摘要
乳癌對於世界上的女性一直都是常見的嚴重疾病，約有百分之十的乳癌患者是遺傳性的乳癌。影響遺傳性乳癌最劇烈的基因是BRCA1及BRCA2，約佔了所有遺傳性乳癌的80﹪。當女性的BRCA1或BRCA2基因具有遺傳上的突變，則會在年輕時增加很大的風險罹患乳癌，而她的家人也會因此可能增加風險罹患乳癌。本研究取得高雄榮總六個乳癌家族病患及家屬的血液，分別進行聚合酶連鎖反應(PCR)及自動核酸序列分析(automatic DNA sequencing)，發現其中五個家族的BRCA1或BRCA2基因有造成胺基酸改變的單一鹼基突變。第一個家族(A家族)在BRCA2基因exon11上發現有A5885C (Gln1886Pro)胺基酸改變的單一鹼基突變及A4806G (Thr1526)胺基酸不變的單一鹼基突變。第二個家族(F家族)在BRCA1基因上發現有三個胺基酸改變的單一鹼基突變位在同一條染色體的exon11上，分別是C2731T (Pro871Leu)、A3232G (Glu1038Gly)、和A3667G (Lys1183Arg)，在同一條染色體的同一個基因的同exon上發生突變這是非常少有的現象。第三個家族(E家族)在BRCA1基因上發現有兩個胺基酸改變的單一鹼基突變分別是A3232G (Glu1038Gly在exon 11)和A4956G (Ser1613Gly在exon 16)及兩個胺基酸不變的單一鹼基突變分別是T2430C (Leu771)和T4427C (Ser1436)。第四及第五個家族(C家族和D家族)在BRCA1基因的exon 16上發現有一個胺基酸改變的單一鹼基突變A4956G (Ser1613Gly)及一個胺基酸不變的單一鹼基突變T4427C (Ser1436)。第六個家族(B家族)目前只在BRCA2基因上發現有一個胺基酸不變的單一鹼基突變T4035C (Val1269)。這些missense mutations所造成胺基酸的改變，可能致使BRCA1及BRCA2蛋白質結構的不穩定，使其功能受損而無法作用，而導致乳癌的發生。F家族的三個missense mutations，有可能是一種地區性遺傳乳癌基因的型態之一，可以當作篩選乳癌方向的參考。

Abstract
Breast cancer is a common malignancy affecting women around the world. Approximately 10 percent of breast cancer patients have a hereditary form of the disease. Women with an inherited alteration in one of the BRCA1 and BRCA2 genes have an increased risk of developing these cancers at a young age (before menopause), and often have multiple family members with the disease. A total of 6 families with multiple cases of breast cancer were identified from southern Taiwan, and five of these families were found to have missense mutations in the BRCA1 or BRCA2 genes. One novel missense mutation of A5885C (Gln1886Pro), as well as new silent mutation of A4806G (Thr1526), in the exon 11 of the BRCA2 gene was found in one(A) family. The second(F) family was found to have three missense mutations of C2731T (Pro871Leu), A3232G (Glu1038Gly) and A3667G (Lys1183Arg) in the exon 11 of the BRCA1 gene. It is very unusual to have three previously reported BRCA1 mutations in the same family and these three mutations are located on the same chromosome. Two missense mutations of A3232G (Glu1038Gly) in exon 11 and A4956G (Ser1613Gly) in exon 16, as well as silent mutations of T2430C (Leu771) and T4427C (Ser1436), of the BRCA1 gene were found in the third(E) family. The missense mutation of A4956G (Ser1613Gly) in exon 16, as well as silent mutation of T4427C (Ser1436), of the BRCA1 are found in the fourth(C) and fifth(D) family. The sixth(B) families were found to possess only one silent mutation of T4035C (Val1269) in the BRCA2 gene. The amino acid changes might cause the protein structure unstable and these could explain the moderate role of BRCA mutations in the pathogenesis of breast cancer.

目錄 ( Contents )
中文摘要……………………………………………………………..... Ⅰ
英文摘要……………………………………………………………..... Ⅲ
英文縮寫表…………………………………………………………..... Ⅳ
壹、緒論………….……………………..…………………………..…. 1
一、 簡介………………………………………………………….. 1
二、 乳癌…………………………………………….……………. 3
三、 乳癌的病理分類…………………………………………….. 4
四、 引起乳癌發生的危險因子……………………………….…. 5
五、 遺傳性乳癌的基因危險因子……………………………….. 8
六、 BRCA1和BRCA2的功能………………..………………….… 9
七、 BRCA1和BRCA2的突變………………..……………….….. 12
貳、研究目的………………………………………………………… 13
參、材料與方法……………………………………………………… 14
一、 血液檢體的取得……………………………………….…... 14
二、 實驗方法……………..………………….….……………… 14
(一) 基因體DNA之萃取………………………………..….…… 14
(二) 引子合成與聚合酶連鎖反應…………………..….………. 17
(三) PCR產物的純化…………………..….…………………… 18
(四) 質體的建構………………..….……………………………. 20
(五) 自動核酸序列定序………………..….……………………. 22
肆、結果……………………………………………………………... 27
一、 遺傳性乳癌家族的選擇…………………………………… 27
二、 乳癌病人的臨床特徵…..…………………………….……. 27
三、 乳癌的家族史……………………………………….….…... 28
四、 DNA定序及基因選殖分析………………………….…..… 30
伍、討論……………………………………………………….……... 34
陸、結論………..…………..…….……………………………….….. 38
柒、參考文獻………………………………..………………….……. 39
表……..…………………….……………………………...…………. 44
表一、高雄榮總六個遺傳性乳癌家族………………………… 44
表二、六個乳癌家族病患病史………………………………… 45
表三、本實驗乳癌病人BRCA1及BRCA2基因的突變……… 46
表四、台灣地區家族性乳癌BRCA1的突變…………..……… 47
表五之一、台灣地區家族性乳癌BRCA2的突變….………..... 48
表五之二、台灣地區家族性乳癌BRCA2的silent突變………. 49
表六、本實驗所發現的突變在世界上被發現的次數……….... 50
表七、本實驗發現的突變所造成的胺基酸改變影響……….... 51
圖……..…………………….………………………………………….. 52
圖一、A家族族譜……………...…………………………….… 52
圖二、B家族族譜……………………………………………… 52
圖三、C家族族譜……………………………………………… 53
圖四、D家族族譜……………………………………………… 53
圖五、E家族族譜……………………………………………… 54
圖六、F家族族譜……………………………………………… 54
圖七、病人AII 2在BRCA2上的突變…………………..……… 55
圖八、病人AII 2在BRCA2上的突變……..…………………… 56
圖九、病人BII 2在BRCA2上的突變………………………….. 57
圖十、病人CII 1在BRCA1上的突變…………………..…….... 58
圖十一、病人CII 1在BRCA1上的突變……………………….. 59
圖十二、病人CII 1的女兒CII 1在BRCA1上的突變…………. 60
圖十三、病人DII 2在BRCA1上的突變……………………..… 61
圖十四、病人DII 2在BRCA1上的突變……………...…….….. 62
圖十五、病人EIII 4在BRCA1上的突變…………..…………... 63
圖十六、病人EIII 4在BRCA1上的突變…………………………. 64
圖十七、病人EIII 4的妹妹EIII 5和EIII 7在BRCA1上的突變…. 65
圖十八、病人EIII 4在BRCA1上的突變………………………… 66
圖十九、病人EIII 4在BRCA1上的突變……………………….... 67
圖二十、病人EIII 4的姊妹EIII 1, EIII 2, EIII 3, EIII 5, EIII 6和EIII 7在BRCA1上的突變……….…………………. 68
圖二十一、病人FII 1在BRCA1上的突變……………………….. 69
圖二十二、病人FII 1在BRCA1上的突變……….……...……...... 70
圖二十三、病人FII 1在BRCA1上的突變………………….….… 71
圖二十四、病人FII 1的兒子在BRCA1上的突變……….………. 72
圖二十五、病人FII 1的親戚FII 5, FIII4, FIII10和FIII11 在BRCA1上的突變………………….……………. 73
附錄……..…………………….……………………………………….. 74
附表一、BRCA1和BRCA2基因的特性……………………….… 74
附表二、BRCA1基因的exons位置……………………………… 75
附表三、BRCA2基因的exons位置………………………...….… 76
附表四、BRCA1基因的PCR primers………………………..……. 77
附表五、BRCA1的exon11的PCR primers………………………… 79
附表六、BRCA2基因的PCR primers……………………………… 81
附圖一、BRCA1及BRCA2的coding regions…………...………… 85

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