Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford Centre for Diabetes, Endocrinology and Metabolism (OCDEM), Churchill Hospital, Oxford, UK.
Oxford Medical Genetics Laboratory, Oxford University Hospitals NHS Trust, Oxford, UK.
J Bone Miner Res. 2021 Jan;36(1):100-109. doi: 10.1002/jbmr.4156. Epub 2020 Sep 15.
Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the occurrence of parathyroid, pancreatic and pituitary tumors, and is due to mutations in the coding region of the MEN1 gene, which encodes menin. We investigated a family with identical twins that had MEN1, with different MEN1 tumors. DNA sequence analysis of the MEN1 coding region had not identified any abnormalities and we hypothesized that deletions and mutations involving the untranslated regions may be involved. Informed consent and venous blood samples were obtained from five family members. Sanger DNA sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses were performed using leukocyte DNA. This revealed a heterozygous 596bp deletion (Δ596bp) between nucleotides -1087 and -492 upstream of the translation start site, located within the MEN1 5' untranslated region (UTR), and includes the core promoter and multiple cis-regulatory regions. To investigate the effects of this 5'UTR deletion on MEN1 promoter activity, we generated luciferase reporter constructs, containing either wild-type 842bp or mutant 246bp MEN1 promoter, and transfected them into human embryonic kidney HEK293 and pancreatic neuroendocrine tumor BON-1 cells. This revealed the Δ596bp mutation to result in significant reductions by 37-fold (p < 0.0001) and 16-fold (p < 0.0001) in luciferase expression in HEK293 and BON-1 cells, respectively, compared to wild-type. The effects of this 5'UTR deletion on MEN1 transcription and translation were assessed using qRT-PCR and Western blot analyses, respectively, of mRNA and protein lysates obtained from Epstein-Barr-virus transformed lymphoblastoid cells derived from affected and unaffected individuals. This demonstrated the Δ596bp mutation to result in significant reductions of 84% (p < 0.05) and 88% (p < 0.05) in MEN1 mRNA and menin protein, respectively, compared to unaffected individuals. Thus, our results report the first germline MEN1 5'UTR mutation and highlight the importance of investigating UTRs in MEN1 patients who do not have coding region mutations. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
多发性内分泌腺瘤病 1 型（MEN1）是一种常染色体显性遗传病，其特征是甲状旁腺、胰腺和垂体肿瘤的发生，这是由于 MEN1 基因编码区的突变，该基因编码 menin。我们研究了一个家族，该家族中有同卵双胞胎患有 MEN1，但 MEN1 肿瘤不同。对 MEN1 编码区的 DNA 序列分析未发现任何异常，我们假设涉及非翻译区的缺失和突变可能参与其中。从五名家庭成员中获得了知情同意和静脉血样。使用白细胞 DNA 进行 Sanger DNA 测序和多重连接依赖性探针扩增（MLPA）分析。结果显示，在翻译起始位点上游的-1087 到-492 核苷酸之间存在杂合性 596bp 缺失（Δ596bp），该缺失位于 MEN1 5'非翻译区（UTR）内，包括核心启动子和多个顺式调控区。为了研究该 5'UTR 缺失对 MEN1 启动子活性的影响，我们构建了包含野生型 842bp 或突变型 246bp MEN1 启动子的荧光素酶报告基因构建体，并将其转染入人胚肾 HEK293 和胰腺神经内分泌肿瘤 BON-1 细胞。结果显示，与野生型相比，Δ596bp 突变使 HEK293 和 BON-1 细胞中的荧光素酶表达分别显著降低 37 倍（p <0.0001）和 16 倍（p <0.0001）。通过 Epstein-Barr 病毒转化的淋巴母细胞系获得的受影响和未受影响个体的 mRNA 和蛋白质裂解物的 qRT-PCR 和 Western blot 分析，分别评估了该 5'UTR 缺失对 MEN1 转录和翻译的影响。结果显示，与未受影响的个体相比，Δ596bp 突变使 MEN1 mRNA 和 menin 蛋白分别显著降低 84%（p <0.05）和 88%（p <0.05）。因此，我们的结果首次报道了生殖系 MEN1 5'UTR 突变，并强调了在没有编码区突变的 MEN1 患者中研究 UTR 的重要性。
