- 學位論文
腫瘤抑制基因p53, BRCA1, BRCA2於台灣乳癌之分子流行病學研究
Molecular epidemiology studies of tumor suppression genes p53, BRCA1& BRCA2 in Taiwanese breast cancer
摘要
P53基因突變存在於許多人類的癌症當中， p53的基因突變分析可以提供分子流行病學的線索，以進一步探討腫瘤形成的病理原因。p53的基因核苷酸序列上，由於位於codon 72上單一鹼基對的不同，因而產生兩個常見的基因多形性：核苷酸序列CCC，轉譯出脯氨酸(proline)；或者是CGC，轉譯出精氨酸(arginine)。另外在乳癌腫瘤中也常可見到BRCA1(位於染色體17q21) 或BRCA2基因 (位於染色體13q12–q13) 上的對偶基因部分片段缺失，顯示BRCA1或BRCA2基因的體突變（somatic mutation）應在乳癌腫瘤發生過程中扮演著重要角色，但BRCA1或BRCA2在乳癌中的體突變（somatic mutation）仍一直被認為是少見的。在過去的研究中，大部分的研究都是針對BRCA1或BRCA2在遺傳性或家族性乳癌中的突變分析，少有研究著重於BRCA1或BRCA2在散發性乳癌中的體突變分析，而這可能是造成BRCA1或BRCA2基因的體突變（somatic mutation）低發生率的原因之一。此篇研究目的是分別調查台灣乳癌患者中的 p53基因突變分析及基因多型性、BRCA1及BRCA2部分基因片段突變分析，探討這些基因變異的臨床意義及p53基因多型性與BRCA1部分基因片段突變的關係，以了解此多形性是否改變BRCA1突變病患罹患乳癌的機率，並比較不同區域及人種間基因變異的差異性。我們利用聚合酶鏈鎖反應-單鏈構像多態性 (PCR-SSCP)分析法，分別篩選分析P53基因exon 5-8、內涵轉譯啟動子的BRCA1 exon2及影響BRCA2基因重要功能，內涵RAD51交互作用區域BAC也是BRCA2上最大的外顯子exon 11 的突變狀態，再加以直接定序確認突變位點。同時我們也用RFLP方法從周邊血液顆粒球檢驗p53codon72上基因多型性分布的狀態，腫瘤及血液樣品是取自 119到175位不等經乳房切除的乳癌病人。在 119個乳癌病例中，共發現 22例中有 26個 p53基因上的突變 (18.5%)。在這些突變當中，78% (20/26)為點突變，其中大多數是置換突變 (missense mutation，15/20，佔 75%)，其餘的 22% (6/26)為移碼突變 (frameshift mutation)。在我們的研究中，P53的突變與否，與乳癌的臨床病理特徵，包括 HER2的狀態，並無顯著關聯。 此外我們的結果也發現了在P53基因突變中，顛換取代突變(transversion)的頻率明顯高於轉換取代突變 (transition)，為 15/21 (71.4%) 比上 6/21 (28.6%)，其中又以 GC?彪G 的顛換取代突變佔大多數 (6/15, 40%)。我們鑑定的P53突變熱點分別為密碼子（codon）167, 185, 186, 210, 265 及295，這些在文獻上均鮮少被提及。另外在175位病人中，有21個乳癌檢體發生BRCA1基因突變(21/175,12%)。在這之中，84.6% (22/26)的BRCA1突變發生在外顯子2的拼接域（splicing region）。五個重複出現的突變發生在IVS1和核苷酸-20和-63，而每個核苷酸則有三個突變。與臺灣乳癌臨床病理特徵的相關性分析顯示，BRCA1突變和病理第2級相關(p=0.034)。另外有十五位病人 (15/175, 8.6%)有共二十個BRCA2外顯子 (exon)11的突變。大部份的突變是點突變 (point mutation，19/20， 95%)，除了一例為核苷酸插入（nucleotide insertion），這些突變有80% (16/20) 是錯義突變（missense mutations）。經由BIC資料庫的搜尋後發現，本實驗發現的都是新的突變。三個突變重覆出現在密碼子（codon）1904，另外在密碼子（codon）1907, 1936, 1937 和1968都各重覆出現兩個突變。進一步分析這些突變與臨床病理組織間的關係發現，BRCA2突變與原位癌較為相關 (p=0.038)，與低組織形態分級則在相關邊緣(p=0.072)。此外，共有三個案例在實驗的部位有多重突變的現象，其中一例合併有淋巴結轉移。而Arg/Arg、Arg/Pro及Pro/Pro三種p53 codon72基因多型性在170位乳癌病患的分布情形分別為29位帶有 Pro/Pro (17.1%)，83位帶有Arg/Pro (48.8%) 及 58位帶有Arg/Arg （34.1%），總計帶有Arg 與Pro對偶基因的比例為59％比上41％。我們的結果基本上與之前的結果一致，顯示相較於歐美地區乳癌，亞洲地區的乳癌病患中帶有較高比例的Pro allele（亞洲40.6%比上歐美26.4%）。另外在Arg/Arg、Arg/Pro及Pro/Pro三種p53codon72基因多型性中，BRCA1突變與Arg/Pro基因型存在明顯關聯性﹝81％比44.3％；P = 0.018﹞。我們的研究結果顯示 p53、BRCA1與BRCA2基因突變與台灣乳癌的致癌機轉有關，在乳癌的發病機理扮演著重要的角色，尤其是BRCA1與BRCA2基因突變現象和某些特定組織病理特徵相關。再者p53基因突變中，高頻率的 G:C到 C:G顛換取代突變顯示，在台灣的乳癌的成因中，外在的環境因素（導致基因突變）可能比基因內在的突變過程，扮演更重要的角色。此外伴有BRCA1突變的乳癌有較高頻率的Arg/Pro allele，而這意味著p53 Arg72Pro多形性在惡性轉化中可能扮演篩選的角色，它可能構成乳癌的危險因子之一，特別是在已帶有BRCA1突變的乳腺組織。
並列摘要
Abstract p53 gene mutation is a very frequent event in many human cancers. Analysis of p53 gene mutations can also provide clues to the etiology of tumor formation. The wild-type p53 has two common polymorphic variants from a single-base-pair substitution at codon 72, where either CCC encodes proline or CGC encodes arginine. Somatic mutation of BRCA1 or BRCA2 have been thought to be rare in breast cancers, though common allelic deletions in the BRCA1 locus (17q21) or BRCA2 locus (13q12-q13) imply an important role of somatic mutation in these tumors. Reasons of the reported rare incidence could be related to very few studies focusing on the mutational analysis of BRCA1or BRCA2 in sporadic tumors. The present study was conducted to investigate the p53 mutations, p53 codon 72 polymorphism, BRCA1 and BRCA2 mutations in patients with breast cancer from Taiwan. We also compare the genetic abnormalities between different areas and ethnicities. Tumor and blood samples from different cohorts including 119 patients to 175 patients respectively undergoing mastectomy for breast cancer were evaluated. The mutational statuses of the p53 gene (exons 5–8), exon 2 of BRCA1 which containing the translation initiation codon and BRCA2 gene in exon11, the largest exon harboring the RAD51 interacting BRC domains which are critical for BRCA2 function were screened by polymerase chain reaction-single strand conformation polymorphism analysis followed by direct sequencing. The p53 codon 72 allele status was also examined by restriction fragment length polymorphism (RFLP) from PMN cell of blood. Of all 119 cases of breast carcinoma, 26 mutations of the p53 gene were found in 22 cases (18.5%). Among these mutations, 78% (20/26) were point mutations with the majority of those being missense mutations (75%, 15 of 20 mutations) and the other 22% (6/26) were frameshift mutations. No significant correlation between p53 mutations and clinicopathological features was found, including HER2 status. Moreover, our results disclosed distinct mutation spectra in excess transversions to transitions (15/21, 71.4% vs. 6/21, 28.6%) with GC to CG dominant (6/15, 40%). Mutation hot spots we identified at codons 167, 185, 186, 210, 265 and 295 have rarely been documented in the literature. In BRCA1 mutation analysis, there were 25 mutations of BRCA1 gene in 21 cases (21/175, 12%). Among them, 84.6% (22/26) of BRCA1 mutations occurred at splicing region of exon 2. Five recurrent mutations occurred in IVS1 and in nucleotides -20 and -63 each harbored 3 mutations respectively. Interestingly, the mutation of BRCA1 was associated with pathological grade 2 (p=0.034). As for BRCA2 mutation status, there were 20 mutations of BRCA2 gene in exon 11 in 15 cases (15/175, 8.6%). Most mutations we identified were point mutation (19/20, 95%), except for one nucleotide insertion. Furthermore, among these mutations, missense mutations comprised 80% (16/20) of the BRCA2 mutations. All mutations we found were novel mutation after searched in the BIC database. There were three recurrent mutations at codon 1904; and two recurrent mutations at 1907, 1936, 1937 and 1968, respectively. The mutations were associated with ductal carcinoma in situ (PZ0.038) and borderline with low histological grade (PZ0.072). Besides, there were three cases possessing multiple mutations in the region we studied and one of them demonstrated aggressive lymph node metastasis. For the p53 codon72 polymorphism, there were 29 Pro/Pro (17.1%), 83 Arg/Pro (48.8%) and 58 Arg/Arg (34.1%) with the allele frequencies 0.59 for the Arg72 and 0.41 for Pro72 allele respectively. Our results are consistent with that there was more Pro72 allele (40.6% in Asian vs. 26.4% in Caucasian) and twice the incidence of p53 codon 72 Pro/Pro homozygotes (18% in Asian vs. 8% in Caucasian) among the Asian population. Furthermore when further correlation of the polymorphism with the BRCA1 mutation status , the BRCA1 somatic mutation associated with the Arg/Pro genotype significantly (81% vs. 44.3%; P = 0.018). Our findings showed that p53, BRCA1 and BRCA2 genes mutations might contribute to the pathogenesis of breast carcinoma in Taiwan. Furthermore, the events of Somatic mutations in BRCA1 and BRCA2 genes were associated with some specific histopathological features. In addition, the different mutation spectrum with high transversions in G:C to C:G may imply that the exogenous mutagens outweigh the endogenous processes in breast cancer in patients in Taiwan. The higher frequency of the p53 Arg/Pro genotype in breast cancer with BRCA1 mutation detected in this study in Taiwan suggests a possible selection role of p53 Arg72Pro polymorphism in malignant transformation and may constitute a risk factor for breast cancers, especially in breast cancer with BRCA1 somatic mutation.