Germline mutation landscape of multiple endocrine neoplasia type 1 using full gene next-generation sequencing

in European Journal of Endocrinology
Correspondence should be addressed to D M Lourenço; Email:
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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.

Downloadable materials

  • Supplementary data
  • 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.


     European Society of Endocrinology

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    Flow chart applied to genetic diagnosis of 76 unrelated MEN1 index cases using Targeted Next Generation Sequencing (tNGS) and multiplex ligation-dependent probe amplification (MLPA) assays. MEN1, multiple endocrine neoplasia type 1; F-MEN1, Familial MEN1; S-MEN1, sporadic MEN1; CDKIs, cyclin-dependent kinase inhibitors; NGS assay was based on long-range PCR amplifycation of the full MEN1 open reading frame using MiSeq Illumina platform; MLPA assay was performed to investigate gross deletions in the MEN1, AIP and CDKN1B/p27 genes in tNGS/MEN1 mutation-negative patients; nine MEN1-negative cases by tNGS and MEN1/AIP/p27-negative by MLPA were screened to germline mutations by tNGS addressed to CDKIs (CDKN2B/p15, CDKN2C/p18 CDKN1A/p21, CDKN1B/p27) and AIP genes; circle with full line, positive MEN1-mutation patients identified by MEN1-NGS or MEN1-MLPA assays; circle with dashed line, MEN1 mutation-negative patients by NGS or MLPA assays (MEN1, CDKN1B/p27 and AIP); triangle, patients without germline mutation identified in full open reading frames of the MEN1, CDKN2B/p15, CDKN2C/p18 CDKN1A/p21, CDKN1B/p27 and AIP genes by tNGS and with no gross deletions in MEN1, CDKN1B/p27 and AIP by MLPA assay.

  • View in gallery

    Mutations were identified in intron-exon frontiers and coding region of the MEN1 gene in 60 out of our cohort of 76 unrelated MEN1 index cases by both tNGS and SS (57 cases), and MLPA assay (3 cases). Truncating mutations (frameshift and nonsense) (NGS and SS) and gross MEN1 deletions (MLPA assay) are represented below diagram while non-truncating (missense and in-frame) and splicing mutations are located above; #, new mutations reported in the present study; ##, mutations previously reported from our cohort (8, 18); shadow region corresponds to non-coding regions of the MEN1 gene.


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