常染色体显性遗传传导性耳聋家系基因鉴定及功能分析

发布时间：2018-04-28 11:09

本文选题：遗传性听力损失 + 非综合征型遗传性听力损失　；参考：《中国人民解放军医学院》2014年博士论文

【摘要】：耳聋是威胁人类健康的常见疾病之一，约60%与遗传因素有关。非综合征型常染色体显性遗传性耳聋（DFNA）一直是聋病遗传学研究的热点。但是迄今为止，我们仅仅定位了55个DFNA基因位点，成功克隆出30个DFNA相关基因，且多数贡献于感音神经性耳聋或混合性耳聋。对于先天性外中耳畸形等原因导致的传导性耳聋，由于环境因素的影响和单纯性传导性耳聋大家系资源的缺失，致病基因的分子机制研究进展缓慢。近年来，HiSeq2000为代表的高通量测序技术的诞生和高级生物信息学的快速进步，为耳聋基因鉴定工作井喷式的增长拉开了序幕。本研究正是基于解放军总医院耳鼻咽喉研究所聋病基因库所收集的宝贵遗传资源，秉承并借鉴既往相关研究的成果与经验，采用外显子测序、全基因组重测序、微阵列比较基因组杂交和质谱分析等先进的遗传学研究策略，对中国常染色体显性遗传性传导性耳聋大家系的分子遗传机制进行研究，力求发现中国人群新的耳聋致病基因，并行散发验证及功能分析，从而揭开遗传性传导性耳聋分子发病机理的面纱，为聋病的临床干预和预防提供新的理论依据。第一部分新一代测序技术鉴定常染色体显性遗传传导性耳聋家系致病基因本研究第一部分收集完善传导性耳聋大家系（028家系）临床资料和遗传资源，运用新一代测序技术和高级生物信息学分析手段，探索传导性耳聋相关致病基因。 028家系临床表型为较为单一的先天性传导性听力损失，伴外耳道狭窄、中耳畸形及双侧上睑下垂，不伴有全身骨骼或内脏的畸形和其他组织器官的异常。颞骨三维重建及手术探查证实患者传导性耳聋的原因是先天性中耳畸形--镫骨固定与锤骨头骨性增生伴外固定。课题组前期通过连锁分析和位置候选基因方法没有找到致病基因。选取家系中6名个体（3名患者，3名正常对照）行外显子测序，所得数据与人类基因组数据库比对，筛选病例组特有的罕见变异，预测候选单核苷酸多态(single nucleotide polymorphism,SNP)和插入缺失突变（Indel）并在家系内验证，但未能发现与表型共分离的致病基因。考虑到外显子测序的局限性，课题组进一步选取家系中3名个体（2名患者，1名正常对照）进行更为全面的全基因组重测序，以期发现非编码区突变或拷贝数变化（CNV）等基因组结构的变异（SV）。分别运用质谱分析和基于sanger测序的长片段聚合酶链式反应（long PCR）方法对全基因组重测序所得大量候选SNP、Indel和SV进行验证。由于海量数据验证耗时较长，课题组同时采用基于高通量芯片平台的微阵列比较基因组杂交技术（array-CGH）对1名患者基因组DNA进行分析，幸运的在染色体2p21区域内发现存在大片段的杂合缺失。而该区域恰好也位于前期连锁分析结果LOD值大于1的区间，一定程度上得到了连锁分析定位数据的支持。随后我们重点分析全基因组重测序该区域数据，并设计缺失断点PCR在家系内验证，成功找到了与028家系表型共分离的基因型变异--位于chr2:45199520-45259001区域内长达59482bp片段的杂合缺失，其产生的机制可能与Alu序列插入致染色体重组相关。该缺失片段内仅包含1个已知基因--C基因，既往研究认为C基因可能作为转录因子参与到耳和肾的发育，但并没有报道直接与耳聋相关。因此考虑C基因可能是贡献于该传导性耳聋家系的致病基因。第二部分C基因在散发传导性耳聋患者中的筛查及发育生物学功能分析本研究第二部分收集与028家系表型近似的中耳或外耳畸形患者进行散发验证，同时探讨C基因家族进化和发育生物学功能，，以期进一步在功能学和遗传学角度证实C基因是传导性耳聋相关致病基因。选取门诊收集的203例散发传导性耳聋患者，运用缺失断点PCR的方法验证chr2:45199520-45259001区域内是否存在大片段杂合缺失。同时使用sanger测序对203例散发传导性耳聋患者和50例正常人进行C基因2个外显子的筛查，验证是否存在可能致病的SNP。遗憾的是，我们并没有在散发患者中发现与028家系类似的大片段的杂合缺失，针对C基因外显子的筛查也仅在10名患者和1名正常人中发现均携带有1个同义突变，该位点是ncbi公布的已知SNP位点。 C基因是哺乳动物特有的Homeobox转录因子，全长291aa，包含两个外显子，可作为Hoxa2的直接下游基因，共同参与第二鳃弓的发育。Hoxa2缺失可以导致C基因表达上升，从而在第二鳃弓位置形成一套复制的砧骨和锤骨，与第一腮弓正常来源的听骨呈镜像对称分布。C基因纯合缺失的小鼠不能存活，肾脏发育没有明显异常，第一和第二鳃弓来源的部分骨骼发育表现出温和缺陷。C基因的同源基因Six1，参与形成耳和肾发育过程中重要的基因调控网络Eya-Six-Pax，Six1突变可削弱Eya1-Six1的相互作用，引起鳃耳肾综合征（BOR）。本研究运用新一代测序技术，结合array-CGH和高级生物信息学分析手段，攻克了困扰课题组长达六年之久的传导性耳聋大家系（028家系）发病机制的难关，成功找到了与该家系表型相关的基于染色体结构的变异，并有幸发现了可能贡献于传导性耳聋的新基因-C基因。尽管对C基因的散发人群验证并未找到足够的遗传学证据，但是系统的发育生物学功能分析支持C基因与中耳发育及结构密切相关。本研究为耳聋新基因的发现提供了一整套行之有效的研究方法体系，同时为下一步行动物模型的功能验证提供了足够的理论依据。课题组今后的工作：继续收集与028家系表型一致的散发病例；完善Taqman探针方法，对散发样本行定量PCR进一步验证CNV；构建C基因动物模型进行功能学的研究。
[Abstract]:Deafness is one of the common diseases that threaten human health, about 60% are related to genetic factors. Non syndrome autosomal dominant hereditary deafness (DFNA) has been a hot spot in the genetic research of deafness. But so far, we have only located 55 DFNA gene loci and successfully cloned 30 DFNA related genes, and most of them contribute to the sensorineural God. Sexual deafness or mixed deafness. The molecular mechanism of the pathogenic gene is progressed slowly because of the causes of congenital deafness, such as external and middle ear deafness, due to the influence of environmental factors and the lack of human resources in the simple conduction deafness.
In recent years, HiSeq2000 has been the prelude to the birth of high throughput sequencing technology and the rapid progress of advanced bioinformatics. This study is based on the valuable genetic resources collected by the deafness gene bank of the otorhinolaryngology Institute of the General Hospital of the PLA, adhering to and drawing on previous related research. The results and experience, using exons sequencing, complete genome resequencing, microarray comparison of genomic hybridization and mass spectrometry, and other advanced genetic research strategies, study the molecular genetic mechanism of Chinese autosomal dominant hereditary conductive deafness, and try to find new deafness genes in Chinese population. It provides a new theoretical basis for clinical intervention and prevention of deafness.
Part one: new generation sequencing technology for identification of pathogenic genes in families with autosomal dominant hereditary hearing loss
The first part of this study collects the clinical data and genetic resources for the improvement of conductive deafness (028 families), and uses a new generation of sequencing and advanced bioinformatics to explore the related genes associated with deafness.
The clinical phenotype of the 028 families is a single congenital conduction hearing loss with external auditory canal stenosis, middle ear malformation and bilateral ptosis, without abnormality of the whole body bone or viscera and other tissues and organs. Three dimensional reconstruction of the temporal bone and surgical exploration proved that the cause of the patient's conduction deafness is congenital middle ear deafness - stapes fixation. The study group failed to identify the pathogenic genes by linkage analysis and location candidate gene methods.
The exons were sequenced in 6 individuals (3 patients and 3 normal controls) in the family. The data were compared with the human genome database, and the rare mutations in the case group were screened. The candidate single nucleotide polymorphisms (single nucleotide polymorphism, SNP) and the insertion deletion mutation (Indel) were predicted and verified in the family, but they were not found to be associated with the phenotype. Taking into account the limitations of exon sequencing, the group further selected 3 individuals (2 patients and 1 normal controls) in the family to complete complete genome resequencing, in order to find the genomic structure variation (SV), such as non coding region mutation or copy number change (CNV), and the use of mass spectrometry analysis and Sanger Long sequencing polymerase chain reaction (long PCR) was used to verify the large number of candidate SNP, Indel and SV obtained from genome wide sequencing.
Due to the long time-consuming verification of massive data, the group uses microarray comparative genomic hybridization (array-CGH) based on high throughput chip platform (array-CGH) to analyze the genomic DNA of 1 patients. Fortunately, there is a large fragment of heterozygous deletion in the chromosome 2p21 region. The region is also located at the result of the early linkage analysis LOD. The interval of value greater than 1 has been supported to some extent by the linkage analysis location data. Then we focus on the analysis of the whole genome resequencing data and design the missing breakpoint PCR in the family system and successfully found the genotypic variation that is co separated from the 028 lineages - in the region of the chr2:45199520-45259001 region as long as 59482bp slices. The loss of heterozygosity in the segment may be associated with the insertion of Alu sequences into chromosome recombination. The deletion fragment contains only 1 known gene --C genes. Previous studies suggest that the C gene may be involved in the development of the ear and kidney as a transcription factor, but it is not reported directly related to the deafness. Therefore, the C gene may contribute to the transmission of the gene. The pathogenic gene of the pedigree of the deafness.
The second part is the screening and developmental biological function analysis of C gene in patients with sporadic conduction deafness.
In the second part, the second part of the study collects the distribution of the middle ear or external ear malformation in 028 families, and discusses the phylogenetic and developmental biological functions of the C gene, in order to further confirm that the C gene is a leading deafness related gene in the functional and genetic perspective.
203 cases of sporadic conductive deafness were collected from the outpatient department, and the deletion of PCR was used to verify the absence of large fragment heterozygosity in the chr2:45199520-45259001 region. At the same time, 203 cases of sporadic deafness and 50 normal persons were screened by Sanger sequencing to verify the possible pathogenicity of the 2 exons of the C gene. SNP. regrets that we did not find heterozygous deletion similar to 028 lines in the sporadic patients. Screening for C exons was also found in only 10 patients and 1 normal people with 1 synonymous mutations, which were known as the known SNP sites published by NCBI.
The C gene is a mammal specific Homeobox transcription factor, full length 291aa, including two exons, which can be used as the direct downstream gene of Hoxa2, participating in the development of the.Hoxa2 deletion of the second branchial arch, which can lead to the increase of the expression of the C gene, thus forming a set of replicating anvil and malleus at the position of the second branchial arch, and the auditory bone of the normal source of the first bows. The mice of the homozygous deletion of the mirror symmetrical distribution of.C gene can not survive and have no obvious abnormalities in the kidney development. The development of the part of the first and second branchial arches shows a homologous gene Six1 of the mild defect.C gene, which is involved in the formation of an important gene regulation network Eya-Six-Pax in the development of the ear and kidney, and the Six1 mutation can weaken Eya1-Six1. The interaction caused the branchial and renal syndrome (BOR).
In this study, a new generation of sequencing technology, combined with array-CGH and advanced bioinformatics analysis, has been used to overcome the difficulties in the pathogenesis of the six year long conductive deafness (028 families). The new gene -C gene for conductive deafness. Although there is no sufficient genetic evidence for the distribution of the C gene, the systematic developmental biological function analysis supports the close correlation between the C gene and the development and structure of the middle ear. This study provides a whole set of effective research methods for the discovery of the new genes for the deafness. The functional validation of the next walking animal model provides sufficient theoretical basis.
The future work of the group: continue to collect sporadic cases that are consistent with the phenotype of 028 lines, improve the Taqman probe method, and further verify the CNV by quantitative PCR for sporadic samples, and construct the functional study of the C gene animal model.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R764.43

【参考文献】