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目的建立一种可靠、准确的植入前遗传学诊断方法,可广泛用于各种类型的脊肌萎缩症。方法采用多重置换扩增(MDA)对单细胞进行全基因组扩增,等位基因特异性扩增SMN1外显子7进行脊肌萎缩症(SMA)致病基因检测及采用与SMN基因紧密连锁的12个短串联重复序列(STR)进行SMA的单体型分析。结果共进行了80单个淋巴细胞及63个单卵裂球的MDA。MDA扩增效率在单淋巴细胞中为97.5%(78/80),在单卵裂球为96.8%(61/63)。在单淋巴细胞及单卵裂球MDA产物中,总的PCR扩增效率和等位基因脱扣率(ADO)为分别为96.2%(587/610)和8.7%(52/597),95.5%(722/756)和9.9%(54/547)。在单淋巴细胞MDA产物中,SMN1外显子7的PCR结果均与外周血结果相符,而单个卵裂球MDA产物中,有一个PCR扩增失败,扩增效率为98.4%(60/61)。结论该方法可用于常见的缺失型的SMA的PGD,而且可以应用于突变型的为SMA的PGD,为进一步的临床应用奠定了基础。
Objective To establish a reliable and accurate preimplantation genetic diagnosis method that can be widely used in various types of spinal muscular atrophy. Methods Multiplexed amplification (MDA) was used to amplify the whole genome of single cells. Allele specific amplification of SMN1 exon 7 was used to detect the pathogenic genes of spinal muscular atrophy (SMA) and was closely linked with the SMN gene Twelve short tandem repeats (STRs) were used for haplotype analysis of SMA. Results A total of 80 single lymphocytes and 63 single blastomere MDA. The efficiency of MDA amplification was 97.5% (78/80) in single lymphocytes and 96.8% (61/63) in single blastomeres. The total PCR amplification efficiency and allele transfer rate (ADO) were 96.2% (587/610) and 8.7% (52/597), respectively, in single lymphocytes and single blastomere MDA products, with 95.5% (722/756) and 9.9% (54/547). In the single lymphocyte MDA product, the PCR results of SMN1 exon 7 are consistent with the results of peripheral blood. However, one PCR amplification failed in a single blastomere MDA product and the amplification efficiency was 98.4% (60/61) . Conclusion This method can be applied to PGD of common SMA without SMA, and can be applied to PGD with SMA as mutant, which lays the foundation for further clinical application.