两个新的RHD等位基因的分子研究及家系分析

Molecular study of two novel RHD alleles and pedigree analysis

目的对新发现的2个RHD等位基因进行分型，并对其家系成员进行分型研究。方法应用单克隆抗体检测Rh血型抗原。对新发现的等位基因再用基因分型技术检测RHD基因型，扩增先证者及家系成员RHD基因10个外显子，作直接测序分析；并用序列特异性引物聚合酶链反应（sequence specific primerpolymerase chain reaction，PCR—SSP）技术检测先证者2的家系成员。结果2例先证者均为RhD阴性。先证者1测序分析显示为RHD78delC，家系调查发现先证者1妹妹的Rh表现型和测序结果与先证者一致；先证者2测序分析为第4外显子520G／A，第8外显子1080始缺失10个碱基，家系调查的测序结果显示先证者的RHD520G〉A＋1080 del 10基因来源于母亲。2个新的等位基因（RHD78 delC、RHD520G〉A＋1080 del 10）已被GenBank受理（GQ477180、GU362076）。结论这两个新发现的RHD等位基因为RHD假基因，家系调查显示均可以稳定遗传。

Objective To study the seggregation of two novel RHD alleles in Chinese pedigrees. Methods The Rh antigens of the samples were identified by using monoclonal antibodies. The 10 exons of the RHD gene for the 2 probands and their family members were amplified separately and sequenced. The parents of proband 2 were analyzed by sequence specific primer-polymerase chain reaction （SSP-PCR）. Results The two probands were RhD negative and the RHD was D/d type. After alignment with the nucleotide sequence in GenBank, a deletion of nucleotide C at position 78 in exon 1 of proband 1 was detected, and her sister also had the deletion, which was confirmed by sequencing. The sequencing results of proband 2 showed a 10 nucleotide deletion in exon 8 as well as a RHD 520 G^A mutation in exon 4. The results of SSP-PCR and sequencing showed that the proband＇s mother also carried RHD 520 G〉A and RHD 1080 del 10 mutation, which was transmitted to proband 2. The sequences of the novel alleles have been submitted to GenBank （accession No. GQ477180 and GU362076）. Conclusion The two novel RHD alleles, RHD 78delC and RHD 520 G〉A＋1080 del 10, were both pseudo genes and stably transmitted.