Multiple Endocrine Neoplasia Type 1 (MEN1) 5′UTR Deletion, in MEN1 Family, Decreases Menin Expression

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 0 untranslated region (UTR), and includes the core promoter and multiple cis-regulatory regions. To investigate the effects of this 5 0 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 0 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 0 UTR mutation and highlight the importance of investigating UTRs in MEN1 patients who do not have coding region mutations.

MENIN‐mediated regulation of gastrin gene expression and its role in gastrinoma development

“…The coding region of the MEN1 locus spans exons 2-10 and maps to chromosome 11q13. 40,46 Exon 1 lies upstream of the ATG translational start codon located in Exon 2. The 3′ region downstream of the stop codon in exon 10 is transcribed but not translated.…”

“…The 2.8 kb transcript encodes two isoforms of MENIN that generate 615 (NP 000235.2) and 610 (NP 570711.1) amino acid residue proteins, respectively. 48 These MEN1 variants differ only in the transcriptional start site and exon splicing; 46,49 however, the transcript encoding 615 amino acids has not been identified in any normal tissues or malignancies.…”

“…The coding region of the MEN1 locus spans exons 2–10 and maps to chromosome 11q13 40,46 . Exon 1 lies upstream of the ATG translational start codon located in Exon 2.…”

Genetics of hereditary forms of primary hyperparathyroidism

“…Germline mutations in other CDKIs have been found in 0.5–1% of patients, including CDKN1A (p21), CDKN2C (p18), and CDKN2B (p15) [ 40 ]. Genetic analysis for MEN1 mutations usually involves sequencing the coding region of MEN1 (exons 2–10); however, mutations involving the promoter region (for example, a 596 bp deletion in the MEN1 5′UTR) have been reported in a MEN1 kindred with no MEN1 mutation in the coding region [ 41 ]. The significance of this deletion was tested in vivo, which reported ~ 80% reduction in MEN1 mRNA and ~ 80% reduction in menin protein expression [ 41 ].…”

“…Genetic analysis for MEN1 mutations usually involves sequencing the coding region of MEN1 (exons 2–10); however, mutations involving the promoter region (for example, a 596 bp deletion in the MEN1 5′UTR) have been reported in a MEN1 kindred with no MEN1 mutation in the coding region [ 41 ]. The significance of this deletion was tested in vivo, which reported ~ 80% reduction in MEN1 mRNA and ~ 80% reduction in menin protein expression [ 41 ]. Other causes of mutation-negative MEN1 syndrome may include the chance co-occurrence of two endocrine tumors without an underlying germline predisposition syndrome or a germline mutation in a gene not commonly screened for as part of a MEN1 panel (e.g., aryl hydrocarbon receptor-interacting protein ( AIP OMIM: 605,555) mutations in familial isolated pituitary adenoma) with the co-occurrence of sporadic PHPT [ 42 ].…”