SNP rs311103於紅血球Xga和CD99抗原表現的關聯

Abstract

XG血型系統包含了兩種抗原，Xga以及CD99抗原。Xga為表現在紅血球的表面抗原，此抗原分成Xg(a+)及Xg(a-)兩種表現型；CD99為表現在多種細胞的表面抗原，此抗原分成CD99-High (CD99H)及CD99-Low (CD99L)兩種表現型。在過去研究發現，CD99及Xga抗原在紅血球上的表現有著一致性的關聯；女性紅血球細胞Xga抗原呈現Xg(a+)時，連結的CD99表現型呈現高的情況(CD99H)；Xga抗原呈現Xg(a-)，連結的CD99表現型呈現低的情況(CD99L)。但是，男性紅血球表面上呈現了第三種表現型，也就是當Xga抗原呈現Xg(a-)，連結的CD99表現型可能呈現高的情況(CD99H)或是低(CD99L)的情況。在此種現象還無法被解釋的同時，使我們對於Xga及CD99抗原於紅血球表現的分子遺傳機制感到好奇。 本研究室首先取不同個體血液樣品進行研究，依據其Xga及CD99抗原表現型，分類成上述的三種類型。從樣品中挑選出16人gDNA以次世代定序(NGS)進行染色體大區域定序，範圍包含XG及CD99兩個基因約570kb，並排列出此區域之單一核甘酸多型性(single nucleotide polymorphism, 簡稱SNP)。透過分析結果發現位於CD99及XG基因間的SNP rs311103(G/C) 的基因型吻合三種表現型的關聯，因此判斷此SNP可能對於兩個基因的調控有很大的關連。 先前的研究發現，在紅血球細胞中Xga及CD99抗原表現型與mRNA表現量有關。以類/非紅血球細胞株作為細胞模型，將帶有SNP rs311103的片段利用報導基因的方式進行實驗，並以PROMO軟體預測會與此rs311103[G]而不是rs311103[C]產生親和性結合的轉錄因子，從中挑選出與血球相關的轉錄因子，包括GATA family of transcription factors (GATAs, GATA1~GATA6，共六個)及Lymphoid enhancer binding factor 1 (LEF1) 進行後續的實驗。 根據報導基因法、EMSA及ChIP實驗中，我們觀察到rs311103[G]與轉錄因子GATA1 和/或 GATA2促進基因轉錄表現，其中以GATA1有較高的專一性結合能力。為了確定基因調節系統上，轉錄因子藉由遠程調控基因的表現，我們分別建構XG或CD99基因的啟動子，並將帶有SNP rs311103重複片段接上報導載體，確認基因啟動子與rs311103區域間是否有相互的調控。同時在過去研究發現GATA1/2調控基因的表現會與TAL1共同調控，兩者之間形成一聚合體而共同調控基因的表現，因而後續我們以點突變的方式去證實先前的研究。本研究結果證實了SNP rs311103對於Xga / CD99血型表現型於紅血球的特異性，並揭開了其抗原形成的分子機制。

XG blood group system comprises two antigens, Xga and CD99. The Xga antigen express on the membranes of red blood cells (RBCs), and it can classify into Xg(a+) and Xg(a-) phenotype. The CD99 antigen express on membranes of many tissue cells, and it can classify into CD99-high (CD99H) and CD99-Low (CD99L) phenotype. According to past study, the Xga and CD99 antigens show a unique and sex-specific phenotypic relationship on the surface of RBCs. Among females and males, the Xg(a+) phenotype is associated with the CD99H phenotype. On the other hand, Xg(a–) females show an association with the CD99L phenotype; however, by way of contrast, Xg(a–) males, in addition to possibly having the CD99L phenotype, may also have the CD99H phenotype. There was no answer to explain why caused this phenomenon, so it attracted us to study what the mechanism of regulation between XG and CD99 genes. We initially conducted a pilot study involving targeted next-generation sequencing of the 570-kb genomic sequences relevant to XG and CD99 from 16 individuals with different Xga/CD99 phenotypes. This was followed by a larger-scale association study that demonstrated an association between SNP rs311103 and the Xga/CD99 blood groups. It has been found that the reticulocytes from Xg(a+)/CD99H individuals have a higher level of the XG and CD99 (MIC2) transcripts than the reticulocytes from Xg(a–)/CD99L individuals. Different genomic segment that encompasses polymorphic rs311103 was introduced into erythroid /non-erythroid lineage cell lines by reporter assay, and the polymorphic rs311103 genomic regions were analyzed using PROMO program to identify potential transcription factor binding motifs. Putative binding motifs for the GATA binding protein family and for lymphoid enhancer binding factor 1 (LEF1) were identified within the rs311103[G] region, but not within rs311103[C] region. Follow-up investigations included ectopic expression of various GATA factors, EMSA, and ChIP to show that the erythroid GATA1 factor is able to bind specifically to the rs311103[G] region and markedly stimulates the transcriptional activity of the rs311103[G] segment. In order to determine the details of the control system, especially their involvements in the co-regulation the XG and CD99 expressions and in the long-range interaction between the rs311103[G] genomic region and the CD99 promoter. Other study showed that GATA1/2 and TAL1 could co-regulate genes expression, the site-directed mutagenesis were conduct to know interaction between them. The present findings identify the genetic basis of the erythroid-specific Xga/CD99 blood group phenotypes and reveal the molecular background to their formation.