Positional Cloning of Putative Tumor Suppressor Genes for Liver Cancer on Chromosome 4q: Genomic and Genetic Approaches

The main purpose of this study is to find out the putative tumor suppressor gene(s) (TSG) on chromosome 4q, which may significantly contribute to the human hepatocarcinogenesis. Three approaches were proposed and we already got several preliminary results described as followed. To precisely define the region containing the putative TSGs, we approached by physical mapping and family based association/linkage analysis. Currently, we have finished the physical mapping in 149 pairs of HCC and their corresponding nontumorous tissues with 49 microsatellite markers of 1-2 cM marker density around 4q21~4q25. The results showed 101 out of 149 HCCs (66%) with allelic imbalance (AI) in this region. However, most cases showed aberrations extending outward of the whole region. Only 8 cases showed limited AI within this interval and the common LOH region has been identified within two intervals with genetic distance less than 10 cM each. The results of association analysis between 4q LOH and several clinical parameters indicated the presence of significant association among 4q LOH, HBV infection, and the elevation of serum alpha-fetoprotein (AFP). It suggests that 4q LOH may involve in the HBV-related hepatocarcinogenesis and the elevation of AFP may be one of the phenotype resulted by the possible synergistic effect of factors underlying the carcinogenic pathway. In the approach of family based linkage or association analysis, we have conducted the genotyping analysis in 250 HCC families (with 806 individuals). Currently, we have finished the genotyping with 62 markers covering the 4q11 to 4q28. The genotyping results have been transformed to the format suitable for TDT (in case parent triads) and linkage analysis (in multiplex HCC families). This part of patient collection and data analysis were collaborated with Dr. Yu MW at the School of Public Health of NTU. Te results indicated several chromosome regions with significant P values (< 0.05), including 4q22, 4q26, and 4q31. The distance of the regions are all less than 10 cM and supported by more than one neighboring markers. Intriguingly, one of the marker loci locates in the common LOH region defined by our deletion mapping, 4q21-23, supporting the region containing a putative TSG significantly associated with HBV related HCC. For the candidate gene approach and the subsequent functional analysis of plausible candidate genes, we have identified a possible candidate TSG, FAP-1. It showed several characteristics as a plausible TSG, including down-regulation in 50% of HCC and promoter hypermethylation in many HCCs. Its apoptosis promoting activity in Huh7 hepatoma cell line further supported its plausible role as the target TSG and warrants further consolidation.染色體4q 之對偶缺失好發於將近70%之肝細胞癌，此發現顯示此染色體區間可能存在一與肝癌之形成密切相關之抑癌基因。事實上已有多個證據支持此抑癌基因之存在及重要性：證據或者來自微細胞染色體轉移之功能補償研究；或者來自comparative genomics 之研究。因此尋找位於此染色體上之抑癌基因，將會對大部分肝癌之共同致癌機轉提供重要訊息。此計畫之主要目標即設定為尋找4q 上之抑癌基因，並希望在未來能對肝癌之診斷、預防、及治療提供重要線索。為了達到這個目標，本計劃提出三種不同的研究策略，均已具初步成果。 首先，我們將定義出含有抑癌基因之染色體區間。首先，我們將遵循傳統之位置選殖方法(positional cloning)，亦即利用微衛星標記進行對偶缺失分析，去找出最小共同缺失區間。另一方面我們將採用家族鏈結(Family linkage analysis)及家族相關分析(family association analysis)，來檢測涵蓋染色體4q 多型性標記是否與肝細胞癌之發生有所相關。目前我們利用49 個多型標記(4q21~4q25)已完成149 對肝癌及其相對正常組織之對偶缺失分析，結果顯示101例肝癌(66%)具有4q 染色體缺失。然大部分缺失均涵蓋大片斷染色體區間，其中只有8 例存 在較小之缺失區間，因而可定義出2 個共同缺失區間(< 10 cM)。進一步還發現染色體4q 之缺失與B 型肝炎病毒之感染，以及甲型胎兒蛋白之升高(＞400 ng/mL)有顯著相關。 在家族鏈鎖及相關分析方面，我們共計利用62 個多型標記(4q11~4q28)已完成250 個肝癌家族(共806 人)之分析。我們已針對基因型分析結果進行TDT 及家族鏈鎖分析(此部分病人家 族之收集及資料分析為與台灣大學公衛學院于明暉老師合作)，結果顯示3 個染色體區間具顯著意義，包括4q22，4q26，及4q31。其中4q22 與共同缺失區間重疊，進一步顯示此區間可 能存在一抑癌基因。 至於候選基因之研究，我們已發現一個可能之候選基因，FAP-1，於此染色體區間內，此基因之表現在將近50%的肝癌有顯著下降之趨勢，且其啟動子具甲基化之情形，我僴將進一步在未來針對此基因進行功能性分析