C型肝炎病毒核心蛋白基因多形性之臨床病理研究

Abstract

研究背景 慢性C型是引發肝硬化、肝癌的重要原因。C肝病毒基因轉譯出結構蛋白與非結構蛋白，其中結構蛋白分為核心蛋白及外套膜蛋白，核心蛋白已知會與許多人體細胞蛋白交互作用。進而影響細胞發炎反應、甚至致癌。此外，核心蛋白可以增加轉殖鼠之胰島素抗性，進而引發糖尿病。由於C肝病毒複製缺乏校正功能，基因容易產生變異。之前的研究顯示，若是C肝病毒核心蛋白胺基酸第70處由Glutamine取代Arginine（R70Q）及第91 處由Methionine取代Leucine（L91M），可能會使得患者接受干擾素合併雷巴威林治療之療效降低，較不易達到快速病毒學反應（RVR）及持續性病毒學反應（SVR）。而日本學者研究也發現C肝病毒核心蛋白胺基酸變異與胰島素抗性有關。針對核心蛋白變異進行研究，可更加了解C肝病毒干擾素抗藥性機轉及與胰島素抗性之相關性。 研究假說 本研究假設感染C型肝炎1b基因型病毒之核心蛋白胺基酸序列變異如R70Q及L91M之患者，於接受干擾素合併雷巴威林治療達到持續性病毒性反應（SVR）比例較低，且合併有較高的胰島素抗性。 研究方法 收集慢性C型肝炎基因型1b患者42 位，接受長效型干擾素合併雷巴威林治療24週，於治療前、第四週及治療完成後六個月檢測血清病毒量，紀錄是否達到RVR及SVR。治療前收集病患基本資料如年齡、性別、BMI，並抽血檢驗血球計數、空腹血糖、胰島素濃度等生化檢驗。病患簽署同意書後，進行肝臟穿刺取得肝組織，由病理學家針對肝纖維化及肝細胞脂肪堆積給予評分。利用PCR方式，將C肝病毒核心蛋白基因放大，取得DNA序列後進行比對，以檢測C肝病毒核心蛋白是否發生R70Q及L91M變異。利用統計分析軟體進行資料分析。 結果 C肝病毒核心蛋白胺基酸70若發生R70Q/H變異，與達到RVR及SVR的比例似乎無關，但R70Q/H變異的患者合併並嚴重胰島素抗性（HOMA-IR≧3.5）比例明顯較未變異組高（60% vs. 26%，P=0.047）。若C肝病毒核心蛋白胺基酸91處發生L91M變異，合併嚴重胰島素抗性比例也較高（56% vs. 25%，P=0.059）。若將R70Q/H與L91M變異與否做比較，雙重野生組達到RVR（72% vs. 42%，P=0.517）及SVR（77.8% vs. 62.5%，P=0.333）似乎較高，而變異組之胰島素抗性明顯高於雙重野生組（7.02±.43 vs. 2.19±1.26，P=0.01）。另多變項分析可知低病毒量者達到SVR機會為高病毒量者的8.7倍（95%CI 1.854-47.540，P=0.013），核心蛋白胺基酸70及91處發生變異亦為合併嚴重胰島素抗性的危險因子。 結論 C型肝炎病毒核心蛋白胺基酸序列第70個胺基酸若是發生R70Q/H變異，及合併第91個胺基酸發生L91M變異較野生型病毒之胰島素抗性（HOMA-IR）有明顯上升，但與血清病毒量、肝臟纖維化程度無關。起始血清病毒量較低、肝臟纖維化程度較輕、年齡≦50歲的病患，接受干擾素合併治療達到SVR機會較高。

Background Hepatitis C virus (HCV) infection is an important cause of liver cirrhosis and hepatocellular carcinoma（HCC）worldwide. HCV belongs to the Hepacivirus genus of the Flaviviridae family. It is a spherical particle, 55 to 65 nm in diameter, with an envelope, and consists of the genome of a 9.6-kb plus strand RNA. It has short untranslated regions at both the 5’- and 3’- termini, and the middle region, which accounts for about 95% of the whole genome, encodes a precursor protein consisting of ~3,010 amino acid residues. The coding regions of viral structural proteins such as core as well as E1 and E2 are located at the N-terminus, while the non-structural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B are situated at the middle region near the C-terminus. Among these viral proteins, core protein reacts with human proteins and bears multiple regulatory functions including the influence on inflammation and induction of HCC. Besides, it can induce insulin resistance in transgenic mice studies. Previous clinical studies from Japan suggested that patient with amino acid substitutions in HCV core region 70 of Glutamine for Arginine（R70Q）and 91 of Methionine for Leucine（L91M）have lower rates of rapid virologic response (RVR) and sustained virologic response (SVR), and HCV core gene polymorphisms are related to the development of insulin resistance in patients with chronic HCV infection. Materials and Methods We collected 42 Taiwanese chronic hepatitis C patients with genotype 1b infection. All of them received combination therapy of pegylated interferon alfa-2a plus ribavirin for 24 weeks. Blood samples were collected for complete blood count, fasting glucose, fasting insulin and liver enzymes, pre-treatment HCV RNA. Liver biopsy was also performed to determine the stage of hepatic fibrosis and steatosis. Serum HCV RNA level was assayed at week 4 and 24 of therapy to identify RVR and SVR. HCV core gene polymorphisms of amino acid 70 and 91 were analysed by direct sequencing after PCR amplification. Statistical analysis on the relationship among virologic response to antiviral therapy, insulin resistance and HCV core gene polymorphisms were done. Results Patients with HCV core gene R70Q/H substitution had a higher frequency of more severe insulin resistance（HOMA-IR≧3.5）than those without（60% vs. 26%，P=0.047）. Patients with L91M substitution also had similar results（56% vs. 25%，P=0.059）. Patients with double wild-type sequences had a higher SVR rate（77.8% vs. 62.5%，P=0.333) and lower insulin resistance（HOMA-IR 2.19±1.26 vs. 7.02±7.43）than those with non-double wild-type sequences. Multivariate analysis suggested that patients with lower viral load tended to achieve a higher SVR rate. In addition, HCV core gene L91M and/or R70Q/H substitution tended to have more severe insulin resistance（Odds ratio 15.598, 95%CI 2.202-110.480, P=0.006.） Conclusions HCV core gene R70Q and /or L91M substitutions are associated with more severe insulin resistance. HCV genotype 1b patients with lower viral load, younger age and lower fibrosis score have a higher likelihood to achieve SVR to combination therapy.