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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。
Purpose Alport syndrome (AS) is the most common hereditary kidney disease with poor prognosis and no specific and effective treatment. X-linked dominant (XLAS) is its major genetic mode, due to mutations in COL4A5 gene or COL4A5 and COL4A6 two gene mutations. Through the two XLAS pedigree genetic counseling and genetic diagnosis, and one of the pedigree implementation of prenatal genetic diagnosis, AS prenatal genetic diagnosis methods and clinical value. Methods After detailed genetic counseling between two XLAS pedigrees, nested PCR was used to amplify the entire coding region of COL4A5 mRNA in peripheral blood lymphocytes to screen for mutations in two pedigrees. Then, the corresponding exons of COL4A5 were amplified from genomes DNA levels further confirm the mutation. Prenatal genetic diagnosis of fetal cells by amniotic fluid cDNA and DNA levels to detect fetal mutations, Y chromosome sex determination by PCR gene sex combined with SRY karyotype analysis of fetal sex, through three X chromosome microsatellite markers (AR, DXS6797 and DXS6807) except for the possibility of maternal cell contamination in amniotic fluid. Results A mutation screening showed that there was a new mutation in the pedigree. There was a total of 10 bases in GTATGATGGG missing from 2696 to 2705 in COL4A5, but the mothers’ peripheral blood DNA did not carry this deletion mutation. Puncture and prenatal genetic diagnosis. Family 4 COL4A5 gene 4271 G was replaced by A, 1424 G> E, other family members, including pregnant mothers are carrying this mutation. Amniotic fluid puncture and prenatal genetic diagnosis were performed on two pregnant women of the family. Amniotic fluid cell cDNA and DNA levels showed no fetal mutation, PCR amplification of SRY gene and amniotic fluid cell karyotype analysis showed that the fetus is male. A linkage analysis revealed no maternal cell contamination in amniotic fluid cells and indirectly prompted the fetus to inherit from the mother the X chromosome carrying the normal COL4A5 allele. Conclusion The detection of COL4A5 gene mutation based on the peripheral blood lymphocyte cDNA level can be quickly used in the screening of pedigree gene mutations in the prenatal diagnosis of XLAS, which is conducive to objective genetic counseling. Prenatal gene diagnosis combined with cDNA and DNA levels Fetal gene mutation more accurate and credible, PCR amplification of SRY gene can quickly identify the sex of the fetus, X chromosome microsatellite marker linkage analysis can not only exclude the amniotic fluid maternal cell contamination, and can indirectly prove that the fetus carries the disease-causing alleles.