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目的试图建立一种准确、可靠的单细胞诊断脊肌萎缩症(spinal muscular atrophy,SMA)的方法,以用于SMA的临床胚胎植入前遗传学诊断(preimplantation genetic diagnosis,PGD)。方法以不同基因型的单个淋巴细胞为研究对象,采用了二重巢式PCR同时扩增SMN基因外显子7(包括内含子7)、外显子8其产物经多重引物延伸-DHPLC法检测的诊断SMA方法。结果在对200个各种基因型单细胞的检测实验中,新PGD诊断方法的PCR总扩增率达97%,SMN1基因外显子7、8同时扩增率为92%,各位点的扩增率为90%~98%;设立的40个空白对照没有一个扩增阳性,所有SMN1基因纯合缺失的标本没有一例SMN1基因外显子7或8扩增阳性;各位点的准确诊断率(即为准确分型率)为90%~97%;二个位点的同时准确诊断率在正常组、携带组、患病组分别为92%、88%、94%,平均为92%;所有操作步骤约需8h完成。结论新建立的单细胞诊断SMA方法是一种准确、可靠、快速、敏感的方法,可直接应用于临床对有生育SMA患儿风险的夫妇行PGD,以获得健康婴儿。
OBJECTIVE: To establish an accurate and reliable single-cell method for the diagnosis of spinal muscular atrophy (SMA) for the clinical preimplantation genetic diagnosis (PGD) of SMA. Methods A single lymphocyte of different genotypes was used as a study object. Double nested PCR was used to amplify exon 7 of SMN gene (including intron 7). Exon 8 was extended by multiple primer-DHPLC Diagnostic SMA method for detection. Results In the detection of 200 single-cell genotypes, the total PCR amplification rate of the new PGD diagnostic method was 97%, while that of the SMN1 gene exon 7 and 8 was 92%. The amplification of each site The rate of increase was 90% -98%. None of the 40 blank controls were positive for amplification. None of the SMN1 gene homozygous deletion samples showed a positive amplification of exon 7 or 8 of SMN1 gene. The exact diagnosis rate That is, the correct typing rate) was 90% -97%. The accuracy of simultaneous diagnosis of the two sites was 92%, 88%, 94% in the normal group, 92% in the diseased group, with an average of 92% Operation steps take about 8h to complete. Conclusion The newly established single cell diagnosis of SMA is an accurate, reliable, rapid and sensitive method that can be directly applied to clinical PGD for couples with risk of having childbirth in order to obtain healthy infants.