台灣第二型多發性內分泌腫瘤的遺傳診斷與諮詢之流程建立

Abstract

第二型多發性內分泌腫瘤(MEN 2)是一種自體顯性遺傳疾病，乃是RET原致癌基因發生突變所致，此病具有高度甲狀腺髓質癌（MTC）的遺傳外顯率。至今，我們對於特定RET突變（基因型）和MTC侵犯性及其他內分泌腫瘤（表現型）之間的關聯性，已經有相當的了解。可以因為RET基因6個exon（10、11、13、14、15、16）上的突變熱點發生改變，分別是位於RET的cysteine-rich或tyrosine kinase domain上，而導致三種不同的臨床亞型：MEN 2A、MEN 2B和家族性MTC。本篇論文研究的目的，是為了了解在台灣MEN 2的家族中，RET原致癌基因發生germ-line mutation的情況，一窺台灣MEN 2的家族遺傳型態，以便評估對於已經出現病徵的患者和正常帶原者，給予適當的醫療介入和遺傳諮商的價值。我們收集了20個有MTC患者的家族，總共61位家屬，並且萃取其白血球的DNA。將這些疑似帶原者和偶發性MTC受試者的檢體，利用PCR和核酸定序法，來分析RET原致癌基因與MEN 2有關的7個exons上的突變，包括exon 8、10、11、13、14、15和16。其中3個MEN 2Ａ家族中的10位成員，檢測出帶有exon 11 codon 634突變型，1個C > R及2個C > F。另一「未分類型」家族的2位家屬，則帶有exon 10的C620F突變型。有一位指標個案，雖然檢測出帶有exon 11的C634W突變型，但是因為其他家屬拒絕接受檢查，因此無法分辨是否為遺傳性或de novo MEN 2A。此外，還檢測出3位de novo個案，包括2位MEN 2B和1位MEN 2A，他們分別帶有M918T和C634R突變型，但是沒有在其父母和兄弟姊妹中發現相同的突變型。其餘12位MTC患者因為沒有在我們所檢測的7個exon上發現有意義的突變，因此將其歸類為偶發性MTC。我們發現，凡是有家族病史或是能利用臨床診斷作出MEN 2分型的患者，都可以找到RET germ-line mutation。但是，如果患者的臨床症狀為isolated MTC，且已知無相關家族病史，或是沒有合併其他內分泌症狀者，都無法在我們所檢測的7個exon上找到有意義的突變。所以，我們的分析結果顯示，所有的MTC患者都應該接受RET原致癌基因的篩檢，以便利用分子生物檢驗法來偵測潛在或de novo的MEN 2或家族性MTC。目前，germ-line基因檢測已經成為MEN 2患者醫療決策的基礎，同時也應用於無症狀帶原者的早期偵測。MEN 2不但爲癌症早期防治和帶原者突變型，建立出一套獨特的分子診斷模式，也同時藉此突顯出了遺傳諮商在癌症治療中的價值。

Multiple endocrine neoplasia type 2 (MEN 2) is caused by autosomal dominant gain-of-function mutations in the RET proto-oncogene which has a high penetrance for medullary thyroid carcinoma (MTC). So far, the associations between specific RET mutations (genotype) and the aggressiveness of MTC and variations of other endocrine neoplasia (phenotype) are well expounded. Hot spot mutations in six exons (10, 11, 13, 14, 15, and 16) located in either cysteine-rich or tyrosine kinase domains of RET cause one of three distinctive clinical subtypes: MEN 2A, MEN 2B, and familial MTC. In this study, we propose to elucidate the germ-line RET proto-oncogene mutations and subtypes in Taiwanese subjects with MTC, and evaluate the value of appropriate medical interventions and genetic counseling in afflicted patients and asymptomatic gene carriers. DNA was extracted from the peripheral blood leukocytes of 61 members of 20 unrelated families as having individuals affected by MTC. The suspected carriers and apparently sporadic MTC cases were tested for MEN 2-associated germ-line mutations by polymerase chain reaction (PCR)-based sequencing of the RET gene exons, including 8, 10, 11, 13, 14, 15, and 16. Ten family members in the three MEN 2A kindreds had mutations in codon 634 of exon 11, 1 C > R and 2 C > F. Two family members of one unclassified kindred had the C620F mutation of exon 10. An index case had the C634W germ-line mutation but her family refused the test, could not be defined as hereditary or de novo MEN 2A. Additionally we found 3 de novo cases, including 2 MEN 2B and 1 MEN 2A, with the M918T and C634R mutations, respectively. These mutations were not detected in either of their parents or siblings. The other 12 MTC cases were defined as sporadic MTC whose DNA did not carry any non-synonymous mutations in the 7 exons. We found that all MTC patient with family history or with the other phenotypes of MEN 2 had RET germ-line mutations. For patients with isolated MTC without family history and other endocrine syndromes, we did not find any non-synonymous RET germ-line mutations among the 7 exons. Our findings suggest that all patients with MTC should be screened for the RET proto-oncogene by molecular analysis in order to detect occult or de novo MEN 2 or familial MTC. To date, germ-line genetic testing has become the basis for therapeutic decisions in MEN 2 affected patients and can facilitate the early presymptomatic detections of gene carriers. MEN 2 gives a unique model for early prevention and cure of cancer and for risk stratification of carriers by genetic diagnosis. This provides important insight into the prominent benefits of genetic counseling in cancer therapy.