巢氏PCR扩增强直性肌营养不良基因在植入前诊断中的局限性

目的I型强直性肌营养不良(myotonic dystrophy 1,DM1)是由于肌强直蛋白激酶基因(DMPK)3`端非翻译区的(CTG)n异常扩增导致。探讨用巢氏PCR法直接扩增该基因在植入前诊断的可行性。方法0.5%低熔点琼脂糖分离来自DMPK(CTG)100的DM1患者的单个精子,巢氏PCR法扩增DMPK基因。结果106例精子标本中53例扩增产物电泳为单条带,但48例产物显示为多条带,扩增产物长度介于(CTG)10至(CTG)30之间。结论利用低熔点琼脂糖可有效分离单个精子进行检查,而巢氏PCR在单细胞水平上直接扩增较多(CTG)n的DMPK基因时存在困难,应用于植入前诊断时容易造成误诊。

The limitation of nested PCR in amplifying dystrophia myotonica protein kinase (DMPK) gene in preimplantation diagnosis

Objective Myotonic dystrophy type one (DM1) is the most common inherited neuro-muscular disease in adult. The genetic aberration is an amplified trinucleotide repeat, CTG triplet, in the 3-prime untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) gene. Since anticipation is commonly seen during intergeneration transmission, DM1 is among the diseases requiring preimplantation diagnosis in Europe and America. This study aimed to discuss the possibility of amplifying directly the DMPK(CTG)n from a single cell by nested PCR.Method We used single sperm as the source of DNA to exclude the influence brought by allele drop out (ADO). Semen sample came from a 40-year-old DM1 male with mild symptoms and approximate DMPK(CTG)100 and DMPK(CTG)10 in his lymphocytes. Single sperm was separated with the use of low-melting-point agarose gels, following by DNA distraction and DMPK(CTG)n gene amplification by nested PCR. PCR products were measured by electrophoresis. Results 101 of 106 sperms had been amplified respectively. 53 samples appeared single-band after electrophoresis while 48 were more than one band. Lengths of the products ranged from that of DMPK(CTG)10 to DMPK(CTG)30.Conclusion Using low-melting-point agarose gel is an effective way of separating sperm. Amplifying DMPK(CTG)n from single cell by nested PCR has other difficulties than allele-drop-out and might cause misdiagnosis in preimplantation diagnosis.