本文關(guān)鍵詞： 遺傳性球形紅細(xì)胞增多癥 紅細(xì)胞膜蛋白缺陷 基因突變　出處：《廣西醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：背景與目的遺傳性球形紅細(xì)胞增多癥(hereditary spherocytosis, HS)是一種以貧血、黃疸、脾大為臨床特征的溶血性貧血。世界各地均有HS報(bào)道,在北歐和北美地區(qū),該病的發(fā)病率可達(dá)1/2000。約75%的HS表現(xiàn)為常染色體顯性遺傳,約25%的HS表現(xiàn)為常染色體隱形遺傳或新生突變。SPTA1、 SPTB、ANK1、SLC4A1和EPB42基因突變導(dǎo)致α收縮蛋白、β收縮蛋白、錨蛋白、帶3蛋白、帶4.2蛋白缺陷或異常。當(dāng)α收縮蛋白、β收縮蛋白、錨蛋白、帶3蛋白、帶4.2蛋白的其中一種或多種蛋白缺陷時(shí)可造成紅細(xì)胞膜骨架與脂質(zhì)雙層間的垂直聯(lián)接減弱,細(xì)胞膜以微囊泡形式丟失,紅細(xì)胞由正常雙凹圓盤狀變?yōu)榍蛐�。HS的診斷通常需結(jié)合患者的病史,臨床表現(xiàn),家族史以及實(shí)驗(yàn)室檢查。Paula H. B. Bolton-Maggs等在2011年發(fā)表的歐美HS診療指南中指出有HS家族史,有貧血、黃疸、脾大典型臨床癥狀,實(shí)驗(yàn)室檢查有球形紅細(xì)胞增多、網(wǎng)織紅細(xì)胞增多、MCHC增高的患者無需進(jìn)一步檢查即可診斷。然而,在實(shí)際工作中發(fā)現(xiàn)相當(dāng)一部分HS患者并無以上所有的診斷依據(jù),這些HS患者常被誤診漏診。SDS-PAGE是診斷HS的經(jīng)典方法,該方法可以確定HS患者為何種膜蛋白缺陷。然而SDS-PAGE敏感性較差,Mariagabriella Mariani等曾對300例HS進(jìn)行研究,發(fā)現(xiàn)11%的HS用SDS-PAGE無法確定膜蛋白缺陷類型�；蛟\斷技術(shù)的快速發(fā)展為HS的基因診斷帶來了新的方向。目前已發(fā)現(xiàn)HS存在移碼突變、堿基置換突變、插入突變以及缺失突變等類型。然而至今尚未發(fā)現(xiàn)HS的熱突變位點(diǎn)。方法本研究收集5個(gè)HS患者及其家系的外周血,提取紅細(xì)胞膜蛋白,利用SDS-PAGE分析患者紅細(xì)胞膜蛋白缺陷類型；提取患者外周血DNA,設(shè)計(jì)并合成相應(yīng)引物,通過PCR擴(kuò)增以得到目的產(chǎn)物,使用DNA直接測序方法,與NCBI數(shù)據(jù)庫內(nèi)標(biāo)準(zhǔn)序列進(jìn)行比對,探明基因突變形式。然后對其家屬進(jìn)行相關(guān)基因突變位點(diǎn)驗(yàn)證,以了解患者的突變形式,初步探討HS患者分子發(fā)病機(jī)制。結(jié)果患者1為帶4.2蛋白完全缺陷,EPB42基因3號外顯子發(fā)生g.5935 GA雜合突變�；颊�2為帶3蛋白部分缺陷,SLC4A1基因4號外顯子發(fā)生g.6505CA雜合突變和SLC4A1基因4號內(nèi)含子發(fā)生g.6557 TC雜合突變�；颊�3帶3蛋白為部分缺陷,SLC4A1基因4號外顯子發(fā)生g.6505 CA雜合突變�；颊�4未發(fā)現(xiàn)蛋白有明顯缺陷,DNA測序表明患者的SPTB基因29號內(nèi)含子發(fā)生g.55554AT雜合突變�；颊�5為帶3蛋白部分缺陷,SLC4A1基因5號外顯子發(fā)生g.7367 TG雜合突變。結(jié)論1. SDS-PAGE對于紅細(xì)胞膜蛋白完全缺陷的HS患者有很好的診斷價(jià)值,但是對紅細(xì)胞膜蛋白部分缺陷的HS患者敏感性低。2.重視家系調(diào)查和應(yīng)用MSCV等檢查指標(biāo)可以提高HS的診斷效率。3.HS突變位點(diǎn)分布散在,同一個(gè)家系的HS患者表現(xiàn)為相同的突變方式。
[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 偽 -contractile protein, 尾 -contractile protein, anchor protein, band 3 protein. When 偽 -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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R556.6;R440

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