Moutou Céline, Gardes Nathalie, Viville Stéphane
Service de Biologie de la Reproduction-SIHCUS-CMCO, CHU de Strasbourg, Schiltigheim Cedex, France.
Prenat Diagn. 2003 Aug;23(8):685-9. doi: 10.1002/pd.676.
The main difficulty in developing a molecular diagnosis of spinal muscular atrophy (SMA) resides in the specific genomic structure of the locus. Indeed, two highly homologous survival motor neurone genes, SMN1 and SMN2, are present at the locus. The detection of the homozygous deletion of exons 7 and 8 of the SMN1 gene, which is present in 90 to 98% of the patients, is based on methods highlighting 1 of the 8 nucleotidic mismatches existing between these 2 genes. In order to offer preimplantation genetic diagnosis (PGD) for SMA, we developed a new allele-specific amplification method. The main disadvantage of our previously described strategy resided in the possibility of diagnosing, in case of amplification failure, an unaffected embryo as affected. We present here a new PGD-SMA method. We established the conditions for three different duplex PCRs, allowing the specific detection of the SMN1 gene and one polymorphic marker, either D5S629, D5S1977, or D5S641. Of the 60 to 90 single cells tested, the PCR efficiency varied from 98 to 100% with a complete genotype obtained in a range between 81 and 87% with a global allele drop-out rate of 9%. Such a test was used to perform 1 PGD cycle for which 7 embryos could be analysed. All the embryos were fully diagnosed, six as unaffected and one as affected. Four embryos were transferred, but no pregnancy ensued.
开展脊髓性肌萎缩症（SMA）分子诊断的主要困难在于该基因座的特定基因组结构。实际上，该基因座存在两个高度同源的存活运动神经元基因，即SMN1和SMN2。90%至98%的患者存在SMN1基因第7和8外显子的纯合缺失，其检测基于突出这两个基因之间存在的8个核苷酸错配中1个的方法。为了提供SMA的植入前基因诊断（PGD），我们开发了一种新的等位基因特异性扩增方法。我们之前描述的策略的主要缺点在于，在扩增失败的情况下，有可能将未受影响的胚胎诊断为受影响。我们在此介绍一种新的PGD-SMA方法。我们确定了三种不同的双重PCR条件，可特异性检测SMN1基因和一个多态性标记，即D5S629、D5S1977或D5S641。在测试的60至90个单细胞中，PCR效率在98%至100%之间，完整基因型的获得率在81%至87%之间，总体等位基因脱扣率为9%。这样的检测用于进行1个PGD周期，可分析7个胚胎。所有胚胎均得到完全诊断，6个为未受影响，1个为受影响。移植了4个胚胎，但未成功妊娠。
