Abstract
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).
Highlights
Sanger DNA sequencing of this region of the Multiple endocrine neoplasia type 1 (MEN1) 50UTR further confirmed the deletion to be 596 bp in size, and to be located between nucleotides −1087 and −492 upstream of the MEN1 gene translation start site (c.−590_−24+29del; Fig. 2B). This deleted region contained the whole of exon 1, the minimal core promoter region and multiple cis-regulatory and initiator elements (Fig. 1B; Supplemental Fig. 1)
This 50UTR 596 bp deletion was not found in 75,464 individuals, which comprised 74,180 participants of the 100KGP, 135 MEN1 patients who had pathogenic MEN1 variants, and 1149 patients with MEN1-associated tumors, thereby demonstrating an allele frequency of 0% for this deletion in these cohorts
We report the first MEN1 mutation that does not involve an abnormality within the protein-coding region, but instead occurs in the promoter region and comprises a 596 bp deletion of the minimal core promoter within the MEN1 50UTR (Figs. 1 and 2; Supplemental Fig. 1)
Summary
Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the combined occurrence of parathyroid, pancreatic and pituitary tumors.[1,2] The majority (>95%) of MEN1 patients will develop parathyroid tumors, 35% to 75% of patients will develop neuroendocrine tumors (NETs) of the pancreas (PNETs),
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