Molecular genetical analysis of the epitopes on Rh blood group antigen and establishment of genetic examination system in RH genes.

Rh血型抗原表位的分子遗传学分析及RH基因遗传检测体系的建立。

• 批准号: 11557036
• 负责人: KAJII Eiji
• 金额: $ 8.64万
• 依托单位: Jichi Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11557036/
• 关键词: Rh blood system; RH gene; RHAG gene; promoter; enhancer; epitope; variant; genetic analysis

We determined the entire nucleotide sequences of both RHD and RHCE genes. Based on the data of these nucleotide sequences, we analyzed the genomic structure of both RH genes in detail. Various short tandem repeats (STRs) and many interspersed nuclear elements, for instance, Alu sequences were identified in both genes. By the alignment analysis of both RH genes, we confirmed multiple recombinations within 2〜3kb in introns 1 and 2. It was speculated that intron 2 in the RHCE (C) gene was produced by two recombinations between RHCE (c) and RHD genes and the following insertion of Alu-Sx.It was supported that Rh50 glycoprotein, which constituted the core of the Rh complex along with Rh polypeptide, showed the erythroid-specific expression. For the purpose of explaining the mechanism of the regulation for the expression in the RHAG gene, we identified and cloned an erythroid specific major DNaseI hypersensitive site (HS). HS located about 10kb upstream from the translation start site of the RHAG gene. A short core enhancer sequence of 195bp that corresponded with the major HS and possessed position- and orientation-independent enhancer activity in K562 cells. Homology plot analysis indicated that the enhancer region was conserved in human and mouse. The GATA motifs bound GATA-1 and mutagenesis analysis revealed that the proximal one is critical for the enhancing activity.In Rh blood system, molecular genetical analysis of various Rh variants smoothly developed. As for partial D variant, we were able to identify new type. Furthermore we proved that weak D variant possessed the qualitative and quantitative change of the RhD antigen.

我们确定了RHD和RHCE基因的全部核苷酸序列。基于这些核苷酸序列的数据，我们详细分析了这两个RH基因的基因组结构。在这两个基因中发现了多种短串联重复序列（STR）和许多散布的核元件，例如Alu序列。通过两个RH基因的比对分析，我们证实了在内含子1和2的2 ~ 3 kb内的多个重组。推测RHCE（C）基因的内含子2是由RHCE（c）与RHD基因的两次重组及随后的插入物--为了阐明RHAG基因表达的调控机制，我们鉴定并克隆了一个红系特异性的主要DNaseI超敏位点（HS）。HS位于RHAG基因翻译起始位点上游约10 kb处。一个195 bp的短核心增强子序列，对应于主要的HS，并在K562细胞中具有位置和方向独立的增强子活性。同源性分析表明，增强子区域在人和小鼠中是保守的。加塔基序与加塔-1结合，突变分析表明，近端基序是提高活性的关键。至于部分D变异，我们能够识别新的类型。进一步证明弱D变异体具有RhD抗原的质和量的变化。

项目成果

Iwamoto S, Suganuma H, Kamesaki T, Omi T, Okuda H, Kajii E.: "Cloning and characterization of erythroid-specific DNase I-hypersensitive site in human rhesus-associated glycoprotein gene."J Biol Chem. 275. 27324-27331 (2000)

Iwamoto S、Suganuma H、Kamesaki T、Omi T、Okuda H、Kajii E.：“人恒河猴相关糖蛋白基因中红系特异性 DNase I 超敏位点的克隆和表征。”J Biol Chem。

梶井英治: "Annual Review血液1999(高久史麿,宮崎澄雄,斎藤英彦,溝口秀昭,坂田洋 一編)温式自己免疫性溶血性貧血の自己抗原"中外医学社. 5 (1999)

Eiji Kajii：“年度回顾血液 1999（Fumimaro Takahisa、Sumio Miyazaki、Hidehiko Saito、Hideaki Mizoguchi、Yoichi Sakata 编辑）温自身免疫性溶血性贫血的自身抗原”Chugai Igakusha 5 (1999)。

Okuda H, Suganuma H, Tsudo N, Omi T, Iwamoto S, Kajii E.: "Sequence Analysis of the Spacer Region between the RHD and RHCE Genes."Biochem Biophys Res Commun. 263(2). 378-383 (1999)

Okuda H、Suganuma H、Tsudo N、Omi T、Iwamoto S、Kajii E.：“RHD 和 RHCE 基因之间间隔区的序列分析”。Biochem Biophys Res Commun。

Fujiwara H, Okuda H, Omi T, Iwamoto S, Tanaka Y, Takahashi J, Tani Y, Minakami H, Araki S, Sato I, Kajii E.: "The STR polymorphisms in intron 8 may provide information about the molecular evolution of RH haplotypes."Hum Genet. 104. 301-306 (1999)

Fujiwara H、Okuda H、Omi T、Iwamoto S、Tanaka Y、Takahashi J、Tani Y、Minakami H、Araki S、Sato I、Kajii E.：“内含子 8 中的 STR 多态性可能提供有关 RH 分子进化的信息

Omi T,Okuda H,Iwamoto S,Takahashi J,Tanaka M,Tani Y,Fraser RH,Seno T,Kajii E.: "Detection of Rh23 in the partial D phenotype associated with the D (Va) category."Transfusion. 40. 256-258 (2000)

Omi T、Okuda H、Iwamoto S、Takahashi J、Tanaka M、Tani Y、Fraser RH、Seno T、Kajii E.：“在与 D (Va) 类别相关的部分 D 表型中检测 Rh23。”输血。