KCNQ4基因在台灣地區非症候群聽障之研究

Study of the KCNQ4 gene of patients with non-syndromic deafness in Taiwan

蘇清泉(Ching-Chyuan Su)

指導教授 ： 李宣佑

中山醫學大學/醫學院/醫學研究所/博士(2007年)

https://doi.org/10.6834/CSMU.2007.00073

摘要

聽障可因遺傳基因突變或環境因素，或兩者兼之引起的。約有1/1000嬰兒在出生時或在小孩早期(即語言學習前時期prelingual period)罹患重度聽障(severe or profound)，在已開發國家約有60%個案是遺傳因素所造成的。到目前為止已知有59個基因發生突變會造成遺傳性聽障，各基因的致病機制不盡相同，非常複雜。本論文主要探討耳蝸內影響離子濃度的恆定性之KCNQ4基因突變與造成聽障的致病機制。藉由分析聽力正常和聽障的人來建立一個台灣地區KCNQ4基因的多形性和突變之資料庫，以瞭解KCNQ4基因突變在非症候群聽障所佔的比例，可作為聽障遺傳諮詢參考；另外藉由KCNQ4基因功能的探討來了解該基因在聽力形成中所扮演的角色。
本研究的結果，在185位非症候群聽障患者和100位聽力正常人的KCNQ4基因篩檢中，發現14個多型性(polymorphism)和3位聽障患者在KCNQ4基因發生突變，其突變所佔的比例為1.62% (3/185)。此三個突變點包括1個missense 突變 546C>G/wt (F182L)和2個Silent 突變 648C>T/wt (R216R)及1503C>T/wt (T501T)，其所佔的比例分別為0.54% (1/185)。F182是位於KCNQ4蛋白的S3 domain，且高度保留在各種動物的KCNQ4蛋白上。同時我們也利用了神經網絡預測系統(neural network prediction system)來分析silent突變是否會影響到mRNA的改變。在這分析中發現648C>T/wt (R216R)的silent突變會產生一個新的donor site。結果造成66個核苷酸的缺失，因而產生不完全的KCNQ4蛋白，可能造成蛋白質功能的缺失。
在KCNQ4功能研究，以顯微注射方法將正常KCNQ4 的cRNA注入 Xenopus oocytes和ionomycin (攜帶Ca2+的化合物)來研究KCNQ4鉀離子管道的功能，其結果發現當加入ionomycin時，可以提高KCNQ4鉀離子管道的電流為未加時的1.7倍。然而直接增加細胞內Ca2+濃度並不會提高KCNQ4鉀離子管道的電流。因此我們認為KCNQ4鉀離子管道的電流受Ca2+濃度的影響，並不是在細胞內Ca2+的流動，而可能是作用在細胞內KCNQ4蛋白厭水性(hydrophobic)的部位。另外，我們利用HeLa細胞來研究正常或突變的KCNQ4蛋白在細胞內之表現位置，本研究中共分析了KCNQ4基因的四個突變點(F182L、W276S、G321S 和T501T)。結果發現KCNQ4基因W276S、G321S 和T501T轉殖的HeLa細胞，其突變蛋白都和正常的KCNQ4蛋白一樣被送到細胞膜上。相對的F182L轉殖的HeLa細胞，其變異蛋白都堆積在細胞核周圍並無法被送到細胞膜。
綜合以上結果，本研究對KCNQ4基因在非症候群遺傳性聽障的成因和機制有更深入的瞭解，將可提供產前或產後基因檢查更精確的診斷，以利於聽障之預防或未來基因治療之發展。

關鍵字

非症候群性聽障 ； 基因突變 ； 功能研究

並列摘要

Hearing loss, caused by gene mutations and environmental factors, is a common sensory disorder in the human population. In the developed countries, the incidence of congenital hearing loss is estimated at 1 in 1000 births, of which approximately 60% cases are attributed to genetic factors. To date, 59 auditory genes have been identified, some of which are those involved in K+ recycling and maintenance. KCNQ4, a hearing associated gene, is part of the KCNQ family and the translated protein is part of the voltage gated potassium channel. However, in Taiwan, the data of KCNQ4 gene are still insufficient; therefore, further research is worthy to conduct. The overall goal of this thesis is to establish the genetic basis for the screening, diagnosis, and pathogenesis studies of KCNQ4 of non-syndromic deafness in Taiwan. In this study, we aim to determine the mutation sited of the KCNQ4 gene by using comparative genetic analysis between genomic DNA from normal individuals and hearing-impaired patients; and to understand etiology of KCNQ4 mutants in nonsyndromic deafness.
We present data of mutation analysis of KCNQ4 from 185 unrelated Taiwanese probands with non-syndromic hearing loss. The analysis revealed three novel KCNQ4 mutations and many polymorphisms. The prevalence of KCNQ4 gene mutations in this study was 1.62 % (3/185). The mutations include a missense mutation (F182L) and two silent mutations (R216R and T501T). The F182L missense mutation was located in the S3 domain of KCNQ4. The F182 residue of KCNQ4 is highly conserved in KCNQ4 among various species and is less conserved in all members of the KCNQ family. In addition, although R216R is a silent mutation and does not alter the content of amino acid residue, the neural network prediction system revealed that it can potentially create a novel splice donor site during transcription. This mutation might affect the protein structure of KCNQ4 and consequently the normal function of the K+ channel.
KCNQ4 channels expressed in Xenopus oocytes were measured using two-electrode voltage clamp. The significant increase of KCNQ4 outward current induced by ionomycin (calcium salt) is about 1.7-fold of control current amplitude at +60 mV. In contrast, direct cytoplasmic injection of calcium medium (up to 1 mM calcium, 50 nl) did not mimic the effect of ionomycin. In conclusion, the effect of ionomycin on enhancement of KCNQ4 current is independent of intracellular calcium mobilization and possibly acts on intramembrane hydrophobic site of KCNQ4 protein expressed in Xenopus oocytes.
Four mutations (F182L, W276S, G321S and T501T) of KCNQ4 gene were investigated in the subcellular localization of HeLa cell. Of the four mutations, W276S, G321S and T501T mutant resulted in a protein that was localized to the membrane. In contrast, the F182L mutation resulted in proteins with impaired trafficking and localized in perinuclear vesicles.
In summary, our study provides information for understanding the importance of genetic factors in nonsyndromic deafness of Taiwanese and that may be of use in the improvement of genetic diagnosis and genetic counseling for the families of hearing loss in Taiwan.

並列關鍵字

nonsyndromic deafness ； KCNQ4 ； mutation ； functional study

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