1 Unidade de Endocrinologia Genetica UEG, Laboratorio de Endocrinologia Celular e Molecular LIM-25, Disciplina de Endocrinologia
2 Unidade de Paratireoide, Laboratorio de Cirurgia Vascular e da Cabeça e Pescoço LIM-28, Disciplina de Cirurgia de Cabeça e Pescoço, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, Sao Paulo, Brazil
3 Division of Metabolism, Department of Internal Medicine, Endocrinology and Diabetes, University of Michigan, Ann Arbor, Michigan, USA
4 Endocrinology Division, Federal University of Sao Paulo (UNIFESP), São Paulo, Brazil
5 Endocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), Bethesda, Maryland, USA
6 Vall d’Hebron Institute of Oncology, Barcelona, Spain
7 Disciplina de Endocrinologia, Instituto do Cancer do Estado de Sao Paulo ICESP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, Sao Paulo, Brazil
Correspondence should be addressed to D M Lourenço; Email: firstname.lastname@example.org
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.
Supplementary Table 1. Primers design, size and chromosomic localization of long-range PCR products covering coding and non-coding regions of the MEN1, AIP and CDKNIs genes.
Supplementary Table 2. Comparison between MEN1-targeted NGS expected and obtained yields with the two runs* selected for ideal metric validation and differences in metrics of coverage/read depth between these runs considering the full MEN1 gene or the MEN1 gene CDS.
Supplementary Table 3. Estimated costs of MEN1 genetic testing by Sanger Sequencing (SS) and Next-generation Sequencing (NGS), considering two different scenarios (maximal efficiency and under demand operation). Undoubtedly, estimated costs may largely vary depending on several factors, thus values should be adapted for local conditions.