5个遗传性球形红细胞增多症家系红细胞膜蛋白缺陷类型及基因突变谱研究

发布时间：2018-03-01 20:16

本文关键词： 遗传性球形红细胞增多症红细胞膜蛋白缺陷基因突变　出处：《广西医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：背景与目的遗传性球形红细胞增多症(hereditary spherocytosis, HS)是一种以贫血、黄疸、脾大为临床特征的溶血性贫血。世界各地均有HS报道,在北欧和北美地区,该病的发病率可达1/2000。约75%的HS表现为常染色体显性遗传,约25%的HS表现为常染色体隐形遗传或新生突变。SPTA1、 SPTB、ANK1、SLC4A1和EPB42基因突变导致α收缩蛋白、β收缩蛋白、锚蛋白、带3蛋白、带4.2蛋白缺陷或异常。当α收缩蛋白、β收缩蛋白、锚蛋白、带3蛋白、带4.2蛋白的其中一种或多种蛋白缺陷时可造成红细胞膜骨架与脂质双层间的垂直联接减弱,细胞膜以微囊泡形式丢失,红细胞由正常双凹圆盘状变为球形。HS的诊断通常需结合患者的病史,临床表现,家族史以及实验室检查。Paula H. B. Bolton-Maggs等在2011年发表的欧美HS诊疗指南中指出有HS家族史,有贫血、黄疸、脾大典型临床症状,实验室检查有球形红细胞增多、网织红细胞增多、MCHC增高的患者无需进一步检查即可诊断。然而,在实际工作中发现相当一部分HS患者并无以上所有的诊断依据,这些HS患者常被误诊漏诊。SDS-PAGE是诊断HS的经典方法,该方法可以确定HS患者为何种膜蛋白缺陷。然而SDS-PAGE敏感性较差,Mariagabriella Mariani等曾对300例HS进行研究,发现11%的HS用SDS-PAGE无法确定膜蛋白缺陷类型。基因诊断技术的快速发展为HS的基因诊断带来了新的方向。目前已发现HS存在移码突变、碱基置换突变、插入突变以及缺失突变等类型。然而至今尚未发现HS的热突变位点。方法本研究收集5个HS患者及其家系的外周血,提取红细胞膜蛋白,利用SDS-PAGE分析患者红细胞膜蛋白缺陷类型；提取患者外周血DNA,设计并合成相应引物,通过PCR扩增以得到目的产物,使用DNA直接测序方法,与NCBI数据库内标准序列进行比对,探明基因突变形式。然后对其家属进行相关基因突变位点验证,以了解患者的突变形式,初步探讨HS患者分子发病机制。结果患者1为带4.2蛋白完全缺陷,EPB42基因3号外显子发生g.5935 GA杂合突变。患者2为带3蛋白部分缺陷,SLC4A1基因4号外显子发生g.6505CA杂合突变和SLC4A1基因4号内含子发生g.6557 TC杂合突变。患者3带3蛋白为部分缺陷,SLC4A1基因4号外显子发生g.6505 CA杂合突变。患者4未发现蛋白有明显缺陷,DNA测序表明患者的SPTB基因29号内含子发生g.55554AT杂合突变。患者5为带3蛋白部分缺陷,SLC4A1基因5号外显子发生g.7367 TG杂合突变。结论1. SDS-PAGE对于红细胞膜蛋白完全缺陷的HS患者有很好的诊断价值,但是对红细胞膜蛋白部分缺陷的HS患者敏感性低。2.重视家系调查和应用MSCV等检查指标可以提高HS的诊断效率。3.HS突变位点分布散在,同一个家系的HS患者表现为相同的突变方式。
[Abstract]:Background & objective hereditary spheroid spherocytosis (HSS) is a hemolytic anemia characterized by anemia, jaundice and splenomegaly. HS has been reported all over the world and has been reported in Northern Europe and North America. The incidence of the disease is as high as 1 / 2000. About 75% of HS is autosomal dominant, and about 25% of HS is autosomal stealthy or new mutation. SPTA1, SPTA1, SPTBANK1, SLC4A1 and EPB42 gene mutations lead to 伪 -contractile protein, 尾 -contractile protein, anchor protein, band 3 protein. When 伪 -contractile protein, 尾 -contractile protein, anchor protein, protein with 3 proteins, one or more of the proteins with 4.2 protein are defective, the vertical connection between erythrocyte membrane skeleton and lipid bilayer can be weakened. The cell membrane was lost in the form of microvesicles, and the diagnosis of erythrocyte from normal double concave disc to spherical. HS usually combined with the patient's history and clinical manifestation. Family history and laboratory examination. Paula H. B. Bolton-Maggs et al., in the guidelines for the diagnosis and treatment of HS published in Europe and the United States in 2011, pointed out that there was a family history of HS, anemia, jaundice, typical clinical symptoms of splenomegaly, and laboratory examination of spherical erythrocytosis. Patients with elevated reticulocyte polycythemia MCHC can be diagnosed without further examination. However, it is found that a considerable number of HS patients do not have all the above diagnostic evidence. These HS patients are often misdiagnosed and missed. SDS-PAGE is a classic method for the diagnosis of HS. This method can determine what kind of membrane protein defect in HS patients. However, Mariagabriella Mariani and others have studied 300 cases of HS. It was found that 11% HS could not determine the type of membrane protein defect by SDS-PAGE. The rapid development of gene diagnosis technology has brought a new direction for HS gene diagnosis. At present, it has been found that HS has frameshift mutation and base replacement mutation. However, no heat mutation sites have been found in HS. Methods in this study, erythrocyte membrane proteins were extracted from peripheral blood of 5 patients with HS and their families. The type of erythrocyte membrane protein defect was analyzed by SDS-PAGE, the peripheral blood DNA of patients was extracted, the corresponding primers were designed and synthesized, the target product was obtained by PCR amplification, and the standard sequence was compared with the standard sequence in NCBI database by DNA direct sequencing method. Find out the mutation form of the gene. Then verify the mutation site of the related gene to understand the mutation form of the patient. Results the heterozygosity of exon 3 of EPB42 gene and exon 4 of exon 3 of EPB42 gene with 4.2-protein complete defect in patient 1 and exon 4 of SLC4A1 gene with partially defective protein in patient 2 were investigated preliminarily. Results: G. 6505CA heterozygosity occurred in patient 1 with complete protein defect EPB42 gene and exon 3 with partially defective SLC4A1 gene in patient 2. G. 6557TC heterozygosity was found in intron 4 of SLC4A1 gene. The mutation of G. 6505 CA heterozygosity in exon 4 of SLC4A1 gene was partially defective in patients with 3 proteins. G. 55554AT heterozygosity was found in intron 29 of SPTB gene and g. 7367 TG heterozygous mutation occurred in exon 5 of SLC4A1 gene with 3 protein partial defect in patient 5. Conclusion 1. SDS-PAGE has a good diagnostic value for HS patients with complete erythrocyte membrane protein defect. However, the sensitivity of HS patients with partial erythrocyte membrane protein deficiency is low. Paying attention to family investigation and applying MSCV can improve the diagnostic efficiency of HS. 3. The distribution of HS mutation sites is scattered, and the same mutation pattern is found in HS patients in the same family.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R556.6;R440

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