本文關(guān)鍵詞： MYH9相關(guān)綜合征 非肌肉肌球蛋白 巨血小板減少癥 基因突變 基因測序　出處：《廣西醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：背景:MYH9相關(guān)綜合征(Myosin heavy chain 9-Related Disorders,MYH9-RD)是MYH9基因突變導致的,具有耳聾、腎炎、白內(nèi)障、凝血障礙等一種或多種臨床癥狀的遺傳病。因MYH9-RD屬于罕見病,它的血象與特發(fā)性血小板減少性紫癜(Idiopathic thrombocytopenic purpura,ITP)相似,醫(yī)院常將MYH9-RD誤診為ITP,做不必要的激素治療甚至是脾切除手術(shù)。因此對疑似MYH9-RD患者進行基因診斷十分必要。目的:本研究通過MYH9基因測序,從基因水平上對誤診為ITP的一家系患者作出確診,找到致病突變并分析基因型與表型的關(guān)系,對家系患者進行遺傳學分析。方法:采集家系患者和健康對照者外周血,檢測血常規(guī)。分別用光學顯微鏡和透射電鏡觀察患者中性粒細胞、血小板及其細胞器的形態(tài)結(jié)構(gòu)特點。根據(jù)NCBI參考序列設(shè)計PCR擴增MYH9蛋白編碼區(qū)的引物,對MYH9基因序列和突變片段進行一代測序、克隆測序和酶切實驗,確定突變位點和類型。并進行線粒體全基因組測序?qū)ふ壹膊∠嚓P(guān)SNP。最后使用計算軟件預測突變蛋白對機體的影響和序列保守性分析。結(jié)果:8名家系患者的血常規(guī)檢測有“巨大血小板、血小板數(shù)量減少和中性粒細胞含包涵體”的MYH9-RD三聯(lián)征,其中一人的血小板數(shù)量在正常值的下限內(nèi)(122×109/L)。患者有皮膚易挫傷、紫癜消退緩慢等癥狀,但臨床表現(xiàn)略有不同。基因測序結(jié)果表明,家系所有8例患者的MYH9基因c.5803delG雜合缺失,導致移碼突變,造成非肌肉肌球蛋白重鏈IIA(nonmuscle myosin heavy chain IIA,NMMHC-IIA)的羧基端非螺旋尾部的蛋白截短,共涉及26個氨基酸變異(p.Ala1935Profs*13)。而家系內(nèi)和非家系健康人均無此突變。透射電鏡結(jié)果顯示,患者的血小板內(nèi)細胞器含量差異大,其中開發(fā)管道數(shù)量與血小板體積成比例增加。PROVEAN軟件預測變異蛋白對機體有害(score=-3.67-2.5),Mutation Taster計算表明變異區(qū)域的氨基酸序列在哺乳動物中是部分保守,預測變異能引起疾病。結(jié)論:家系中共有8名MYH9-RD患者,突變c.5803delG與疾病共分離,是該家系患者的致病突變。NMMHC-IIA的異常磷酸化可能是導致MYH9-RD患者血小板一直處于激活狀態(tài)的重要原因�；颊叩呐R床表現(xiàn)支持國際上對MYH9基因型與表型關(guān)系的研究結(jié)論:發(fā)生在NMMHC-IIA蛋白羧基端尾部的突變產(chǎn)生的癥狀較輕。預計患者不會出現(xiàn)嚴重的腎病、白內(nèi)障或耳聾,但不排除老年期發(fā)病的可能。該突變p.Ala1935Profs*13是一個新發(fā)現(xiàn)的突變,國際上尚未見報道。
[Abstract]:Background: MYH9 syndrome (Myosin heavy chain 9-Related Disorders, MYH9-RD) is a mutation in the MYH9 gene, with deafness, nephritis, cataract, blood clotting disorders and genetic disease or a variety of clinical symptoms. Because MYH9-RD is a rare disease, its blood and idiopathic thrombocytopenic purpura (Idiopathic thrombocytopenic purpura. ITP) is similar to that of MYH9-RD hospital often misdiagnosed as ITP, do not need hormone therapy or even splenectomy. Therefore the gene diagnosis of patients with suspected MYH9-RD is necessary. Objective: To study the MYH9 gene sequencing, from gene level to misdiagnosis of a pedigree with ITP to find the relationship between diagnosis, pathogenic mutation and the analysis of genotype and phenotype and genetic analysis of familial patients. Methods: to collect the families of patients and healthy controls. Peripheral blood routine test respectively by optical microscope and transmission The observation of neutrophils in patients with electron microscopy, morphological characteristics and platelet organelles. According to the reference design primer NCBI amplified PCR sequences encoding MYH9 protein, a generation of sequencing of the MYH9 gene sequence and mutation, cloning, sequencing and restriction enzyme digestion experiment, determine the mutation sites and types. And mitochondrial genome sequencing to find finally, using the calculation software of disease related SNP. mutant protein prediction effects on the body and sequence conservation analysis. Results: 8 patients with family blood test "to reduce the number of giant platelets, platelet and neutrophil inclusions containing" MYH9-RD triad, one of the number of platelets in the lower limit of the normal value in (122 x 109/L). Patients with skin purpura subsided slowly to contusion, and other symptoms, but clinical manifestations were slightly different. The sequencing results showed that the family of all 8 patients with MYH9 gene C.5 803delG LOH, leading to frameshift mutation, resulting in non muscle myosin heavy chain IIA (nonmuscle myosin heavy chain IIA, NMMHC-IIA) of the C-terminal tail of the non helical protein truncation, involving a total of 26 amino acid mutation (p.Ala1935Profs*13). And within family and non family per capita Xi Jian Kang no mutation. The TEM results show the difference, organelles content of platelets in patients with large, including the number of platelet and development pipeline proportional to the volume variation prediction of protein on the body harmful increase of.PROVEAN software (score=-3.67-2.5), Mutation Taster calculations indicate that the amino acid sequence variation region in mammals is conservative, prediction of variation can cause diseases. Conclusion: there are 8 families MYH9-RD patients, c.5803delG mutation cosegregated with the disease, patients with abnormal phosphate pathogenic mutations in.NMMHC-IIA in this family may be a result of platelets in patients with MYH9-RD Direct is an important reason for the activation status. The conclusion of study on MYH9 genotype phenotype correlation in the clinical manifestations of patients with international support: the occurrence of mutations in the NMMHC-IIA protein C-terminal tail of the expected mild symptoms. Patients don't have serious kidney disease, cataract or deafness, but not to the exclusion of old age may be the mutation. P.Ala1935Profs*13 is a newly discovered mutation, the world has not yet been reported.

【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R596

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本文編號：1495453