Zhang Hong-wen, Ding Jie, Wang Fang, Yang Hui-xia
Department of Pediatrics, Peking University First Hospital, Beijing 100034, China.
Zhonghua Er Ke Za Zhi. 2007 Jul;45(7):484-9.
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.
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.
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 amniocytes 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.
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.
Alport综合征（AS）是一种以血尿和进行性肾衰竭为特征的进行性肾脏疾病。X连锁显性是该综合征的主要遗传形式，约占病例的80%，由COL4A5基因突变引起。目前尚无已证实能有效改善AS预后的治疗方法，因此早期诊断甚至产前诊断非常重要。
本研究采用巢式PCR扩增两个中国X连锁显性Alport综合征（XLAS）家系外周血淋巴细胞的整个编码序列mRNA，检测COL4A5突变，然后进行中国首例XLAS的产前诊断。对羊水细胞进行基于cDNA水平和基于DNA水平的胎儿突变分析。通过PCR扩增SRY以及核型分析确定胎儿性别。通过连锁分析排除母源细胞污染。
第一个家系的先证者存在缺失突变，位于COL4A5第32外显子的2696 - 2705 del gtatgatggg，但母亲未携带该突变（新发突变）。第二个家系COL4A5基因第46外显子的4271位发生G到A的替换（c.G4271A），母亲也携带此突变。经过遗传咨询，只有第二个家系接受产前诊断。基于羊水细胞cDNA水平和羊水细胞基因组DNA水平的产前诊断均显示胎儿未携带与母亲相同的突变。PCR扩增SRY以及核型分析显示为男性胎儿。X染色体多态性微卫星标记的连锁分析显示羊水细胞中无母源细胞污染。
cDNA水平和DNA水平分析均可提高产前诊断的准确性和可靠性。在胎儿性别确定方面，PCR扩增SRY比核型分析更快。连锁分析有助于检测羊水细胞中的母源细胞污染。
