探討人類Xga/CD99與P1/P2的血型形成之分子遺傳機制

Abstract

Part I. XG血型是人類36種血型系統之一，由Xga及CD99兩種抗原組成。Xga抗原分佈於紅血球表面，分成兩種表現型，Xg(a+)及Xg(a-)。CD99抗原廣佈於各種細胞表面，在紅血球上的表現量具有高低差異，分成CD99High及CD99Low。過去研究指出，CD99抗原在紅血球表面表現量的多寡與Xga抗原表現與否有著相互連結的關係之外，CD99抗原及Xga抗原的表現也存在著性別上的差異。在男性與女性的紅血球上具有Xg(a+)表現型者，CD99皆為高表現(CD99High)；若是Xg(a-)表現型者，則是CD99低表現(CD99Low)。但是在男性紅血球上會出現第三種表現型Xg(a-)表現型者，CD99卻是高表現(CD99High)。對於紅血球上Xga及CD99抗原的表現差異，先前的研究指出可能是透過兩者基因轉錄層面上的調控而改變其表現量，但其中的分子遺傳機制尚未明瞭。 為了闡明這個機制，我們實驗室抽取78人血液樣本的genomic (g)DNA進行研究，將樣本區分成三種表現類型(Xg(a+)/CD99High; Xg(a-)/CD99Low; Xg(a-)/CD99High)。接著從樣本中選取16人[Xg(a+)/CD99High男女各4人; Xg(a-)/CD99Low男女各4人]，針對可能為Xga及CD99調控區域大約570 Kb的範圍進行大規模次世代定序(next generation sequencing, NGS)，並對此區域的單一核甘酸多型性(Single nucleotide polymorphism, 簡稱SNP)進行分析。透過分析結果發現SNP rs311103[G/C]的基因型符合Xga及CD99表現型的分佈;Xg(a+)/CD99High表現型男女rs311103的基因型為[G/G]或[G/C]，而Xg(a-)/CD99Low表現型男女rs311103的基因型為[C/C]，因此我們推測此SNP可能與XG及CD99基因表現量的調控相關。在我們報導基因法檢測的結果證實rs311103[G]片段會大量促進基因轉錄的表現量，但在rs311101[C] 片段則不具有刺激基因轉錄的能力，而且扮演enhancer角色的rs311103[G]片段只會專一於紅血球型細胞促進基因的轉錄。接著我們進一步探討rs311103[G]片段是否透過與轉錄因子的結合進而調控目標基因的轉錄能力。程式預測分析的結果顯示轉錄因子GATA family (GATA1- GATA 6)與LEF-1可能與rs311103[G]片段結合。在報導基因法分析各個轉錄因子所造成的轉錄影響中，我們發現GATA1及GATA2對於rs311103[G]片段皆會促使報導基因轉錄的表現量再大幅地上升。接著我們更進一步利用凝膠電泳分析(electrophoretic mobility shift assay, EMSA)證實rs311103[G]片段與GATA1及GATA2產生直接的結合。不過，在使用直接由紅血球細胞株萃取出的細胞核物質進行凝膠電泳分析(EMSA)實驗中，卻只有GATA1對於rs311103[G] 片段具有專一結合力，GATA2則沒有。對於GATA1及GATA2與rs311103[G] 片段實際在紅血球細胞株中的結合能力，我們利用染色質免疫沉澱(chromatin immunoprecipitation, CHIP)的方法去檢測細胞內真實的結合力。而實驗結果證實，在紅血球細胞株實驗中，只有GATA1對於rs311103[G] 片段具有專一性的結合力。我們的研究結果闡明紅血球上的Xga與CD99抗原表現相互連結兩者之間的遺傳分子機制形成的途徑。 Part II. P1/PK血型系統中的P1及PK抗原是兩種表現在紅血球細胞表面的醣抗原，這兩種抗原的表現存在著個體差異。依據個體間P1抗原的表現，可以區分為P1及P2兩種表現型:(1) P1表現型:包含P1及P抗原(P抗原為PK抗原的下游產物)；(2) P2表現型:只包含P抗原。從過去研究中得知這P1及PK抗原的形成與A4GALT基因相關，A4GALT基因生成酵素α-1,4-galactosyltransferase (α4GalT)，為一種半乳糖基轉移酶，具有催化P1及PK抗原合成的能力。過去研究也證實P1/P2表現型和A4GALT基因的表現量具有相關性，P1表現型的A4GALT基因的表現量高於P2表現型。於是找出調控A4GALT基因進而影響P1抗原生成的因素有助於了解P1/P2表現型形成的分子機制。 在實驗室先前的研究已經發現，兩個位於A4GALT基因1號內含子裡的SNP rs2143918 (SNP5) 及rs5751348 (SNP6)，對於A4GALT基因表現量的高低與P1/P2表現型的成因具有很重要的關聯。因此我們在本研究中進一步去探討這兩個SNP是透過甚麼分子機制去調控P1-A4GALT 和P2-A4GALT兩個等位基因的表現。首先，我們利用電腦軟體預測分析可能會與兩個SNP結合的轉錄因子。接著，由報導基因分析法(reporter assay)的結果中得知，EGR1轉錄因子會促進報導基因載體的轉錄活性之外，其主要可能是針對P1-A4GALT SNP6區域去做結合並影響轉錄的活性，而導致P1-A4GALT 和 P2-A4GALT兩個等位基因在紅血球中具有高低不同的轉錄表現量。而Electrophoretic mobility shift assay (EMSA)的實驗結果也進一步證實EGR1轉錄因子直接與P1-A4GALT SNP6區域專一的結合。除此之外，表現EGR1轉錄因子在異型核子HT-29細胞中的實驗結果也發現，EGR1轉錄因子確實會促使細胞核中的P1-A4GALT的heterogeneous nuclear RNA (hnRNA)的表現量上升。綜合我們過去與現在的研究，我們發現rs5751348 (SNP6) 對於P1-A4GALT及P2-A4GALT基因表現量的高低具有重要的影響，我們更進一步證實這個表現量的差異是透過EGR1轉錄因子與A4GALT基因上不同基因型的rs5751348 (SNP6)做結合所導致的。此分子遺傳機制所影響的P1-A4GALT及P2-A4GALT基因表現量的高低闡明了造成P1與P2不同血型的成因。

Part I. Xga and CD99 are two antigens belong to the human Xg blood group system and express on the surface of red blood cells (RBCs). The expression levels of Xga and CD99 on RBCs are highly correlated and this correlation also relative to the sex-specific phenotype. Among the RBCs in both females and males, the Xg(a+) is associated with the CD99High phenotype. Interestingly, the Xg(a-) RBCs in females express CD99Low, but Xg(a-) RBCs in males show both CD99High and CD99Low phenotypes. According to the previous studies, the expressions of Xga and CD99 are determined by transcriptional regulation. However, the molecular and genetic mechanisms remain unclear. In order to elucidate the mechanism, we analyzed the genomic areas between XG and CD99 by next generation sequencing (NGS). We collected the genomic (g)DNA from Taiwanese and divided the samples into three groups according to the expression levels of Xga and CD99: 1) Xg(a+)/CD99High in male and female; 2) Xg(a-)/CD99Low in male and female; 3) Xg(a-)/CD99High in male. Our result demonstrated that a single nucleotide polymorphism (SNP) rs311103 shows an association with Xga/CD99 blood group. The genotypes of rs311103[G] and [C] were associated with Xg(a+)/CD99High and Xg(a-)/CD99Low phenotypes, respectively. Moreover, the rs311103[G] strongly enhanced the transcriptional activity, but not in the case of rs311103[C]. Notably, this regulatory mechanism is specifically in the red blood cell linages but not other cell types. Furthermore, the transcription database analysis and in vitro experiment demonstrated that the transcription factor GATA1 binds to the region with the rs311103[G] genotype, which subsequently promotes the transcription level in erythroid cell lines. In this study, we clarified the phenotypic regulation between Xga and CD99, and elucidated the molecular genetic mechanism of the erythroid-specific Xga/CD99 blood group formation. Part II. P1 and PK are carbohydrate antigens on the surface of red blood cells (RBCs) in the P1/PK blood group system, and the expression level of these antigens varies among individuals. According to the expression level of the P1 antigen, blood types are divided into two groups: (1) the P1 phenotype, containing the P1 and P antigens (the P antigen is the downstream product of the PK antigen); (2) P2 phenotype, containing only the P antigen. Previous studies demonstrated that the synthesis of P1 and PK antigens is determined by the enzymatic activity of an α-1,4-galactosyltransferase (α4GalT), and this enzyme is encoded by the A4GALT gene. Moreover, the phenotypic polymorphism of the P1/P2 blood groups result from the different quantities of P1 antigen expression, that is associated with the different expression levels of the A4GALT gene, in P1 and P2 RBCs. However, the molecular mechanism for the formation of the P1/P2 blood groups remains unclear. Our previous investigations demonstrated that two SNPs, rs2143918 (SNP5) and rs5751348 (SNP6), located in intron 1 of the A4GALT gene were associated with the P1/P2 polymorphic phenotype, which is regulated by the different expression levels of the A4GALT gene. Following our previous identification, we further explored the detailed molecular mechanism associated with these SNPs in the formation of different P1/P2 phenotypes. First, in silico analysis predicted the potential transcription factors that may bind to the SNP5 and SNP6 regions of the P1-A4GALT allele. The following transcriptional activity determined in reporter assay experiments indicated that EGR1 elevated the transcriptional activity of the reporter construct bearing the SNP6 region with P1-A4GALT genotype, but such transcriptional activity was absent in the reporter construct bearing the SNP5 region with the P1-A4GALT genotype. Moreover, results of electrophoretic mobility shift assays (EMSA) validated the specific binding of EGR1 to the SNP6 segments with the P1-A4GALT genotype in vitro, and P1-A4GALT and P2-A4GALT allelic expression analysis further demonstrated that EGR1 specifically induce the expression of P1-A4GALT allele in vivo. Taken together, the results of our previous investigation demonstrated that two SNPs, rs2143918 (SNP5) and rs5751348 (SNP6), were associated with the P1/P2 blood groups, which result from variations in P1-A4GALT and P2-A4GALT differential expression, and we further demonstrated that the differential expression levels of the P1-A4GALT and P2-A4GALT genes result from the differential binding of EGR1 to the A4GALT SNP6 genomic region with the different genotypes. Through these investigations, we elucidated the detailed molecular mechanism for the formation of P1/P2 blood group polymorphic phenotypes.