prenatal diagnosis and genetic counseling of X-linked Alport syndrome in China

目的 Alport 综合征(AS)是最常见的遗传性肾脏疾病,预后较差,无特异有效的治疗。X 连锁显性(XLAS)是其主要遗传方式,因 COL4A5基因突变或 COL4A5和 COL4A6两个基因突变所致。通过对两个 XLAS 家系进行遗传咨询和基因诊断,并对其中一个家系实施产前基因诊断,探讨AS 产前基因诊断的方法及临床应用价值。方法对两个 XLAS 家系进行详细的遗传咨询后,采用巢式 PCR 扩增外周血淋巴细胞 COL4A5 mRNA 的整个编码区序列筛查两个家系的基因突变,然后扩增COL4A5相应的外显子从基因组 DNA 水平进一步证实突变。产前基因诊断通过羊水细胞 cDNA 和DNA 两个水平来检测胎儿突变情况,通过 PCR 扩增 Y 染色体性别决定基因 SRY 联合核型分析检测胎儿性别,通过三个 X 染色体微卫星标记(AR、DXS6797和 DXS6807)连锁分析除外羊水中母体细胞污染的可能。结果突变筛查显示家系一为新发突变,先证者 COL4A5第2696～2705位缺失GTATGATGGG 共10个碱基,但母亲外周血基因组 DNA 不携带该缺失突变,通过遗传咨询,该家系未进行羊水穿刺和产前基因诊断。家系二 COL4A5基因第4271位 G 被 A 取代,1424位 G>E,家系其他患者包括孕母均携带该突变。对家系二孕母进行了羊水穿刺和产前基因诊断。羊水细胞 cDNA 和DNA 水平检测均显示胎儿无突变,PCR 扩增 SRY 基因和羊水细胞核型分析均显示胎儿为男性。连锁分析显示羊水细胞中无母体细胞污染,且间接提示胎儿从母体遗传了携带正常 COL4A5等位基因的X染色体。结论基于外周血淋巴细胞 cDNA 水平的 COL4A5基因突变检测技术可快速用于 XLAS产前基因诊断时家系基因突变筛查,有利于客观进行遗传咨询;产前基因诊断时联合 cDNA 和 DNA两个水平检测胎儿基因突变情况更准确可信,PCR 扩增 SRY 基因可以快速鉴定胎儿性别,X 染色体微卫星标记连锁分析不但可以排除羊水中母体细胞污染,而且可以间接证明胎儿是否携带致病等位基因。

Objective Alport syndrome （AS） is a progressive renal disease characterized by hematuria and progressive renal failure. X-linked dominance is the major inheritance form of the syndrome, accounting for almost 80% of the cases, caused by mutations in COL4A5 genes. There is currently no effective treatment that has been shown to favorably affect the outcome of AS, so early diagnosis and even prenatal diagnosis is very important. Methods In this study mutation of COL4A5 was detected by amplifying the entire coding sequence mRNA of peripheral blood lymphocytes using nested PCR in two Chinese X-linked dominant Alport syndrome （XLAS） families, then the first prenatal diagnosis of XLAS in China was performed. Mutation analysis of the fetus was performed on both cDNA-based level and DNA- based level of amniocytes. Fetus sex was determined by PCR amplification of SRY as well as karyotypes analysis. Maternal cells contamination was excluded by linkage analysis. Results There was a deletion mutation in the proband of the first family, 2696-2705 del gtatgatggg in the 32 exon of COL4A5, but the mother did not carry the mutation （de novo）. There was a G to A substitution at position 4271 in exon 46 of COL4A5 gene （c. G4271A） in the second family, the mother also carried this mutation. After genetic counselling, only the second family accepted prenatal diagnosis. Both amnioeytes cDNA level and amniocytes genomic DNA level based prenatal diagnosis showed that the fetus did not carry the same mutation as the mother. PCR amplification of SRY and karyotypes analysis showed a male fetus. Linkage analysis of X chromosome polymorphic microsatellite markers showed that there was no MCC in amniocytes. Conclusion Both cDNA level and DNA level analysis could enhance the accuracy and reliability of prenatal diagnosis. PCR amplification of SRY was faster than karyotypes analysis in the fetal sex determination. Linkage analysis was useful in the detection of maternal cells contamination in amniocytes.