以次世代定序鑑定台灣地區嗜鉻細胞瘤及副神經節瘤之致病變異位點

Identifying Causative Genetic Variants of Pheochromocytoma and Paraganglioma by Next-Generation Sequencing (NGS) in Taiwan

周毓慈(Yuh-Tsyr Chou)

指導教授 ： 楊偉勛

共同指導教授 ： 陳沛隆

國立臺灣大學/醫學院/分子醫學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201904043

摘要

嗜鉻細胞瘤(pheochromocytoma, PCC)和副神經節瘤(paraganglioma, PGL)為神經內分泌腫瘤的一種，分別長在腎上腺或是腎上腺外的嗜鉻細胞中。這樣的腫瘤大部分是良性的，但由於過度增生的嗜鉻細胞會不斷分泌兒茶酚胺(catecholamine)，而兒茶酚胺會使心跳加速、心臟收縮力增強、血壓上升等，長期兒茶酚胺過高會導致高血壓、心律不整、中風而增加患者的死亡率。約有三分之一的嗜鉻細胞瘤/副神經節瘤患者帶有遺傳性的致病基因變異，所以本研究的目的為建立一個次世代定序(next-generation sequencing, NGS)的基因檢測平台，幫助這些患者找出致病基因變異位點。
根據前人的研究，我們設計了一個32個基因群的檢測平台，包含12個最常見的致病基因(RET, VHL, NF1,FH, SDHA, SDHAF2, SDHB, SDHC, SDHD, MAX, TMEM127, EGLN1(PHD2))、10個體細胞變異基因(ARNT(HIF1β), ATRX, BRAF, CSDE1, EPAS1(HIF1α), FGFR1, HRAS, IDH1, SETD2, TP53)、3個融合基因 (BRAF, MAML3, NGFR)及其它7個曾被報導過的致病基因(CDKN2A , H3F3A, IDH2, KMT2D, MDH2C, MERTK, MET)。我們利用Illumina MiSeq平台產出雙端定序(paired-end)序列，經由軟體分析產出變異位點，比對資料庫(gnomAD, ExAC, Taiwan Biobank)的等位基因频率篩選可能的點位，最後藉由2015年ACMG的準則判斷變異位點的致病性。
本研究結果顯示，我們在41個家族中找到12個家族帶有遺傳性的致病變異位點，檢出率約為29%，與前人研究的33%相近。這12個家族帶的遺傳性致病變異位點由7個不同的變異位點組成，其中有7個家族帶有相同的致病變異位點(SDHD, c.3G>C, p.M1X, NM_003002)，暗示這個能是一個創立者效應(founder effect)，但還需要一後續的實驗來驗證這個說法。帶有SDHB及SDHD致病變異位點的患者在收案的3年內都看到了轉移現象，與過去文獻中轉移機率最高的基因SDHB稍有不同。另外，在TMEM127及RET基因上的變異位點，因其族群基因頻率低、軟體預測為可能影響蛋白質功能及缺乏完整研究，故值得做相關功能性試驗以釐清其致病性。

關鍵字

嗜鉻細胞瘤 ； 副神經節瘤 ； 基因檢測 ； 世代定序

並列摘要

Pheochromocytoma (PCC) and paraganglioma (PGL) are neuroendocrine tumors arising from adrenal and extra-adrenal chromaffin cells respectively. They mostly present benign, yet show high morbidity and mortality due to the overproduction of catecholamine, which leads to hypertension, arrhythmia and even ischemia stroke. About one thirds of PCC/PGL are caused by germline genetic variants; therefore, here we established a NGS panel to detect possible disease-causing variants. After literature review, we aimed at the top 12 PCC/PGL causative genes (RET, VHL, NF1,FH, SDHA, SDHAF2, SDHB, SDHC, SDHD, MAX, TMEM127, EGLN1(PHD2)), 10 somatic mutation genes (ARNT(HIF1β), ATRX, BRAF, CSDE1, EPAS1(HIF1α), FGFR1, HRAS, IDH1, SETD2, TP53), 3 fusion genes (BRAF, MAML3, NGFR) and other 7 genes (CDKN2A , H3F3A, IDH2, KMT2D, MDH2C, MERTK, MET) which were reported relative to PCC/PGL. Paired-end reads were generated from Illumina MiSeq platform and analyzed with in-house pipeline (BWA-MEM, Picard SortSam, MarkDuplicates, GATK-BQSR and ANNOVAR). Variants were filtering according to the allele frequencies in gnomAD, ExAC and Taiwan Biobank, and the pathogenicity interpretation were facilitated by disease/gene database, scientific literature and the 2015 ACMG Guidelines. In this study, we sequenced 41 probands and yielded approximately 29% diagnosis rate with identifying 7 different disease-causing variants and several variants of unknown significant (VUS). Among the results, seven unrelated probands carried a same causative variant in SDHD (c.3G>C, p.M1X, NM_003002), which implied a founder mutation in Taiwan. Three different variants of SDHB and SDHD in nine unrelated probands showed metastasis in our study. Variants in TMEM127 and RET showed low allele frequency or absent in population database and predicted to affect protein function deserved further functional study.

並列關鍵字

pheochromocytoma ； paraganglioma ； genetic testing ； next-generation sequencing (NGS)

參考文獻

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