PCR测序技术在ABO亚型鉴定和丙酮酸激酶缺乏症诊断中的应用

发布时间：2018-05-22 19:12

本文选题：ABO血型系统 + 亚型　；参考：《上海交通大学》2015年硕士论文

【摘要】：ABO血型系统是临床输血医学和移植医学中最重要的血型系统,ABO血型的各种亚型造成临床血型鉴定困难,某些情况下可能使患者无法及时接受输血治疗或器官移植,延误治疗时机,甚至严重威胁患者生命。丙酮酸激酶缺乏症是一种常见的红细胞酶缺陷性疾病,主要引起慢性非球形红细胞溶血性贫血,为常染色体隐性遗传。PCR测序采用传统的Sanger测序法,其本身所具有的准确性高、易于标准化操作等优点使其被广泛的应用于临床检验诊断,大大提高了诊断的特异性和灵敏度。本研究主要是通过PCR测序技术对临床工作中遇到的ABO血型系统亚型及单基因遗传病给予基因诊断。通过收集先证者临床信息,对先证者临床表型进行分析,收集患者外周血进行DNA的提取,设计引物进行PCR扩增、测序,进而利用生物信息学软件对先证者基因型进行分析,实现PCR技术在临床检验诊断中的应用。在对一例B(A)亚型的研究中,发现该患者ABO基因存在多个变异,其第6外显子区存在261del和c.297AG二处杂合变异,第7外显子区存在c.526CG、c.640AG、c.646TA、c.657CT、c.681GA、c.703GA、c.771CT、c.796CT、c.803GC、c.829GA、c.930GA十一处杂合变异,以A101等位基因为标准序列进行比较,对照Blood Group Antigen Gene Mutation Database进行ABO等位基因突变分析,判断患者血型为B(A)04/O06血型。在对一例丙酮酸激酶缺乏症的研究中,发现先证者PKLR基因的7号外显子和9号外显子分别存在c.941TC和c.1183GC的复合杂合突变,先证者父亲和姐姐均为c.1183GC杂合突变,先证者母亲为c.941TC杂合突变,蛋白分析预测软件表明两个突变均引起蛋白结构的改变及酶活力的降低。
[Abstract]:The ABO blood group system is the most important blood group system in clinical transfusion medicine and transplantation medicine. It is difficult for clinical blood group identification because of various subtypes of ABO blood group. In some cases, patients may not be able to receive blood transfusion therapy or organ transplantation in time. Delay in treatment, and even a serious threat to patients' lives. Pyruvate kinase deficiency is a common disease of erythrocyte enzyme deficiency, which mainly causes chronic non-spherical erythrocyte hemolytic anemia. It is widely used in clinical laboratory diagnosis because of its advantages such as easy to standardize operation, which greatly improves the specificity and sensitivity of diagnosis. In this study, genetic diagnosis of ABO blood group system subtype and single gene hereditary disease was carried out by PCR sequencing technique. By collecting the clinical information of proband, analyzing the clinical phenotype of proband, extracting DNA from peripheral blood of patients, designing primers for PCR amplification, sequencing, and then using bioinformatics software to analyze the genotype of proband. To realize the application of PCR technology in clinical laboratory diagnosis. In the study of a ABO subtype, it was found that there were several mutations in the ABO gene of this patient. The heterozygotes of 261del and c.297AG were found in exon 6, and the heterozygosity in exon 7 was c. 526 CGCnc. 640AGC646 TAAc. 657 CTA c. 681GAc. 773GAc 771CTC. 796CTU. 803GCU. 829GAC930GA. 930GA 11 heterozygotes were found in exon 6. The A101 allele was used as the standard sequence and the ABO allele mutation analysis was performed in the control Blood Group Antigen Gene Mutation Database. The patient's blood group was identified as B(A)04/O06 blood group. In a case of pyruvate kinase deficiency, it was found that exon 7 and exon 9 of the PKLR gene of the proband had complex heterozygous mutations of c.941TC and c.1183GC, respectively. The father and sister of the proband were both c.1183GC heterozygosity mutations. The mother of the proband was a c.941TC heterozygosity mutation. The software of protein analysis and prediction showed that both mutations caused the change of protein structure and the decrease of enzyme activity.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R457.11;R440

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