目的:本研究旨在探讨中国输血患者Jk(a-b-)表型的分子机制。
背景:已经报道了许多与Jk(a-b-)表型有关的不同突变类型。然而,剪接位点突变相对较少,缺乏相关的功能验证。
方法:在本研究中,血液样本采集自一名具有Jk(a-b-)表型的输血患者.使用常规血清学方法进行血清分型。使用聚合酶链反应扩增JK基因的外显子序列和编码区,并直接测序。为了进行小基因剪接分析,将内含子突变序列克隆到pSPL3剪接报告载体中.在HEK293T细胞中进行剪接报告小基因测定。
结果:通过血清学测试鉴定了血液样品的Jk(a-b-)表型。测序结果显示样品具有新的纯合剪接位点突变JK*02N(NM_015865.7:c.663+3A>C)。进一步分析,包括cDNA测序和小基因剪接分析,证实了新的剪接位点突变导致外显子跳跃。有趣的是,通过两种方法获得不同数量的被跳过的外显子。
结论:本研究揭示了中国人群中与Jk(a-b-)表型相关的新型纯合剪接位点突变。我们的结果强调了体外功能方法小基因剪接测定的重要性,同时也承认与cDNA测序相比其潜在的局限性。
OBJECTIVE: This study aimed to investigate the molecular mechanism of the Jk(a-b-) phenotype in a Chinese transfusion patient.
BACKGROUND: Many different mutation types relating to Jk(a-b-) phenotype have been reported. However, the splice-site mutation is relatively rare and the related functional verification is lacking.
METHODS: In this study, the blood sample was collected from a transfusion patient with the Jk(a-b-) phenotype. Serotyping was performed using routine serological methods. The exons sequences and coding regions of the JK gene were amplified using polymerase chain reaction and directly sequenced. To perform a minigene splicing assay, the intronic mutation sequences were cloned into a pSPL3 splice reporting vector. The splicing reporter minigene assay was performed in HEK 293T cells.
RESULTS: The Jk(a-b-) phenotype of the blood sample was identified through serological testing. Sequencing results revealed that the sample had a novel homozygous splice-site mutation JK*02N (NM_015865.7: c.663+3A>C). Further analysis, including cDNA sequencing and minigene splicing assay, confirmed that the novel splice-site mutation resulted in exon skipping. Interestingly, different numbers of exons being skipped were obtained by the two methods.
CONCLUSIONS: This study revealed a novel homozygous splicing-site mutation associated with the Jk(a-b-) phenotype in Chinese population. Our results emphasise the importance of the in vitro functional method minigene splicing assay, while also acknowledging its potential limitations when compared to cDNA sequencing.