應用高解析熔點法分析遺傳性疾病

Molecular Diagnosis of Genetic Diseases by High-Resolution Melting Analysis

余志強(TZE-KIONG ER)

指導教授 ： 鐘育志

共同指導教授 ： 張建國 ； 張慧秋(HUI-CHIU CHANG)

高雄醫學大學/醫學院/醫學研究所/博士(2011年)

摘要

近年來於基因體學中，研究特定點突變對於疾病的相關聯性以及判定遺傳性疾病基因型成為一重要的方向。因此，在生命科學研究領域中必須積極尋找一個可以快速篩選突變的方法。高解析熔點法(High-resolution melting，简稱HRM)是一門新興的分析技術並且可以在節省時間及成本的前提下進行。HRM是一種相對於傳統的突變分析而言，操作步骤大大簡化而且檢體經過PCR擴增後直接進行HRM分析，無需再轉移至其他裝置做進一步分析，真正實現了同一管的閉管操作(close-tube)，降低了污染的風險。基於其易用性、簡單操作、成本低、高敏感性、高特異性，HRM已經成為首選的基因突變篩選技術。本研究主要目的為開發HRM分析基因突變，其中包括JAK2 V617錯義突變和ETFDH基因突變。希望藉由HRM這個分析方法可以做為臨床上基因診斷的依據。

詹納斯氏激酶2(Janus kinase 2, JAK2)是一種酪氨酸激酶，主要是扮演傳達訊息的角色。當細胞膜上的受體接受到細胞激素或其他成長因子，如紅血球生成素、血小板生成素及顆粒球生長激素時，JAK2會活化，並藉由一系列的磷酸化反應與訊息傳遞及轉錄活化的傳遞路徑，將訊號傳遞至細胞核內，藉以調節細胞的增生或暫時中止增生。 JAK2基因第14個外顯子的1849核苷酸位置G改變成T導致JAK2蛋白的617位置的氨基酸位置由苯丙氨酸(phenyalanine)替代纈氨酸(valine) (V617F)。目前， JAK2 V617F基因突變常發生在骨髓增生性疾病(myeloproliferative disorders, MPDs)，例如血小板增多症(essential thrombocytosis)，真性紅細胞增多症(polycythemia vera)，原發性骨髓纖維化(primary myelofibrosis)。利用分子診斷鑑定特殊的MPDs，尤其是JAK2 V617F，可以更進一步了解疾病的預後與治療方向。我們研究的目的主要是利用HRM鑑別JAK2 V617F基因突變。我們的研究結果顯示，利用HRM在MPDs患者可以測定百分之五以上的JAK2 V617F mutant allele。HRM與擴增受阻突變系統聚合酶鏈反應(amplification-refractory mutation system, ARMS)技術互相比較其結果一致。總而言之，HRM是一個快速和有效的分析方法以檢測 JAK2 V617F錯義突變。

多發性醯基輔酶A去氫酶缺乏(Multiple Acyl-CoA Dehydrogenase Deficiency, MADD)或戊二酸血症第二型，是一種體隱性遺傳疾病，因粒線體電子傳遞系統和脂肪酸代謝缺陷所造成的。最近，ETFDH基因突變是核黃素反應 MADD的主要成因。本研究主要目的是利用HRM分析ETFDH基因突變。我們的研究結果顯示，高分辨率熔點法可以分析已知ETFDH基因突變檢測 其中包括(c.250G>A、c.380T>A、c.524G>T)和一個新突變點(c.1831G>A)。我們估計其中c.250G>A為台灣MADD的熱點突變(hotspot mutation)，其發生率大約為 1:125（0.8%）。總而言之，我們成功地應用HRM可以準確分析ETFDH基因突變。由於核黃素反應 MADD(Riboflavin-responsive MADD, RR-MADD)通常是早期診斷早期治療，因此ETFDH基因突變分析對於RR-MADD病患之早期診斷扮演了重要的角色。

我們所發現的ETFDH基因突變點中有兩個突變(p.Ala84Thr和p.Phr128Ser)位於蛋白結構的黃素腺鹼二核脢酸(Flavin Adenine Dinucleotide, FAD)結合結構區。但是，根據ETF：QO的三級結構預測，這兩種氨基酸並沒有直接與 FAD相互作用。因此，我們利用分子動力學(Molecular Dynamics, MD)探討野生型及突變型之間的差異。除了利用MD，我們亦利用正模分析法(Normal mode analysis, NMA)探討突變位點和FAD結合結構域的動態相關性。利用MD和NMA，我們發現 p.Ala84Thr和p.Phe128Ser突變，可能會影響 FAD的結合位點附近的蛋白質結構因而影響ETF:QO蛋白質的活化。NMA研究顯示目前一些已被發表的致病突變點與 FAD的結合位點高度相關性。根據目前的研究結果，我們相信ETFDH基因突變所造成的胺基酸改變，可能影響FAD結合的穩定性而造成MADD。

關鍵字

高解析熔點法 ； 詹納斯氏激酶2 ； 多發性醯基輔酶A去氫酶缺乏 ； 黃素腺鹼二核脢酸 ； 分子動力學 ； 正模分析法

並列摘要

Identifying and understanding genetic variation between populations and individuals is an important aim in the field of genomics. The unique genetic profile of an individual confers susceptibility to a given trait or disease. Therefore, there is a rapidly growing interest in feasible methods for mutation screening in life science research. High-resolution melting (HRM) analysis represents the next generation of mutation scanning technology and offers considerable time and cost savings prior to other screening method. HRM is a novel, homogeneous, close-tube, post-PCR method, enabling researchers to analyze genetic variations in PCR amplicons. Samples can be discriminated according to their sequence, length, GC content or strand complementarity. Based on its ease of use, simplicity, flexibility, low cost, nondestructive nature, high sensitivity, and specificity, HRM analysis is quickly becoming the tool of choice to screen patients for pathogenic variants. Here we briefly discuss the establishment of HRM analysis for mutation screening including JAK2 V617 missense mutation and ETFDH gene mutation. Taken together, HRM analysis can be used for high-throughput mutation screening for research, as well as for molecular diagnostic and clinical purposes.

Janus kinase 2 (JAK2) is a tyrosine kinase involved in the cytokine signaling of several growth factors such as erythropoietin and thrombopoietin in normal and neoplastic cells. The G to T exchange at nucleotide 1849 in exon 14 of the JAK2 gene leads to a substitution of valine with phenylalanine at the amino acid position 617 (V617F) of the JAK2 protein. Currently, the occurrence of the JAK2 V617F mutation is well recognized in myeloproliferative disorders (MPDs), such as essential thrombocytosis, polycythemia vera, and primary myelofibrosis. The identification of molecular lesions specific to the myeloproliferative neoplasms, in particular JAK2 V617F, has broadened understanding of the common features within these disorders and has advanced diagnostic, prognostic, and therapeutic tools. The aim of our study was to assess the value of the HRM analysis using real-time polymerase chain reaction (PCR) (Lightcycler® 480; Roche Applied Science) for identifying the JAK2 V617F missense mutation. Our results showed that up to 5% of the JAK2 V617F mutation was successfully detected in patients with MPD using HRM analysis. The results proved 100% comparable to those obtained by ARMS assay. In conclusion, the HRM analysis is a rapid and effective technique for the detection of JAK2 V617F missense mutation.

Multiple acyl-CoA dehydrogenase deficiency (MADD) or glutaric aciduria type II is an autosomal recessive disease caused by defects in mitochondrial electron transfer system and metabolism of fatty acid. Recently, ETFDH mutations were reported to be major causes of riboflavin-responsive MADD. The present study is aimed at screening ETFDH mutations by HRM analysis. Our results showed that HRM analysis proved to be feasible in detecting 3 known (c.250G>A, c.380T>A, c.524G>T) and 1 novel (c.1831G>A) ETFDH mutations. Each mutation could be readily and accurately identified in the difference plot curves. We estimated the carrier frequency of the hotspot mutation, c.250G>A, in the Taiwanese population to be 1:125 (0.8%). In summary, HRM analysis can be successfully applied to screen ETFDH mutations. Since riboflavin-responsive MADD is often treatable, especially with mutations in ETFDH, identifying ETFDH mutations is crucial for these patients.

Interestingly, two of the mutations (p.Ala84Thr and p.Phr128Ser) are located in the FAD-binding domain; however, the two amino acids do not have direct interactions with FAD according to the predicted 3D structure of ETF:QO. Therefore, to explore the effects of the mutations on ETF:QO dynamics, molecular dynamics (MD) simulations of the wild type (WT) and mutant type (MT) ETF:QO in the same model environment were compared. Besides the MD simulations, an alternative method, normal mode analysis (NMA), for studying protein motions was used to analyze the dynamic correlations between the mutation sites and the FAD-binding motif. Using molecular dynamics (MD) simulations and normal mode analysis (NMA), we found that the p.Ala84Thr and p.Phe128Ser mutations are most likely to alter the protein structure near the FAD binding site as well as disrupt the stability of the FAD binding required for the activation of ETF:QO. Intriguingly, NMA revealed that several reported disease-causing mutations in the ETF:QO protein show highly correlated motions with the FAD-binding site. Based on the present findings, we conclude that the changes made to the amino acids in ETF:QO are likely to influence the FAD-binding stability.

並列關鍵字

High-resolution melting ； Janus kinase 2 ； Multiple acyl-CoA dehydrogenase deficiency ； Flavin adenine dinucleotide ； Molecular dynamics ； Normal mode analysis

參考文獻

Chapter 1

[1] Nguyen-Dumont T, Calvez-Kelm FL, Forey N and et al. Description and validation of high-throughput simultaneous genotyping and mutation scaning by high-resolution melting curve analysis. Hum Mutat 2009;30:884-90.

[2] Santos PC, Soares RA, Nascimento RM and et al. SLCO1B1 rs4149056 polymorphism associated with statin-induced myopathy is differently distributed according to ethnicity in the Brazilian general population: Amerindians as a high risk ethic group. BMC Med Genet 2011;12136.

[3] Chang CC, Lin PC, Lin CH and et al. Rapid identification of CYP2C8 polymorphism by high resolution melting analysis. Clin Chim Acta 2012;413:298-302.

[4] Tai CL, Liu MY, Yu HC and et al. The use of high resolution melting analysis to detect Fabry mutations in heterozygous females via dry bloodspots. Clin Chim Acta 2012;413:422-7.