高分辨率溶解曲线分析技术在胆汁淤积性患儿ABCB11基因突变筛查中的应用

发布时间：2018-03-24 20:27

本文选题：ABCB11基因　切入点：肝内胆汁淤积　出处：《南京大学》2014年硕士论文

【摘要】：背景与目的：胆汁淤积是婴儿肝炎综合征最常见的表型之一,病因多种多样,其中由各种基因突变引起的遗传因素是仅次于胆道闭锁引起胆汁淤积的第二大病因。人类ABCB11基因编码表达一种称作胆盐输出泵的膜蛋白,其功能是将肝细胞内的胆盐分泌到毛细胆管中,是影响胆汁分泌最重要的因素。ABCB11基因突变直接影响到胆盐输出泵的表达量以及功能,与多种胆汁淤积性肝脏疾病相关,在儿科疾病当中,以进行性家族性肝内胆汁淤积症2型为多见。许多基因突变检测技术目前已被运用到临床进行遗传性疾病的基因诊断,使得进行性家族性肝内胆汁淤积症等遗传代谢性疾病可从婴儿肝炎综合征中辨别出来,从而得到确诊。目前基因诊断主要是对疾病的关联基因的编码外显子和(或)启动子区域及其他区域进行聚合酶链反应后将产物用Sanger法直接测序。直接测序是突变检测的金标准,但存在着成本高、操作复杂等问题,对于拥有众多外显子的致病基因而言,直接测序法效率低下、成本高等问题显得尤为突出。因此,创建一种价格低廉、高效、简单方便、而又牢靠的检测方法是解决问题的关键。高分辨率熔解曲线分析技术是新近发展的以价格低廉、高效简便为特点的基因突变检测工具,本研究拟建立以高分辨率熔解曲线分析技术与直接测序相配合的方法对胆汁淤积性患儿的ABCB11基因进行突变筛查,为临床基因诊断提供廉价高效的突变检测方法。研究方法：对20例临床疑似进行性家族性肝内胆汁淤积症2型患儿的ABCB11基因的27个编码外显子及其剪接区域运用高分辨率熔解曲线分析技术进行突变筛查,并测序验证。测序结果运用Mutation Taster进行分析,新发现的错义突变用SIFT、PolyPhen-2、SNPsGO进行致病性预测,同时比较人类与6种哺乳动物(黑猩猩、小鼠、大鼠、兔、狗、牛)胆盐输出泵蛋白的氨基酸序列,判断错义突变是否发生在进化保守区。对于ABCB11基因上常见的2个单核苷酸多态性V444A和A1028A,应用高分辨率熔解曲线分析技术筛查这20例胆汁淤积患儿和200个正常儿童,分析其是否与胆汁淤积发病有关。结果：在20例患儿中共检测到14种变异,包括7种单核苷酸多态性(rs3815675、rs4148777、rs2287616、rs183390670、rs2287622、rs118109635、 rs497692),6种错义突变(c.1009TC、c.1415AG、C.2086C＞T、c.2792AC、 c.3392AT、c.3593AG)和1种无义突变(c.2782CT)。其中,错义突变c.1009TC、C.2086C＞T、c.2792AC、c.3392AT、c.3593AG为首次发现的致病突变。综合分析临床资料及突变检测结果,4例患儿可诊断为进行性家族性肝内胆汁淤积症2型。V444A和A1028A的等位基因频率在正常儿童中分别为74.5%、67.2%,在胆汁淤积患儿中分别为80%、75%,胆汁淤积患儿的V444A和A1028A的等位基因频率与正常儿童比较均无统计学意义。结论：对反复发作、黄疸持久不退的胆汁淤积症患儿有必要做基因诊断。高分辨率熔解曲线分析技术结合直接测序可对PFIC2患儿进行高效廉价的基因诊断。对V444A和A1028A的等位基因频率的筛查表明在正常儿童及胆汁淤积患儿V444A和A1028A多态性分布无明显差异。
[Abstract]:Background and objective: cholestasis is one of the most common phenotype of infant hepatitis syndrome, a variety of causes, including genetic factors caused by various mutations after biliary atresia is the second leading cause of cholestasis. An expression called bile salt export pump membrane protein of human ABCB11 gene encoding, its function is to make bile salt the liver cells secreted into the bile, bile secretion is the most important factor.ABCB11 gene mutation directly affects the expression of bile salt output and pump function, associated with a variety of cholestatic liver disease in the pediatric disease, with progressive familial intrahepatic cholestasis type 2 is rare. Many gene mutation detection technology has been applied to clinical gene diagnosis of hereditary diseases, the progressive familial intrahepatic cholestasis of inherited metabolic diseases from infants Hepatitis syndrome identified, so as to obtain the diagnosis. The genetic diagnosis is mainly related to the disease gene encoding exons and (or) by polymerase chain reaction promoter region and other regions will be characterized by Sanger direct sequencing method. Direct sequencing is the gold standard for mutation detection, but there is a high cost the problems of complicated operation, the pathogenic gene has many exons, direct sequencing of low efficiency, high cost is particularly prominent. Therefore, the creation of a low price, high efficiency, simple and convenient, and reliable detection method is the key to solve the problems of high resolution melting analysis technology is newly developed to low price, simple and efficient for the characteristics of the mutation testing tool, this study intends to establish high resolution melting curve analysis method combined with direct sequencing technology on cholestatic bile Children with ABCB11 gene mutation screening, mutation detection methods provide cheap and efficient for clinical gene diagnosis. Methods: 27 encoding of 20 cases with suspected progressive familial intrahepatic cholestasis in children with type 2 ABCB11 gene exons and splicing region using high resolution analysis technology for mutation screening rate of melting curve, and sequencing. The sequencing results were analyzed by Mutation Taster, the newly discovered missense mutations in SIFT, PolyPhen-2, SNPsGO and pathogenic prediction, and compare the 6 kinds of mammals and humans (chimpanzee, mice, rats, rabbits, dogs, cattle) amino acid sequence of bile salt export pump protein, determine whether missense mutation in the evolutionary conserved region. For the common ABCB11 gene 2 single nucleotide polymorphisms of V444A and A1028A, the application of high resolution melting analysis the screening of 20 cases of bile siltation and 200 children A normal children, and analyze whether it is associated with bile siltation. Results: in 20 patients were detected in 14 variants, including 7 single nucleotide polymorphisms (rs3815675, rs4148777, rs2287616, rs183390670, rs2287622, rs118109635, rs497692), 6 missense mutations (c.1009TC, c.1415AG, C.2086C, T, c.2792AC, c.3392AT 1, c.3593AG) and nonsense mutation (c.2782CT). Among them, missense mutations in c.1009TC, C.2086C, T, c.2792AC, c.3392AT, c.3593AG for the first time found mutations. The mutation and comprehensive analysis of the clinical data of test results, 4 cases of children can be diagnosed as progressive familial intrahepatic cholestasis type 2.V444A and A1028A the allele frequencies in normal children were 74.5%, 67.2%, 80% respectively in cholestasis, with 75% allele frequency in children with normal cholestasis of children with V444A and A1028A were not statistically significant. Theory: recurrent jaundice persistence of cholestasis were necessary for gene diagnosis. High resolution melting curve analysis can be used in gene diagnosis of PFIC2 with high efficiency and low cost technique combined with direct sequencing. The screening of V444A and A1028A allele frequencies showed that in normal children and children with cholestasis V444A and A1028A polymorphism had no significant difference.

【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R725.7;R440

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