Rafael A Carvalho, Betsaida Urtremari, Alexander A L Jorge, Lucas S Santana, Elisangela P S Quedas, Tomoko Sekiya, Viviane C Longuini, Fabio L M Montenegro, Antonio M Lerario, Sergio P A Toledo, Stephen J Marx, Rodrigo A Toledo and Delmar M Lourenço Jr
Loss-of-function germline MEN1 gene mutations account for 75–95% of patients with multiple endocrine neoplasia type 1 (MEN1). It has been postulated that mutations in non-coding regions of MEN1 might occur in some of the remaining patients; however, this hypothesis has not yet been fully investigated.
To sequence for the entire MEN1 including promoter, exons and introns in a large MEN1 cohort and determine the mutation profile.
Methods and patients
A target next-generation sequencing (tNGS) assay comprising 7.2 kb of the full MEN1 was developed to investigate germline mutations in 76 unrelated MEN1 probands (49 familial, 27 sporadic). tNGS results were validated by Sanger sequencing (SS), and multiplex ligation-dependent probe amplification (MLPA) assay was applied when no mutations were identifiable by both tNGS and SS.
Germline MEN1 variants were verified in coding region and splicing sites of 57/76 patients (74%) by both tNGS and SS (100% reproducibility). Thirty-eight different pathogenic or likely pathogenic variants were identified, including 13 new and six recurrent variants. Three large deletions were detected by MLPA only. No mutation was detected in 16 patients. In untranslated, regulatory or in deep intronic MEN1 regions of the 76 MEN1 cases, no point or short indel pathogenic variants were found in untranslated, although 33 benign/likely benign and three new VUS variants were detected.
Our study documents that point or short indel mutations in non-coding regions of MEN1 are very rare events. Also, tNGS proved to be a highly effective technology for routine genetic MEN1 testing.
Viviane C Longuini, Delmar M Lourenço Jr, Tomoko Sekiya, Osorio Meirelles, Tatiana D Goncalves, Flavia L Coutinho, Guilherme Francisco, Luciana H Osaki, Roger Chammas, Venancio A F Alves, Sheila A C Siqueira, David Schlesinger, Michel S Naslavsky, Mayana Zatz, Yeda A O Duarte, Maria Lucia Lebrão, Patricia Gama, Misu Lee, Sara Molatore, Maria Adelaide A Pereira, Raquel S Jallad, Marcello D Bronstein, Malebranche B Cunha-Neto, Bernardo Liberman, Maria Candida B V Fragoso, Sergio P A Toledo, Natalia S Pellegata and Rodrigo A Toledo
To date, no evidence of robust genotype–phenotype correlation or disease modifiers for multiple endocrine neoplasia type 1 (MEN1) syndrome has been described, leaving the highly variable clinical presentation of patients unaccounted for.
As the CDKN1B (p27) gene causes MEN4 syndrome and it is transcriptionally regulated by the product of the MEN1 gene (menin), we sought to analyze whether p27 influences the phenotype of MEN1-mutated patients. The cohort consisted of 100 patients carrying germline MEN1 gene mutations and 855 population-matched control individuals.
Genotyping of the coding p27 c.326T>G (V109G) variant was performed by sequencing and restriction site digestion, and the genotypes were associated with clinical parameters by calculating odds ratios (ORs) and their 95% CIs using logistic regression.
There were significant differences in p27 V109G allele frequencies between controls and MEN1-mutated patients (OR=2.55, P=0.019, CI=1.013–5.76). Among patients who are ≥30 years old carrying truncating MEN1 mutations, the T allele was strongly associated with susceptibility to tumors in multiple glands (three to four glands affected vs one to two glands affected; OR=18.33; P=0.002, CI=2.88–16.41). This finding remained significant after the Bonferroni's multiple testing correction, indicating a robust association. No correlations were observed with the development of MEN1-related tumors such as hyperparathyroidism, pituitary adenomas, and enteropancreatic and adrenocortical tumors.
Our study suggests that the p27 tumor suppressor gene acts as a disease modifier for the MEN1 syndrome associated with MEN1 germline mutations. If confirmed in independent patient cohorts, this finding could facilitate the management of this clinically complex disease.