杜氏肌营养不良症(DMD)的植入前遗传学诊断

Preimplantation genetic diagnosis of duchenne muscular dystrophy (DMD)

目的对杜氏肌营养不良症(duchenne muscular dystrophy,DMD)进行植入前遗传学诊断(preim-plantation genetic diagnosis,PGD),阻断DMD患儿的出生。方法针对患者的DMD基因的48号外显子缺失位点采用巢式PCR分别对患者和携带者的单个淋巴细胞、行体外授精-胚胎移植治疗的健康志愿捐献者的单个卵裂球细胞进行扩增,建立稳定的经单细胞基因诊断DMD的方法。再对在该中心进行超排和体外授精-胚胎移植治疗的DMD携带者的胚胎活检后完成PGD,根据诊断结果选择健康的优质的胚胎移植入子宫。结果携带者的单个淋巴细胞的PCR扩增成功率为90.5%(95/105),健康志愿捐献者的单个卵裂球细胞PCR扩增成功率为85.7%(54/63),假阳性率为0(0/28)。分别对3例DMD携带者施行了植入前遗传学诊断,病例1移植了2枚未受累的优质胚胎,且成功妊娠单胎并已于2005年4月分娩了1个健康的女婴;病例2移植了2枚优良胚胎,不幸的是未能妊娠。病例3诊断为未受累的仅有的2枚胚胎因胚胎质量差而未能移植。结论该组采用的方案可对DMD基因的48号外显子缺失突变的DMD家庭进行PGD,达到了阻断DMD患儿出生的目的。

[ Objective ] To carry out preimplantation genetic diagnosis （PGD） for duchenne muscular dystrophy （DMD）, and prevent the birth of affected infant with DMD. [Method] Amplifying the DNA of single lymphocytes from the patient and the carrier of DMD, of single blastomeres from healthy volunteer female donor treated by in-vit- ro fertilization and embryo transplantation （IVF-ET） with nested-PCR detected the deleted exon 48 within dystrophin gene, so that established a stable method of diagnosing DMD by single cell. Then PGD was accomplished with biopsied blastomeres from cleavage-stage embryos of the carrier of DMD in IVF-ET at our reproductive center, and the unaffected high quality embryos were chosen according to the result of PGD and were transferred into the uterus of the DMD carrier. [Results] Successful amplification rates of exon 48 locus of dystrophin gene in single lymphocytes from the carrier and single blastomeres from healthy volunteer female donor were 90.5 %（95/105） and 85.7% （54/63）, respectively. The false positive rate is 0 （0/28）. 3 cycles of PGD from the DMD carrier was accomplished, which resulted in transfer of 2 unaffected high quality embryoes in patient 1 and the singleton pregnancy ensued, finally a healthy female infant was born at April, 2005. 2 unaffected good quality embryoes were transferred to patient 2. Unfortunately, no pregnancy ensued. In patient 3, only 2 unaffected poor quality embryoes were obtain, no transplantation ensued. [ Conclusions] The protocol was suitable for the PGD of DMD family with the deletion mutation of exon 48 and had attained the purpose of preventing the birth of affected infant with DMD.