一先天性并指（趾）多指（趾）家系的临床表型和HOXD13基因突变研究

发布时间：2018-05-31 15:48

本文选题：SPD + HOXD13　；参考：《吉林大学》2012年硕士论文

【摘要】：背景及目的：先天性并指(趾)多(趾)畸形（SPD）是常见的常染色体显性遗传疾病。典型的SPD表现包括3、4手指和4、5足趾并指（趾），伴有完全或不完全的并指(趾)内的指(趾)的重复，该畸形常表现为外显率不全和表现度的多样化。HOXD13基因是公认的可导致先天性并多指畸形的重要的候选基因。在人类中，HOX基因被认为编码一个高度保守的转录因子家族，对胚胎发育起重要的作用。HOX基因簇分布在7、17、12、2号染色体的特定区域上，位于第2号染色体HOXD基因簇5’末端的是HOXD13基因，它包含两个外显子，第1外显子区域易导致多聚丙氨酸链延展突变，第2外显子区域易导致碱基缺失及错义突变、无义突变。EPHA7是比较明确的受HOXD13基因调节的下游基因之一,在胚胎组织广泛表达，调节神经系统的发育、组织分化等多个方面, HOXD13基因通过结合启动子从而调节EPHA7下游基因的转录表达。本论文通过对中国一先天性并指(趾)多(趾)畸形（SPD）家系的临床表型探索及分子遗传学研究，进一步明确先天性并指畸形的分型，分析可能导致其异常畸形的发病原因，探讨致病基因在胚胎发育过程中的作用机制。方法：对中国吉林省一例四代并多指(趾)畸形家系进行调查，总结其家系患者的临床表型，绘制家系图谱，分析遗传学特征，同时收集家系成员外周静脉血，，提取静脉血DNA，设计HOXD13外显子引物，PCR扩增目的基因，将扩增后产物直接测序，分析有无突变。同时针对EPHA7下游基因采用双荧光素酶报告基因检测对新发现的基因突变做进一步的功能实验研究。结果：通过分析家系图谱，该患者符合常染色体显性遗传。总结该家系患者临床表现，符合先天性并指多指畸形特点。通过分子遗传学研究，在本家系所有患者中均发现HOXD13基因第2外显子区域发现一个新的错义突变：c.893GA (p.R298Q),即编码区第893位碱基由鸟嘌呤（G）突变为腺嘌呤（A），使得HOXD13基因第298位氨基酸（同源盒结构域区域第31位）由CGG突变为CAG（R31Q），导致精氨酸被谷氨酰胺替代。同时在家系一例临床表现正常的患者中发现同样的基因突变，显示其为隐性携带者，提示该家系的外显率不全。我们同时对突变体进行双荧光酶报告基因功能实验，结果显示对比野生型HOXD13基因，突变型HOXD13（R31Q）对下游EPHA7基因启动子的转录活性明显降低。结论： 1.本家系显示出外显率不全和并指畸形表现度的多样化，丰富了SPD的临床表型。 2.本家系研究新发现的HOXD13基因第2外显子的错义突变，导致氨基酸的改变，从而影响HOXD13与DNA的结合，引起HOXD13对下游基因的转录结合能力减弱，推测该突变可能是导致并指多指畸形的分子机制之一。
[Abstract]:Background and purpose: Congenital syndactyly polydeformity (SPD) is a common autosomal dominant genetic disease. Typical SPD findings include 3 fingers with 4 fingers and 4 with 5 toes and fingers with complete or incomplete duplication of the fingers (toes) within the fingers (toes). HOXD13 gene is recognized as an important candidate gene for congenital polydactyly deformity. In humans, the HOX gene is thought to encode a highly conserved family of transcription factors that play an important role in embryonic development. HOXD13 gene is located at the 5'end of HOXD gene cluster on chromosome 2. It contains two exons. The region of exon 1 easily leads to polyalanine chain extension mutation, and exon 2 region easily leads to base deletion and missense mutation. Nonsense mutation. EPHA7 is one of the downstream genes regulated by HOXD13 gene. It is widely expressed in embryonic tissues and regulates the development of nervous system. In tissue differentiation and other aspects, HOXD13 gene regulates the transcription and expression of downstream EPHA7 gene by binding to promoter. In this paper, the clinical phenotype and molecular genetics of a Chinese family with congenital syndactyly polydactyly (SPD) were investigated, and the types of congenital syndactyly were further determined, and the possible causes of the abnormal deformity were analyzed. To explore the mechanism of pathogenicity gene in embryonic development. Methods: A family of four generations with polydigital deformity was investigated in Jilin Province, China. The clinical phenotypes of the patients were summarized, family maps were drawn, genetic characteristics were analyzed, and peripheral venous blood was collected at the same time. HOXD13 exon primer was designed to amplify the target gene. The amplified product was sequenced directly and the mutation was analyzed. At the same time, double luciferase reporter gene detection for downstream EPHA7 gene was used to further study the new gene mutation. Results: The pedigree map showed that the patient was autosomal dominant inheritance. The clinical manifestations of this family are conformed to congenital syndactyly deformity. Through molecular genetic research, A new missense mutation in exon 2 of HOXD13 gene was found in all patients of this family. A new missense mutation was found in the region of exon 2 of the HOXD13 gene. A new missense mutation was found, namely, the 893 base of the coding region mutated from guanine G) to adenine An, resulting in the 298th amino acid (homology box) of the HOXD13 gene. The region of domain 31) mutated from CGG to CAGN R31QN, resulting in arginine being replaced by glutamine. At the same time, the same gene mutation was found in a family of patients with normal clinical manifestations, indicating that it was a recessive carrier, indicating that the outlier rate of the pedigree was not complete. At the same time, we performed a double fluorescence reporter gene function experiment on the mutant. The results showed that the transcriptional activity of the mutant HOXD13 / R31Q to the downstream EPHA7 gene promoter was significantly lower than that of the wild type HOXD13 gene. Conclusion: 1. The clinical phenotype of SPD was enriched by the diversity of phenotype of syndactyly. 2. The missense mutation in exon 2 of HOXD13 gene was found in our family, which resulted in the change of amino acid, which affected the binding of HOXD13 to DNA and weakened the transcriptional binding ability of HOXD13 to downstream gene. It is speculated that this mutation may be one of the molecular mechanisms leading to polydactyly deformity.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R726.8

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