以家猪为模型解析先天性外耳发育畸形的遗传机制
发布时间：2019-02-24 20:35
【摘要】：人类小耳畸形是一类发病率较高的先天性外耳缺陷疾病，常常伴有听力障碍，严重影响了患者的容貌和正常的社会交往。此病的成因非常复杂，人们对其遗传基础知之甚少。家猪是研究人类疾病的理想模型，耳型也是家猪的重要品种特征。因此，本研究以家猪为模型开展外耳发育缺陷遗传机制的研究。本研究前期构建了二花脸×沙子岭猪外耳缺陷的F2近交群体，F2中部分仔猪的外耳具有先天性畸形，与人类小耳畸形的临床表型一致。根据近交群体的系谱信息和患病仔猪在F2群体中的分离情况，我们推测该疾患呈常染色体单基因隐性遗传模式。 在此基础上，我们对该近交群体前4胎F2共47个个体进行了全基因组60K SNP芯片扫描。扫描结果的全基因组关联分析在SSC18上检测到了唯一的信号峰，50个达到基因组显著水平的SNP（P=2.73×10-6）位于4.9Mb的区域内。全部11个患病个体共享一段5.0Mb的IBD片段。表型正常F2个体的断点重组分析将影响此疾病的因果突变最终精细定位在2.0Mb的片段内，此区段内含有17个注释基因，其中包括HOXA家族的11个基因。 选择1个F2患病个体及其亲本，针对覆盖精细定位区间的2.6Mb区域，开展目标区域捕获重测序，最终筛选到了15个候选因果突变。通过来源广泛且耳朵表型正常的643个个体及二花脸×沙子岭F2近交系的全部103个个体的判型分析，揭示出位于HOXA1基因编码区的c.451GTC是导致家猪先天性外耳发育畸形的因果突变，，该突变仅存在于沙子岭群体中。HOXA1突变导致翻译提前终止，该蛋白缺少了homeobox结构域，改变了HOXA1蛋白的生物学功能。为了进一步在全基因组范围内明确因果突变在表达水平上对其他基因造成的影响，将14.25日龄的两个患病个体和两个正常个体胚胎组织的RNA分别组池，开展RNA-Seq试验。我们共筛选到了337个差异表达基因（DEGs），其中许多与外耳发育有关。随机选择10个DEGs，利用Realtime PCR实验验证了RNA-Seq试验的可靠性。337个DEGs的GO、Pathway及IPA的基因网络分析表明DEGs富集在药物代谢、脂代谢、细胞组装、癌症、有机体畸形及神经系统的发育和功能等方面。 在此基础上，利用ToppGene、Endeavour和SUSPECTS生物信息学软件对337个候选基因进行优先排序。选择HOXA1，HOXC4，PDX1，NKX2-8，EVC2，FGF1，FGFR3和CTCF基因在146份散发的人小耳畸形患者中进行外显子测序，搜寻到4个可能的致病突变位点，即EVC2基因上的错义突变p.1094DN，HOXA1基因的缺失突变p.65-67HHHdel和其3’UTR区内的g.2613GA和g.2944delinsGT突变。这4个候选因果突变的破坏程度经预测均显示为可能致病。本项研究成果为最终解析人类小耳畸形的致病机理提供了重要借鉴。
[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.
【学位授予单位】：江西农业大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R764.71;R-332
本文编号：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.
【学位授予单位】：江西农业大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R764.71;R-332
【参考文献】
相关期刊论文 前1条
1 王海龙;杨时昕;杨玉;郭晋宣;赵威;;胶原蛋白海绵复合BMP-2修复兔耳软骨缺损的实验研究[J];中国医疗前沿;2012年07期
本文编号：2429907
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