Haptoglobin 於分子生物學及流行病學之研究

Abstract

第一部分摘要 Haptoglobin (Hp) 具有兩個對偶基因 (allele) 為Hp 1 及Hp 2 ， 可產生三種表現型 (phenotype : 1-1、2-1及2-2)。臨床上認為不同表現型與發炎相關病症有關連性。同合配子 (homozygote) 之Hp1-1或2-2分別產生α1β或α2β之mRNA。本研究突破性發現，於人類周邊血液單核細胞進行Hp表現型鑑定時，發現Hp2-2型竟會產生α1β mRNA，不同於已知只有Hp1-1型產生α1β mRNA。預料之外產生α1β mRNA具有一個獨特變異，在第431位置缺少一個核苷酸 (腺嘌呤，adenine)，於轉譯成氨基酸序列時，隨即於第439至441位置提前產生一個終止密碼子 (premature stop codon，UGA)，以致Hp2-2無法形成完整Hp1-1蛋白質。本研究發現具有變異之α1β mRNA會經由nonsense-mediated mRNA degradation (NMD) 機制使mRNA快速降解，以防止細胞產生具有危害性之截斷式蛋白質。同時，發現於α2β mRNA構形上會形成stem-loop結構，造成會splicing產生α1β mRNA。故本研究認為Hp2-2型個體試圖利用alternative splicing機制以產生Hp1-1分子，卻因為一個核苷酸的缺失而無法成功產生Hp1-1蛋白質。這項發現可解釋為何在血液中Hp2-2型濃度相較於其他表現型之濃度為低 (Hp2-2 < 2-1 < 1-1)。 第二部分摘要 肝癌為前三高發生率癌症之一，高居國內十大癌症死因前2名。近年於日、法、英、美等國，肝癌發生率具有上升趨勢，由於肝癌早期臨床症狀不明顯造成診斷困難，使得病患預後情況不甚良好。 Haptoglobin (Hp) 是一種抗發炎反應之蛋白分子，當肝臟受損害、發炎時，肝臟將會大量產生Hp，故可反映出肝臟是否處於損傷狀態，因此Hp為診斷肝臟相關疾病之潛力標的。本研究著重探討Hp不同表現型 (1-1, 2-1, and 2-2) 及表現量於肝癌病患與各種臨床指數及表現之關連性，包括腫瘤大小、腫瘤數目、alpha fetoprotein (AFP)及alanine aminotransferase (ALT)。首次發現Hp 2-2肝癌患者中Hp濃度較高者具有顯著高死亡率且預後情形不甚良好，而其他表現型患者並無顯著差異。另有重大發現為肝癌病患Hp濃度與腫瘤之大小(P<0.05)、數目(P<0.05)、AFP(P<0.05)皆呈正相關性，而Hp濃度與ALT呈負相關(P<0.05)。故測定Hp表現型及濃度將來可應用於肝癌早期診斷及治療上。Hp於肝癌之致病機制及預後扮演具影響力之角色。

Part I Abstract Human haptoglobin (Hp) phenotypes (1-1, 2-1, and 2-2) are genetically determined by two alleles: Hp1 and Hp2. Clinically, these phenotypes are associated with the inflammation related diseases. Homozygote subjects Hp 1-1 or 2-2 is defined by their allele producing α1β or α2β mRNA, respectively. Remarkable interestingly, using peripheral blood mononuclear cells for Hp genotyping, we show that Hp 2-2 subjects were able to produce α1β mRNA thought to be only present in Hp 1-1 subjects. The unexpected α1β mRNA (nucleotides 1-1043), however, was found not to make a complete copy of Hp 1-1 protein owing to a single nucleotide deletion at nucleotide 431 (A), which immediately resulted in a premature stop codon (UGA) at nucleotide 439 - 441. This mutant α1β mRNA was identified rapidly degraded by nonsense-mediated mRNA degradation pathway for decreasing its mRNA levels and preventing production of truncated protein in the cells. We also identified that a stem-loop formation in the conformation of α2β mRNA that might be responsible for its splicing into α1β. Therefore, we postulate that Hp2-2 subjects attempt to make Hp1-1 molecules through the alternative splicing mechanism, but fail to accomplish it by a single nucleotide deletion. This finding may explain why the Hp concentrations of Hp 2-2 subjects are differently lower than that of other phenotypes (Hp2-2 < 2-1 < 1-1). Part II Abstract Liver cancer is the third leading cause of cancer mortality worldwide and ranks first or second in Taiwan. Recently, the incidence of liver cancer has been found to be increasing in countries such as Japan, France, the United Kingdom, and the United States, while the overall prognosis of liver cancer patients remains dismal because of the late clinical presentation and diagnosis. Plasma haptoglobin (Hp), an anti-inflammatory protein, is elevated in response to liver injury, infection and malignancy, as a potential biomarker in the diagnosis of liver disease. In the present study, we investigated the relationship of three phenotypes of Hp (namely 1-1, 2-1, and 2-2) and their concentrations in hepatoma patients with the clinical biochemical factors, including tumor size, tumor number, alpha fetoprotein (AFP), and alanine aminotransferase (ALT). We identified that Hp 2-2 patients with high levels of Hp had significantly higher mortality and unfavorable prognosis than those with other Hp types. There was a significant positive correlation between Hp levels and liver tumor size (P<0.05), tumor number (P<0.05), and AFP (P<0.05), but was negative with ALT (P<0.05). Therefore, Hp phenotype and its concentrations become an essential determinant in patients with hepatoma. Hp may play a provocative role in the pathogenesis and prognosis of hepatoma.