单基因遗传性疾病的胚胎种植前遗传学诊断

Preimplantation Genetic Diagnosis for Single Gene Defects

因在绝大多数的单基因遗传性疾病(SGD)中基因突变是异质性的,所以对大多数SGD病例行胚胎种植前遗传学诊断(SGD-PGD)时都需个别设计。为此,设计就成为SGD-PGD的重要一环。由于SGD的遗传模式(常染色体显性和隐性,性染色体连锁显性和隐性)和突变种类(点突变,大小片段插入和丢失,重复顺序数超常等)不同,应有相应的不同设计技巧和策略。笔者通过设计实例,向读者介绍了不同的设计技巧和策略。现今SGD-PGD的最基本实验室操作还是用聚合酶链反应(PCR)把单细胞(卵裂细胞)靶DNA片段扩增,然后诊断扩增后所得片段。设计最重要的一步就是对PCR所用引物的设计。设计还应包括单细胞PCR的特殊操作规定,DNA限制性内切酶酶切点设计(因在点突变时,常需用限制性酶内切后片段长度多态性分析方法作诊断)做诊断结果分析预估,确定最后一轮PCR的两个引物之一为荧光标记引物。成功的SGD-PGD工作需要有权威性的领导核心组织,协调,对团队的统管和支持,这也是成批地开展SGD-PGD的先决条件。而且,只有在遗传流行病学工作者、各医院儿科、各专门疾病研究所和分子生物学基因诊断科室的密切配合下,才能对多种SGD全面开展SGD-PGD。

Because in overwhelming majority of single gene defects（SGD） the gene mutations are heterozygous,it is necessary for most pre-implantation genetic diagnosis（PGD） of SGD（SGD-PGD） to design individually.Thus,the designing becomes very important one of the steps in SGD-PGD.Since the heredity pattern（autosomal dominant and recessive,X-linked dominant and recessive） and the mutation type（point mutation,large or small fragment insertion and deletion,repeat expansion and so on） are different in cases,we should have the corresponding designing tactics and strategy.Through different designing examples,the author will introduce to readers different design tactics and strategies.Nowadays the basic technique link of SGD-PGD is still the amplification for target DNA fragment from single cells（blastomeres） with DNA polymerase chain reaction（PCR）,and after then to diagnose the obtained target fragment.The most important step of designing is design of primers that will be used in PCR.At same times the designing should also include to set special PCR protocol for single cell,design restriction enzyme cut site（s）[because for most point mutations,restriction fragment length polymorphism（RFLP） method is commonly used for diagnosis],make the analysis pre-estimate of diagnosis result,and determine which primer of the two in the last round of PCR should be fluorescence-labelled.Any success SGD-PGD requires an authority core of leadership to organize,coordinate,manage and support the whole team,which is also the precondition to serve SGD-PGD in groups.Moreover,only when there is close coordination among the inheritance epidemiology workers,the paediatric departments of various hospitals,some special disease research institutes and the groups of molecular biology gene diagnosis,SGD-PGD can be served for various SGDs comprehensively.