臺灣博碩士論文加值系統

人類最常見的感覺性缺陷為聽障，而造成聽障的原因有很多，其中包括遺傳基因突變、環境因素或兩者兼之。聽障可依是否合併身體其它器官的異常，區分為症候群聽障及非症候群聽障，非症候群聽障佔先天性聽障的70 % 。本論文主要探討的是與非症候群聽障有關之基因-TMPRSS3。
在本研究中一共收集120位聽力正常人和190位聽障孩童對TMPRSS3進行基因篩檢，結果突變之盛行率總計為7.37%，其中含1.59% 的239 G→A/wt (R80H)、551 T→C/wt (L184S)、1253 C→T/wt (A418V)等三個錯意突變(missense mutation)；1.59% 的621 T→C/wt、933 C→T/wt、1269 C→T/wt等三個靜默突變(silent mutation)；及4.19%在intron中的四個突變點： -75 A→G/wt、 205+38 C→T/wt、 1194+15 C→A/wt、1347+11 C→T/wt。另外還發現-78 T→A、 -36 G→C、 157 G→A (V53I)、 447-13 A→G、 453 G→A (V151V)、617-4 ins AT、 681 G→A (Q227Q)、 757 A→G (I253V)、 763 G→T (A255A)、 945 G→A (T315T)、 952+18 T→C、 1048+56 C→T、 1121 A→G (D374G)、 1122 C→T (D374D)、 1335 C→T (H445H)、 1367 G→A、1399+19 A→G等共17種多型性(polymorphism)的變異。
進一步為了探討TMPRSS3 突變後造成功能的影響，所以建構突變型TMPRSS3 239 G→A (R80H)、 551 T→C (L184S)、 1253 C→T (A418V)的質體，利用sGASP(secretory genetic assay for site-specific proteolysis)系統，將建構好的突變型TMPRSS3質體送入缺乏suc2-的酵母菌KSY01中，當substrate 被具有特異性的protease切斷時，invertase 會被釋放到細胞質中，再分泌至細胞外將蔗糖分解成葡萄糖及果糖。因此TMPRSS3的功能可藉由轉殖後的酵母菌是否可在含有蔗糖的選擇性固態培養基上生長為判斷。結果發現TMPRSS3 R80H、L184S 及A418V等突變型在選擇性固態培養基上的生長率各為 94.12%、 90.98% 及88.04%，因此我們認為這三個突變點R80H、L184S 及A418V 並沒有影響到TMPRSS3蛋白水解的功能。

Hearing loss is the most common sensory defect in humans. The cause of this disease is multifactorial and includes both genetic and environmental factors. Non-syndromic autosomal recessive deafness accounts for about 70% cases of congenital hereditary hearing loss.
We have analyzed TMPRSS3 from 120 unrelated normal Taiwanese individuals and 190 Taiwanese patients with prelingual deafness. The prevalence of TMPRSS3 mutation appeared to be 7.37%. Two categories of the mutations of TMPRSS3 gene carried by the deaf patients were found. Three were missense mutations of 239G→A/wt (R80H), 551T→C/wt (L184S), and 1253C→T/wt (A418V), which account for 1.59% of all mutations found. Three were silent mutations of 621T→C/wt (C207C), 933C→T/wt (A311A), and 1269C→T/wt (I423I), which are 1.59% of all mutations identified. There were also four mutations found in introns, including -75 A→G/wt, 205+38 C→T/wt, 1194+15 C→A/wt, and 1347+11 C→T/wt. These account for 4.19% of all mutations found. We have also identified polymorphisms of TMPRSS3 gene including -78T→A, -36G→C, 157G→A (V53I), 447-13A→G, 453G→A (V151V), 617-4 insAT, 681G→A (Q227Q), 757A→G (I253V), 763G→T (A255A), 945G→A (T315T), 952+18T→C, 1048+56C→T, 1121A→G (D374G), 1122C→T (D374D), 1335C→T (H445H), and 1367G→A.
To determine the functional importance of TMPRSS3 mutations, we constructed plasmids carrying TMPRSS3 mutations of 239 G→A (R80H), 551 T→C (L184S) and 1253 C→T (A418V). These plasmids are capable of expressing mutant TMPRSS3 in yeast cell. The function of TMPRSS3 can be examined by the sGASP (secretory genetic assay for site-specific proteolysis) system. In such system, the growth of yeast cells with sucrose as a sole carbon source depends on the ability of functional TMPRSS3 to site-specifically cleave its substrate such that invertase moiety can be released from Golgi membrane to periplasmic space allowing conversion of sucrose into glucose and fructose. The ratio of the number of colonies on sucrose versus glucose plate reflects the function of TMPRSS3. The results showed that TMPRSS3 mutations of R80H, L184S, and A418V gave the ratio of 94.12%, 90.98%, and 88.04% respectively. We concluded that those three TMPRSS3 mutations of R80H, L184S, and A418V do not impair its proteolytic activity.

目錄

中文摘要…………………………………………………………………1
英文摘要…………………………………………………………………3
第一章 序論(Introduction)………………………………………… 5
第二章 材料方法(Materials and Methods)…………………………16
第三章 結果(Result)…………………………………………………30
第四章 討論(Discussion)……………………………………………35圖表……………………………………………………………40
第五章 參考文獻(References)………………………………………62
第六章 附錄(Appendixes)……………………………………………65
試劑配方………………………………………………………73

Aggarwal R, Saeed SR (2005) The genetics of hearing loss. Hosp Med 66(1): 32-6
Ahmed ZM, Li XC, Powell SD, Riazuddin S, Young TL, Ramzan K, Ahmad Z, Luscombe S, Dhillon K, MacLaren L, Ploplis L, Shotland LI, Ives E, Riazuddin S, Friedman TB, Morell RJ, Wilcox ER (2004) haracterization of a new full length TMPRSS3 isoform and identification of mutant alleles responsible for nonsyndromic recessive deafness in Newfoundland and Pakistan. BMC Med Genet 24;5:24
Ben-Yosef T, Wattenhofer M, Riazuddi S, Ahmed ZM, Scott HS, Kudoh J, Shibuya K, Antonarakis SE, Bonne-Tamir B, Radhakrishna U, Naz S, Ahmed Z, Riazuddin S, Pandya A, Nance WE, Wilcox ER, Friedman TB, Morell RJ (2001),Novel mutations of TMPRSS3 in four DFNB8/B10 families segregating congenital autosomal recessive deafness. J Med Genet 38(6):396-400
Bitner-Glindzicz M (2002) Hereditary deafness and phenotyping in humans. Br Med Bull 63:73-94
DeStefano AL, Cupples LA, Arnos KS, Asher JH, Jr, Baldwin,C.T., Blanton S, Carey ML, da Silva EO, Friedman TB, Greenberg J, Lalwani AK, Milunsky A, Nance WE, Pandya A, Ramesar RS, Read AP, Tassabejhi M, Wilcox ER, Farrer LA (1998) Correlation between Waardenburg syndrome phenotype and genotype in a population of individuals with identified PAX3 mutations. Hum Genet 102:499-506
Fortnum H, Davis A (1997) Epidemiology of permanent childhood hearing impairment in Trent Region, 1985-1993. Br J Audiol 31:409-46
Gietz D, Jean AS, Woods RA, Schiestl RH (1992) Improved method for high efficiency transformayion of intact yeast cells. Nucleic Acids Res. 20:1425
Guipponi M, Vuagniaux G, Wattenhofer M, Shibuya K, Vazquez M, Dougherty L, Scamuffa N, Guida E, Okui M, Rossier C, Hancock M, Buchet K, Reymond M, Hummler E, Marzella PL, Kudoh J, Shimizu N, Scott HS, Antonarakis SE, Rossier BC (2002) The transmembrane serine protease (TMPRSS3) mutated in deafness DFNB8/10 activates the epithelial sodium channel (ENaC) in vitro. Hum Mol Genet 11(23):2829-36
Hooper JD, Clements JA, Quigley JP, Antalis TM (2001) Type Ⅱ transmembrane serine protease. Insights into an emerging class of cell surface proteolytic enzymes. J Biol Chem 276:857-60
Kalatzis V, Petit C (1998) The fundamental and medical impacts of recent progress in research on hereditary hearing loss. Hum Mol Genet 7:1589-97
Kim SY, Park D, Oh M, Sellamuthu S, Park WJ (2002) Detection of site-specific proteolysis in secretory pathways. Biochem Biophys Res Commun 296:419-24
Lee YJ, Park D, Kim SY, Park WJ (2003) Pathogenic mutations but not polymorphisms in congenital and childhood onset autosomal recessive deafness disrupt the proteolytic activity of TMPRSS3. J Med Genet 40:629-31
Lim DJ, Kalinec F (1998) Cell and molecular basis of hearing. Kidney IntSuppl 65:S104-13
Masmoudi S, Antonarakis SE, Schwede T, Ghorbel AM, Gratri M, Pappasavas MP, Drira M, Elgaied-Boulila A, Wattenhofer M, Rossier C, Scott HS, Ayadi H, Guipponi M (2001) Novel missense mutations of TMPRSS3 in two consanguineous Tunisian families with non-syndromic autosomal recessive deafness. Hum Mutat 18(2):101-8
Mese G, Londin E, Mui R, Brink PR, White TW (2004) Altered gating properties of functional Cx26 mutants associated with recessive non-syndromic hearing loss. Hum. Genet. 115:191-99
Morton NE (1991) Genetic epidemiology of hearing impairment. Ann N Y Acad Sci 630:16-31
Morton CC (2002) Genetic, genomics and gene discovery in the auditory system. Hum. Mol. Genet 11:1229-40
Nance WE, Sweeney A (1975) Symposium on sensorineural hearing loss in children: early detection and intervention.Genetic factors in deafness of early life. Otolaryngol Clin North Am 8:19-48
Petit C (1996) Genes responsible for human hereditary deafness: symphony of a thousand. Nat Genet 14:385-91
Resendes BL, Williamson RE, Morton CC (2001) At the speed of sound: gene discovery in the auditory system. Am J Hum Genet 69:923-35
Scott DA, Wang R, Kreman TM, Andrews M, McDonald JM, Bishop JR, Smith RJH, Karniski LP, Sheffield VC (2000) Functional differences of the PDS gene product are associated with phenotypic variation in patients with Pendred syndrome and non-syndromic hearing loss (DFNB4). Hum. Molec. Genet. 9: 1709-15
Scott HS, Kudoh J, Wattenhofer M, Shibuya K, Berry A, Chrast R, Guipponi M, Wang J, Kawasaki K, Asakawa S, Minoshima S, Younus F, Mehdi SQ, Radhakrishna U, Papasavvas MP, Gehrig C, Rossier C, Korostishevsky M, Gal A, Shimizu N, Bonne-Tamir B, Antonarakis SE (2001) Insertion of beta-satellite repeats identifies a transmembrane protease causing both congenital and childhood onset autosomal recessive deafness. Nature Genet 27:59-63
Tasaki I, Spyropoulos CS (1959) Stria vascularis as source of endocochlear potential. J Neurophysiol 22:149-55
Teubner B, Michel V, Pesch J, Lautermann J, Cohen-Salmon M, Sohl G, Jahnke K, Winterhager E, Herberhold C, Hardelin JP, Petit C, Willecke K (2003) Connexin30 (Gjb6)-deficiency causessevere hearing impairment and lack of endocochlear potential. Hum Mol Genet 12:13-21
Tekin M, Arnos KS, Pandya A (2001) Advances in hereditary deafness. Lancet 358:1082-90
Van CG, Smith RJ (2000) Maternally inherited hearing impairment 12. Clin Genet 57(6): 409
Veske A, Oehlmann R, Younus F, Mohyuddin A, Muller-Myhsok B, Mehdi SQ, Gal A (1996) Autosomal recessive non-syndromic deafness locus (DFNB8) maps on chromosome 21q22 in a large consanguineous kindred from Pakistan. Hum Mol Genet 5:165-168
Wangemann P, Liu J, Marcus DC (1995) Ion transport mechanisms responsible for K+ secretion and the transepithelial voltage across marginal cells of stria vascularis in vitro. Hear Res 84:19-29
Wattenhofer M, Di lorio MV, Rabionet R, Dougherty L, Pampanos A, Schwede T, Montserrat-Sentis B, Arbones ML, Iliades T, Pasquadibisceglie A, D’Amelio M, Alwan S, Rossier C, Dahl HH, Petersen MB, Estivill X, Gasparini P, Scott HS, Antonarakis SE (2002) Mutations in the TMPRSS3 gene are a rare cause of childhood nonsyndromic. J Mol Med 80:124-31
Wattenhofer M, Sahin-Calapoglu N, Andreasen D, Kalay E, Caylan R, Braillard B, Flower-Jaeger N, Reymond A, Rossier BC, Karaguzel A, Antonarakis SE (2005) A novel TMPRSS3 missense mutation in a DFNB8/10 family prevents proteolytic activation of the protein. Hum Genet 117:528-535
Willecke K, Eiberger J, Degen J, Eckardt D, Romualdi A, Guldenagel M, Deutsch U, Sohl G (2002) Structural and functional diversity of connexin genes in the mouse and human genome. Biol Chem 383:725-37
Yasunaga S, Grati M, Cohen-Salmon M, El-Amraoui A, Mustapha M, Salem N, El-Zir E, Loiselet J, Petit C (1999)A mutation in OTOF, encoding otoferlin, a FER-1-like protein, causes DFNB9, a nonsyndromic form of deafness. Nature Genet. 21: 363-69