本文選題：溶血性貧血 + 基因突變��； 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：研究目的:通過(guò)對(duì)38例溶血性貧血(Hemolytic Anemia,HA)患兒進(jìn)行病因?qū)W分析,研究?jī)和疕A的病因及發(fā)病機(jī)制,為難治性溶血性貧血病因診斷、疾病治療及預(yù)后指導(dǎo)提供理論依據(jù)。并通過(guò)對(duì)特殊病例的進(jìn)一步分析,為建立切實(shí)有效的遺傳性難治性溶血性貧血的診斷方法提供依據(jù),為遺傳性難治性溶血性貧血的病因診斷提供優(yōu)化途徑。研究方法:對(duì)2016年1月至2016年12月長(zhǎng)海醫(yī)院住院的38例HA患兒進(jìn)行詳細(xì)的病史采集及體格檢查,并通過(guò)常規(guī)檢查、抗人球蛋白試驗(yàn)(Coomb’s試驗(yàn))、骨髓穿刺術(shù)等相關(guān)實(shí)驗(yàn)室檢查,初步對(duì)HA患兒病因進(jìn)行診斷。對(duì)常規(guī)檢查手段不能明確病因的患兒,行溶血疾病檢查對(duì)紅細(xì)胞膜、酶學(xué)及血紅蛋白進(jìn)行檢測(cè),如仍病因不明或臨床反復(fù)發(fā)作的患兒,采用二代基因測(cè)序技術(shù)對(duì)其中15例患兒271個(gè)已知貧血相關(guān)基因及位點(diǎn)進(jìn)行測(cè)序,并對(duì)測(cè)序數(shù)據(jù)進(jìn)行核苷酸插入或缺失(insertion and deletion,InDel)分析和單核苷酸多態(tài)性(single nucleotide polymorphisms,SNP)分析,對(duì)篩選出的所有的與疾病相關(guān)的SNPs、lnDels使用Sanger一代測(cè)序技術(shù)進(jìn)行重復(fù)驗(yàn)證。同時(shí),為了研究患兒突變基因來(lái)源和遺傳學(xué)機(jī)制,對(duì)部分貧血患兒針對(duì)該病進(jìn)行家系突變基因序列分析。研究結(jié)果:通過(guò)對(duì)38例HA患兒進(jìn)行病因?qū)W分析及常規(guī)檢查、溶血疾病檢查發(fā)現(xiàn),38例患兒中,由感染因素誘發(fā)溶血的患兒18例(18/38,47.4%,包括EB病毒感染2例,肺炎支原體感染2例),自身免疫性溶血性貧血(AIHA)患兒11例(11/38,28.9%),紅細(xì)胞膜病6例(6/38,),酶學(xué)異常1例(1/38,2.6%),血紅蛋白病2例(2/38,5.3%%)。對(duì)15例通過(guò)常規(guī)檢查手段病因不明的遺傳性難治性溶血性貧血患兒采用二代測(cè)序技術(shù)對(duì)其271個(gè)己知貧血相關(guān)基進(jìn)行深度測(cè)序,發(fā)現(xiàn)11例患兒存在致病性單基因或多基因突變,突變位點(diǎn)包括12個(gè)錯(cuò)義突變,3個(gè)移碼突變,1個(gè)無(wú)義突變,陽(yáng)性檢出率達(dá)73%;其中13個(gè)基因突變位點(diǎn)是國(guó)內(nèi)外文獻(xiàn)未報(bào)道的新發(fā)突變位點(diǎn),并且所有的突變位點(diǎn)都導(dǎo)致了保守的氨基酸殘基及序列發(fā)生改變。采用MaxEntSca0分析算法和程序,經(jīng)Provean、SIFT、Polyphen2、Mutationtaster軟件預(yù)測(cè)基因突變的潛在致病風(fēng)險(xiǎn)。結(jié)論:對(duì)遺傳性難治性溶血性貧血患兒基因檢測(cè)結(jié)果顯示,單基因位點(diǎn)及復(fù)合位點(diǎn)基因突變是兒童遺傳性難治性溶血性貧血的常見病因。同時(shí),二代基因測(cè)序技術(shù)作為遺傳性難治性溶血性貧血的一種具有高效性且特異性高的檢查手段,為其提供了較為有效的病因?qū)W診斷,并為該病的疾病復(fù)發(fā)風(fēng)險(xiǎn)評(píng)估、遺傳咨詢方面提供依據(jù)。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R725.5

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