李啟明臺灣大學：臨床藥學研究所胡馨Hu, HsinHsinHu2007-11-272018-07-092007-11-272018-07-092006http://ntur.lib.ntu.edu.tw//handle/246246/55408背景：中國心臟衰竭病患對於β-腎上腺素受體拮抗劑之耐受劑量較西方國家低，藥物對不同病患之間的療效亦具有差異性。雖然曾有研究指出，β1-腎上腺素受體基因密碼子49及389之多型性可能造成β-腎上腺素受體拮抗劑耐受度及療效之不同，其對偶基因之分布在中國人和西方人之間並無差異。目前尚無亞洲人β1-腎上腺素受體基因全長定序之研究。 目的：本研究欲探討中國人之β1-腎上腺素受體基因中是否有其他位置發生基因多型性，導致我們與西方人對β-腎上腺素受體拮抗劑耐受度之不同；並探討其他對於β-腎上腺素受體拮抗劑的耐受度及療效之預測因子。 方法：我們針對69位目前正在使用，或曾經使用過carvedilol, metoprolol succinate,或bisoprolol之心臟衰竭病人之血液樣本進行β1-腎上腺素受體基因全長定序。此外，我們回溯性地收集病人開始服用β-腎上腺素受體拮抗劑之前，以及增加至穩定劑量後之各種臨床參數(例如病史、心臟衰竭原因、併用藥物、左心室射出分率等等)以進行分析。 結果：不同心臟衰竭原因之病人對於β-腎上腺素受體拮抗劑之耐受劑量有顯著的差異(p = 0.047)。擴張性心肌病變患者之耐受劑量較瓣膜性心臟病患顯著為高(分別為14.589 ± 8.882及5.357 ± 7.594 毫克/天，p = 0.018)，亦傾向高於冠狀動脈心臟病患者(9.544 ± 5.558毫克/天，p = 0.054)。多元逐步迴歸分析顯示擴張性心肌病變(B = 4.852, p = 0.007)為β-腎上腺素受體拮抗劑耐受劑量之顯著預測因子；而擴張性心肌病變(B = 9.015, p = 0.011)及服藥前之左心室射出分率(B = - 0.503, p < 0.001)為左心室收縮功能改善之顯著預測因子。次族群分析(26位服藥前左心室射出分率≦35%之冠狀動脈心臟病及擴張性心肌病變患者)顯示低劑量之carvedilol (12.464 ± 7.977 毫克/天)可顯著改善病患之左心室射出分率(13.49 ± 16.66 %，p < 0.001)。我們並未在β1-腎上腺素受體基因之轉錄區發現新的基因多型性；密碼子49及389之對偶基因分布與文獻報告相符，並且與β-腎上腺素受體拮抗劑耐受性及療效之間沒有顯著關聯。 結論：低劑量之β-腎上腺素受體拮抗劑即可對中國心臟衰竭的病人帶來助益。此外，擴張性心肌病變患者可耐受較高劑量的β-腎上腺素受體拮抗劑；而擴張性心肌病變及服藥前左心室射出分率較低之病患，其服藥後左心室收縮功能改善之幅度較大。我們並未在β1-腎上腺素受體基因之轉錄區發現新的基因多型性；而密碼子49及389之對偶基因分布亦與文獻報告相符。Background: The tolerated doses of β-blockers in Chinese heart failure (HF) patients were substantially lower than those wildly used in the Western countries, and the therapeutic response was heterogeneous. Although the mechanism responsible for the various tolerability and response was often attributed to β1-adrenoceptor (β1-AR) genetic polymorphisms at codon 49 and 389, their allele distribution was similar between Chinese and Caucasian, and β1-AR genetic full length sequencing has not been performed in Asian population. Objective: This study aimed to examine whether there are novel polymorphic loci in the β1-AR gene responsible for the difference in tolerability to β-blockers between Chinese and Caucasian HF patients, and to assess for other clinical predictors of tolerability and therapeutic response to β-blockers in Chinese population. Methods: We performed full length sequencing for β1-AR in 69 patients who had the diagnosis of chronic HF and were using, or have used, one of the three β-blockers carvedilol, metoprolol succinate, and bisoprolol. Clinical parameters including comorbidities, HF etiology, concurrent medication, left ventricular ejection fraction (LVEF) before the initiation and at the stable dose of β-blocker were retrieved from the medical records or inquiries into the patients. Results: Tolerated carvedilol stable dose was significantly different by HF etiology (p = 0.047). The stable dose of carvedilol for patients with dilated cardiomyopathy (DCMP) as the etiology of HF was higher than that of valvular heart disease (VHD) patients (14.589 ± 8.882 and 5.357 ± 7.594 mg/day, respectively, p = 0.018), and also tended to be higher than that of coronary artery disease (CAD) patients (9.544 ± 5.558 mg/day for CAD, p = 0.054). Multiple stepwise regression revealed that DCMP (B = 4.852, p = 0.007) was the only significant predictor of stable carvedilol dose; DCMP (B = 9.015, p = 0.011) and baseline LVEF (B = - 0.503, p < 0.001) appeared to be the significant predictors of LVEF improvement. In a subgroup analysis of 26 patients with CAD or DCMP (baseline LVEF≦35%), low-dose carvedilol (12.464 ± 7.977 mg / day) produced a significant improvement in LVEF (13.49 ± 16.66 %, p < 0.001). Neither novel variants other than the reported codon 49 and codon 389 within the coding region, nor correlation between β1-AR gene diplotypes and tolerability or response was found. The allele distributions of codon 49 and 389 were similar to those reported previously in the Chinese and Caucasian populations. Conclusion: Low-dose β-blocker therapy improved left ventricular function in Chinese patients with HF. Moreover, DCMP patients tolerated better to β-blocker, and those with DCMP as the etiology of HF or those had lower LVEF at baseline gained a greater improvement. For the β1-AR gene polymorphism, neither novel variant within the coding region, nor a different distribution pattern of alleles at codon 49 and codon 389 was noted.Contents -------------------------------------------------------------------------- i Table Contents --------------------------------------------------------------------------------- iii Figure Contents -------------------------------------------------------------------------------- iv 摘要 ------------------------------------------------------------------------------- v Abstract ------------------------------------------------------------------------ viii Chapter 1 Introduction --------------------------------------------------- 1 Section 1 Review of Literature ----------------------------------------------------------- 1 1 Chronic Heart Failure and β-blocker Therapy -------------------------------------- 1 2 Pharmacological Properties of the Three β-blockers Available for Clinical Use in Heart Failure Patients ----------------------------------------------- 3 3 β1-Adrenoceptor Polymorphisms and β-blocker Therapy ------------------------- 4 Section 2 Research Motivation ----------------------------------------------------------- 7 Section 3 Research Purpose --------------------------------------------------------------- 8 Chapter 2 Materials and Methods -------------------------------------- 9 Section 1 Study Population ---------------------------------------------------------------- 9 Section 2 Study Design -------------------------------------------------------------------- 9 Section 3 DNA Preparation -------------------------------------------------------------- 10 Section 4 Polymerase Chain Reaction ------------------------------------------------- 12 Section 5 Polymerase Chain Reaction Product Purification ------------------------ 14 Section 6 Sequence Analysis ------------------------------------------------------------ 15 Section 7 Statistical Analysis ------------------------------------------------------------ 15 Chapter 3 Results --------------------------------------------------------- 17 Section 1 β1-Adrenoceptor Gene Polymorphisms ------------------------------------ 17 Section 2 Tolerability --------------------------------------------------------------------- 18 1 Carvedilol Stable Dose as a Categorical Variable -------------------------------- 18 2 Carvedilol Stable Dose as a Continuous Variable -------------------------------- 18 (1) Relationships between Heart Failure Etiology and Carvedilol Dose ------ 18 (2) Multiple Regression -------------------------------------------------------------- 18 Section 3 Response ---------------------------------------------------------------------- 20 1 Comparison with the MOCHA Trial ------------------------------------------------ 20 2 Relationship between Tolerability and Response --------------------------------- 20 3 Multiple Regression ------------------------------------------------------------------- 21 Section 4 Relationships between β1-Adrenoceptor Genetic Polymorphisms and Carvedilol Dose and Left Ventricular Ejection Fraction Improvement --------------------------------------------- 22 Chapter 4 Discussion ----------------------------------------------------- 23 Section 1 Relationships between Gene Polymorphisms and Clinical Observation ---------------------------------------------------------- 23 Section 2 Tolerability -------------------------------------------------------------------- 24 Section 3 Response ---------------------------------------------------------------------- 26 1 Comparison with MOCHA Trial ----------------------------------------------------- 26 2 Dilated Cardiomyopathy as a Predictor of Left Ventricular Ejection Fraction Improvement ------------------------------------------------------ 28 3 Baseline Left Ventricular Ejection Fraction as a Predictor of Left Ventricular Ejection Fraction Improvement ---------------------------------------- 29 Section 4 Limitations -------------------------------------------------------------------- 30 Chapter 5 Conclusion ---------------------------------------------------- 31 References ---------------------------------------------------------------------- 32 Table Contents Table 1. Primers for polymerase chain reaction ------------------------- 41 Table 2. Primers for sequencing ------------------------------------------- 41 Table 3. Primers for forward confirmation ----------------------------- 41 Table 4. Baseline characteristics stratified by carvedilol tolerability - 42 Table 5. Genotypes and diplotypes of β1-adrenoceptor gene stratified by carvedilol tolerability ----------------------------- 44 Table 6. Regression analysis for carvedilol dose ------------------------ 45 Table 7a. Comparison of the tolerability and response to carvedilol between the present study and the MOCHA trial ------------- 46 Table 7b. Responses to carvedilol of the subgroup of patients compared with the MOCHA trial ------------------------------- 47 Table 8a. Response to carvedilol therapy according to tolerability ---- 48 Table 8b. Response to carvedilol therapy according to tolerability ---- 49 Table 9. Regression analysis for left ventricular ejection fraction improvement ---------------------------------- 50 Table 10. Baseline characteristics stratified by diplotypes -------------- 51 Figure Contents Figure 1. Electrophoresis of the polymerase chain reaction products- 53 Figure 2. Electrophoresis of the purified polymerase chain reaction products ------------------------------------------ 53 Figure 3. β1-adrenoceptor map -------------------------------------------- 54 Figure 4. Chromatograms of the β1-adrenoceptor gene polymorphisms at codon 49 and 389 -------------------------- 55 Figure 5. Chromatogram of a new β1-adrenoceptor gene polymorphism in the 3’ untranslated region ------------------ 56 Figure 6. Carvedilol stable dose by etiology ----------------------------- 57 Figure 7. Relationship between β1-adrenoceptor diplotype and carvedilol stable dose -------------------------------------------- 58 Figure 8. Relationship between β1-adrenoceptor diplotype and improvement in left ventricular ejection fraction ------------ 59579355 bytesapplication/pdfen-US心臟衰竭拮抗劑β1-腎上腺素受體基因多型性耐受度療效Heart failureβ-blockerβ1-adrenoceptorgene polymorphismtolerabilitytherapeutic responsetexthttp://ntur.lib.ntu.edu.tw/bitstream/246246/55408/1/ntu-95-R93451008-1.pdf