本文選題：遺傳性聽力損失 + 非綜合征型遺傳性聽力損失�。� 參考：《中國人民解放軍醫(yī)學(xué)院》2014年博士論文

【摘要】：耳聾是威脅人類健康的常見疾病之一，約60%與遺傳因素有關(guān)。非綜合征型常染色體顯性遺傳性耳聾（DFNA）一直是聾病遺傳學(xué)研究的熱點。但是迄今為止，我們僅僅定位了55個DFNA基因位點，成功克隆出30個DFNA相關(guān)基因，且多數(shù)貢獻于感音神經(jīng)性耳聾或混合性耳聾。對于先天性外中耳畸形等原因?qū)е碌膫鲗?dǎo)性耳聾，由于環(huán)境因素的影響和單純性傳導(dǎo)性耳聾大家系資源的缺失，致病基因的分子機制研究進展緩慢。 近年來，HiSeq2000為代表的高通量測序技術(shù)的誕生和高級生物信息學(xué)的快速進步，為耳聾基因鑒定工作井噴式的增長拉開了序幕。本研究正是基于解放軍總醫(yī)院耳鼻咽喉研究所聾病基因庫所收集的寶貴遺傳資源，秉承并借鑒既往相關(guān)研究的成果與經(jīng)驗，采用外顯子測序、全基因組重測序、微陣列比較基因組雜交和質(zhì)譜分析等先進的遺傳學(xué)研究策略，對中國常染色體顯性遺傳性傳導(dǎo)性耳聾大家系的分子遺傳機制進行研究，力求發(fā)現(xiàn)中國人群新的耳聾致病基因，并行散發(fā)驗證及功能分析，從而揭開遺傳性傳導(dǎo)性耳聾分子發(fā)病機理的面紗，為聾病的臨床干預(yù)和預(yù)防提供新的理論依據(jù)。 第一部分新一代測序技術(shù)鑒定常染色體顯性遺傳傳導(dǎo)性耳聾家系致病基因 本研究第一部分收集完善傳導(dǎo)性耳聾大家系（028家系）臨床資料和遺傳資源，運用新一代測序技術(shù)和高級生物信息學(xué)分析手段，探索傳導(dǎo)性耳聾相關(guān)致病基因。 028家系臨床表型為較為單一的先天性傳導(dǎo)性聽力損失，伴外耳道狹窄、中耳畸形及雙側(cè)上瞼下垂，不伴有全身骨骼或內(nèi)臟的畸形和其他組織器官的異常。顳骨三維重建及手術(shù)探查證實患者傳導(dǎo)性耳聾的原因是先天性中耳畸形--鐙骨固定與錘骨頭骨性增生伴外固定。課題組前期通過連鎖分析和位置候選基因方法沒有找到致病基因。 選取家系中6名個體（3名患者，3名正常對照）行外顯子測序，所得數(shù)據(jù)與人類基因組數(shù)據(jù)庫比對，篩選病例組特有的罕見變異，預(yù)測候選單核苷酸多態(tài)(single nucleotide polymorphism,SNP)和插入缺失突變（Indel）并在家系內(nèi)驗證，但未能發(fā)現(xiàn)與表型共分離的致病基因�？紤]到外顯子測序的局限性，課題組進一步選取家系中3名個體（2名患者，1名正常對照）進行更為全面的全基因組重測序，以期發(fā)現(xiàn)非編碼區(qū)突變或拷貝數(shù)變化（CNV）等基因組結(jié)構(gòu)的變異（SV）。分別運用質(zhì)譜分析和基于sanger測序的長片段聚合酶鏈?zhǔn)椒磻?yīng)（long PCR）方法對全基因組重測序所得大量候選SNP、Indel和SV進行驗證。 由于海量數(shù)據(jù)驗證耗時較長，課題組同時采用基于高通量芯片平臺的微陣列比較基因組雜交技術(shù)（array-CGH）對1名患者基因組DNA進行分析，幸運的在染色體2p21區(qū)域內(nèi)發(fā)現(xiàn)存在大片段的雜合缺失。而該區(qū)域恰好也位于前期連鎖分析結(jié)果LOD值大于1的區(qū)間，一定程度上得到了連鎖分析定位數(shù)據(jù)的支持。隨后我們重點分析全基因組重測序該區(qū)域數(shù)據(jù)，并設(shè)計缺失斷點PCR在家系內(nèi)驗證，成功找到了與028家系表型共分離的基因型變異--位于chr2:45199520-45259001區(qū)域內(nèi)長達59482bp片段的雜合缺失，其產(chǎn)生的機制可能與Alu序列插入致染色體重組相關(guān)。該缺失片段內(nèi)僅包含1個已知基因--C基因，既往研究認為C基因可能作為轉(zhuǎn)錄因子參與到耳和腎的發(fā)育，但并沒有報道直接與耳聾相關(guān)。因此考慮C基因可能是貢獻于該傳導(dǎo)性耳聾家系的致病基因。 第二部分C基因在散發(fā)傳導(dǎo)性耳聾患者中的篩查及發(fā)育生物學(xué)功能分析 本研究第二部分收集與028家系表型近似的中耳或外耳畸形患者進行散發(fā)驗證，同時探討C基因家族進化和發(fā)育生物學(xué)功能，，以期進一步在功能學(xué)和遺傳學(xué)角度證實C基因是傳導(dǎo)性耳聾相關(guān)致病基因。 選取門診收集的203例散發(fā)傳導(dǎo)性耳聾患者，運用缺失斷點PCR的方法驗證chr2:45199520-45259001區(qū)域內(nèi)是否存在大片段雜合缺失。同時使用sanger測序?qū)?03例散發(fā)傳導(dǎo)性耳聾患者和50例正常人進行C基因2個外顯子的篩查，驗證是否存在可能致病的SNP。遺憾的是，我們并沒有在散發(fā)患者中發(fā)現(xiàn)與028家系類似的大片段的雜合缺失，針對C基因外顯子的篩查也僅在10名患者和1名正常人中發(fā)現(xiàn)均攜帶有1個同義突變，該位點是ncbi公布的已知SNP位點。 C基因是哺乳動物特有的Homeobox轉(zhuǎn)錄因子，全長291aa，包含兩個外顯子，可作為Hoxa2的直接下游基因，共同參與第二鰓弓的發(fā)育。Hoxa2缺失可以導(dǎo)致C基因表達上升，從而在第二鰓弓位置形成一套復(fù)制的砧骨和錘骨，與第一腮弓正常來源的聽骨呈鏡像對稱分布。C基因純合缺失的小鼠不能存活，腎臟發(fā)育沒有明顯異常，第一和第二鰓弓來源的部分骨骼發(fā)育表現(xiàn)出溫和缺陷。C基因的同源基因Six1，參與形成耳和腎發(fā)育過程中重要的基因調(diào)控網(wǎng)絡(luò)Eya-Six-Pax，Six1突變可削弱Eya1-Six1的相互作用，引起鰓耳腎綜合征（BOR）。 本研究運用新一代測序技術(shù)，結(jié)合array-CGH和高級生物信息學(xué)分析手段，攻克了困擾課題組長達六年之久的傳導(dǎo)性耳聾大家系（028家系）發(fā)病機制的難關(guān)，成功找到了與該家系表型相關(guān)的基于染色體結(jié)構(gòu)的變異，并有幸發(fā)現(xiàn)了可能貢獻于傳導(dǎo)性耳聾的新基因-C基因。盡管對C基因的散發(fā)人群驗證并未找到足夠的遺傳學(xué)證據(jù)，但是系統(tǒng)的發(fā)育生物學(xué)功能分析支持C基因與中耳發(fā)育及結(jié)構(gòu)密切相關(guān)。本研究為耳聾新基因的發(fā)現(xiàn)提供了一整套行之有效的研究方法體系，同時為下一步行動物模型的功能驗證提供了足夠的理論依據(jù)。 課題組今后的工作：繼續(xù)收集與028家系表型一致的散發(fā)病例；完善Taqman探針方法，對散發(fā)樣本行定量PCR進一步驗證CNV；構(gòu)建C基因動物模型進行功能學(xué)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.

【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R764.43

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