以家豬為模型解析先天性外耳發(fā)育畸形的遺傳機(jī)制
發(fā)布時間：2019-02-24 20:35
【摘要】：人類小耳畸形是一類發(fā)病率較高的先天性外耳缺陷疾病，常常伴有聽力障礙，嚴(yán)重影響了患者的容貌和正常的社會交往。此病的成因非常復(fù)雜，人們對其遺傳基礎(chǔ)知之甚少。家豬是研究人類疾病的理想模型，耳型也是家豬的重要品種特征。因此，本研究以家豬為模型開展外耳發(fā)育缺陷遺傳機(jī)制的研究。本研究前期構(gòu)建了二花臉×沙子嶺豬外耳缺陷的F2近交群體，F(xiàn)2中部分仔豬的外耳具有先天性畸形，與人類小耳畸形的臨床表型一致。根據(jù)近交群體的系譜信息和患病仔豬在F2群體中的分離情況，我們推測該疾患呈常染色體單基因隱性遺傳模式。 在此基礎(chǔ)上，我們對該近交群體前4胎F2共47個個體進(jìn)行了全基因組60K SNP芯片掃描。掃描結(jié)果的全基因組關(guān)聯(lián)分析在SSC18上檢測到了唯一的信號峰，50個達(dá)到基因組顯著水平的SNP（P=2.73×10-6）位于4.9Mb的區(qū)域內(nèi)。全部11個患病個體共享一段5.0Mb的IBD片段。表型正常F2個體的斷點重組分析將影響此疾病的因果突變最終精細(xì)定位在2.0Mb的片段內(nèi)，此區(qū)段內(nèi)含有17個注釋基因，其中包括HOXA家族的11個基因。 選擇1個F2患病個體及其親本，針對覆蓋精細(xì)定位區(qū)間的2.6Mb區(qū)域，開展目標(biāo)區(qū)域捕獲重測序，最終篩選到了15個候選因果突變。通過來源廣泛且耳朵表型正常的643個個體及二花臉×沙子嶺F2近交系的全部103個個體的判型分析，揭示出位于HOXA1基因編碼區(qū)的c.451GTC是導(dǎo)致家豬先天性外耳發(fā)育畸形的因果突變，，該突變僅存在于沙子嶺群體中。HOXA1突變導(dǎo)致翻譯提前終止，該蛋白缺少了homeobox結(jié)構(gòu)域，改變了HOXA1蛋白的生物學(xué)功能。為了進(jìn)一步在全基因組范圍內(nèi)明確因果突變在表達(dá)水平上對其他基因造成的影響，將14.25日齡的兩個患病個體和兩個正常個體胚胎組織的RNA分別組池，開展RNA-Seq試驗。我們共篩選到了337個差異表達(dá)基因（DEGs），其中許多與外耳發(fā)育有關(guān)。隨機(jī)選擇10個DEGs，利用Realtime PCR實驗驗證了RNA-Seq試驗的可靠性。337個DEGs的GO、Pathway及IPA的基因網(wǎng)絡(luò)分析表明DEGs富集在藥物代謝、脂代謝、細(xì)胞組裝、癌癥、有機(jī)體畸形及神經(jīng)系統(tǒng)的發(fā)育和功能等方面。 在此基礎(chǔ)上，利用ToppGene、Endeavour和SUSPECTS生物信息學(xué)軟件對337個候選基因進(jìn)行優(yōu)先排序。選擇HOXA1，HOXC4，PDX1，NKX2-8，EVC2，F(xiàn)GF1，F(xiàn)GFR3和CTCF基因在146份散發(fā)的人小耳畸形患者中進(jìn)行外顯子測序，搜尋到4個可能的致病突變位點，即EVC2基因上的錯義突變p.1094DN，HOXA1基因的缺失突變p.65-67HHHdel和其3’UTR區(qū)內(nèi)的g.2613GA和g.2944delinsGT突變。這4個候選因果突變的破壞程度經(jīng)預(yù)測均顯示為可能致病。本項研究成果為最終解析人類小耳畸形的致病機(jī)理提供了重要借鑒。
[Abstract]:Human microauricular malformation is a kind of congenital external ear defects with high incidence, which is often accompanied by hearing impairment, which seriously affects the appearance of patients and normal social communication. The causes of the disease are so complex that little is known about its genetic basis. Domestic pig is an ideal model for studying human disease, and ear type is also an important variety characteristic of domestic pig. Therefore, the genetic mechanism of external ear defects in domestic pigs was studied. In this study, a F2 inbred population with external ear defects in Erhualian 脳 Shailing pigs was constructed. Some of the piglets in F2 had congenital deformity of the external ear, which was consistent with the clinical phenotype of human microauricular malformation. According to the pedigree information of inbred population and the segregation of sick piglets in F2 population, we speculate that the disease is an autosomal single gene recessive genetic pattern. On the basis of this, we scanned the whole genome of 47 F _ 2 individuals in the first 4 embryos of the inbred population by 60 K SNP microarray. A unique signal peak was detected on SSC18 by the whole genome association analysis of scanning results, and 50 SNP (Pu 2.73 脳 10-6), which reached the level of genomic significance, were located in the region of 4.9Mb. All 11 individuals shared a 5.0Mb IBD fragment. The breakpoint recombination analysis of the phenotypic normal F2 individuals resulted in the precise localization of causal mutations affecting the disease into the 2.0Mb fragment, which contained 17 annotated genes, including 11 genes in the HOXA family. A F2 patient and its parents were selected to carry out the target region capture and resequencing for the 2.6Mb region covering the fine location interval. Finally, 15 candidate causality mutations were screened. Based on the analysis of 643 individuals from a wide range of sources with normal ear phenotype and all 103 individuals of Erhualian 脳 Shaoling F2 inbred lines, It is revealed that c.451GTC located in the coding region of the HOXA1 gene is a causal mutation leading to congenital external ear deformity in domestic pigs, which only exists in the Shaheling population. The HOXA1 mutation leads to the early termination of translation, and the protein lacks the homeobox domain. It changed the biological function of HOXA1 protein. In order to further clarify the effect of causality mutation on other genes in the whole genome, RNA-Seq tests were carried out in the RNA group of two diseased individuals and two normal embryo tissues at 14.25 days of age. We screened 337 differentially expressed genes (DEGs), many of which were related to ear development. Ten DEGs, were selected randomly to verify the reliability of RNA-Seq test by Realtime PCR. The gene network analysis of GO,Pathway and IPA of 337 DEGs showed that DEGs was enriched in drug metabolism, lipid metabolism, cell assembly, cancer, and so on. The deformity of organism and the development and function of nervous system. On this basis, 337 candidate genes were prioritized by ToppGene,Endeavour and SUSPECTS bioinformatics software. HOXA1,HOXC4,PDX1,NKX2-8,EVC2,FGF1,FGFR3 and CTCF genes were selected for exon sequencing in 146 sporadic patients with microauricular malformation. Four possible pathogenetic mutation sites were found, namely the missense mutation in the EVC2 gene, p. 1094 DN. HOXA1 gene deletion mutation p.65-67HHHdel and its 3'UTR region g.2613GA and g.2944delinsGT mutation. The damage degree of the four candidate causality mutations was predicted to be possible pathogenicity. The results of this study provide an important reference for the final analysis of the pathogenic mechanism of human microauricular malformation.
【學(xué)位授予單位】：江西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R764.71;R-332
本文編號：2429907
[Abstract]:Human microauricular malformation is a kind of congenital external ear defects with high incidence, which is often accompanied by hearing impairment, which seriously affects the appearance of patients and normal social communication. The causes of the disease are so complex that little is known about its genetic basis. Domestic pig is an ideal model for studying human disease, and ear type is also an important variety characteristic of domestic pig. Therefore, the genetic mechanism of external ear defects in domestic pigs was studied. In this study, a F2 inbred population with external ear defects in Erhualian 脳 Shailing pigs was constructed. Some of the piglets in F2 had congenital deformity of the external ear, which was consistent with the clinical phenotype of human microauricular malformation. According to the pedigree information of inbred population and the segregation of sick piglets in F2 population, we speculate that the disease is an autosomal single gene recessive genetic pattern. On the basis of this, we scanned the whole genome of 47 F _ 2 individuals in the first 4 embryos of the inbred population by 60 K SNP microarray. A unique signal peak was detected on SSC18 by the whole genome association analysis of scanning results, and 50 SNP (Pu 2.73 脳 10-6), which reached the level of genomic significance, were located in the region of 4.9Mb. All 11 individuals shared a 5.0Mb IBD fragment. The breakpoint recombination analysis of the phenotypic normal F2 individuals resulted in the precise localization of causal mutations affecting the disease into the 2.0Mb fragment, which contained 17 annotated genes, including 11 genes in the HOXA family. A F2 patient and its parents were selected to carry out the target region capture and resequencing for the 2.6Mb region covering the fine location interval. Finally, 15 candidate causality mutations were screened. Based on the analysis of 643 individuals from a wide range of sources with normal ear phenotype and all 103 individuals of Erhualian 脳 Shaoling F2 inbred lines, It is revealed that c.451GTC located in the coding region of the HOXA1 gene is a causal mutation leading to congenital external ear deformity in domestic pigs, which only exists in the Shaheling population. The HOXA1 mutation leads to the early termination of translation, and the protein lacks the homeobox domain. It changed the biological function of HOXA1 protein. In order to further clarify the effect of causality mutation on other genes in the whole genome, RNA-Seq tests were carried out in the RNA group of two diseased individuals and two normal embryo tissues at 14.25 days of age. We screened 337 differentially expressed genes (DEGs), many of which were related to ear development. Ten DEGs, were selected randomly to verify the reliability of RNA-Seq test by Realtime PCR. The gene network analysis of GO,Pathway and IPA of 337 DEGs showed that DEGs was enriched in drug metabolism, lipid metabolism, cell assembly, cancer, and so on. The deformity of organism and the development and function of nervous system. On this basis, 337 candidate genes were prioritized by ToppGene,Endeavour and SUSPECTS bioinformatics software. HOXA1,HOXC4,PDX1,NKX2-8,EVC2,FGF1,FGFR3 and CTCF genes were selected for exon sequencing in 146 sporadic patients with microauricular malformation. Four possible pathogenetic mutation sites were found, namely the missense mutation in the EVC2 gene, p. 1094 DN. HOXA1 gene deletion mutation p.65-67HHHdel and its 3'UTR region g.2613GA and g.2944delinsGT mutation. The damage degree of the four candidate causality mutations was predicted to be possible pathogenicity. The results of this study provide an important reference for the final analysis of the pathogenic mechanism of human microauricular malformation.
【學(xué)位授予單位】：江西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R764.71;R-332
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 王海龍;楊時昕;楊玉;郭晉宣;趙威;;膠原蛋白海綿復(fù)合BMP-2修復(fù)兔耳軟骨缺損的實驗研究[J];中國醫(yī)療前沿;2012年07期
本文編號：2429907
本文鏈接：http://sikaile.net/yixuelunwen/yank/2429907.html
最近更新
教材專著
