应用高分辨率熔解曲线分析技术快速、准确检测TMPRSS6基因突变

发布时间：2018-08-27 09:33

【摘要】：背景与目的铁是人体内含量最丰富的一种必需元素,存在于所有细胞内。在体内除主要参与血红蛋白的合成和氧的输送外,还参与体内的一些生物化学的过程,包括线粒体的电子传递、儿茶酚胺代谢及DNA的合成。此外,参加三羧酸循环的酶和辅酶约半数含有铁或需要铁的存在。因此,铁代谢平衡被破坏会造成人体多方面功能的紊乱。人体内铁的含量在正常情况下维持在一个狭窄的范围内,铁缺乏和铁过载均会引起相对应的疾病。铁缺乏可以引起缺铁性贫血和儿童神经系统的疾病,铁过载可以引起血色病。铁缺乏在全球范围内超过5亿人,导致最常见的疾病为缺铁性贫血。缺铁性贫血中有一种类型被称为铁不应性缺铁性贫血(iron-refractory iron deficiency anemia, IRIDA),患者特点为先天性小细胞低色素性贫血,平均血红细胞体积低,血清铁和铁传递蛋白饱和度低,经口服铁剂以后血液学指标没有改善,但静脉注射的铁剂治疗有部分效果。有学者发现此病是一种常染色体隐性遗传病,是患者跨膜丝氨酸蛋白酶6(Transmembrane serine protease 6, TMPRSS6或称Matriptase-2)基因突变导致血清中铁调素(hepcidin,或称肝抗菌肽)含量增高所致。跨膜丝氨酸蛋白酶6是一种Ⅱ型细胞膜丝氨酸蛋白酶,最早在2005年被克隆并且定位。有学者认为跨膜丝氨酸蛋白酶6与人体铁代谢平衡密切相关。跨膜丝氨酸蛋白酶6通过控制体内铁调素的水平来调节体内铁的代谢平衡。铁调素是肝脏内产生的一种荷尔蒙,能够调节消化道内铁的吸收和巨噬细胞内铁的释放。铁调素与细胞膜铁释放通道蛋白结合,使其内化并且在溶酶体内被水解,从而阻止消化道细胞和巨噬细胞内铁释放到血清中。人体内铁调素表达增高可以使血清铁含量下降,导致缺铁性贫血；铁调素表达降低则可以使血清铁含量增高,导致体内铁过载。编码跨膜丝氨酸蛋白酶6的TMPRSS6基因是位于22号染色体长臂1区2带的基因。TMPRSS6基因突变导致铁不应性缺铁性贫血的病例在2008年被报道。迄今为止,国外已经有很多TMPRSS6基因突变导致铁不应性缺铁性贫血的报道。同时,一些学者认为TMPRSS6基因与铁过载也有联系。中国人群与其他民族人群遗传背景存在差异,中国人TMPRSS6基因突变的与其他民族之间也可能存在差异。根据PubMed等文献检索数据库的检索结果,未见有关中国人群TMPRSS6基因突变导致缺铁性贫血的报道。据研究,我国儿童铁元素缺乏的病例所占比例较高。高分辨率熔解曲线分析(High-resolution melting, HRM)技术是一种基于PCR的突变检测方法,该方法通过检测DNA双链序列之间的解链差异来判断序列之间的差异。双链DNA分子的解链受一些因素的影响,包括：片段长度、序列的GC含量和GC分布等。当片段在一定条件下,单个碱基的变化对片段的解链温度有影响,通过高分辨率和灵敏度的检测技术可以检测出来。一个纯合子样品经过PCR扩增之后得到的是同源双链,G：C和A：T之间单个碱基的变化的纯合突变对片段的解链温度有影响。杂合子样品经过PCR变性、复性之后,其组分包含四种双链分子：两种同源双链、两种异源双链。PCR之后,PCR产物结合上了染料,开始升温之前PCR的荧光强度很高。逐渐升高温度,随着温度的升高,DNA双链逐渐解开,结合上的染料被释放,荧光值逐渐减少,得到荧光强度随着温度的升高而变化的曲线,即熔解曲线。当片段一定的情况下,单个碱基的差异能反映在解链温度上,表现在熔解曲线的差异。它是一种低成本、高通量、快速、高灵敏度的检测基因突变的方法。本研究主要是通过定点诱变技术与基因克隆技术来构建TMPRSS6基因的已知突变,并针对TMPRSS6基因的所有外显子及剪接位点来设计高分辨率熔解曲线分析检测方法,并用该检测方法筛检中国人群中缺铁性贫血病例以期找到中国人TMPRSS6基因的相关数据。本研究旨在为进一步阐明中国人铁不应性缺铁性贫血的分子机制,为临床上该类疾病的诊断、预防和治疗提供参考,同时也为TMPRSS6基因突变导致的其他铁代谢异常疾病的分子机制的研究提供借鉴。材料与方法 1.标本：共收集145例缺铁性贫血病例(男性41例、女性104例；年龄：18.3±14.9岁)的血清及对应的全血标本。采用标准的饱和酚/氯仿法从外周血中提取基因组DNA。 2.血液学分析：血常规、血红蛋白含量分析、铁参数指标分析由珠海市妇幼保健医院完成。用“人铁调素hepcidin酶联免疫分析”试剂盒检测血清标本中铁调素(hepcidin)含量。 3.分子分析方法： (1)针对国外人群报道的与缺铁性贫血有关的TMPRSS6基因突变采用大引物PCR方法定点诱变和基因克隆方法：首先通过设计定点诱变引物,采用PCR技术直接从人基因组DNA样品中获取目的基因——含某一突变的基因,然后运用经典的基因工程技术将其克隆至pTA2-T载体,转化的宿主菌为大肠杆菌DH5a。每个克隆重组子中插入的目的基因进行DNA测序以验证基因突变型。 (2)针对TMPRSS6基因的所有外显子及剪接位点设计高分辨率熔解曲线分析的引物,构建TMPRSS6基因的高分辨率熔解曲线分析方法。 4.统计学分析：用建立的TMPRSS6基因高分辨率熔解曲线分析方法对构建的包含TMPRSS6基因已知突变的人工突变体进行稳定性分析,采用统计学分析方法,以确定该方法的稳定性与可靠性。使用的统计学分析软件为SPSS13.0. 5.标本筛检：对所收集的缺铁性贫血标本进行TMPRSS6基因突变筛查。 6.实验结果的综合分析、总结。结果运用大引物PCR定点诱变方法人工构建包含TMPRSS6基因已知突变的突变体,并将诱变得到的片段克隆到pTA2T-载体后进行测序,所有克隆均鉴定为所需突变,诱变成功率100%。建立的针对TMPRSS6基因的高分辨率熔解曲线分析方法能够快速、准确地区分已知突变序列和野生型序列。收集145例缺铁性贫血标本,对其进行血液学分析、检测血清铁调素(hepcidin)含量,共筛选出26例铁调素表达水平高的标本,对这26例标本进行TMPRSS6基因的高分辨率熔解曲线分析检测,未发现已知突变,亦未发现新突变,只检测到3个已知的单核苷酸多态性(single nucleotide polymorphism, SNP)。结论人类营养不良主要以微量元素缺乏为主。人体内铁元素缺乏是当今世界最流行的营养性问题之一,缺铁性贫血是全世界发病率最高的营养性疾病之一。由于体内铁元素不平衡导致的疾病正日益受到关注。高分辨率熔解曲线分析技术是近几年来兴起的基因突变检测技术。这种检测方法不受突变碱基位点与类型的局限,无需序列特异性探针,在PCR结束后直接运行高分辨率熔解检测,即可完成对样品基因型的分析。这种方法因其操作简便、快速,使用成本低,结果准确,并且实现了真正的闭管操作而受到普遍的关注。本课题研究中建立的针对TMPRSS6基因的高分辨率熔解曲线分析检测方法能够快速、准确地将TMPRSS6基因突变序列与野生型序列区分,并且实验的重复性和再现性好、稳定性高,方法可靠。本研究建立的基于PCR的高分辨率熔解曲线分析TMPRSS6基因突变诊断方法是一种高通量、高灵敏度、自动化、快速、准确、经济的突变检测技术。近年来,TMPRSS6基因突变导致铁不应性缺铁性贫血的病例不断被报道,但并没有对TMPRSS6基因进行人群突变筛查的研究。本研究中,利用高分辨率熔解曲线分析技术对中国人缺铁性贫血病例标本进行突变筛查,没有发现已知突变,这也说明了中国人TMPRSS6基因突变的携带率相对较低。目前,国内对缺铁性贫血的病因分析主要认为是铁摄入的量过少或铁丢失的量过多造成的,较少涉及到遗传学病因的分析。根据PubMed等文献检索数据库的检索结果,未见有关中国人群TMPRSS6基因突变导致缺铁性贫血的报道。因此对缺铁性贫血的遗传学病因的研究具有重要的意义和价值。本研究为TMPRSS6基因的检测建立了一种简便、经济、快速、高效和灵敏的检测方法,这为TMPRSS6基因突变导致的一类疾病的基因诊断和产前诊断提供了一种可选择的工具。本研究对进一步阐明中国人铁不应性缺铁性贫血的分子机制,对临床上该病的诊断、预防和治疗具有重要的参考价值,同时也为TMPRSS6基因突变导致的其他铁代谢异常疾病的分子机制的分析提供了借鉴。
[Abstract]:Background and purpose
Iron is one of the most abundant essential elements in the body and exists in all cells. In addition to its main role in the synthesis of hemoglobin and oxygen transport, it also participates in some biochemical processes in the body, including mitochondrial electron transport, catecholamine metabolism and DNA synthesis. Iron deficiency and iron overload can cause corresponding diseases. Iron deficiency can cause iron deficiency anemia and neurological disorders in children. Iron deficiency is the most common cause of iron deficiency anemia worldwide. One type of iron deficiency anemia is called iron refractory iron deficiency anemia (IRIDA), characterized by congenital small cell hypochromic anemia, with an average of hemoglobin. Low cell volume, low serum iron and transferrin saturation, after oral iron did not improve hematological indicators, but intravenous iron therapy has some effect. Some scholars found that the disease is an autosomal recessive inherited disease, is a transmembrane serine protease 6 (TMPRSS6 or Matriptase). E-2) mutation of the gene results in an increase in serum hepcidin.
Transmembrane serine protease 6, a type II cell membrane serine protease, was first cloned and localized in 2005. Some scholars believe that transmembrane serine protease 6 is closely related to the balance of iron metabolism in human body.
Transmembrane serine protease 6 regulates the balance of iron metabolism by controlling the level of ferritin in the body. Fermitin is a hormone produced in the liver that regulates iron absorption in the digestive tract and iron release from macrophages. Fermitin binds to the iron release channel protein of the cell membrane, internalizes it and is hydrolyzed in the lysosome. Increased expression of ferritin in the human body can reduce serum iron content and lead to iron deficiency anemia; decreased expression of ferritin can increase serum iron content and lead to iron overload in the body.
The TMPRSS6 gene encoding transmembrane serine protease 6 is located in the 2-band region of the long arm 1 of chromosome 22. The TMPRSS6 gene mutation causing iron deficiency anemia was reported in 2008. So far, there have been many reports of iron deficiency anemia caused by TMPRSS6 gene mutation abroad. RSS6 gene is also associated with iron overload. There are differences in genetic background between Chinese and other ethnic groups, and there may be differences between Chinese and other ethnic groups. In China, the proportion of children with iron deficiency is relatively high.
High-resolution melting (HRM) technique is a PCR-based mutation detection method, which detects the differences between DNA double-stranded sequences by detecting the differences of the unstranded DNA sequences. A homozygote sample was amplified by PCR and the homozygous mutation of a single base between G:C and A:T affected the temperature of the fragment. The heterozygote sample was denatured and renatured by PCR, and its components consisted of four double-stranded molecules: two homologous double-stranded, two heterologous double-stranded. After PCR, the PCR products were bound to dyes. The fluorescence intensity of PCR was very high before the beginning of warming. When the fragment is fixed, the difference of the single base can be reflected in the temperature of the chain breaking, which shows the difference of the melting curve. It is a low-cost, high-throughput, rapid and highly sensitive method to detect gene mutation.
In this study, the known mutations of TMPRSS6 gene were constructed by site-directed mutagenesis and gene cloning techniques, and a high resolution melting curve analysis method was designed for all exons and splicing sites of TMPRSS6 gene. The method was used to screen iron deficiency anemia cases in Chinese population in order to find TMPRSS6 gene. Gene related data.
The purpose of this study is to elucidate the molecular mechanism of iron deficiency anemia in Chinese, to provide reference for the diagnosis, prevention and treatment of iron deficiency anemia, and to study the molecular mechanism of other iron metabolism disorders caused by TMPRSS6 mutation.
Materials and methods
1. Specimens: A total of 145 cases of iron deficiency anemia (41 males, 104 females; age: 18.3 (+ 14.9 years) were collected from serum and corresponding whole blood samples. Genomic DNA was extracted from peripheral blood by standard saturated phenol/chloroform method.
2. Hematological analysis: Blood routine, hemoglobin content analysis, iron parameter analysis were completed by Zhuhai Maternal and Child Health Hospital. The content of hepcidin in serum samples was detected by "hepcidin ELISA kit".
3. molecular analysis methods:
(1) The site-directed mutagenesis and gene cloning of TMPRSS6 gene mutation related to iron deficiency anemia reported by foreign people were studied by using large primer PCR. Firstly, the target gene, containing a mutant gene, was obtained directly from human genomic DNA samples by designing site-directed mutagenesis primers and PCR technology, and then classical gene was used. It was cloned into pTA2-T vector and transformed into E.coli DH5a. The target gene inserted in each cloned recombinant was sequenced to verify the mutation.
(2) Primers were designed to analyze the exons and splicing sites of TMPRSS6 gene by high resolution melting curve analysis, and a high resolution melting curve analysis method for TMPRSS6 gene was constructed.
4. Statistical analysis: The stability of the constructed artificial mutant containing TMPRSS6 gene mutation was analyzed by the established high resolution melting curve analysis method, and the stability and reliability of the method were determined by statistical analysis method. The statistical analysis software was SPSS13.0.
5. sample screening: screening for TMPRSS6 gene mutations in the samples collected from iron deficiency anemia.
6. comprehensive analysis and conclusion of the experimental results.
Result
A mutant containing TMPRSS6 gene mutation was constructed by site-directed PCR with large primers. The mutant fragment was cloned into pTA2T-vector and sequenced. All the clones were identified as the required mutation with 100% mutagenesis power.
The established high resolution melting curve analysis method for TMPRSS6 gene can quickly and accurately distinguish the known mutation sequence from the wild type sequence.
145 samples of iron deficiency anemia were collected for hematological analysis and serum ferritin (hepcidin) levels were detected. A total of 26 samples with high levels of ferritin expression were screened out. High resolution melting curve analysis of TMPRSS6 gene was performed on these 26 samples. No known mutations were found and no new mutations were found. Only three known single mutations were detected. Single nucleotide polymorphism (SNP).
conclusion
Iron deficiency is one of the most prevalent nutritional problems in the world today. Iron deficiency anemia is one of the most common nutritional diseases in the world. Diseases caused by iron imbalance in the body are attracting increasing attention.
High-resolution melting curve analysis is a new technique for gene mutation detection in recent years. This method is not limited by mutation base sites and types, and does not require sequence-specific probes. Rapid, low cost, accurate results and the realization of true closed tube operation have received widespread attention.
The high resolution melting curve analysis method for TMPRSS6 gene established in this study can quickly and accurately distinguish TMPRSS6 gene mutation sequence from wild type sequence, and the repeatability and reproducibility of the experiment is good, the stability is high, and the method is reliable. Gene mutation diagnosis is a high throughput, high sensitivity, automatic, rapid, accurate and economical mutation detection technology.
In recent years, cases of iron deficiency anemia caused by mutation of TMPRSS6 gene have been reported continuously, but there has been no study on population mutation screening of TMPRSS6 gene. In China, the mutation rate of TMPRSS6 gene is relatively low.
At present, the etiological analysis of iron deficiency anemia in China is mainly attributed to low iron intake or excessive iron loss, and less to the analysis of genetic causes. According to the retrieval results of PubMed and other literature retrieval databases, there are no reports about iron deficiency anemia caused by TMPRSS6 gene mutation in Chinese population. The study of genetic etiology of anemia is of great significance and value.
This study establishes a simple, economical, rapid, efficient and sensitive method for the detection of TMPRSS6 gene, which provides an alternative tool for gene diagnosis and prenatal diagnosis of a class of diseases caused by TMPRSS6 gene mutation.
This study has important reference value for further elucidating the molecular mechanism of iron deficiency anemia in Chinese, and for the diagnosis, prevention and treatment of iron deficiency anemia. It also provides a reference for the molecular mechanism analysis of other iron metabolism disorders caused by TMPRSS6 gene mutation.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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