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目的 研究KIR 2DL4基因测序分型中序列及分型结果“异常”的原因。方法 2016年5月对KIR 2DLA基因测序分型时检出的2例杂合碱基位置峰高不平衡、判定为模棱两可结果或无完全匹配分型结果的标本,采集新鲜外周血样,提取mRNA,反转录成cDNA后,进行cDNA分子克隆和单体型测序;同时采用荧光定量法检测基因组DNA中2DL4基因的拷贝数。结果 分子克隆和单体型测序表明,1例标本携带正常的KIR 2DL4~*00102,~*00501及~*011等位基因;另一标本中检出了KIR 2DL4~*00102,~*00501及~*00602等位基因。2例标本中均检出了3种不同的KIR 2DL4等位基因,无新等位基因检出。荧光定量PCR实验证实这2例标本KIR 2DL4拷贝数均为3个。结论人类KIR单体型上KIR 2DL4基因的拷贝数存在多样性,当出现杂合碱基位置峰高不平衡、无与之完全匹配分型结果,并非由新等位基因所致。
Objective To study the causes of “abnormalities” in sequencing and typing of KIR 2DL4 gene sequencing. Methods In May 2016, two cases of heterozygous base positions detected at the sequencing of KIR 2DLA gene were unbalanced in peak height, and were judged to be ambiguous or incompletely matched. The fresh peripheral blood samples were collected and mRNA was extracted. Reverse transcription into cDNA, cDNA molecular cloning and haplotype sequencing; at the same time using fluorescent quantitative detection of genomic DNA 2DL4 gene copy number. Results The results of molecular cloning and haplotype sequencing showed that one sample carried the normal KIR 2DL4 ~ * 00102, ~ * 00501 and ~ * 011 alleles, while the other samples detected KIR 2DL4 ~ * 00102, ~ * 00501 and ~ * 00602 allele. Three different KIR 2DL4 alleles were detected in 2 cases without any new allele. Fluorescent quantitative PCR experiments confirmed these two specimens KIR 2DL4 copy number were 3. Conclusion The diversity of KIR 2DL4 genes in human KIR haplotypes is quite different. When heterozygous base positions are imbalanced in peak heights, there is no perfect match typing result, which is not caused by the new allele.