Study on gene expression control and pathophysiological mechanism of red cell membrane protein 4.2 in normal and disorders of hereditary hemolytic anemia

遗传性溶血性贫血正常及异常状态下红细胞膜蛋白4.2基因表达调控及病理生理机制研究

• 批准号: 09670164
• 负责人: KANZAKI Akio
• 金额: $ 1.98万
• 依托单位: KAWASAKI MEDICAL SCHOOL
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670164/
• 关键词: Protein 4.2 gene; Isoform; Protein 4.2 deficiency; Erythroblast; Band 3 gene; Red cell membrane protein; Protein expression; Gene analysis

The following results were obtained for the recent two years (1997-1999)1.Protein 4.2 (P4.2) anomalies in heredirary hemolytic anemia P4.2 anomalies were identified by quantitative membrane protein analysis from 179 patients in 8Okindreds with hereditary hemolytic anemias. These cases were divided into two types ; i.e., (1) a qualitative anomaly (P4.2 doublet Nagano), and (2) a quantitative anomaly. In qualitative P4.2 anomaly, a point mutation (R488H) was identified on the P4.2 gene in P4.2 doublet Nagano with abnormal P4.2 (72/74kD). In quantitative P4.2 anomalies, mutant genes were detected ; i.e., (1) Mild to moderate P4.2 deficiencies : B3 Fukuoka : G 130R, B3 Kagoshima : 56, 1 nt. del. , B3 Fukuyama I : 112-113, 2nt. del., B3 Fukuyama II : 183, 1 nt. ins., B3 Yamagata : G455R, B3 Okinawa : G714R, B3 Tochigi : R760W, 83 IKumamoto : R760Q B3 Nara : R808H, B3 Nagoya : T837R, B3 Philadelphia : T837M, all mutations were detected on B3 gene. (2) P4.2 complete deficiency : P4.2 mutation … More of the Nippon type (A 142T).2.P4.2 expression in human erythroblasts The expression of P4.2 in normal human erythroid cells was studied utilizing erythroblasts(Ebl) from bone marrow and cultured erythroid cells. P4.2 was first detected in orthochromatic Ebl. Among the various major membrane proteins, the expression of P4.2 was the latest. P4.2 gene mRNA was expressed in early Ebl. During normal erythroid maturation, the expression of seven different P4.2 gene products was observed by Southern blot analysis. Therefore, it can be speculated that P4.2 is expressed after the cytoskeletal network has been constructed and assembled with integral proteins in the membrane lipid bilayer.3.The methylation status of the promoter region of the band3(B3)gene in P4.2 complete deficiency Normally, the 15 CpG sites in the promoter region were almost fully methylated in the B3 gene. In contrast, in P4.2 complete deficiency, three distinct CpG sites(G,K &L) were totally or nearly totally unmethylated in the B3 genes.Although no significant changes of the status of methylation were observed in P4.2 genes.Therefore, the state of methylation might be altered in the disease states probably by changes of the comformation of the DNAs. Less

近两年(1997-1999)的研究结果如下： 1.遗传性溶血性贫血的蛋白质4.2(P4.2)异常通过定量膜蛋白分析，对80个家族的179名遗传性溶血性贫血患者进行了P4.2异常的鉴定。这些案例分为两种类型；即，(1) 定性异常（P4.2 双峰长野），以及 (2) 定量异常。在定性 P4.2 异常中，在具有异常 P4.2 (72/74kD) 的 P4.2 双联体 Nagano 的 P4.2 基因上发现了点突变 (R488H)。在定量P4.2异常中，检测到突变基因；即，(1) 轻度至中度 P4.2 缺陷：B3 福冈：G 130R，B3 鹿儿岛：56, 1 nt。德尔。 ，B3 福山 I：112-113，2nt。 del.，B3 福山 II：183，1 nt。 ins.，B3山形：G455R，B3冲绳：G714R，B3栃木：R760W，83熊本：R760Q B3奈良：R808H，B3名古屋：T837R，B3费城：T837M，所有突变均在B3基因上检测到。 (2) P4.2 完全缺乏：Nippon 型 (A 142T) 的 P4.2 突变…更多。 2.P4.2 在人成红细胞中的表达 利用来自骨髓和培养的红细胞的成红细胞 (Ebl) 研究了正常人红细胞中 P4.2 的表达。 P4.2首先在正色Ebl中检测到。在各种主要膜蛋白中，P4.2的表达最晚。 P4.2基因mRNA在早期Ebl中表达。在正常红细胞成熟过程中，通过Southern印迹分析观察到七种不同的P4.2基因产物的表达。因此，可以推测P4.2是在细胞骨架网络构建完成并与膜脂双层中的整合蛋白组装后表达的。3.P4.2完全缺失时band3(B3)基因启动子区的甲基化状态正常情况下，B3基因启动子区的15个CpG位点几乎完全甲基化。相比之下，在P4.2完全缺乏的情况下，B3基因中的三个不同的CpG位点（G、K和L）完全或几乎完全未甲基化。尽管在P4.2基因中没有观察到甲基化状态的显着变化。因此，甲基化状态可能在疾病状态下通过DNA构象的变化而改变。较少的

项目成果

Inoue,T.: "Homozygous missense mutation (band 3 Fukuoka:G130R):a mild form of hereditary spherocytosis with near-normal band 3 bontent and minimal changes of membrane ultrastructure despite moderate protein 4.2 deficiency." Brit.J.Haematol.102. 932-939 (1

Inoue,T.：“纯合错义突变（带 3 福冈：G130R）：一种轻度形式的遗传性球形红细胞增多症，尽管存在中度蛋白质 4.2 缺陷，但带 3 的强度接近正常，膜超微结构的变化最小。”

Kanzaki, A., Hayette, S., Morle, L., Inoue, F., Matsuyama, R., Inoue, T., Yawata, A., Wada, H., Vallier, A., Alloisio, N., Yawata, Y., Delaunay, J.: "Absence of protein 4.2 with partial deficiency of band 3 in hereditary spherocytosis : Compound heterozyg

Kanzaki, A.、Hayette, S.、Morle, L.、Inoue, F.、Matsuyama, R.、Inoue, T.、Yawata, A.、Wada, H.、Vallier, A.、Alloisio, N.、

Inoue, T., kanzaki, A., Kaku, M., Yawata, A., Takezono, M., Okamoto, N.Wada, H., Sugihara, T., Yamada, O., Katayama, Y., Nagata, N., Yawata, Y.: "Homozygous missense mutation (band 3 Fukuoka : G130R) : a mild form of hereditary spherocytosis with near-nor

井上 T.、神崎 A.、加来 M.、八幡 A.、竹园 M.、冈本 N.Wada, H.、杉原 T.、山田 O.、片山 Y.、永田

Ayumi Yawata: "A Markedly Disrupted Skeletal Network With Abnormally Distributed Intramembrane Particles in Complete Protein 4.1-Deficient Red Blood Cells(Allele 4.1 Madrid):Implication Regarding a Critical Role of Protein 4.1 in Maintenance of the Integr

Ayumi Yawata：“完全蛋白质 4.1 缺陷的红细胞（等位基因 4.1 马德里）中的骨骼网络显着破坏，膜内颗粒分布异常：关于蛋白质 4.1 在维持完整性中的关键作用的暗示

Akio Kanzaki: "Molecular and genetic characteristics in Japanese patients with hereditary spherocytosis: Frequent band 3 mutations and rarer ankvrin mutations" Blood. 90・10. Suppl2 6b (1997)

Akio Kanzaki：“日本遗传性球形红细胞增多症患者的分子和遗传特征：频繁的带 3 突变和罕见的 ankvrin 突变”Suppl2 6b (1997)。