行政院國家科學委員會專題研究計畫成果報告
大腸直腸癌之基因與環境交互作用
計畫編號:NSC 91-2314-13-002-345
執行期限:91 年 8 月 1 日至 92 年 7 月 31 日
主持人:梁金銅 台大醫院 外科
中文摘要 大腸直腸癌的致癌過程牽涉到「基因與環境的交互作用」。本研究室以早發性大腸直 腸癌(發病年齡小於 40 歲者)為模型來進一步闡明「基因與環境」交互作用的機轉。 我們先前的研究指出發病年齡小於 40 歲與大於 60 歲者相較,其有較高的機會出現 MSI-H (high-frequency microsatellite instability),其比例分別為 29.4﹪與 6.3﹪(p<0.001)。 然而進一步追蹤這二組病患的家族史卻顯示二組並無差別,分別是 17.4﹪與 14.3﹪ (p=0.859)。此結果意味著早發性大腸直腸癌的產生是由於環境的作用,而非來自遺 傳。在本研究中我們共收集 54 位年齡小於 40 歲的早發性大腸直腸癌且表現 MSI-H 的病 患作分析。實驗步驟包括核酸誤配基因(mismatch repair genes)hMSH2 與 hMLH1的西方點墨法檢驗,dHPLC 與直接定序方法分析 hMLH1 的基因突變,以及分析 hMLH1
promoter region 的過度甲基化情況(hypermethy lation)。 我們發現西方點墨法中 hMLH1
不表現的機率 63.0﹪(n=34),而 hMSH2不表現的機率為 3.7﹪(n=2)。以 dHPLC
及直接定序的分法分析 hMLH1基因(n=34)發現基因突變率為 5.6﹪(n=3),其餘 31
位中我們發現 hMLH1基因之 promoter region 的過度甲基化現象在 28 位病患(51.9﹪)
出現,而其餘 3 位則未知其突變情況。此一研究結果顯示台灣地區早發性大腸直腸癌的 產生是透過 MSI 的路徑,而 hMLH1 promoter region 的過度甲基化是造成 MSI 的主要機
轉,此意味著早發性大腸直腸癌的產生主要仍是環境的變動造成。 關鍵字:早發性大腸直腸癌、微小衛星不穩定、過度甲基化
英文摘要:
The colorectal tumorigenesis involves the interaction between environmental and genetic alterations. In this study, we further clarify this mechanism, i.e., the gene-environment interaction, based on the previous study that early onset colorectal cancers (age of disease onset less than 40 years old) tended to present with high fuquency microsatellite instability (MSI-H), as compared with late onset counterparts (age of disease onset more than 60 years old), with 29.4% and 6.3%, respectively (p<0.001). However, further tracing family history showed that there was no significant difference between these 2 groups of patients, with the incidence of a positive family history of cancer 17.4% and 14.3%, respectively (p=0.0859). This result implied that the early-onset colorectal cancers in Taiwan result predominantly from environmental rather than familial germline alteration. In the present study, we further
Western blotting of mismatch repair genes hMSH2 and hMLH1, and dHPLC analysis, direct
sequencing, and hypermethylation analysis of hMLH1 gene. We found that hMLH1protein was
negative in 63.0% (n=34), and hMSH2protein was negative in 3.7% (n=2), during the Western
blotting analysis. dHPLC and direct gene sequencing showed that genetic mutation of hMLH1
gene was present in 3 patients (5.6%), 28 (51.9%) of the remaining 31 patients presented with hypermethylation on the promoter region of hMLH1, whereas the mechanism for altmations of
hMLH1 in the other 3 patients was unknown. This result implied that a subset of early onset of colorectal cancer was through the carcinogenic pathway of MSI, and the hypermethylation of hMLH1 promoter represented a major mechanism for the presentation of MSI-H in the
early-onset colorectal cancers of Taiwan. Therefore, we concluded that environmental factors might be more important than genetic factors for the tumorigenesis of early onset colorectal cancer in Taiwan.
Key word: early onset colorectal cancer, MSI-H, hypermethylation.
前言/目的/文獻探討: 台灣地區大腸直腸癌近年來有明顯上升,其原因不外乎環境變動與家族遺傳因素造 成。環境的變動包括飲食習慣的改變及生活習慣的西洋化等。至於遺傳因素則包括遺傳 性非息肉大腸直腸癌(HNPCC)以及家族性腺性息肉(FAP)等生殖因子(germline) 的改變造成。過去的研究一直以為早發性大腸直腸癌(發病年齡小於 40 歲者)是遺傳 因子造成的成分較多,而其致癌基轉是因為 microsatellite instability,而其下游基因包括 TGFβ typeⅡ receptor,等一系列基因便表現異常,以致此種早發型的癌症便進展特別 快速,此一假說似乎與我們在臨床上所觀察到年輕型大腸直腸癌在診斷時多屬晚期者不 謀而合。再者,年輕型的大腸直腸癌所佔比例甚少(約佔所有大腸直腸癌病例的 6%而 已),這是否與 HNPCC 或 FAP 發生比例(約佔所有大腸直腸癌的 4-5%)相符合?這 些臨床觀察讓人強烈懷疑早發性大腸直腸癌是家族遺傳因子造成。 本研究目的主要在探討年輕型大腸直腸癌的 hMLH1與 hMSH2基因的變化情況,我們 收集54位具 MSI-H 的早發性大腸直腸癌,其家族史經詳細收集,而 hMSH2,hMLH1 經 Western blots 分析,並以 dHPLC 及直接基因定序,以及甲基化分析等方式,來明瞭 這些基因的變化情況,此舉將有助於明瞭家族遺傳因子與環境因子的變動在年輕型大腸 直腸癌的致癌角色。 研究方法:
1. 所有54位早發性大腸直腸癌均以5個 molecular markers(包括 BAT25,BAT26, D2S123,D5S346,D17S250)確定為 MSI-H 者。
2. 將這些病患的家族史至少追蹤三代,並期家族史的歸類分成 Grade 0, 1, 2, 3(詳細見 研究成果之表一)。
表一. Comparisons of Family History and Molecular Biologic Features Between Younger
(<40 years) and Older (≧60years) Colorectal Cancer Patients
Younger Patients, n(%) Older Patients, n(%) p-value
13-19 yrs 20-29 yrs 30-39 yrs
n=2 n=22 n=102 Total Family History* 0 1 17 86 104 (82.5) 277 ( 8 5 . 7 ) Ⅰ 0 2 8 10 ( 7.9) 21 ( 6 . 5 ) 0.859 Ⅱ 0 2 6 8 ( 6.4) 16 ( 5 . 0 ) Ⅲ 1 1 2 4 ( 3.2) 9 ( 2 . 8 ) MSI Status* MSS MSI-L MSI-H 0 1 1 9 3 10 66 10 26 75 14 37 (59.5) (11.1) (29.4) 108 10 8 ( 8 5 . 7 ) ( 7 . 9 ) ( 6 . 4 ) P<0.001 P53 Overexpression 1 7 41 49 (38.9) 67 ( 5 3 . 2 ) P=0.023 K-ras Mutation 1 7 45 53 (42.1) 56 ( 4 4 . 4 ) 0.703 LOH of DCC + 0 7 33 40 (31.8) 48 ( 3 8 . 1 ) 0.517 - 1 5 22 28 (22.2) 28 ( 2 2 . 2 ) non-informative 1 10 47 58 (46.0) 50 ( 3 9 . 7 ) a. The total number of younger patients was 126, whereas the denominator of the older
patients for the analysis of family history and molecular biologic features was 323 and 126, respectively.
b. Family history was graded as follows: 0= no family history of colorectal cancer or extracolonic cancers related to hereditary nonpolyposis colorectal cancers (HNPCC), such as endometrium, ovary, stomach, pancreas, ureter, and renal pelvis cancers; Ⅰ= relative with HNPCC-related extracolonic cancers; Ⅱ= relative with colorectal cancer who does not fulfill the modified Amsterdam criteria; and Ⅲ= nuclear family fulfills the modified Amsterdam criteria for HNPCC [22].
* Family history of cancer and MSI-H were significantly correlated with the younger subcategories within the young age group of less than 40 years, with p=0.017 and p=0.015, respectively, by Chi-square test.
4. 以 dHPLC 及 direct sequencing 的方法分析 hMLH1基因之突發情況(詳見結果欄之圖 二)。
圖一
圖二
5. 以Kane's method 分析hMLH1 promoter region的hypermethylation情況(詳見結果欄 之圖三)。 結果與討論: 1. 早發性大腸直腸癌(發病年齡小於40歲者)與晚發性大腸直腸癌相較,其有較高 之機會發生 MSI-H,但就出現家族史的比例而言,則兩組差不多(表一)。 2.具MSI-H的早發性大腸直腸癌中(n=54),hMLH1不表現的機會佔 63.0%(n=34), 而hMSH2不表現的機會佔3.7%(n=2)。此結果乃依Western blot的分析定出(圖 一)。此結果意味著hMLH1的不表現在早發型大腸直腸癌中此hMSH2的不表現重 要。 3.dHPLC與直接基因定序的方法發現hMLH1之基因突變只佔所有病例的5.6%(n=3), 此結果意味著家族遺傳因子在早發性大腸直腸癌的角色似乎不像想中重要(圖二)。 4.以Kane’s method 分析hMLH1 promoter region的hypermethylation情況發現此一現
象佔所有病例的 51.9%(n=28),此一結果顯示具MSI-H的早發性大腸直腸癌大部 分是因為hMLH1的epigenetic changes,即promoter region的過度甲基化造成,而這
些epigenetic changes主要是受環境因子,如環境污染、飲食、或生活習慣的西洋化
造成。
本研究主要針對二種mismatch repair genes 進行研究,然mismatch repair genes 為數 眾多,以其表現亦牽涉到許多cofactors,值得進一步研究,將此研究成果與西方研究成 果相互比對,將可增進大腸直腸癌至癌機轉的進一步闡明。
參考資料:
1.Liang J.-T. et al. Clinicopathological and molecular biological features of colorectal cancer in patients less than 40 years of age. Br J Surg 2003: 90: 205-214.
2.Hypermethylation of hMLH1 promoter represents a major mechanism for the presentation of MSI-H in the early-onset colorectal cancers of Taiwan (in submitting).