Diagnosis of mosaic mutations in the MEN1 gene by next generation sequencing

in European Journal of Endocrinology
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  • 1 Univ. Lille, Inserm, UMR-S 1172, Team “Mucins, Epithelial Differentiation and Carcinogenesis”, JPARC - Jean-Pierre Aubert Research Center, Lille, France
  • 2 CHU Lille, Service de Biochimie et Biologie moléculaire « Hormonologie, Métabolisme-Nutrition, Oncologie », Lille, France
  • 3 Department of Endocrinology, Diabetes and Nutrition, University Hospital (CHU) and University of Bordeaux, Bordeaux, France
  • 4 CH Valenciennes, Service d’endocrinologie, Valenciennes Cedex, France
  • 5 Univ. Lille, UMR995-LIRIC (Lille Inflammation Research International Center), Lille, France

Correspondence should be addressed to A Tabarin; Email: antoine.tabarin@chu-bordeaux.fr

(A Tabarin and M-F Odou contributed equally to the study)

We recently read with interest the review article of Persani et al. (1) who highlighted the occurrence of unexpected results obtained by next generation sequencing (NGS) with the identification of novel candidate genes or variants in non-coding regions. In complement to their review, we report herein for the first time two cases of MEN1 mosaic mutations identified only by NGS, a finding that emphasizes the usefulness of this tool in current endocrine practice.

Case 1

The proband was a 68-year-old man diagnosed at the age of 45 years with primary hyperparathyroidism (PHPT) in the presence of renal lithiasis and mild hypercalcemia (2.9 mmol/L). Two years after surgical removal of a parathyroid adenoma, a prolactinoma was diagnosed during the investigation of sexual impotence. After 2 years of medical treatment, a 11 mm prolactinoma was surgically removed. This pathological association and the recurrence of PHPT led to the hypothesis of MEN1. MEN1 gene analysis was performed by Sanger sequencing, which came up negative. Further investigations were carried out by NGS (Illumina, Miseq). A splicing mutation was then identified in the intron 7 of MEN1 gene, c.1050-3C>A, that could lead to the skip of exon, with a proportion of 15% of mutated alleles. This sequence variation could be validated since the depth of sequencing was 998 reads, which is over the threshold of 700 reads recommended by Izawa et al. (2) to detect mosaic mutation at the level of 1%.

Case 2

The proband presented at the age of 32 years with PHPT due to multiglandular parathyroid hyperplasia, facial angiofibromas and bilateral adrenal tumors that turned out to be adrenocortical carcinomas (Weiss score = 3 and 5). Sanger sequencing detected the presence of a heterozygous nonsense mutation in exon 5 of MEN1 gene (c.794G>A; p.(Trp265*)) possibly responsible for biosynthesis of a truncated protein (Fig. 1A). Systematic familial genetic sequencing failed to identify the mutation in her parents, including her 57-year-old father who had a previous history of nephrolithiasis (Fig. 1B). Contrasting with his negative genetic analysis, endocrine investigations revealed undiagnosed PHPT responsible for mild hypercalcemia (2.8 mmol/L). Remission was obtained after subtotal parathyroidectomy revealing four hyperplastic parathyroid glands. During the follow-up, a distal pancreatectomy had to be performed at the age of 66 years for a growing grade 2 non-functioning neuroendocrine tumor (NET) of the pancreatic tail of 25 mm in size associated with multiple NET of 2 to 7 mm in size. Before this clinical presentation, initial genetic investigations were completed by NGS which allowed the detection of the same mutation than in the proband but at the level of 6% of mutated alleles (total depth of sequencing: 1027 reads) (Fig. 1C and D).

Figure 1
Figure 1

Genetic analysis in case n°2. (A) MEN1 Sanger sequencing of the proband: c.794G>A p.(Trp265*) heterozygous. (B) MEN1 Sanger sequencing of the father: no sequence variation detected. (C) MEN1 NGS sequencing of the father: mosaicism at the level of 6% of mutated alleles for c.794G>A p.(Trp265*). (D) Detail of the reads obtained by NGS.

Citation: European Journal of Endocrinology 180, 2; 10.1530/EJE-18-0852

Mosaic mutations implied in the development of inheritable disease are increasingly revealed by NGS that can detect mosaic mutation up to 1% of mutated alleles if the number of reads is over 700 (2), whereas Sanger sequencing has a sensitivity around 15% (3) and fails to detect mosaic mutation below this threshold. Mosaic mutations detected by NGS only have recently been described in various diseases associated with endocrine tumors such as VHL disease (4) and paraganglioma (5). However, to the best of our knowledge, a mosaic mutation in the MEN1 gene had only been reported in a congress abstract (6). The two cases reported herein further demonstrate that NGS, in addition to the advantage of simultaneous analysis of several endocrine diseases susceptibility genes, is a useful tool to detect mosaic mutation in MEN1 and identify causal event in initial negative situation.

The finding that patients described herein developed a macroprolactinoma and a grade 2 NET suggests that mosaic mutations are not associated with mild phenotype. The same conclusions have been made by Coppin et al. in VHL (4) and by Spier et al. (3) who demonstrated that the level of mosaicism for APC gene was not correlated with disease severity in colon polyposis. In conclusion, MEN1 mosaicism should be added to the rare CDKN1B mutations (7) as a cause of multiple endocrine neoplasia. Although its prevalence among suspected MEN1 syndrome phenocopies genetically unsolved remains to be determined, our observations suggest that the use of NGS may increase the detection of genetic events responsible for endocrine tumors and improve clinical management and genetic counseling. Our data also suggest that re-sequencing the MEN1 gene by NGS could be useful in patients bearing suggestive association of endocrine tumors and in whom MEN1 gene sequencing was previously negative using the Sanger technique.

Declaration of interest

Antoine Tabarin is an Editorial Board Member of European Journal of Endocrinology. Antoine Tabarin was not involved in the review or editorial process for this paper, on which he is listed as an author. The other authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this article.

Funding

This research did not receive any specific grant from any funding agency in the public commercial or not-for-profit sector.

References

  • 1

    Persani L, de Filippis T, Colombo C & Gentilini D. Genetic diagnosis of endocrine diseases by NGS: novel scenarios and unpredictable results and risks. European Journal of Endocrinology 2018 179 R111R123. (https://doi.org/10.1530/EJE-18-0379)

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  • 2

    Izawa K, Hijikata A, Tanaka N, Kawai T, Saito MK, Goldbach-Mansky R, Aksentijevich I, Yasumi T, Nakahata T & Heike T et al. Detection of base substitution-type somatic mosaicism of the NLRP3 gene with >99.9% statistical confidence by massively parallel sequencing. DNA Research 2012 19 142152. (https://doi.org/10.1093/dnares/dsr047)

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    • Export Citation
  • 3

    Spier I, Drichel D, Kerick M, Kirfel J, Horpaopan S, Laner A, Holzapfel S, Peters S, Adam R & Zhao B et al. Low-level APC mutational mosaicism is the underlying cause in a substantial fraction of unexplained colorectal adenomatous polyposis cases. Journal of Medical Genetics 2016 53 172179. (https://doi.org/10.1136/jmedgenet-2015-103468)

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  • 4

    Coppin L, Grutzmacher C, Crépin M, Destailleur E, Giraud S, Cardot-Bauters C, Porchet N & Pigny P. VHL mosaicism can be detected by clinical next-generation sequencing and is not restricted to patients with a mild phenotype. European Journal of Human Genetics 2014 22 11491152. (https://doi.org/10.1038/ejhg.2013.279)

    • Search Google Scholar
    • Export Citation
  • 5

    Buffet A, Smati S, Mansuy L, Menara M, Lebras M, Heymann MF, Simian C, Favier J, Murat A & Cariou B et al. Mosaicism in HIF2A- related polycythemia-paraganglioma syndrome. Journal of Clinical Endocrinology and Metabolism 2014 99 E369E373. (https://doi.org/10.1210/jc.2013-2600)

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    • Export Citation
  • 6

    Farook S, Kannappan D, Kenz S, Lalloo F, Trainer P & Brabant G. MEN-1 mosaic: the founder of a family. Endocrine Abstracts 2011 25 P196.

  • 7

    Molatore S & Pellegata NS. The MENX syndrome and p27: relationships with multiple endocrine neoplasia. Progress in Brain Research 2010 182 295320. (https://doi.org/10.1016/S0079-6123(10)82013-8)

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    Genetic analysis in case n°2. (A) MEN1 Sanger sequencing of the proband: c.794G>A p.(Trp265*) heterozygous. (B) MEN1 Sanger sequencing of the father: no sequence variation detected. (C) MEN1 NGS sequencing of the father: mosaicism at the level of 6% of mutated alleles for c.794G>A p.(Trp265*). (D) Detail of the reads obtained by NGS.

  • 1

    Persani L, de Filippis T, Colombo C & Gentilini D. Genetic diagnosis of endocrine diseases by NGS: novel scenarios and unpredictable results and risks. European Journal of Endocrinology 2018 179 R111R123. (https://doi.org/10.1530/EJE-18-0379)

    • Search Google Scholar
    • Export Citation
  • 2

    Izawa K, Hijikata A, Tanaka N, Kawai T, Saito MK, Goldbach-Mansky R, Aksentijevich I, Yasumi T, Nakahata T & Heike T et al. Detection of base substitution-type somatic mosaicism of the NLRP3 gene with >99.9% statistical confidence by massively parallel sequencing. DNA Research 2012 19 142152. (https://doi.org/10.1093/dnares/dsr047)

    • Search Google Scholar
    • Export Citation
  • 3

    Spier I, Drichel D, Kerick M, Kirfel J, Horpaopan S, Laner A, Holzapfel S, Peters S, Adam R & Zhao B et al. Low-level APC mutational mosaicism is the underlying cause in a substantial fraction of unexplained colorectal adenomatous polyposis cases. Journal of Medical Genetics 2016 53 172179. (https://doi.org/10.1136/jmedgenet-2015-103468)

    • Search Google Scholar
    • Export Citation
  • 4

    Coppin L, Grutzmacher C, Crépin M, Destailleur E, Giraud S, Cardot-Bauters C, Porchet N & Pigny P. VHL mosaicism can be detected by clinical next-generation sequencing and is not restricted to patients with a mild phenotype. European Journal of Human Genetics 2014 22 11491152. (https://doi.org/10.1038/ejhg.2013.279)

    • Search Google Scholar
    • Export Citation
  • 5

    Buffet A, Smati S, Mansuy L, Menara M, Lebras M, Heymann MF, Simian C, Favier J, Murat A & Cariou B et al. Mosaicism in HIF2A- related polycythemia-paraganglioma syndrome. Journal of Clinical Endocrinology and Metabolism 2014 99 E369E373. (https://doi.org/10.1210/jc.2013-2600)

    • Search Google Scholar
    • Export Citation
  • 6

    Farook S, Kannappan D, Kenz S, Lalloo F, Trainer P & Brabant G. MEN-1 mosaic: the founder of a family. Endocrine Abstracts 2011 25 P196.

  • 7

    Molatore S & Pellegata NS. The MENX syndrome and p27: relationships with multiple endocrine neoplasia. Progress in Brain Research 2010 182 295320. (https://doi.org/10.1016/S0079-6123(10)82013-8)

    • Search Google Scholar
    • Export Citation