摘要
目的：探讨多重置换扩增（multiple displacement amplification，MDA）结合短串联重复序列多态性在植入前遗传学诊断（preimplantation genetic diagnosis，PGD）中的应用。方法：采用多重置换扩增对单细胞进行全基因组扩增，针对单基因疾病和人类白细胞抗原（human leukocyte antigen,HLA）配型选择位于致病基因内部或两侧的短串联重复序列位点进行单体型分析，同时对致病基因进行直接检测；针对染色体数量及结构异常或非整倍体筛查，选择位于相关染色体上的短串联重复序列位点进行等位基因数分析。结果：进行了以下5类12种类型植入前遗传学诊断的临床应用：（1）单基因疾病：脊髓性肌萎缩症、杜氏肌营养不良、X-连锁慢性肉芽肿、石骨症、骨软骨发育不全、Ｘ-连锁免疫缺陷综合征；（2）同时合并两种单基因疾病：α-和β-双重地中海贫血；（3）染色体罗氏易位；（4）单基因疾病结合HLA配型：β-地中海贫血、杜氏肌营养不良；（5）单基因疾病合并染色体异常：α-地中海贫血合并染色体罗氏易位、α-地中海贫血合并性染色体数目异常。对35个家系共进行了44个周期的PGD，共47个移植周期。MDA扩增成功率为96.4%（374/388），后续PCR扩增效率为97.1%，等位基因脱扣（allele drop out，ADO）率为12.6%（波动于0~47.5%），总体诊断效率为94.6%（367/388）。47个移植周期共移植91个胚胎，种植率为30.7%（28/91）。临床妊娠率为47.7%/PGD周期（21/44），共诞生了20个健康婴儿（12例单胎，4例双胎），5例继续妊娠。结论：采用多重置换扩增结合短串联重复序列多态性可建立一个通用的植入前遗传学诊断平台，其很大程度上缩短了新病种植入前遗传学诊断体系的研发时间，而且可同时进行两种或两种以上遗传学状态的诊断，也提高了诊断效率，有利于进一步拓宽植入前遗传学诊断的适应征。
To explore the application of multiple displacement amplification（MDA） combined with short tandem repeats （STRs） in preimplantation genetic diagnosis （PGD）. Methods： MDA was applied to amplify the whole genome of a single cell and to retrieve and assemble the highly heterogeneous STR loci among human population. Haplotype analytic system was established with aiming at diagnosis of the single gene diseases by selecting the STR loci located within the pathogenic genes or on both bounding sides of the pathogenic genes. At the same time, allele specific amplification, PCR-reverse dot-blotting hybridization methods and gene sequencing methods were employed for direct detection of the pathogenic genes. The STR loci located at related chromosomes were selected to carry out allele number analysis on the basis of chromosome number and structural abnormality. Results： In the study, 12 PGD systems were set up including 6 different monogenic diseases （spinal muscular atrophy, Duchenne muscular dystrophy, X-linked chronic granulomatous disease, osteopetrosis, achondroplasia, X-linked severe combined immunodeficiency）, Robertsonian translocations, αthalassemia combined with Robertsonian translocation, α- and β-double thalassemia, β- thalassemia with HLA typing and DMD with HLA typing. Then 44 PGD cycles were performed for 35 couples with different kinds of inherited diseases, which resulted in 20 healthy liveborns （12 singletons and 4 twins） and 5 ongoing pregnancies. The clinical pregnancy rate was 47.7% （21/44） per PGD cycle. The overall diagnostic rate was 94.6% （367/388）. The MDA failed in 3.6% （14/388） single blastomeres. The amplification rate of the subsequent PCR was 97.1% and the average allele drop out （ADO） rate was 12.6% （range： 0-47.5%）. Conclusion： The application of MDA combined with STRs provided a generic PGD approach for different genetic disorders, especially for simultaneous diagnosis of two or more hereditary statuses. The method could greatly shorten the time of developing PGD system of new diseases, which broadens the indications of PGD.
出处
《北京大学学报（医学版）》
CAS
CSCD
北大核心
2013年第6期852-858,共7页
Journal of Peking University:Health Sciences
基金
广东省自然科学基金博士启动项目(S2013040014613
S2012040006785)资助~~
关键词
植入前诊断
基因扩增
微卫星重复
Preimplantation diagnosis, Gene amplification, Microsatellite repeats
