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標題: | 遺傳性聽損之基因流行病學及分子病理學暨其臨床應用 Genetic epidemiology and molecular pathology of hereditary hearing impairment and the clinical applications |
作者: | Chen-Chi Wu 吳振吉 |
指導教授: | 許權振,陳培哲 |
關鍵字: | 遺傳性聽損,基因流行病學,SLC26A4基因,PDS基因,大前庭導水管,Pendred氏症候群,GJB2基因,Cx26基因,粒線體12S rRNA基因,人工耳蝸植入手術,SNaPshot技術,胚胎著床前基因診斷, Hereditary hearing impairment,Genetic epidemiology,SLC26A4 gene,PDS gene,Enlarged vestibular aqueduct,Pendred syndrome,GJB2 gene,Cx26 gene,Mitochondrial 12S rRNA gene,Cochlear implantation,SNaPshot technique,Pre-implantation genetic diagnosis, |
出版年 : | 2010 |
學位: | 博士 |
摘要: | 研究背景及目的
特發性(原因不明)感覺神經性聽損係臨床上相當常見的疾病,而最近的研究證實,基因變異是導致特發性感覺神經性聽損的重要成因之一。最近數年來,學界已經發現至少四十多個基因與特發性感覺神經性聽損的發生有關(即遺傳性聽損)。遺傳性聽損基因及其突變,常因種族不同而存在著很大的差異,易言之,吾人臨床上並無法直接採用國外的研究結果,來作為遺傳諮詢的準則,若要提供病患及其家屬正確的資訊及診斷,顯然必須仰賴國人自行完成一大規模且完整的基因流行病學調查,而以此一檢體庫為基礎,吾人日後亦可致力於新耳聾基因的找尋或其他研究。同時,一些常見耳聾基因變異之致病機制,諸如:GJB2基因的p.V37I變異之致病性、SLC26A4基因變異之基因型與表現型之關連性、SLC26A4基因變異如何導致聽損、以及影響粒腺體m.1555A>G突變臨床表現型之因素等,皆有待吾人釐清,以助於吾人了解病人聽損發生之原因,進而針對該原因研擬治療方針。 而如同其他單基因遺傳疾病,過去幾年來遺傳性聽損的研究,也逐漸地被應用於臨床醫學以幫助病人及其家屬。然而,從臨床研究者的角度觀之,吾人自不宜安於現狀、故步自封,而應致力於新臨床應用之研究或研發,以使更多病人得以受惠於基因醫學的新進步。而隨著我們對於耳聾基因變異流行病學更精確的掌握,以及對於耳聾基因變異致病機制更深入的了解,若能結合其他領域醫學的較新進步,如內耳之人工耳蝸手術、基因定型技術、以及生殖科技等,應能從中研究出聽損基因檢測之臨床新應用。 研究材料與方法 本研究分兩大部分執行:第一部分為遺傳性聽損之基因流行病學及分子病理學研究;第二部分為遺傳性聽損基因檢測之臨床應用。 於第一部分之遺傳性聽損之基因流行病學及分子病理學研究,本研究先由建立一大規模之特發性感覺神經性聽損基因檢體庫著手,並從中研究國人聽損基因流行病學;其次,再分就三個最常見之耳聾基因,即GJB2基因、SLC26A4基因與粒線體12S rRNA基因之基因變異,研究其致病機制: 1. 遺傳性聽損之基因流行病學研究:於一大規模之聽損世代中,進行基因檢測,並比較常見遺傳性聽損基因突變之機構間及地區間差異。 2. SLC26A4基因變異及其致病機制:於大前庭導水管家族,分析SLC26A4基因型與表現型之關連性,搜尋僅帶單一SLC26A4基因突變或未帶突變之初始受試者是否帶有SLC26A4基因促進區(promoter)及FOXI1基因之基因變異,並進行單套型分析以尋找隱藏突變之可能位置;同時,培育帶有Slc26a4基因國人最常見之c.919-2A>G突變之基因置換鼠,以研究SLC26A4基因變異之致病機轉。 3. GJB2基因變異及其致病機制:分別於特發性感覺神經性聽損與老年性聽損等二研究世代,藉由分析比較GJB2基因p.V37I變異之對偶基因頻率及基因型分布,以釐清該變異與聽損發生之相關性;同時,亦於HeLa細胞進行轉染實驗,並培育帶有p.V37I變異之基因置換鼠,以研究其致病機轉。 4. 粒線體12S rRNA基因變異及其致病機制:分析影響粒線體12S rRNA基因m.1555A>G突變表現型之因子,包括粒線體基因體之單套群、m.1555A>G之突變負荷、細胞核內可能影響粒線體表現的基因、以及抗生素暴露等因素。 於第二部分之遺傳性聽損基因檢測之臨床應用研究,本研究則區分下列三子題,研究聽損基因檢測之新應用: 1. 以基因檢測結果預測人工耳蝸植入手術術後成果:研究接受人工耳蝸植入手術病童之術後長期語音聽知覺表現,分析比較各預後因子(包括基因檢測結果、手術年齡、人工耳蝸使用期間、術前殘存聽力及影像學檢查結果等)對於人工耳蝸植入手術術後聽能創建效果好壞的影響。 2. 研發高效率低成本之基因診斷工具:使用SNaPshot此一基本技術,設計一套可同時檢測約20-40個耳聾基因突變位點的檢測方法,並應用該檢測方法於一新的特發性感覺神經性聽損世代,以確認此一診斷工具的效力。 3. 胚胎著床前聽損基因診斷:針對帶有特定耳聾基因變異的家族,設計胚胎著床前基因診斷技術,以助其生育聽力正常之胎兒。 結果 第一部分之遺傳性聽損之基因流行病學及分子病理學研究 1. 遺傳性聽損之基因流行病學研究:本研究建立了一個大規模國人特發性感覺神經性聽損的基因庫,也釐清了國人聽損基因流行病學的大致輪廓—國人的基因變異仍以GJB2、SLC26A4及粒線體12S rRNA基因等三個耳聾基因最為常見,並證實了來自不同機構的病人,由於其聽力特徵亦不相同,將導致基因流行病學研究結果的歧異。 2. SLC26A4基因變異及其致病機制:國人大前庭導水管病人中,八成以上可歸因於SLC26A4基因變異,而進一步之表現型-基因型關連性研究,則暗示仍有未被檢測出之SLC26A4基因變異;另外,於本研究所培育出之Slc26a4基因c.919-2A>G突變之基因置換鼠Slc26a4tm1Dontuh/tm1Dontuh,表現型可觀察到重度聽損、平衡能力差、前庭導水管擴大等與人類疾病類似之表徵。 3. GJB2基因變異及其致病機制:對偶基因頻率、病人基因型分佈與家族樹分析顯示,p.V37I變異與特發性感覺神經性聽損以及老年性聽損均顯著相關,而於HeLa細胞進行之實驗則顯示該變異可能為影響蛋白質功能之突變。然而p.V37I變異同型合子臨床上聽損程度差異極大,甚至可能完全正常,顯示應有其他因素調控其聽力表徵。 4. 粒線體12S rRNA基因變異及其致病機制:國人特發性感覺神經性聽損家族中,粒線體m.1555A>G突變之盛行率約為3%,而於多病例家族,其盛行率更高達11%。抗生素暴露、m.1555A>G突變之突變負荷及細胞核相關基因之基因變異,皆非決定國人m.1555A>G突變臨床表徵之重要因子;反之,某些單套群,如單套群F,似乎與家族之低外顯率相關。 第二部分之遺傳性聽損基因檢測之臨床應用研究 1. 以基因檢測結果預測人工耳蝸植入手術術後成果:基因檢測結果及影像學結果為影響人工耳蝸植入術後成效最重要的兩個指標。以影像學上內聽道狹窄之有無評估人工耳蝸之預後,可預測出術後成效不佳之病例;而以基因診斷預測預後,則可預測出術後成效良好之病例。兩者恰可相輔相成,合併使用,將有助於吾人於人工耳蝸植入術前,更精準地預測病人術後成效。 2. 研發高效率低成本之基因診斷工具:本研究研發之SNaPshot assay,可於2次SNaPshot反應,完成國人最常見之20個耳聾基因變異點位之掃描。將該項基因檢測技術應用於診斷214個聽損家族則發現其正確率不亞於傳統之基因定序,且具效率高、成本低、可彈性調整點位與易於推廣等優點。 3. 胚胎著床前聽損基因診斷:本研究首先以GenomiPhi technology和primer extension mini-sequencing等方法,針對一SLC26A4基因c.919-2A>G突變同型合子病童及其父母,研發並校正單一細胞c.919-2A>G突變點檢測技術。其後,以人工生殖技術,誘導排卵並進行體外受精,於胚胎培育成八細胞期時,進行胚胎切片以作單一細胞基因診斷。於第二次PGD療程,本研究成功使病童母親受孕,並於2009年5月生育聽力完全正常之嬰兒,而完成世界首例報告。 結論 本研究過去幾年來所進行的遺傳性聽損之基因流行病學及分子病理學研究,已使我們對於國人特發性感覺神經性聽損之常見基因變異、基因型與表現型之關連性、以及其致病機制,有了基本的了解。而最近幾個常見耳聾基因變異基因置換鼠之成功培育,不僅有助於我們釐清各基因變異之致病機制,亦將成為我們未來研究新治療方針之礎石。當然,本研究也注意到有些尚未解決的課題,有待吾人進行更深入的研究。 本研究發現,聽損之基因檢測結果不僅可用來預測人工耳蝸手術術後成果,基因流行病學資料的建立,也使得吾人得以研發高效率、低成本的基因檢測工具來推廣基因檢測並幫助更多病人。近年來,本研究也嘗試結合其他領域醫學的較新進展,以研究聽損基因檢測之臨床新應用:聽損之基因檢測與生殖科技的結合,使得吾人臨床上得以扮演更積極的角色,而能夠於生命的最始期即能提供聽損家族其所需要的幫助。 PART I. Genetic epidemiology and molecular pathology of hereditary hearing impairment Outlines 1. Genetic epidemiology of common deafness genes in Taiwanese 2. SLC26A4 mutations and the pathogenetic mechanisms A. SLC26A4 mutations in humans B. SLC26A4 mutations in mice 3. GJB2 mutations and the pathogenetic mechanisms A. The p.V37I variant vs. idiopathic sensorineural hearing impairment B. The p.V37I variant vs. age-related hearing impairment 4. Mitochondrial 12S rRNA mutations and the pathogenetic mechanisms 1. Genetic epidemiology of common deafness genes in Taiwanese: Prospective mutation screening of three common deafness genes in a large Taiwanese cohort with idiopathic bilateral sensorineural hearing impairment reveals a difference in the results between families from hospitals and those from rehabilitation facilities. Background Molecular genetic analysis provides us with a powerful tool in approaching patients with sensorineural hearing loss of unknown cause. Among a plethora of deafness genes discovered in the past decade, certain genes are more important than others from an epidemiologic perspective. These genes include GJB2, SLC26A4 and mitochondria 12S rRNA gene. Not only do the mutations of these genes demonstrate a universal distribution, but the frequencies of these mutations in the hearing-impaired population are also much higher than the frequencies of other mutations. One of the most important findings that we have learned from past years’ researches is that different ethnic groups showed different mutation spectra for each deafness gene. Consequently, establishment of a database for each ethnic group becomes indispensable, and the ethnic background should be considered whenever genetic counseling for hereditary hearing impairment is performed. Accurate epidemiological data of common deafness genes are essential to improve the efficiency and the cost of molecular diagnosis. They may depend on several factors, including a clear delineation of the source of patients being studied. In the present study, we hypothesize that idiopathic sensorineural hearing-impaired patients of different sources might reveal discrepancies in the epidemiological results of genetic screening, because patients of different sources might demonstrate distinct clinical or audiologic features and thus result in biased selection of subjects. To elucidate the relative importance of mutations of the common deafness genes in the Taiwanese population and to verify our hypothesis, we conducted a prospective project screening mutations in GJB2, SLC26A4 and the mitochondria 12S rRNA gene in families with idiopathic bilateral sensorineural hearing loss, and compared the prevalence of common mutations between families of different sources. Patients and Methods We conducted a prospective project screening mutations in GJB2, SLC26A4 and mitochondria 12S rRNA gene in a total of 420 Taiwanese families with idiopathic bilateral sensorineural hearing loss, of which 325 families were recruited from hospitals and 95 from hearing rehabilitation facilities. Allele frequencies of common mutations in these three genes and distributions of the corresponding genotypes were then compared between the two groups. Results The allele frequencies of variants in SLC26A4, GJB2 and mitochondria 12S rRNA in the probands of the 420 families were 14.4%, 21.7% and 3.8%, respectively. The allele frequency of SLC26A4 mutations in the hospital group was significantly higher than that in the rehabilitation facility group (16.2% vs. 8.4%, chi-square test, p < 0.05); whereas no difference in the frequencies of GJB2 variants and mitochondria 12S rRNA variants was found between the two groups. Distributions of probands classified by SLC26A4 genotypes were also different between the two groups (chi-square test, p < 0.05). Accordingly, a discrepancy in the genetic screening results might exist between different sources of idiopathic hearing-impaired patients. Further analysis of audiological results and construction of a logistic regression model showed that different audiological features, namely hearing levels and hearing loss patterns, might be responsible for the unequal distributions of mutations and probands between the hospital and rehabilitation facility groups. Conclusion The source of patients might affect the results of genetic screening for idiopathic hearing impairment. Patient source should therefore always be labeled with clarity when the results of genetic epidemiological studies for idiopathic hearing impairment are being reported. 2. SLC26A4 mutations and the pathogenetic mechanisms A. SLC26A4 mutations in humans: Phenotypic analyses and mutation screening of the SLC26A4 and FOXI1 genes in 101 Taiwanese families with bilateral nonsyndromic enlarged vestibular aqueduct (DFNB4) or Pendred syndrome. Background Recessive mutations in the SLC26A4 gene are responsible for non-syndromic enlarged vestibular aqueduct (EVA) and Pendred syndrome. However, in some affected families, only one or zero mutated allele can be identified, together with the fact that there is no clear correlation between SLC26A4 genotypes and clinical phenotypes, hampering the accuracy of genetic counseling. To elucidate the genetic composition of non-syndromic EVA and Pendred syndrome, we screened related genomic fragments for genetic variants in a large Taiwanese cohort and analyzed the phenotypic and genotypic results. Patients and Methods We used direct sequencing and quantitative polymerase chain reaction (PCR) to screen 101 families with non-syndromic EVA or Pendred syndrome for mutations in the SLC26A4 coding regions. For those with zero or one mutation detected, genetic variants were then screened in the SLC26A4 promoter and the FOXI1 transcription factor gene. Meanwhile, the phenotypes, including the radiological findings, the presence of goiters and the audiological results were correlated with the genotypes. Haplotypes of the SLC26A4 alleles without mutations detected were also investigated to explore mutations in non-coding regions. Results Mutation screening in the SLC26A4 coding regions detected two mutations in 63 (62%) families, one mutation in 24 (24%) families and no mutation in 14 (14%) families. The radiological findings, the presence of goiters and the audiological results were not different among probands (i.e. index cases of the families) with different SLC26A4 genotypes. Specifically, probands heterozygous for SLC26A4 mutations demonstrated clinical features indistinguishable from those with two mutated alleles, implicating that there might be undetected mutations. However, except for a variant (c.-2554G>A of SLC26A4) with possible pathological consequences, no definite mutation was detected after extensive screening in the SLC26A4 promoter and FOXI1. In other words, most Taiwanese families with non-syndromic EVA or Pendred syndrome might not result from aberrance in the transcriptional control of SLC26A4 by FOXI1. Meanwhile, exploration of undetected mutations in the SLC26A4 non-coding regions revealed 9 divergent haplotypes among the 21 no-mutation-detected SLC26A4 alleles of the c.919-2A>G heterozygotes, indicating that there might be no common and predominant mutations in the SLC26A4 introns. Conclusion There is no significant difference in phenotypes among patients with different SLC26A4 genotypes. The similarity in clinical features between probands with one mutation and those with two mutations and the co-segregation of the mutation with the phenotype in pedigrees indicate the presence of undetected mutations. The transcriptional control of SLC26A4 by FOXI1 appears to contribute minimally to non-syndromic EVA or Pendred syndrome in Taiwanese. The divergence of haplotypes of the no-mutation-detected SLC26A4 alleles excludes the possibility of predominant founder intronic mutations. B. SLC26A4 mutations in mice: Characterization of phenotypes in knock-in mice with SLC26A4 c.919-2A>G mutation. Background Recessive mutations in SLC26A4 are responsible for non-syndromic enlarged vestibular aqueduct (EVA) and Pendred syndrome (PS), two disorders commonly encountered in children with hearing impairment. Among more than 100 SLC26A4 mutations identified to date, c.919-2A>G is by far the most prevalent mutation in Taiwanese, and the second most prevalent in Japanese. In the present study, we established a knock-in mouse model homozygous for the c.919-2A>G mutation, Slc26a4tm1Dontuh/tm1Dontuh, and explored the associated audiological and vestibular phenotypes. Materials and Methods The phenotypes of Slc26a4tm1Dontuh/tm1Dontuh were characterized by behavioral observations, audiologic assessment using auditory brainstem response (ABR) and distortion production otoacoustic emissions (DPOAE), and balance assessment using rotorod testing. Inner ear morphology was investigated using H&E staining, fluorescence confocal microscopy and electron microscopy. Result 45% of the Slc26a4tm1Dontuh/tm1Dontuh mice demonstrated pronounced head tilting and circling behaviors, as well as severely impaired balancing ability on rotorod testing. All Slc26a4tm1Dontuh/tm1Dontuh mice revealed profound hearing loss (> 120 dB SPL) on ABR and absent OAEs at 3 weeks. Histological studies using H&E staining showed enlarged endolymphatic duct and sac, endolymphatic hydrops in the cochlea, and atrophy of stria vascularis. Fluorescence confocal microscopy and electron microscopy demonstrated degeneration of hair cells in the organ of Corti. Conclusion Preliminary characterization of the Slc26a4tm1Dontuh/tm1Dontuh mice revealed phenotypes reminiscent of those observed in the human counterpart. The Slc26a4tm1Dontuh/ tm1Dontuh mice might thus serve as a good animal model for patients segregating SLC26A4 mutations, and can be utilized for therapeutic researches in the future. 3. GJB2 mutations and the pathogenetic mechanisms A. The p.V37I variant vs. idiopathic sensorineural hearing impairment: Pathogenetic role of the deafness-related p.V37I variant of GJB2: evidences from a large clinical cohort, cell-line studies and the knock-in mouse model. Background p.V37I, a GJB2 allele with debatable pathogenicity frequently identified in East Asians, was reported to contribute to mild-to-moderate sensorineural hearing impairment (SNHI). The purpose of the study is to elucidate the pathogenicity of p.V37I through investigations in a clinical cohort, cell-lines and the knock-in mouse model. Methods and Materials A total of 732 unrelated Han Chinese patients with idiopathic non-syndromic SNHI and 1005 Han Chinese controls were enrolled. According to the GENDEAF criteria, 356 were classified as severe-to-profound SNHI, and 376 were classified as mild-to-moderate SNHI. The allele frequencies and genotype distributions of p.V37I were compared among the groups, and the corresponding phenotypes were analyzed. The pathogenetic mechanisms of p.V37I were then investigated in cell-lines by transfecting HCx26wt and the p.V37I variant into HeLa cells. A knock-in mouse model homozygous for p.V37I, Gjb2tm1Dontuh/tm1Dontuh, was also established. Results Allele frequencies of p.V37I in these two groups and the controls were 12.8%, 30.3% and 9.2%, respectively (chi-square test, p < 0.001). Both patients with severe-to-profound SNHI and those with mild-to-moderate SNHI revealed a deviated genotype distribution from estimation (chi-square goodness-of-fit test, both p < 0.001). Homozygosity for p.V37I was identified in 77 (20.5%) and 9 (2.5%) patients with mild-to-moderate and severe-to-profound SNHI, respectively, and co-segregated with the phenotypes of SNHI in 56 kindreds. Further mutation screening in the GJB2 promoter and GJB6 coding regions did not detect sequence variants in the 86 p.V37I homozygotes, excluding the possibility of a near-by mutation in linkage disequilibrium with p.V37I. HeLa cells transfected with p.V37I expressed protein levels comparable to those transfected with HCx26wt, but were less able to form clear junctional plaques and demonstrated a less permeability for intercellular dye transfer. Preliminary characterization of the audiological phenotype in Gjb2tm1Dontuh/tm1Dontuh revealed near-normal hearing levels at 4 weeks. Conclusion Homozygosity for GJB2 p.V37I allele is strongly associated with idiopathic mild-to-moderate sensorineural hearing impairment in Taiwanese, and demonstrates a diverse spectrum of audiologic phenotypes. Further functional genetic studies in cell lines and knock-in mice indicated that p.V37I might mildly impact the function of connexin26. B. The p.V37I variant vs. age-related hearing impairment: The contribution of GJB2 (Connexin26) variant alleles to age-related hearing impairment. Background Age-related hearing impairment (ARHI), the most common form of hearing impairment in humans, is a complex disease caused by an interaction between environmental and genetic factors. It has been postulated that genes leading to monogenic hearing impairment might also be susceptibility genes for ARHI. The purpose of this study is to investigate the association between ARHI and variant alleles of GJB2, of which the mutations are the most important cause of monogenic hearing impairment worldwide. Patients and Methods A total of 1005 Taiwanese volunteers (all were Han Chinese, aged 40 to 80 y) were included in the analyses, and were classified into controls (the 1/3 subjects with better hearing, n=335) or cases (the 1/3 subjects with worse hearing, n=335) according to the Zlow, Z4-tone and Zhigh scores converted from their original frequency-specific hearing thresholds. Seven sequence variants in the coding region of GJB2, including c.79G>A, c.109G>A, c.235delC, c.299_300delAT, c.341A>G, c.368C>A and c.608T>C, were genotyped and then correlated to the audiological phenotypes. Results Among the 7 sequence variants, genotype distributions of the c.109G>A allele were different between cases and controls classified according to the Zlow and Z4-tone scores (Fisher’s exact test, p < 0.05). Further analyses revealed subjects with the AA genotype demonstrated significantly higher Zlow, Z4-tone and Zhigh scores than those with the GA or GG genotype (ANOVA with post hoc Tukey multiple comparison procedure, all p < 0.001). The AA genotype was still significantly associated with ARHI after other non-genetic factors had been adjusted by logistic regression models. Accordingly, the c.109A allele of GJB2 might contribute to ARHI in Han Taiwanese via a recessive mode. Conclusion The association between GJB2 and ARHI supports the hypotheses that genes responsible for monogenic hearing impairment and genes regulating the potassium homeostasis in the inner ear might be susceptibility genes for ARHI. The significant variety in the audiological phenotypes of subjects homozygous for c.109A implicates the presence of other factors modulating the development of ARHI. 4. Mitochondrial 12S rRNA mutations and the pathogenetic mechanisms: Prevalence and clinical features of the mitochondrial m.1555A>G mutation in Taiwanese patients with idiopathic sensorineural hearing loss and association of haplogroup F with low penetrance in three families. Background The m.1555A>G mutation in the mitochondria 12S rRNA gene has been reported to be an important cause of non-syndromic hereditary hearing loss. However, remarkable inter-familial and intra-familial variations in the phenotypes of the mutation preclude precise prognosis during genetic counseling. Hence, this study was performed to explore the factors which might contribute to the differences in the phenotypes, including aminoglycoside exposure, mutation load and mitochondrial DNA (mtDNA) background. Also reported were the prevalence and the clinical features of the m.1555A>G mutation in hearing-impaired Taiwanese patients. Patients and Methods Mutations in the 12S rRNA gene were screened in a panel of 315 unrelated Taiwanese families with idiopathic sensorineural hearing loss. Clinical features in families with m.1555A>G mutation were analyzed, and the roles of aminoglycoside exposure, mutation load and mtDNA background in disease expression were investigated. Penetrance was then compared among families with different mtDNA backgrounds. Results The m.1555A>G mutation was identified in a total of ten (3.2%) families, and was characterized clinically by progressive, post-lingual and bilaterally symmetric sensorineural hearing loss and normal temporal bone radiological results. The m.1555A>G mutation was homoplasmic (i.e. all the mitochondrial DNA carries the mutation) in all matrilineal relatives in these 10 pedigrees. Among the 44 hearing-impaired relatives of the 10 pedigrees, only two recalled definite episodes of aminoglycoside-induced hearing loss. mtDNA backgrounds in these 10 families could be categorized into 6 main haplogroups (A, B, D, F, M7, N*), including three families belonging to haplogroup F, two belonging to haplogroup A, two belonging to haplogroup M7, and three belonging to haplogroups B, N* and D, respectively. Penetrance differed among various haplogroups, and certain haplogroups appeared to be associated with a lower penetrance, like the three haplogroup F families, in which the penetrance ranged from 13 to 33%. Further analysis confirmed a heterogeneous distribution of hearing-impaired subjects among various haplogroups (chi-square test, p = 0.018). Conclusion The mitochondrial m.1555A>G mutation accounted for 3.2% of the Taiwanese families (0% of the simplex families and 11% of multiplex families respectively) with sensorineural hearing impairment of unknown etiology. Since it was identified in a variety of mtDNA backgrounds, the mutation appeared to arise from multiple origins in Taiwanese. As subjects with various haplogroups demonstrated different penetrance, mtDNA background might exert effects on the disease expression of the m.1555A>G mutation. PART II. Novel clinical applications of genetic examination in the field of hereditary hearing impairment Outlines 1. Role of genetic diagnosis in predicting the outcome after cochlear implantation 2. Development of an efficient and low-cost diagnostic tool to promote genetic examination in Taiwanese patients with hearing impairment 3. Application of pre-implantation genetic diagnosis (PGD) to hereditary hearing impairment 1. Role of genetic diagnosis in predicting the outcome after cochlear implantation in children: Genetic diagnosis and imaging results are the predominant predictors determining the speech perception performance outcome after cochlear implantation in Children. Background Research over time has shown that early identification of hearing loss followed by rehabilitation procedures, such as hearing aid usage commencing during the first 6 months of life, significantly increases the level of language development, speech intelligibility and emotional stability. For those who get limited benefits from hearing aids and fail to reach communication milestones because of the severity of their hearing impairment, cochlear implantation has been demonstrated to be an effective intervention and is currently regarded as the treatment of last resort. Bypassing the sensory organ of the inner ear, cochlear implants (CI) activate auditory nerve fibers directly, transmitting auditory signals through the central neural pathway and ultimately yielding speech understanding in the cortex. The performance outcome with CI, however, varies significantly among implantees. A plethora of factors, including age of implantation, duration of implant use, amount of residual hearing, primary mode of communication before operation, and presence of certain inner ear malformations (IEMs) such as narrow internal auditory canal (IAC), have been proposed to influence the outcome. Still, a panoramic prediction of CI results remains unavailable thus far, largely because pediatric SNHI is extremely heterogeneous in its etiology. As an invasive and expensive surgical procedure, identification of the most crucial predictors of CI outcome is of paramount importance, since it may help steer appropriate rehabilitation programs and expectations by clinical workers, schools and families. The purpose of the study is to investigate the roles of genetic diagnosis, image results, as well as other prognostic factors in predicting the long-term outcome with cochlear implant (CI) in children. Patients and Methods The study prospectively enrolled 67 consecutive implantees who had a minimum of 3 years’ experience with the device. Image results of inner ear were obtained, and mutations were screened in 3 genes commonly associated with hearing impairment: GJB2, SLC26A4 and the mitochondria 12S rRNA gene. Speech perception performance, expressed by speech recognition scores of 5 parameters, was compared according to genetic diagnosis and image results, respectively. General linear model was constructed to testify the predicting values of specific genetic and image results after adjusting other prognostic factors. Results Twenty-two (33%) children harbored genetic mutations, including 18 with SLC26A4 mutations and 4 with GJB2 mutations. When classified by image findings, 33 (49%) children revealed inner ear malformations (IEMs), inclusive of 9 with narrow internal auditory canal (IAC) and 24 with other IEMs. Children with SLC26A4 or GJB2 mutations exhibited excellent speech recognition scores, whereas children with narrow IAC performed more poorly as compared to those with other IEMs or those without IEMs. General linear model confirmed that both narrow IAC and SLC26A4 mutations correlated with the speech perception outcome, indicating that genetic diagnosis and image results are the two predominant factors determining the outcome with CI. Conclusion In pediatric CI candidates, both genetic examination and image study might be included in the battery of pre-operative evaluations before proceeding to implantation. 2. Development of an efficient and low-cost diagnostic tool to promote genetic examination in Taiwanese patients with hearing impairment: Application of SNaPshot® multiplex assays for simultaneous multigene mutation screening in patients with idiopathic sensorineural hearing impairment. Background The genetic basis of idiopathic non-syndromic SNHI is particularly heterogeneous, making the efficient molecular diagnosis of individual patients challenging. More than 100 genes are associated with deafness, and to date, at least 46 genes have been identified to cause non-syndromic hereditary hearing impairment (The Hereditary Hearing Loss Homepage, http://webhost.ua.ac.be/hhh/). Although mutations in certain genes including GJB2, SLC26A4 and the mitochondrial 12SrRNA gene have been shown to be much more prevalent than other genes in many populations, comprehensive screening of mutations in these genes using conventional genotyping techniques, such as direct sequencing, remains a laborious task. Based on genetic epidemiological data obtained from previous studies, several high throughput strategies like microarray technology and the Invader assay, have been developed to help screen for mutations. Since both the mutation spectra and the complexity of the mutation spectra in common deafness-associated genes differ among populations, it is possible that each population might require a different strategy for optimal rapid genetic examination. The purpose of the study is to develop a cost-effective and robust genetic diagnostic tool for patients with idiopathic non-syndromic sensorineural hearing impairment. Patients and Methods Twenty common sequence variants in GJB2, SLC26A4, and the mitochondrial 12S rRNA gene were selected based on our previous epidemiological study. These variants were analyzed using the SNaPshot technique. The efficacies of the SNaPshot multiplex assays were determined by using a prospective cohort composed of 214 unrelated Taiwanese patients with idiopathic sensorineural hearing impairment. The results of the assays were compared to the results obtained by direct sequencing. Results We developed a diagnostic technique consisting of two consecutive panels of SNaPshot multiplex assays, with each panel screening 10 common sequence variants. Theoretically, this design can detect more than 98% of the known deafness-associated sequence variants in Taiwanese individuals. A total of 126 (58.9%) patients were diagnosed as having at least one sequence variant using the SNaPshot multiplex assays. In total, the SNaPshot assays yielded an accuracy of more than 99%. Conclusion The strengths of SNaPshot multiplex assays include high accuracy, high sensitivity, high flexibility (the examination panel can be easily expanded for additional mutations), low cost (<10 US dollars per patient), and easy implementation for any institute with a DNA sequencer. Although only 20 to 30 mutations can be examined in two to three runs of the SNaPshot assay, this technology may be suitable for first-pass screening of deafness-associated mutations in populations with a relatively homogeneous ethnic background. 3. Application of pre-implantation genetic diagnosis (PGD) to hereditary hearing impairment: Pre-implantation genetic diagnosis (embryo screening) for enlarged vestibular aqueduct due to SLC26A4 mutation. Background Once the mutations responsible for deafness in a family are confirmed, pre-natal diagnosis (PND) using chorionic villus sampling or amniocentesis can be performed to screen and subsequently terminate an affected pregnancy. However, PND is not an acceptable option to many families for ethical, psychological or religious reasons. For these families, pre-implantation genetic diagnosis (PGD) offers an alternative, with the opportunity of beginning a pregnancy with a disease-free embryo, thus circumventing the need for late diagnosis and termination. PGD has been performed for many monogenic disorders, including cystic fibrosis, beta-thalassemia, myotonic dystrophy, Huntington’s disease and fragile X syndrome. However, the application of PGD to hereditary hearing impairment has not been explored. Patients and Methods In the present study, we reported the development and application of PGD protocols to address enlarged vestibular aqueduct (EVA), which is a common type of hereditary hearing impairment associated with mutations in the SLC26A4 gene. The family requesting PGD had a history of EVA, segregating the SLC26A4 c.919-2A>G mutation. In short, the PGD process was composed of two steps: the development of a single-cell testing protocol and clinical PGD cycles (i.e., selection and implantation of unaffected embryos using the single-cell testing protocol). Results First, protocols for genetic testing in a single cell were established for the c.919-2A>G mutation using GenomiPhi technology and primer extension mini-sequencing. These protocols were validated on single lymphocytes collected from both parents and their affected child. Two clinical PGD cycles were then performed for the parents, with the second cycle successfully leading to a singleton pregnancy. The baby was homozygous for the wild type SLC26A4 allele and revealed a normal audiological phenotype after birth. Conclusion To our knowledge, there has not ever been reported in the literature describing successful PGD in families with genetic hearing impairment. In our opinion, the application of PGD in the field of hereditary hearing impairment involves fewer ethical controversies than other novel applications of PGD and traditional indications for PGD for other monogenic diseases. Therefore, the approach demonstrated in the present study can also be used in a large number of families with other types of hereditary hearing impairment. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8773 |
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