成都地区献血人群RhD阴性个体遗传学背景研究

The molecular background of RhD negative blood donors in Chengdu,China

目的 RhD阴性存在多种变异体,通过研究成都地区RhD阴性献血者,了解其遗传背景。方法 2009年6—11月采集的74947人份全血中获得盐水法初筛阴性标本318例,采用间接抗球蛋白试验(IAT)筛选部分D和弱D,对IAT阴性的标本采用吸收放散试验,筛查Del型,采用序列特异性引物PCR(PCR-SSP)检测各种变异D基因型。检测的Del等位基因包括RHD(M285)、RHD(IVS3+1)、RHD(IVS1+1)、RHD(1227A)、RHD(DelEX9)、RHD(M1I)、RHD(R10W)、RHD(L84P)。吸收放散试验阳性标本使用DelDNA分型试剂盒筛查,未分出型别的标本检测RHD基因1—7和9—10外显子,并对检测到的各种变异D以及疑难标本进行测序分析。结果 318份盐水法初筛阴性标本中检出IAT阳性标本3例,弱D152例,部分D(RhDⅥⅢ)1例。成都地区献血人群中RhD阴性比例为0.42%,表型dccee(56.19%)和dCcee(32.06%)比例高。对可获得的303例IAT阴性标本进行微量吸收放散试验,发现阳性71例。通过DelDNA分型试剂盒检测,RHD(1227A)型57例、RHD(M1I)型1例、无法确定为已知的Del型13例。无法确定的13例标本通过检测RHD基因1—7和9—10外显子,发现11例部分或全部缺失上述外显子,2例含有完整的RHD基因。34例吸收放散阴性含有C或E抗原标本,经检测RHD基因1—7和9—10外显子及1227A,发现RHD(1227A)型9例、部分或全部缺失型25例。血清学试验和分子生物学试验确定的Del型共69例,占IAT筛查RhD阴性的22.77%(69/303)。结论成都地区常规血清学筛查RhD阴性献血人群中RHD基因存在多态性,RHD1227A等位基因是Del型的主要基因类型。吸收放散试验检测Del准确率偏低。

Objective To elucidate the molecular background of RhD negative blood donors in Chengdu. Methods A total of 74 947 blood samples of blood donors were collected from June to November 2009. In order to screen the D variants, 318 Rh D negative samples were tested by immediate spin, indirect antiglobulin test （IAT） and adsorption and elution assay. DNA of D variant samples was analyzed by PCR-SSP. The Del alleles, RHD（M285）, RHD（IVS3＋1）, RHD（IVS1＋1）, RHD（1227A）, RHD（Del EX9）, RHD（M1I）,RHD（R10W） and RHD（L84P） were tested. The negative samples were analyzed for 1—7 and 9—10 RHD exons, and the mutation of D variants were confirmed by gene sequencing method.Results Three positive samples, including 2 weak D15 and 1 RhDⅥⅢ, were found by IAT in 318 samples. The RhD-negative rate was 0.42%. The phenotypes of dccee （56.19%） and dCcee（32.06%） were the predominant phenotypes. In 303 RhD IAT negative samples, 71 positive samples were found using adsorption and elution assay. And 57 RHD（1227A） and 1 RHD（M1I） gene positive samples and 13 gene negative samples were found using Del genotype PCR-SSP. Eleven partial or whole RHD gene deletion and 2 whole RHD gene were found in the 13 negative samples by analyzing the RHD exons 1—7 and 9—10. And 34 samples with C or E, which showed negative result by adsorption and elution assay, were analyzed. Nine RHD（1227A） and 25 partial or whole RHD gene deletion were found using PCR-SSP. A total of 67 Rh Del samples were found using serological and molecular biology methods, and the positive rate was 22.11%（67/303）. The false positive and negative rates of adsorption and elution assay were 15.49%（11/71） and 26.47%（9/34）, respectively. Conclusion The RHD 1227A allele might be an important genetic marker for Rh Del phenotypes in Chengdu blood donors. RHD gene polymorphism exists in Chengdu blood donors. In addition, the accuracy of small volume adsorption and elution assay is poor as compared with others.