软骨发育不全植入前遗传学诊断的方法学研究

目的：针对软骨发育不全（ACH）高危家系的 FGFR3基因的突变类型（c.1138G 〉 A， p.G380R），建立多种快速特异、行之有效的植入前遗传学诊断（PGD）方法，为实施该 ACH 家系的 PGD创造必要的前提条件。方法在确诊该 ACH 患者特定突变类型的基础上，首先用外周血建立各种PGD方法，包括： A.直接测序法； B. ARMS法； C.酶切鉴定法；D. ARMS／RE法。然后对单个卵裂球的全基因组扩增（WGA）产物进行相应方法学的研究。最后，对各种方法进行优化优选。结果 A.直接测序法：正常对照c.1138为G 纯合子，而患者为G／A 杂合峰； B. ARMS 法：正常对照扩增阴性，而患者有445 bp 的特异扩增条带。 C.酶切鉴定法：正常对照酶切后仍为513 bp，而患者酶切后产生205 bp、308 bp、513 bp三条带； D. ARMS／RE法：正常对照扩增阴性，无法做酶切鉴定。而患者有445 bp扩增产物，经SfcI酶切后可产生27 bp和418 bp两个片段。结论本研究已成功建立针对c.1138 G 〉 A 杂合错义突变的直接测序法、ARMS 法、酶切鉴定法和ARMS／RE 法。所建立的4种方法快速、特异，但各有利弊，同时使用可相互弥补，结合STR 连锁分析可把误诊风险降到最低程度。在正常情况下，可在24 h 内完成对ACH高危胚胎的PGD。

The methodological study of preimplantation genetic diagnosis for achondroplasia

Objective To establish quick, specific and effective methods for preimplantation genetic diagnosis （PGD） focusing on the gene mutation （c.1138G 〉 A, p.G380R） of FGFR3 gene which causes the achondroplasia （ACH）, as well as to provide a prerequisite for the implementation of PGD of the ACH family. Methods Based on the confirmed diagnosis of the ACH specific gene mutation, four methods of PGD were established by using the peripheral blood, which including, Direct DNA sequencing,ARMS assay, restriction enzyme digestion assay and ARMS/RE assay. Then the methodology-based study on the whole genome amplification （WGA） product of a single blastomere was done for optimization. Results Direct DNA sequencing analysis： The normal control had a G homozygous peak at 1138th G base point in cDNA while the patient had a G/A heterozygous peak. ARMS assay： The normal control had no amplification products while the patient had a specific band at 445 bp. Restriction enzyme digestion identification assay： The normal control showed a 513 bp band after enzyme digestion, while the patient got three bands with 205 bp, 308 bp and 513 bp, respectively. ARMS /RE assay： The normal control could not been done RE as it is negative amplification. On the contrary, the patient got a 445 bp amplification product, and two fragments of 27 bp and 418 bp were got after SfcI enzyme digestion. Conclusion This study has successfully established the heterozygous missense mutation of c.1138 G 〉 A by using direct DNA sequencing, ARMS assay, restriction enzyme digestion assay and ARMS/RE assay. Each of the four methods is rapid and specific, but each has its advantages and disadvantages. The combined use makes them complement each other and minimizes the risk of misdiagnosis, especially by combining with the STR linkage analysis. Under normal circumstances, the PGD for high-risk fetuses of ACH could be accomplished within 24 hours.