基于下一代目标区域序列捕获测序技术的MEN2早期个体化诊疗策略研究

By integrating pedigrees, analysis of preoperative calcitonin levels, and then screening of RET protein-coding region (mutations/SNPs) based on targeted next-generation sequencing (T-NGS), we aim to (1) generalize the relationship between genotype and phenotype in Chinese multiple endocrine neoplasia type 2 (MEN2)-related RET mutations and/or SNPs; (2) assess the accuracy and feasibility of RET screening by targeted next-generation sequencing, which could be a model for genetic diagnosis in other monogenic diseases with multiple exons and without homologous repetitive sequence within their causative genes; (3) demonstrate the time course, surgical procedures and extent of prophylactic thyroidectomy for Chinese MEN2 patients, and evaluate the utility of cortical sparing surgery for MEN2-related phaeochromocytoma; (4) attempt to establish an refinement and individualized strategy of prevention, early diagnosis and late remedy treatment for Chinese MEN2 patients, to reduce clinic hazards and postoperative complications，improve disease-free survival and promote the overall improvement of the diagnosis and treatment.

通过整合家系调查、基于下一代目标区域捕获测序技术（T-NGS）的RET基因全外显子区域（突变/SNPs）测序和术前降钙素水平等，①勾勒国人RET突变基因型-MEN2表型的关系谱，分析RET基因SNPs在国人MEN 2差异表型中的可能修饰作用；②评估T-NGS检测RET基因的准确性和实用性，为其直接应用于无同源重复序列的单基因遗传病的临床检测提供依据。③探讨国人MEN2的预防性甲状腺切除术的手术时机、手术方式和范围，以及MEN2-PHEO的保留肾上腺皮质功能的肾上腺切除术的运用评估。④初步勾勒国人MEN2的早期诊疗策略规范，推动实施精细化和个体化的诊疗，减少或避免MEN2的临床危害及其术后并发症，最大限度地提高MEN2无病生存率，为最终建立集MEN2的预防、预警和早期诊疗、晚期补救为一体的国人个体化MEN2诊疗平台体系，促进国人MEN2诊治水平的整体提高。

以MEN2和MTC/PHEO为研究对象，采用系统家系调查和TGS技术和Sanger法对目标样本行种系水平RET基因测序，发现和诊治22种RET突变体72个MEN2家系262人次患者：C634F/G/R/S/Y, C609R, C611Y, C618G/R/S/Y, C620R/S, S409Y, L790F, V804M, S891A, M918V, M918T等19种分属于68个家系248例患者：① MEN2A家系数和患者数分别占92.6%（63/68）和98.0%（243/248）：经典型(38.1%,24/63和58.8%,143/243)，CLA型(7.9%,5/63和2.5%,6/243)，HSCR型(1.6%,1/63和0.4%,1/243)，FMTC型(44.4%,28/63和38.3%,93/243)。其中第11外显子占50.8%(32/63)和46.5%(113/243)，第10外显子占20.6%(13/63)和30%(73/243)。其它包括S409Y, L790F, V804M, S891A和M918V。②MEN 2B均为M918T(5/5,100%)，占7.4%(5/68)和2.0%(5/248)。另外4种复合突变分属于4个家系14例患者。RET-G691S增加MEN2-PHEO风险，但与MTC无关。整合RET突变和术前Ctn等检测，对23例无症状MEN2A患者行预防性甲状腺切除术，平均诊断年龄29.7(5.5-74)岁，术前Ctn 65.9(8.45-247) pg/mL，随访3-80个月，发现当ATA-MOD~H患者Ctn<70pg/mL，MTC仍局限于甲状腺内，可避免淋巴结清扫；当MEN2A患者其Ctn正常时，监视随访；建议参考ATA-2015指南推荐在中国实施个体化MEN2-MTC分层外科手术。51例MEN2-PHEO术后总复发风险为1.9 %%，保留肾上腺功能术可明显减少患者肾上腺全切除术后出现的终生激素依赖(100% vs. 21.1%, P <0.05)，应优先选择腹腔镜下术式。利用TGS技术首次运用于MEN2A-PGD并顺利产下国际首例健康婴儿，TGS检测RET突变安全有效。基于RET突变和Ctn等检测的MEN2“7P”诊治策略理念的运用推广，可有效避免或减少MEN2的临床危害。

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