應(yīng)用高分辨率熔解曲線分析技術(shù)快速、準(zhǔn)確檢測(cè)TMPRSS6基因突變
發(fā)布時(shí)間：2018-08-27 09:33
【摘要】：背景與目的 鐵是人體內(nèi)含量最豐富的一種必需元素,存在于所有細(xì)胞內(nèi)。在體內(nèi)除主要參與血紅蛋白的合成和氧的輸送外,還參與體內(nèi)的一些生物化學(xué)的過程,包括線粒體的電子傳遞、兒茶酚胺代謝及DNA的合成。此外,參加三羧酸循環(huán)的酶和輔酶約半數(shù)含有鐵或需要鐵的存在。因此,鐵代謝平衡被破壞會(huì)造成人體多方面功能的紊亂。人體內(nèi)鐵的含量在正常情況下維持在一個(gè)狹窄的范圍內(nèi),鐵缺乏和鐵過載均會(huì)引起相對(duì)應(yīng)的疾病。鐵缺乏可以引起缺鐵性貧血和兒童神經(jīng)系統(tǒng)的疾病,鐵過載可以引起血色病。鐵缺乏在全球范圍內(nèi)超過5億人,導(dǎo)致最常見的疾病為缺鐵性貧血。缺鐵性貧血中有一種類型被稱為鐵不應(yīng)性缺鐵性貧血(iron-refractory iron deficiency anemia, IRIDA),患者特點(diǎn)為先天性小細(xì)胞低色素性貧血,平均血紅細(xì)胞體積低,血清鐵和鐵傳遞蛋白飽和度低,經(jīng)口服鐵劑以后血液學(xué)指標(biāo)沒有改善,但靜脈注射的鐵劑治療有部分效果。有學(xué)者發(fā)現(xiàn)此病是一種常染色體隱性遺傳病,是患者跨膜絲氨酸蛋白酶6(Transmembrane serine protease 6, TMPRSS6或稱Matriptase-2)基因突變導(dǎo)致血清中鐵調(diào)素(hepcidin,或稱肝抗菌肽)含量增高所致。 跨膜絲氨酸蛋白酶6是一種Ⅱ型細(xì)胞膜絲氨酸蛋白酶,最早在2005年被克隆并且定位。有學(xué)者認(rèn)為跨膜絲氨酸蛋白酶6與人體鐵代謝平衡密切相關(guān)。 跨膜絲氨酸蛋白酶6通過控制體內(nèi)鐵調(diào)素的水平來調(diào)節(jié)體內(nèi)鐵的代謝平衡。鐵調(diào)素是肝臟內(nèi)產(chǎn)生的一種荷爾蒙,能夠調(diào)節(jié)消化道內(nèi)鐵的吸收和巨噬細(xì)胞內(nèi)鐵的釋放。鐵調(diào)素與細(xì)胞膜鐵釋放通道蛋白結(jié)合,使其內(nèi)化并且在溶酶體內(nèi)被水解,從而阻止消化道細(xì)胞和巨噬細(xì)胞內(nèi)鐵釋放到血清中。人體內(nèi)鐵調(diào)素表達(dá)增高可以使血清鐵含量下降,導(dǎo)致缺鐵性貧血；鐵調(diào)素表達(dá)降低則可以使血清鐵含量增高,導(dǎo)致體內(nèi)鐵過載。 編碼跨膜絲氨酸蛋白酶6的TMPRSS6基因是位于22號(hào)染色體長(zhǎng)臂1區(qū)2帶的基因。TMPRSS6基因突變導(dǎo)致鐵不應(yīng)性缺鐵性貧血的病例在2008年被報(bào)道。迄今為止,國(guó)外已經(jīng)有很多TMPRSS6基因突變導(dǎo)致鐵不應(yīng)性缺鐵性貧血的報(bào)道。同時(shí),一些學(xué)者認(rèn)為TMPRSS6基因與鐵過載也有聯(lián)系。中國(guó)人群與其他民族人群遺傳背景存在差異,中國(guó)人TMPRSS6基因突變的與其他民族之間也可能存在差異。根據(jù)PubMed等文獻(xiàn)檢索數(shù)據(jù)庫的檢索結(jié)果,未見有關(guān)中國(guó)人群TMPRSS6基因突變導(dǎo)致缺鐵性貧血的報(bào)道。據(jù)研究,我國(guó)兒童鐵元素缺乏的病例所占比例較高。 高分辨率熔解曲線分析(High-resolution melting, HRM)技術(shù)是一種基于PCR的突變檢測(cè)方法,該方法通過檢測(cè)DNA雙鏈序列之間的解鏈差異來判斷序列之間的差異。雙鏈DNA分子的解鏈?zhǔn)芤恍┮蛩氐挠绊?包括：片段長(zhǎng)度、序列的GC含量和GC分布等。當(dāng)片段在一定條件下,單個(gè)堿基的變化對(duì)片段的解鏈溫度有影響,通過高分辨率和靈敏度的檢測(cè)技術(shù)可以檢測(cè)出來。一個(gè)純合子樣品經(jīng)過PCR擴(kuò)增之后得到的是同源雙鏈,G：C和A：T之間單個(gè)堿基的變化的純合突變對(duì)片段的解鏈溫度有影響。雜合子樣品經(jīng)過PCR變性、復(fù)性之后,其組分包含四種雙鏈分子：兩種同源雙鏈、兩種異源雙鏈。PCR之后,PCR產(chǎn)物結(jié)合上了染料,開始升溫之前PCR的熒光強(qiáng)度很高。逐漸升高溫度,隨著溫度的升高,DNA雙鏈逐漸解開,結(jié)合上的染料被釋放,熒光值逐漸減少,得到熒光強(qiáng)度隨著溫度的升高而變化的曲線,即熔解曲線。當(dāng)片段一定的情況下,單個(gè)堿基的差異能反映在解鏈溫度上,表現(xiàn)在熔解曲線的差異。它是一種低成本、高通量、快速、高靈敏度的檢測(cè)基因突變的方法。 本研究主要是通過定點(diǎn)誘變技術(shù)與基因克隆技術(shù)來構(gòu)建TMPRSS6基因的已知突變,并針對(duì)TMPRSS6基因的所有外顯子及剪接位點(diǎn)來設(shè)計(jì)高分辨率熔解曲線分析檢測(cè)方法,并用該檢測(cè)方法篩檢中國(guó)人群中缺鐵性貧血病例以期找到中國(guó)人TMPRSS6基因的相關(guān)數(shù)據(jù)。 本研究旨在為進(jìn)一步闡明中國(guó)人鐵不應(yīng)性缺鐵性貧血的分子機(jī)制,為臨床上該類疾病的診斷、預(yù)防和治療提供參考,同時(shí)也為TMPRSS6基因突變導(dǎo)致的其他鐵代謝異常疾病的分子機(jī)制的研究提供借鑒。 材料與方法 1.標(biāo)本：共收集145例缺鐵性貧血病例(男性41例、女性104例；年齡：18.3±14.9歲)的血清及對(duì)應(yīng)的全血標(biāo)本。采用標(biāo)準(zhǔn)的飽和酚/氯仿法從外周血中提取基因組DNA。 2.血液學(xué)分析：血常規(guī)、血紅蛋白含量分析、鐵參數(shù)指標(biāo)分析由珠海市婦幼保健醫(yī)院完成。用“人鐵調(diào)素hepcidin酶聯(lián)免疫分析”試劑盒檢測(cè)血清標(biāo)本中鐵調(diào)素(hepcidin)含量。 3.分子分析方法： (1)針對(duì)國(guó)外人群報(bào)道的與缺鐵性貧血有關(guān)的TMPRSS6基因突變采用大引物PCR方法定點(diǎn)誘變和基因克隆方法：首先通過設(shè)計(jì)定點(diǎn)誘變引物,采用PCR技術(shù)直接從人基因組DNA樣品中獲取目的基因——含某一突變的基因,然后運(yùn)用經(jīng)典的基因工程技術(shù)將其克隆至pTA2-T載體,轉(zhuǎn)化的宿主菌為大腸桿菌DH5a。每個(gè)克隆重組子中插入的目的基因進(jìn)行DNA測(cè)序以驗(yàn)證基因突變型。 (2)針對(duì)TMPRSS6基因的所有外顯子及剪接位點(diǎn)設(shè)計(jì)高分辨率熔解曲線分析的引物,構(gòu)建TMPRSS6基因的高分辨率熔解曲線分析方法。 4.統(tǒng)計(jì)學(xué)分析：用建立的TMPRSS6基因高分辨率熔解曲線分析方法對(duì)構(gòu)建的包含TMPRSS6基因已知突變的人工突變體進(jìn)行穩(wěn)定性分析,采用統(tǒng)計(jì)學(xué)分析方法,以確定該方法的穩(wěn)定性與可靠性。使用的統(tǒng)計(jì)學(xué)分析軟件為SPSS13.0. 5.標(biāo)本篩檢：對(duì)所收集的缺鐵性貧血標(biāo)本進(jìn)行TMPRSS6基因突變篩查。 6.實(shí)驗(yàn)結(jié)果的綜合分析、總結(jié)。 結(jié)果 運(yùn)用大引物PCR定點(diǎn)誘變方法人工構(gòu)建包含TMPRSS6基因已知突變的突變體,并將誘變得到的片段克隆到pTA2T-載體后進(jìn)行測(cè)序,所有克隆均鑒定為所需突變,誘變成功率100%。 建立的針對(duì)TMPRSS6基因的高分辨率熔解曲線分析方法能夠快速、準(zhǔn)確地區(qū)分已知突變序列和野生型序列。 收集145例缺鐵性貧血標(biāo)本,對(duì)其進(jìn)行血液學(xué)分析、檢測(cè)血清鐵調(diào)素(hepcidin)含量,共篩選出26例鐵調(diào)素表達(dá)水平高的標(biāo)本,對(duì)這26例標(biāo)本進(jìn)行TMPRSS6基因的高分辨率熔解曲線分析檢測(cè),未發(fā)現(xiàn)已知突變,亦未發(fā)現(xiàn)新突變,只檢測(cè)到3個(gè)已知的單核苷酸多態(tài)性(single nucleotide polymorphism, SNP)。 結(jié)論 人類營(yíng)養(yǎng)不良主要以微量元素缺乏為主。人體內(nèi)鐵元素缺乏是當(dāng)今世界最流行的營(yíng)養(yǎng)性問題之一,缺鐵性貧血是全世界發(fā)病率最高的營(yíng)養(yǎng)性疾病之一。由于體內(nèi)鐵元素不平衡導(dǎo)致的疾病正日益受到關(guān)注。 高分辨率熔解曲線分析技術(shù)是近幾年來興起的基因突變檢測(cè)技術(shù)。這種檢測(cè)方法不受突變堿基位點(diǎn)與類型的局限,無需序列特異性探針,在PCR結(jié)束后直接運(yùn)行高分辨率熔解檢測(cè),即可完成對(duì)樣品基因型的分析。這種方法因其操作簡(jiǎn)便、快速,使用成本低,結(jié)果準(zhǔn)確,并且實(shí)現(xiàn)了真正的閉管操作而受到普遍的關(guān)注。 本課題研究中建立的針對(duì)TMPRSS6基因的高分辨率熔解曲線分析檢測(cè)方法能夠快速、準(zhǔn)確地將TMPRSS6基因突變序列與野生型序列區(qū)分,并且實(shí)驗(yàn)的重復(fù)性和再現(xiàn)性好、穩(wěn)定性高,方法可靠。本研究建立的基于PCR的高分辨率熔解曲線分析TMPRSS6基因突變?cè)\斷方法是一種高通量、高靈敏度、自動(dòng)化、快速、準(zhǔn)確、經(jīng)濟(jì)的突變檢測(cè)技術(shù)。 近年來,TMPRSS6基因突變導(dǎo)致鐵不應(yīng)性缺鐵性貧血的病例不斷被報(bào)道,但并沒有對(duì)TMPRSS6基因進(jìn)行人群突變篩查的研究。本研究中,利用高分辨率熔解曲線分析技術(shù)對(duì)中國(guó)人缺鐵性貧血病例標(biāo)本進(jìn)行突變篩查,沒有發(fā)現(xiàn)已知突變,這也說明了中國(guó)人TMPRSS6基因突變的攜帶率相對(duì)較低。 目前,國(guó)內(nèi)對(duì)缺鐵性貧血的病因分析主要認(rèn)為是鐵攝入的量過少或鐵丟失的量過多造成的,較少涉及到遺傳學(xué)病因的分析。根據(jù)PubMed等文獻(xiàn)檢索數(shù)據(jù)庫的檢索結(jié)果,未見有關(guān)中國(guó)人群TMPRSS6基因突變導(dǎo)致缺鐵性貧血的報(bào)道。因此對(duì)缺鐵性貧血的遺傳學(xué)病因的研究具有重要的意義和價(jià)值。 本研究為TMPRSS6基因的檢測(cè)建立了一種簡(jiǎn)便、經(jīng)濟(jì)、快速、高效和靈敏的檢測(cè)方法,這為TMPRSS6基因突變導(dǎo)致的一類疾病的基因診斷和產(chǎn)前診斷提供了一種可選擇的工具。 本研究對(duì)進(jìn)一步闡明中國(guó)人鐵不應(yīng)性缺鐵性貧血的分子機(jī)制,對(duì)臨床上該病的診斷、預(yù)防和治療具有重要的參考價(jià)值,同時(shí)也為TMPRSS6基因突變導(dǎo)致的其他鐵代謝異常疾病的分子機(jī)制的分析提供了借鑒。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346
本文編號(hào)：2206872
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 李迎春;王彪;;缺鐵性貧血的診斷和鑒別診斷[J];中國(guó)實(shí)用鄉(xiāng)村醫(yī)生雜志;2008年06期
2 劉樹芳,馬冠生;輔食添加與嬰幼兒生長(zhǎng)發(fā)育[J];國(guó)外醫(yī)學(xué)(衛(wèi)生學(xué)分冊(cè));2005年01期
3 李娜;郭俊生;秦海宏;;Hepcidin與鐵代謝[J];國(guó)外醫(yī)學(xué)(衛(wèi)生學(xué)分冊(cè));2007年03期
4 莫秋華,楊翠蘭;大引物PCR定點(diǎn)誘變技術(shù)的研究進(jìn)展[J];國(guó)外醫(yī)學(xué).遺傳學(xué)分冊(cè);2003年02期
5 張敏紅,王海寶,杜有功;鐵的生物學(xué)功能與代謝調(diào)節(jié)研究現(xiàn)狀[J];醫(yī)藥導(dǎo)報(bào);2005年10期
6 黃橋梁;胡曉抒;袁寶君;;缺鐵性貧血研究進(jìn)展[J];中國(guó)公共衛(wèi)生;2006年11期
7 傅新暉;鄧艷紅;范新娟;駱衍新;陳典克;王磊;蘭平;劉煥亮;汪建平;;利用高分辨率熔解曲線分析法檢測(cè)結(jié)直腸癌中KRAS基因突變[J];中華普通外科學(xué)文獻(xiàn)(電子版);2009年05期
8 莫秋華,徐湘民,鐘雄霖,劉忠英;一種改進(jìn)的大引物PCR定點(diǎn)誘變方法[J];中華醫(yī)學(xué)遺傳學(xué)雜志;2002年01期
9 張宏麗,姜寶華;肝抗菌肽在鐵代謝及遺傳性血色病中的作用[J];中國(guó)老年學(xué)雜志;2005年10期
10 張時(shí)民;李麗娜;;如何篩檢缺鐵性貧血?[J];中國(guó)臨床醫(yī)生;2006年08期
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