二代基因测序技术在儿童遗传性难治性溶血性贫血中的应用研究

发布时间：2018-05-03 05:22

本文选题：溶血性贫血 + 基因突变　；参考：《第二军医大学》2017年硕士论文

【摘要】：研究目的:通过对38例溶血性贫血(Hemolytic Anemia,HA)患儿进行病因学分析,研究儿童HA的病因及发病机制,为难治性溶血性贫血病因诊断、疾病治疗及预后指导提供理论依据。并通过对特殊病例的进一步分析,为建立切实有效的遗传性难治性溶血性贫血的诊断方法提供依据,为遗传性难治性溶血性贫血的病因诊断提供优化途径。研究方法:对2016年1月至2016年12月长海医院住院的38例HA患儿进行详细的病史采集及体格检查,并通过常规检查、抗人球蛋白试验(Coomb’s试验)、骨髓穿刺术等相关实验室检查,初步对HA患儿病因进行诊断。对常规检查手段不能明确病因的患儿,行溶血疾病检查对红细胞膜、酶学及血红蛋白进行检测,如仍病因不明或临床反复发作的患儿,采用二代基因测序技术对其中15例患儿271个已知贫血相关基因及位点进行测序,并对测序数据进行核苷酸插入或缺失(insertion and deletion,InDel)分析和单核苷酸多态性(single nucleotide polymorphisms,SNP)分析,对筛选出的所有的与疾病相关的SNPs、lnDels使用Sanger一代测序技术进行重复验证。同时,为了研究患儿突变基因来源和遗传学机制,对部分贫血患儿针对该病进行家系突变基因序列分析。研究结果:通过对38例HA患儿进行病因学分析及常规检查、溶血疾病检查发现,38例患儿中,由感染因素诱发溶血的患儿18例(18/38,47.4%,包括EB病毒感染2例,肺炎支原体感染2例),自身免疫性溶血性贫血(AIHA)患儿11例(11/38,28.9%),红细胞膜病6例(6/38,),酶学异常1例(1/38,2.6%),血红蛋白病2例(2/38,5.3%%)。对15例通过常规检查手段病因不明的遗传性难治性溶血性贫血患儿采用二代测序技术对其271个己知贫血相关基进行深度测序,发现11例患儿存在致病性单基因或多基因突变,突变位点包括12个错义突变,3个移码突变,1个无义突变,阳性检出率达73%;其中13个基因突变位点是国内外文献未报道的新发突变位点,并且所有的突变位点都导致了保守的氨基酸残基及序列发生改变。采用MaxEntSca0分析算法和程序,经Provean、SIFT、Polyphen2、Mutationtaster软件预测基因突变的潜在致病风险。结论:对遗传性难治性溶血性贫血患儿基因检测结果显示,单基因位点及复合位点基因突变是儿童遗传性难治性溶血性贫血的常见病因。同时,二代基因测序技术作为遗传性难治性溶血性贫血的一种具有高效性且特异性高的检查手段,为其提供了较为有效的病因学诊断,并为该病的疾病复发风险评估、遗传咨询方面提供依据。
[Abstract]:Objective: to study the etiology and pathogenesis of hemolytic anemiahae in 38 children with hemolytic anemia, and to provide theoretical basis for etiological diagnosis, treatment and prognosis of refractory hemolytic anemia. Through further analysis of special cases, this paper provides a basis for the establishment of effective diagnostic methods for hereditary refractory hemolytic anemia, and provides an optimized approach for the etiological diagnosis of hereditary refractory hemolytic anemia. Methods: from January 2016 to December 2016, 38 cases of HA patients hospitalized in Changhai Hospital were examined with detailed medical history and physical examination. The results of routine examination, anti-human globulin test and CoombSs test, bone marrow puncture, etc. The etiology of children with HA was preliminarily diagnosed. For children whose etiology is not clear by routine examination, hemolytic disease is performed to detect erythrocyte membrane, enzymology and hemoglobin, such as children with unknown etiology or recurrent clinical episodes, Two generation gene sequencing techniques were used to sequence 271 known anemia related genes and loci in 15 children, and the sequencing data were analyzed by insertion or deletion and deletion (deletion and) and single nucleotide polymorphisms (SNPs), and single nucleotide polymorphisms (SNPs) were analyzed by single nucleotide polymorphisms (SNPs). All disease-related SNPs lnDels were repeatedly verified using Sanger generation sequencing technique. At the same time, in order to study the origin and genetic mechanism of mutation gene in children with anemia, the family mutation gene sequence analysis was carried out in some children with anemia. Results: according to the etiological analysis and routine examination of 38 children with HA, among 38 children with hemolytic disease, 18 cases were induced hemolysis by infection factors, including 2 cases of Epstein-Barr virus (EBV) infection. There were 2 cases of mycoplasma pneumoniae infection, 11 cases of autoimmune hemolytic anemia (AIHAA), 6 cases of erythrocyte membrane disease (6 cases), 6 cases of erythrocyte membrane disease (6 cases), 1 case of abnormal enzymology (1 case), 2 cases of hemoglobinopathy (2 cases) and 2 cases of hemoglobinopathy (2 cases). In 15 children with hereditary refractory hemolytic anemia whose etiology was unknown by routine examination, 271 of their known anemia related bases were deeply sequenced by second-generation sequencing technique. It was found that 11 children had pathogenicity single gene mutation or polygene mutation. The mutation sites included 12 missense mutations, 3 frameshift mutations, 1 nonsense mutation and the positive rate of 73 mutation sites, 13 of which were new mutation sites not reported in domestic and foreign literature. And all mutation sites lead to conserved amino acid residues and sequence changes. Using the MaxEntSca0 analysis algorithm and program, the potential risk of gene mutation was predicted by Proveansifen Polyphen2Mutationtaster software. Conclusion: the results of gene detection in children with hereditary refractory hemolytic anemia indicate that single gene locus and complex locus gene mutation are the common etiology of hereditary refractory hemolytic anemia in children. At the same time, as a highly effective and specific method for the detection of hereditary refractory hemolytic anemia, the second generation gene sequencing technique provides an effective etiological diagnosis and a risk assessment for the recurrence of the disease. To provide the basis for genetic counseling.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R725.5

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