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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78977完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林芯? | zh_TW |
| dc.contributor.advisor | Shin-Yu Lin | en |
| dc.contributor.author | 柯盈盈 | zh_TW |
| dc.contributor.author | Ying-Ying Ke | en |
| dc.date.accessioned | 2021-07-11T15:33:43Z | - |
| dc.date.available | 2024-02-28 | - |
| dc.date.copyright | 2018-10-05 | - |
| dc.date.issued | 2018 | - |
| dc.date.submitted | 2002-01-01 | - |
| dc.identifier.citation | 1. Morton, C.C. and W.E. Nance, Newborn hearing screening--a silent revolution. N Engl J Med, 2006. 354(20): p. 2151-64.
2. Smith, R.J., J.F. Bale, Jr., and K.R. White, Sensorineural hearing loss in children. Lancet, 2005. 365(9462): p. 879-90. 3. Goderis, J., et al., Hearing loss and congenital CMV infection: a systematic review. Pediatrics, 2014. 134(5): p. 972-82. 4. Medearis, D.N., Jr., Viral Infections during Pregnancy and Abnormal Human Development. Am J Obstet Gynecol, 1964. 90: p. SUPPL:1140-8. 5. Dollard, S.C., S.D. Grosse, and D.S. Ross, New estimates of the prevalence of neurological and sensory sequelae and mortality associated with congenital cytomegalovirus infection. Rev Med Virol, 2007. 17(5): p. 355-63. 6. Arvin, A.M., et al., Vaccine development to prevent cytomegalovirus disease: report from the National Vaccine Advisory Committee. Clin Infect Dis, 2004. 39(2): p. 233-9. 7. Cannon, M.J. and K.F. Davis, Washing our hands of the congenital cytomegalovirus disease epidemic. BMC Public Health, 2005. 5: p. 70. 8. Hilgert, N., R.J. Smith, and G. Van Camp, Forty-six genes causing nonsyndromic hearing impairment: which ones should be analyzed in DNA diagnostics? Mutat Res, 2009. 681(2-3): p. 189-96. 9. Wu, C.C., et al., Newborn genetic screening for hearing impairment: a preliminary study at a tertiary center. PLoS One, 2011. 6(7): p. e22314. 10. Fowler, K.B., et al., A Targeted Approach for Congenital Cytomegalovirus Screening Within Newborn Hearing Screening. Pediatrics, 2017. 139(2). 11. Vincent, E., et al., Detection of cytomegalovirus in whole blood using three different real-time PCR chemistries. J Mol Diagn, 2009. 11(1): p. 54-9. 12. Gault, E., et al., Quantification of human cytomegalovirus DNA by real-time PCR. J Clin Microbiol, 2001. 39(2): p. 772-5. 13. Metzker, M.L., Sequencing technologies - the next generation. Nat Rev Genet, 2010. 11(1): p. 31-46. 14. Wu, C.C., et al., Application of massively parallel sequencing to genetic diagnosis in multiplex families with idiopathic sensorineural hearing impairment. PLoS One, 2013. 8(2): p. e57369. 15. Chen, S., et al., Targeted Next-Generation Sequencing Successfully Detects Causative Genes in Chinese Patients with Hereditary Hearing Loss. Genet Test Mol Biomarkers, 2016. 20(11): p. 660-665. 16. Shearer, A.E., et al., Comprehensive genetic testing for hereditary hearing loss using massively parallel sequencing. Proc Natl Acad Sci U S A, 2010. 107(49): p. 21104-9. 17. Lanzieri, T.M., et al., Systematic review of the birth prevalence of congenital cytomegalovirus infection in developing countries. Int J Infect Dis, 2014. 22: p. 44-8. 18. Wu, C.C., et al., 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. Audiol Neurootol, 2008. 13(3): p. 172-81. 19. Wu, C.C., et al., Prevalent SLC26A4 mutations in patients with enlarged vestibular aqueduct and/or Mondini dysplasia: a unique spectrum of mutations in Taiwan, including a frequent founder mutation. Laryngoscope, 2005. 115(6): p. 1060-4. 20. Chiu, Y.H., et al., Mutations in the OTOF gene in Taiwanese patients with auditory neuropathy. Audiol Neurootol, 2010. 15(6): p. 364-74. 21. Wu, C.C., et al., Newborn genetic screening for hearing impairment: a population-based longitudinal study. Genet Med, 2017. 19(1): p. 6-12. 22. Chai, Y., et al., Molecular etiology of non-dominant, non-syndromic, mild-to-moderate childhood hearing impairment in Chinese Hans. Am J Med Genet A, 2014. 164A(12): p. 3115-9. 23. Hilgert, N., R.J. Smith, and G. Van Camp, Function and expression pattern of nonsyndromic deafness genes. Curr Mol Med, 2009. 9(5): p. 546-64. 24. Kenneson, A. and M.J. Cannon, Review and meta-analysis of the epidemiology of congenital cytomegalovirus (CMV) infection. Rev Med Virol, 2007. 17(4): p. 253-76. 25. Kimberlin, D.W., et al., Valganciclovir for symptomatic congenital cytomegalovirus disease. N Engl J Med, 2015. 372(10): p. 933-43. 26. Fowler, K.B., et al., Newborn hearing screening: will children with hearing loss caused by congenital cytomegalovirus infection be missed? J Pediatr, 1999. 135(1): p. 60-4. 27. Dahle, A.J., et al., Longitudinal investigation of hearing disorders in children with congenital cytomegalovirus. J Am Acad Audiol, 2000. 11(5): p. 283-90. 28. Jennings, L.J., et al., Guidelines for Validation of Next-Generation Sequencing-Based Oncology Panels: A Joint Consensus Recommendation of the Association for Molecular Pathology and College of American Pathologists. J Mol Diagn, 2017. 19(3): p. 341-365. 29. Simpson, D.A., et al., Molecular diagnosis for heterogeneous genetic diseases with targeted high-throughput DNA sequencing applied to retinitis pigmentosa. J Med Genet, 2011. 48(3): p. 145-51. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78977 | - |
| dc.description.abstract | 兒童聽損基因異常與CMV病毒感染皆會導致感覺神經性聽力損失。台灣常見導致聽損基因之點位如GJB2 c.109G>A, GJB2 c.235delC, SLC26A4 c.919-2A>G, SLC26A4 c.2168, MTRNR1 m.1555及OTOF c.5098等，近幾年合併CMV納入檢測，兩者皆已運用於目前新生兒聽損基因篩檢中。目前利用即時核酸定量分析反應，作為聽損四個基因六個點位與CMV感染之檢測方法，其優點為高靈敏度及快速偵測，提供個案與醫師進行診斷。由於此方式無法進行多基因多點位之檢測，對於聽損高度遺傳異質性之特性，無法提供有更效率之運用。因此，本研究方法利用次世代定序技術平台之轉換，以設計專一性引子對進行多重聚合酶連鎖反應，配合Adapter接合反應，最後利用次世代定序進行23個聽損變異點位分析，提供一套擴大範圍與更快速的檢測方式，於一次性實驗中能同時獲得兩者之檢測結果。我們發現聽損基因與CMV之次世代定序平台結果，與即時核酸定量方法之結果一致，顯示其平台之準確性。另外，利用此平台發現於不同檢體中，有兩個新突變點位SLC26A4 c.1174A>T與SLC26A4 c.1226G>A，此變異點無法利用傳統即時核酸定量方法發現，對於個案可能有不同之臨床表現型。因此，本研究方法利用次世代定序技術擴大範圍與快速檢測，成功地運用於新生兒聽損基因篩檢，亦發現非常規性篩檢之變異點位，可提供醫師進一步診斷，次世代定序技術平台並可作為個案日後家族遺傳諮詢之新方法。 | zh_TW |
| dc.description.abstract | Genetic hearing loss and cytomegalovirus (CMV) infections can lead to sensorineural hearing loss in children. In recent years, CMV and common genetic loci in Taiwan that lead to hearing loss (such as GJB2 c.109G>A, GJB2 c.235delC, SLC26A4 c.919-2A>G, SLC26A4 c.2168, MTRNR1 m.1555, and OTOF c.5098) were incorporated into screening tests, and these are already part of the current genetic screening test conducted for neonatal hearing loss. At present, real-time polymerase chain reaction (PCR) is used as a method for detecting six loci of four hearing loss genes and CMV infections. The advantage of this method lies in its high sensitivity and quick detection capability, which facilitate the diagnostic process for physicians and patients. However, as this method cannot be used to perform multi-gene, multi-locus detection, it is thus unable to be used effectively to test high levels of genetic heterogeneity of hearing loss. Therefore, next-generation sequencing platforms were utilized in this study to design specific primer pairs that can perform multiplex PCR. These pairs were then used in conjunction with the application of the adapter ligation reaction and next-generation sequencing to conduct an analysis of 23 hearing loss variant loci, so as to provide a detection method that can increase the scope and speed of tests, and concurrently generate test results for the two item types in a single test. The hearing loss gene and CMV detection results of the next-generation sequencing platform were found to be consistent with those generated using the real-time PCR method, which indicated the accuracy of the platform. Furthermore, two new mutation loci (SLC26A4 c.1174A>T and SLC26A4 c.1226G>A) were identified during the use of this platform on two specimens, additionally. These loci were not previously detected using the traditional real-time PCR method. Thus, the next-generation sequencing technique utilized in this study was able to increase the scope and speed of tests, and be successfully applied to genetic screening tests for neonatal hearing loss. This unconventional screening approach also revealed variant loci that can assist physicians in making further diagnoses. This next-generation sequencing technique and platform can be used as a new method for genetic counseling in the future. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:33:43Z (GMT). No. of bitstreams: 1 ntu-107-P05448008-1.pdf: 1578851 bytes, checksum: 9ca85ea1cdd76de20059a159ae1c4850 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 口試委員會審定書 I
誌謝 II 中文摘要 III 英文摘要 IV 目 錄 VI 圖目錄.......................................................................................................................VIII 表目錄..........................................................................................................................IX 第一章 緒論 1 1.1 研究背景與動機 1 1.2 感覺神經性聽損（Sensorineural hearing impairment）疾病簡介 2 1.3 巨細胞病毒(Cytomegalovirus, CMV)疾病簡介 4 1.4 檢測技術平台 5 1.5 研究目的 6 第二章 研究方法 8 2.1 台灣新生兒聽損基因篩檢方法 8 2.2 DNA萃取 8 2.3 多重聚合酶連鎖反應（Multiplex Polymerase Chain Reaction：Multiplex PCR） 8 2.4 Multiplex PCR磁珠純化 9 2.5 Adapter接合聚合酶連鎖反應（Adapter Ligation PCR） 9 2.6 Adapter Ligation PCR磁珠純化 10 2.7 DNA濃度定量 10 2.8 DNA片段長度定量 10 2.9 DNA樣品稀釋及變性（DNA diluted and denature） 11 2.10 次世代定序儀器上機（Next-generation sequencing） 11 2.11 生物資訊分析（Bioinformatics） 11 第三章 結果 12 3.1 12個擴增子之DNA濃度與長度測定結果 12 3.2 次世代定序下機結果 12 3.3 聽損基因生物資訊分析結果 12 3.4 CMV生物資訊分析結果 13 第四章 討論 14 4.1 現行聽損基因篩檢之問題 14 4.2 NGS Amplicons實驗探討 14 4.3 Real-Time PCR與NGS平台比較 15 4.4 聽損之遺傳諮詢 16 4.5 聽損檢測之諮詢 17 4.6 綜合討論 17 第五章 參考文獻 19 圖目錄 圖一、現行台灣新生兒的聽損基因篩檢方法，後續本研究以NGS平台進行23個聽損突變點位偵測 22 圖二、NGS實驗流程圖 23 圖三、雜交螢光探針技術（Hybridizing probes） 24 圖四、NGS定序完成後利用生物資訊分析之流程圖 25 圖五、與和平婦幼醫院合作之CMV研究案檢測結果統計（n=1117） 26 圖六、Multiplex PCR及Adapter Ligation PCR進行Bioanalyzer確定產物片段大小之分析結果 27 圖七、利用IGV軟體確認SLC26A4 c.1174A>T與SLC26A4 c.1226G>A之突變位置 28 圖八、利用傳統定序（Sanger sequence）方法進行SLC26A4 c.1174A>T與SLC26A4 c.1226G>A之方法驗證結果 29 圖九、聽損之遺傳諮詢流程 30 圖十、聽損之檢測諮詢流程 31 表目錄 表一、設計12個Amplicons 進行Multiplex PCR之引子序列 32 表二、多重聚合酶連鎖反應12個擴增子之引子對混合物配製條件 36 表三、多重聚合酶連鎖反應Pool 1及Pool 2試劑條件 37 表四、多重聚合酶連鎖反應Pool 1及Pool 2之熱循環參數 38 表五、進行Adapter Ligation PCR反應所使用之Nextera XT index引子序列（illumina） 39 表六、Adapter Ligation PCR反應Pool 1及Pool 2試劑條件 41 表七、Adapter Ligation PCR反應Pool 1及Pool 2之熱循環參數 42 表八、利用NGS平台進行血液與口腔黏膜細胞檢體，於11個Amplicon之平均讀深（coverage） 43 表九、利用校正後計算血液與口腔黏膜細胞檢體每個Amplicon讀深之平均標準差（Standard Deviation） 44 表十、利用NGS平台進行新生兒血片檢體，於11個Amplicon之平均讀深（coverage） 45 表十一、利用校正後計算新生兒血片檢體每個Amplicon讀深之平均標準差（Standard Deviation） 46 表十二、利用NGS平台進行已知和未知之基因型突變點位 47 表十三、利用NGS平台進行CMV已知陽性檢體之分析結果 48 表十四、利用校正後計算CMV Negative、CMV Positive之平均標準差（Standard Deviation） 49 表十五、比較NGS Amplicon平台與Real-Time PCR平台之優缺點 50 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 先天性巨細胞病毒感染 | zh_TW |
| dc.subject | 感覺神經性聽損 | zh_TW |
| dc.subject | 次世代定序 | zh_TW |
| dc.subject | next generation sequencing | en |
| dc.subject | sensorineural hearing loss | en |
| dc.subject | congenital cytomegalovirus infection | en |
| dc.title | 建立次世代定序技術平台應用於新生兒感覺神經性聽損及巨細胞病毒感染篩檢 | zh_TW |
| dc.title | Newborn genetics screening in sensorineural hearing loss and congenital cytomegalovirus infection by next generation sequencing | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 106-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 蘇怡寧;吳振吉 | zh_TW |
| dc.contributor.oralexamcommittee | Yi-Ning Su;Chen-Chi Wu | en |
| dc.subject.keyword | 感覺神經性聽損,先天性巨細胞病毒感染,次世代定序, | zh_TW |
| dc.subject.keyword | sensorineural hearing loss,congenital cytomegalovirus infection,next generation sequencing, | en |
| dc.relation.page | 50 | - |
| dc.identifier.doi | 10.6342/NTU201803444 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2018-08-16 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 分子醫學研究所 | - |
| dc.date.embargo-lift | 2023-10-05 | - |
| 顯示於系所單位： | 分子醫學研究所 | |
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