MYH9相关综合征—家系的遗传学分析

发布时间：2018-02-08 12:34

本文关键词： MYH9相关综合征非肌肉肌球蛋白巨血小板减少症基因突变基因测序　出处：《广西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景:MYH9相关综合征(Myosin heavy chain 9-Related Disorders,MYH9-RD)是MYH9基因突变导致的,具有耳聋、肾炎、白内障、凝血障碍等一种或多种临床症状的遗传病。因MYH9-RD属于罕见病,它的血象与特发性血小板减少性紫癜(Idiopathic thrombocytopenic purpura,ITP)相似,医院常将MYH9-RD误诊为ITP,做不必要的激素治疗甚至是脾切除手术。因此对疑似MYH9-RD患者进行基因诊断十分必要。目的:本研究通过MYH9基因测序,从基因水平上对误诊为ITP的一家系患者作出确诊,找到致病突变并分析基因型与表型的关系,对家系患者进行遗传学分析。方法:采集家系患者和健康对照者外周血,检测血常规。分别用光学显微镜和透射电镜观察患者中性粒细胞、血小板及其细胞器的形态结构特点。根据NCBI参考序列设计PCR扩增MYH9蛋白编码区的引物,对MYH9基因序列和突变片段进行一代测序、克隆测序和酶切实验,确定突变位点和类型。并进行线粒体全基因组测序寻找疾病相关SNP。最后使用计算软件预测突变蛋白对机体的影响和序列保守性分析。结果:8名家系患者的血常规检测有“巨大血小板、血小板数量减少和中性粒细胞含包涵体”的MYH9-RD三联征,其中一人的血小板数量在正常值的下限内(122×109/L)。患者有皮肤易挫伤、紫癜消退缓慢等症状,但临床表现略有不同。基因测序结果表明,家系所有8例患者的MYH9基因c.5803delG杂合缺失,导致移码突变,造成非肌肉肌球蛋白重链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个氨基酸变异(p.Ala1935Profs*13)。而家系内和非家系健康人均无此突变。透射电镜结果显示,患者的血小板内细胞器含量差异大,其中开发管道数量与血小板体积成比例增加。PROVEAN软件预测变异蛋白对机体有害(score=-3.67-2.5),Mutation Taster计算表明变异区域的氨基酸序列在哺乳动物中是部分保守,预测变异能引起疾病。结论:家系中共有8名MYH9-RD患者,突变c.5803delG与疾病共分离,是该家系患者的致病突变。NMMHC-IIA的异常磷酸化可能是导致MYH9-RD患者血小板一直处于激活状态的重要原因。患者的临床表现支持国际上对MYH9基因型与表型关系的研究结论:发生在NMMHC-IIA蛋白羧基端尾部的突变产生的症状较轻。预计患者不会出现严重的肾病、白内障或耳聋,但不排除老年期发病的可能。该突变p.Ala1935Profs*13是一个新发现的突变,国际上尚未见报道。
[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.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R596

【参考文献】