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The RET E616Q Variant is a Gain of Function Mutation Present in a Family with Features of Multiple Endocrine Neoplasia 2A

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Abstract

The REarranged during Transfection (RET) proto-oncogene is a receptor tyrosine kinase involved in growth and differentiation during embryogenesis and maintenance of the urogenital and nervous systems in mammals. Distinct mutations across hotspot RET exons can cause Multiple Endocrine Neoplasia Type 2A (MEN2A) characterised by development of medullary thyroid cancer (MTC), phaeochromocytoma (PCC) and primary hyperparathyroidism (PHPT), with a strong correlation between genotype and phenotype. Here, we report a 42-year-old man presented in the clinic with a unilateral PCC, with subsequent investigations revealing a nodular and cystic thyroid gland. He proceeded to thyroidectomy, which showed bilateral C-cell hyperplasia (CCH) without evidence of MTC. His brother had neonatal Hirschsprung disease (HSCR). Genetic testing revealed the presence of a heterozygous variant of unknown significance (VUS) in the cysteine-rich region of exon 10 in the RET gene (c.1846G>C, p.E616Q), in both affected siblings and their unaffected mother. Exon 10 RET mutations are known to be associated with HSCR and MEN2. Variants in the cysteine-rich region of the RET gene, outside of the key cysteine residues, may contribute to the development of MEN2 in a less aggressive manner, with a lower penetrance of MTC. Currently, a VUS in RET cannot be used to inform clinical management and direct future care. Analysis of RETE616Q reveals a gain of function mutant phenotype for this variant, which has not previously been reported, indicating that this VUS should be considered at risk for future clinical management.

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Acknowledgments

The study has an ethical approval 13/SC/0574 (NRES Committee South Central - Hampshire A) sponsored by Guy’s and St Thomas’ NHS Foundation Trust (study number RJ114/N093) and also registered at King’s College Hospital (study number KCH14-124). All subjects provided written, informed consent for the research study (ethical approval 13/SC/0574).

This work was supported by The Generation Trust (KCL) and Friends of Guy’s Hospital Research Fund.

Author Contributions

We thank the following people for their contributions:

Dr Neil McDonald (The Francis Crick Institute)

Dr Ivan Plaza-Menacho (UCL)

Dr Simon Aylwin, Mr Klaus-Martin Schulte, Dr Dylan Lewis and Professor Alan McGregor (KCH)

Dr Paul Carroll, Dr Hosahalli Mohan, Celia Mills and Teresa Brothwood, Michelle Weston and Dr Christine Patch (GSTFT)

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Authors and Affiliations

  1. Cancer Genetics, Department of Medical and Molecular Genetics, Division of Genetics and Molecular Medicine, King’s College London, London, UK

    William Grey, Rosaline Hulse, Anna Yakovleva, Dilyana Genkova, Ellen Solomon & Louise Izatt

  2. Clinical Endocrinology, King’s College Hospital London, London, UK

    Benjamin Whitelaw

  3. Histopathology, King’s College Hospital London, London, UK

    Salvador J. Diaz-Cano

  4. Clinical Genetics, Guy’s and St Thomas’ NHS Foundation Trust London, Great Maze Pond, London, SE1 9RT, UK

    Louise Izatt

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  1. William Grey
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Correspondence to Louise Izatt.

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Supp. Fig. 1

Foci of C-cell hyperplasia revealed intrafollicular circumferential proliferation of C-cell without nuclear atypia. No solid C-cell nests or stromal invasion were identified (haematoxylin-eosin and immunohistochemical stain for calcitonin, 100×). Method as described by Williams et al. [45]. (PPTX 915 kb)

Supp. Fig. 2

A. In silico analysis of the RET51 protein sequence using the Align-GVGD program (http://agvgd.iarc.fr). Mutations are depicted as solid circles, labelled to the right. GV = Grantham Variation, GD = Grantham Deviation. B. Table summarising GD, GV and predicted class of pathogenicity along the Align-GVGD scale (C0 = weakest pathogenicity, C65 = strongest pathogenicity). (GIF 70 kb)

Supp. Fig. 3

Data for Empty Vector controls only from Fig. 2. The graph shows increasing induction of Luciferase activity when serum, GDNF or both are added for 16 h. (GIF 23 kb)

Supplementary Table 1

Patient features for all heterozygous RET E616Q family members at last clinical evaluation or age at operation. (DOCX 16.4 kb)

Supplementary Table 2

Primers used in this study. Purpose is noted in the final column. (DOCX 18 kb)

Supplementary Table 3

Further hereditary phaeochromocytoma and paraganglioma genes sequenced by NGMS during this study (PPGL) including; HGNC name, symbol, number, OMIM ID and clinical phenotype. The entire RET gene (20 exons) was sequenced during initial investigation and subsequent PPGL gene analysis confirmed the heterozygous RET E616Q variant. In addition MLPA analysis of the following genes was completed: RET, SDHAF2, SDHB, SDHC, SDHD and VHL. (DOCX 15 kb)

Supplementary Table 4

Adapted from ARUP laboratories, University of Utah (www.arup.utah.edu/database/men2). Genetic and disease associated information for mutations generated in this study, including our RET E616Q. N/R, not recorded, N/A, not applicable. (DOCX 15 kb)

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Grey, W., Hulse, R., Yakovleva, A. et al. The RET E616Q Variant is a Gain of Function Mutation Present in a Family with Features of Multiple Endocrine Neoplasia 2A. Endocr Pathol 28, 41–48 (2017). https://doi.org/10.1007/s12022-016-9451-6

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