Rh血型基因差異性的偵測

Abstract

Rh血型系統是在早期發現有別於ABO的另一個重要的血型系統。Rh血型系統主要的抗原是D、C、c、E、e，世界各地分佈著各種不同的Rh血型，其主要的基因型也不盡相同。 Rh血型基因位第一對染色體上，由兩個基因負責:RHD及RHCE，前者負責D抗原，後者是C/c及E/e四個抗原，C/c的抗原決定位是在RHCE基因上的第二個外顯子上，而E/e的抗原決定位在RHCE基因上的第五個外顯子上，另一方面，兩個基因有高度的同源性，因此，在同一條染色體上會因為兩者相配對發生基因轉換，而在RHD基因前後各有一個高同源性的Rhesus Box，會與另一條染色體上的Rhesus Box配對而造成RHD基因的缺失；正因為Rh血型系統如此複雜，所以不同Rh血型的基因型和分子機制都很值得研究。 本研究主要分析的對象是D+C-E-c-e-的病患(個案六、七)、一個Rh陰性的病患(個案八)以及五位正常人的檢體(個案一~五)，探討的目標是Rh的基因型。我們先利用抗原抗體之間的反應分析了血型，接著利用網路系統NCBI找到了RHD及RHCE兩個基因的序列並作出比對找出兩基因以及C/c和E/e抗原多型性的差異點，設計了針對各個外顯子的引子利用定序的方法分析病人及正常人的Rh基因。 如同網路系統預測的兩個基因RHD與RHCE的基因序列，所有正常人的兩個基因序列都與C/c及E/e的抗原多型性分析相符合。在病人的結果中，個案六除了第一外顯子有兩個基因的序列以及第十個外顯子是兩個基因產物都有，其他外顯子都是RHD基因的序列；而個案七除了第一及第九個第一外顯子有兩個基因的序列以及第十個外顯子是兩個基因產物都有，其他外顯子都是RHD基因的序列；個案八則是只有第二個外顯子有兩個基因的序列，其他的外顯子分析都只找到RHCE的基因 針對這次的研究結果，我們可以猜測個案六和七的Rh基因是一個融合基因，個案六的基因型是RHCE-D(2-9)-CE ，而個案七的則是RHCE-D(2-7/8)-CE或是RHCE-D(2-9)-CE，這些融合基因讓它的C/c及E/e抗原表現消失，而個案八則有兩種可能，一是兩個對偶基因上的RHD基因都被刪除，其中一個RHCE基因發了基因轉換，另一個可能是RHD發生了兩次的基因轉換，或發生了大片段的基因刪除，使得個案八的Rh血型是陰性的表現型，未來需要更進一步的分析。

Rh blood type system has found as another important blood system besides ABO blood type system, the most important antigens in Rh blood type system are D, C, c, E, e, the antigen is the reason for many different Rh blood in worldwide. Rh gene locates in chromosome 1, two genes are involved : RHD and RHCE, the first is for antigen D, and the second is for antigen C/c and E/e, the antigen epitope of C/c antigen is Exon2 of RHCE gene, and the antigen epitope of E/e antigen is Exon5 of RHCE gene, In the other hand, two gene are highly homologous, so that in the same allele, two genes can have gene conversion because of the pair between RHCE and RHD gene. Furthermore, upstream and downstream Rhesus box which flank the RHD gene can make the RHD gene lost due to the unequal crossing by Rhesus box between two alleles; and now we know the Rh blood type system is very complicated, and it is worth for us to study the molecular mechanism and the genotype. Our study focuses on the patients (Case 6 & 7) which Rh blood type is D+C-E-c-e-, one Rh-negative patient (Case 8) and five normal sample (Case 1~5) , and my aim is the genotype of Rh gene. First we analyze the blood type by antigen and antibody reaction, then we used the website NCBI to search the RHD and RHCE hole gene sequence and compare it to the difference of two genes and the polymorphism between C/c antigen and E/e antigen. After all, we design some pair of primers which targets on exon to analyze the difference between normal and sample’s Rh gene. As the sequence of RHD and RHCE genes we predict by website, the polymorphism between C/c antigen and E/e antigen are observed in all normal’s genes sequence; in the patient’s result, Case 6’s gene sequence is only RHD gene except Exon 1 and 10 , Case 7’s gene sequence is only RHD gene except Exon 1, 9 and 10, Case 8’s gene sequence is only RHCE gene except Exon 2. According to the study result, we make a hypothesis that Case 6 and 7 are the hybrid gene, the genotype of case 6 is RHCE-D(2-9)-CE, the genotype of case 7 is RHCE-D(2-7/8)-CE or RHCE-D(2-9)-CE , and this is the reason for the C/c and E/e antigen disappear, and case 8, the Rh-negative phenotype is responsible for two possible molecular mechanism, one is two alleles which RHD gene is deleted, and one of alleles is RHCE hybrid gene, another one is gene conversion or gene deletion, and we shall study more in the future.