RHD基因测序方法的建立及其在弱D表型分子鉴定中的应用

目的 建立RHD基因的测序方法并对9例弱D表型作分子鉴定.方法用间接抗人球蛋白试验（IAT）筛选弱表达的D变异体,聚合酶链反应-序列特异性引物（PCR-SSP）方法 扩增RHD基因特异的外显子及其侧翼序列,PCR产物直接序列分析测定核苷酸的变异.结果 用PCR-SSP方法特异扩增RHD基因,50份随机Rh阴性（ccdee）样本呈阴性结果,51例随机Rh阳性样本呈阳性结果.扩增产物测序结果表明,15份代表性Rh阳性单倍体型的RHD基因序列和标准序列一致.用所建立的方法对9份弱D表型样本进行测序分析,发现5份有845G＞A突变,3份有1 227G＞A突变,1例有1013T＞C突变.结论 所建立的RHD基因测序方法可以用于弱D的分子鉴定.9份弱D表型的分子机理得到明确.

Establishment of a direct RHD gene sequencing method and its application in molecular identification of weak D phenotypes

Objectives To establish a direct RHD gene sequencing method and to identify the molecular basis of nine weak D phenotypes.Methods D variants with weak D expression were screened out with an indirect anti-human globulin test (IAT) assay.A polymerase chain reaction-sequence specific primer (PCR-SSP) method was designed to amplifity RHD specific exons and flanking regions.The amplification products were sequenced directly to determine the variations of weak D genes.The sensibility and specificity of the method were evaluated with the amplification and by sequencing randomized Rh-negative and Rh-positive controls.Results In the specific amplification of RHD genes with the PCR-SSP, the negative results were found in 50 random Rh-negative samples (ccdee)while the positive results were found in 51 random Rh-positive samples.RHD sequences in 15 Rh-positive samples with representative haplotypes were consistent with the normal RHD sequence.With the established direct RHD gene sequencing method, 845G>A mutation was found in five weak D individuals, 1227G>A mutation in 3, and 1013T>C mutation in one. Seven of nine weak D phenotypes had a RHD gene deletion while the rest two with RHD 1227A allele did not.Conclusion A new RHD gene sequencing method is established and the molecular basis of nine weak D phenotypes is characterized.