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目的:对先天性脊柱骨骺发育不良提供1种快速分子产前诊断技术。方法:对该患者于孕14周进行羊膜囊穿刺,获取羊水20 ml,提取羊水细胞基因组DNA。选用3个短串连重复(STR)序列位点,进行荧光PCR检测,以排除母源细胞污染。在此基础上,对COL2A1基因的第23外显子进行扩增,并以此为模板,针对COL2A1基因的第23外显子1510G→A的突变进行SSP-PCR。根据是否存在特异性的扩增条带进行判断,对初次扩增的产物测序,以证实巢式SSP-PCR的结果。结果:对D13S 317、D18S51和D21S11位点的STR检测结果排除母源细胞的污染。羊水样本仅在野生型反应管中有扩增产物,表明胎儿是COL2A1基因的第23外显子1510 G的纯合子,未带COL2A1基因的第23外显子1510G→A的突变。测序结果与SSP-PCR结果一致。结论:SSP-PCR技术快速简便、敏感特异,可作为该突变的快速分子诊断。其他已知点突变遗传病的快速分子诊断也可以考虑使用SSP-PCR技术进行。
Objective: To provide a rapid molecular prenatal diagnosis of congenital spinal epiphyseal dysplasia. Methods: Amniotic sac puncture was performed in 14 weeks’ gestation, and 20 ml of amniotic fluid was obtained. Genomic DNA was extracted from amniotic fluid cells. Three short tandem repeat (STR) sequence sites were selected for fluorescent PCR detection to exclude maternal cell contamination. Based on this, we amplified the 23rd exon of COL2A1 gene and used it as a template for the SSP-PCR analysis of the 1510G → A mutation of exon 23 of COL2A1 gene. The primary amplified product was sequenced to confirm the result of nested SSP-PCR, based on the presence of a specific amplification band. Results: The results of STR test on D13S 317, D18S51 and D21S11 loci ruled out the contamination of maternal cells. The amniotic fluid sample contained amplification products only in the wild type reaction tube, indicating that the fetus was a homozygote at position 1510 G of exon 23 of COL2A1 gene and did not carry mutation 1515G → A of exon 23 of COL2A1 gene. Sequencing results were consistent with SSP-PCR results. Conclusion: SSP-PCR is a rapid, simple and sensitive method that can be used as a rapid molecular diagnosis of this mutation. Other molecular diagnostics for the genetic diagnosis of point mutations can also be considered using the SSP-PCR technique.