非症候群聽障TMPRSS3 基因突變及功能研究

Study of the TMPRSS3 gene of patients with non-syndromic deafness

楊舒婷(Shu-Ting Yang)

指導教授 ： 李宣佑

中山醫學大學/醫學院/醫學研究所/碩士(2007年)

https://doi.org/10.6834/CSMU.2007.00124

摘要

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。

關鍵字

聽障 ； 酵母菌

並列摘要

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.

並列關鍵字

deafness ； TNPRSS3

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