肝細胞癌家族4號與13號染色體長臂之遺傳研究

Abstract

背景 肝細胞癌家族聚集研究與分離率分析結果顯示遺傳因子參與家族性肝細胞癌致病機轉。在與B型肝炎相關的肝細胞癌中已發現4號染色體長臂與13號染色體長臂有高比率的缺失（loss-of-heterozygosity）現象，暗示其帶有之遺傳因子可能與B型肝炎相關肝細胞癌有關。本研究利用階段分析策略（hierarchical strategy），以連鎖分析與連鎖不平衡方法進行4號染色體長臂與13號染色體長臂上肝細胞癌易感性基因位置定位。 研究個案與方法 我們以連鎖分析與家族相關性研究方法進行第一階段研究，後續再以一獨立選樣之病例對照樣本進行第二階段的相關性驗證分析。第一階段研究以74個多發病例家族與166個單發病例家族，其中86.7%病例個案為B型肝炎帶原者，進行母數與無母數方法之連鎖分析。相關性研究則是以家族不平衡檢定（pedigree disequilibrium test，PDT）方法進行。第二階段則是以855名B型肝炎表面抗原陽性之肝細胞癌病例與875名B型肝炎表面抗原陽性之對照個案，針對第一階段所發現之可能候選區域內的單核甘多型性標記（single-nucleotide polymorphism，SNP），利用羅吉斯（logistic）與Cox迴歸方法分析其和肝細胞癌發生的相關性及與肝細胞癌或肝硬化死亡相關之存活分析。 結果 連鎖分析分別於4q25（於微衛星標記D4S3240，hetergeneity LOD score=3.14）、13q12.13-13.1、13q14.3-21.31與13q31.1-32.2 (高峰位於131q31.3上的微衛星標記D13S886，MOD score=3.73 ) 發現與肝細胞癌可能相關的連鎖訊息。針對4q25連鎖高峰下6.16-cM範圍的精確定位分析，在包含PAPSS1基因的兩個相鄰的單套體區塊（haplotype block）發現10個與肝細胞癌有顯著相關的單核甘多型性標記（PDT分析結果之empirical P-value介於0.0002~0.0045間）。PAPSS1蛋白質主要是參與體內許多物質的硫酸鹽化（sulfation）。以罹病手足資料進行genotype identity-by-descent sharing test，分析結果顯示，其中6個標記可解釋部分連鎖分析於4q25所發現的連鎖訊息。病例對照研究亦顯示，位於PAPSS1基因區域內的單核甘多型性標記與肝細胞癌發生有顯著的相關性，此一顯著相關性尤見於早發的肝細胞癌病例。此外，針對腫瘤≤2 cm的肝細胞癌病患進行的存活分析發現，帶有由4個單核甘多型性標記所構成的一常見單套體，其存活結果會顯著較差（調整風險比值 [hazard ratio] 為1.71，95％信賴區間為1.10-2.65）。此組成單套體之對偶基因在PDT分析中亦呈現過度傳遞現象（overtransmission）。 結論 位於PAPSS1基因內的遺傳變異在B型肝炎病毒相關之肝細胞癌致病過程，可能扮演一定角色。未來亦應針對13q31.3區域，進行候選基因或精細定位分析研究。

Background Studies of familial aggregation of hepatocellular carcinoma (HCC) and segregation analyses have suggested existence of genetic component in the etiology of familial HCC. High rates of loss-of-heterozygosity on chromosome 4q and 13q have been observed in hepatitis B-related HCC. The objective of this study was to use a hierarchical strategy with linkage and linkage disequilibrium (LD) approaches to identify HCC-susceptibility loci on chromosome 4q and 13q. Subjects and Methods We performed a two-stage genetic study in which linkage and family-based association mapping was followed up by association and replication studies in an independent sample of unrelated cases and controls. For stage 1, data were from 74 multiplex families and 166 singleton families. The majority of the affected individuals (86.7%) in the family sample were hepatitis B surface antigen (HBsAg) carriers. We applied model-based and model-free linkage analyses, as well as the pedigree disequilibrium test (PDT). For stage 2, promising single-nucleotide polymorphisms (SNPs) identified by stage 1 were examined in an independent set of 855 HBsAg-positive cases and 875 HBsAg-positive controls. Logistic and Cox regression analyses were used to study the relationship of SNPs representative of the implicated region with HCC development and with disease-specific survival determined in terms of HCC or cirrhosis deaths. Results We found evidence of linkage to microsatellite markers on 4q25 (peak multipoint heterogeneity LOD score =3.12 at marker D4S3240), 13q12.13-13.1, 13q14.3-21.31, and 13q31.1-32.2 (peak multipoint maximized LOD (MOD) =3.73 at marker D13S886 in 13q31.3). Fine mapping applied to the 4q25 region with SNPs across a 6.16-cM interval under peak resulted in significant association in ten SNPs (empirical P-value range: 0.0002 to 0.0045 from the PDT) from two adjacent haplotype blocks within or flanking the PAPSS1 gene, which encodes an enzyme involved in sulfation of a broad range of compounds. Our affected-sibship data and the genotype identity-by-descent sharing test suggest that six of these ten SNPs can account in part for the observed linkage signal. In the case-control analyses, multiple statistically significant SNP associations were identified in the PAPSS1 gene, particularly for the young-onset cases aged <45 years. In addition, the presence of a common haplotype containing the same alleles at four SNPs, which revealed overtransmission to HCC subjects in the PDT analyses, were associated with poor survival among patients with small tumor present at hospital admission (adjusted hazard ratio= 1.71; 95% confidence interval=1.10 to 2.65). Conclusions Genetic variation in the PAPSS1 gene may play a role in the pathogenesis of HBV-related HCC. Further fine-mapping studies or positional candidate gene studies on 13q31.3 are warranted.