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Accepted Preprint first posted on 16 December 2014 as Manuscript EJE-14-0818
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RET Cys634Arg mutation confers a more aggressive multiple endocrine neoplasia type 2A
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phenotype than Cys634Tyr mutation
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Nuria Valdés1, Elena Navarro2, Jordi Mesa3, Anna Casterás3, Victoria Alcázar4, Cristina
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Lamas5 , Javier Tébar6 , Luis Castaño7, Sonia Gaztambide8, Lluís Forga.9
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1. Department of Endocrinology and Nutrition. Hospital Universitario Central de Asturias, Oviedo, Spain. 2. Department of Endocrinology and Nutrition. Hospital Universitario Virgen del Rocío, Sevilla, Spain. 3. Department of Endocrinology and Nutrition. Hospital de Vall d´Hebron, Barcelona, Spain.
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4. Department of Endocrinology and Nutrition. Hospital Severo Ochoa, Madrid, Spain.
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5. Department of Endocrinology and Nutrition. Complejo Hospitalario Universitario de
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Albacete, Albacete, Spain. 6. Department of Endocrinology and Nutrition. Hospital Universitario Virgen de la Arrixaca, Murcia, Spain. 7. Endocrinology and Diabetes Research Group. Hospital Universitario de Cruces, Barakaldo, Spain. 8. Department of Endocrinology and Nutrition. Hospital Universitario de Cruces, Barakaldo, Spain. 9. Department of Endocrinology. Complejo Hospitalario de Navarra, Pamplona, Spain.
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Corresponding author and requested reprints:
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Dr. Nuria Valdés. Department of Endocrinology and Nutrition. Hospital Universitario Central
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de Asturias. Avda. de Roma s/n, E-33011-Oviedo, Asturias, Spain. Phone: (+34)-985-108000.
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Fax: (+34)-985-107954. E-mail:
[email protected]
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1 Copyright © 2014 European Society of Endocrinology.
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Short tittle: RET C634R mutation is more adverse than C634Y.
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Keywords: Multiple Endocrine Neoplasia Type 2 A, RET C634R mutation, RET C634Y
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mutation, Medullary Thyroid Carcinoma, Pheochromocytoma, Hyperparathyroidism, RET-
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proto-oncogene.
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Word count: 4174
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Abstract
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Objective: Specific germline mutations in the RET proto-oncogene are correlated with clinical
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features in multiple endocrine neoplasia type 2A (MEN 2A); however, data are scarce regarding
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differences in clinical profiles dependent on the type of nucleotide and amino acid substitution
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at the same codon. We aimed to analyse differences in clinical risk profiles and outcomes
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between different amino acid encoded by codon 634.
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Design: The study was retrospective and multicentric.
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Methods: We collected data included in the Spanish Online National Database from MEN 2A
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patients carrying a RET-proto-oncogene mutation on codon 634. The mean follow-up time was
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7.6 ±6.9 years (1−32).
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Results: Patients (173) from 49 unrelated families were C634Y carriers, and 26 patients from 8
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different families had C634R mutation. We found higher penetrance of medullary thyroid
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carcinoma, pheochromocytoma, and hyperparathyroidism (P < 0.001, P = 0.007, P < 0.001,
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respectively) in C634R carriers than in C634Ycarriers. The Kaplan−Meier estimate of
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cumulative lymph node and distant metastases rates showed that these events occurred earlier in
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individuals harbouring the C634R mutation (P < 0.001). A multivariate adjusted Cox regression
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analysis showed that the C634R mutation was an independent factor for persistent/recurrent
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disease (hazard ratio, 3.17; 95% confidence interval: 1.66−6.03; P < 0.001).
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Conclusions: Our results suggest that there could be clinical differences caused by different
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amino acid substitutions at codon 634; specifically, the C634R mutation was associated with a
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more aggressive MEN 2A phenotype than the C634Y mutation.
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Introduction
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Multiple endocrine neoplasia type 2A (MEN 2A) is a rare syndrome characterized by
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the presence of medullary thyroid carcinoma (MTC; 95% of MEN 2A patients),
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pheochromocytoma (PHEO: 30%–50%), and hyperparathyroidism (HPT: 10%–20%) (1,2).
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This syndrome is caused by a germline activation mutation in the REarranged during
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Transfection (RET) proto-oncogene transmitted by an autosomal dominant inheritance (3,4).
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The RET proto-oncogene encodes a transmembrane tyrosine kinase receptor which comprises a
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highly conserved, cysteine-rich, extracellular domain, 2 intracellular tyrosine kinase domains,
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and an intracellular catalytic core (3,5). In the majority of MEN2A families (more than 90%),
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germline mutations affect the cysteine-rich extracellular domain by converting a cysteine into
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another amino acid, and this mutation determines RET spontaneous dimerization and activation
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(1-5). These mutations are located on codon 634 (exon 11) or codons 609, 611, 618, and 620
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(exon 10).
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Genotype-phenotype correlation has been described between specific codon mutation
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and age of onset, penetrance and clinical aggressiveness of MTC (1,2,6,7). Based on these
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findings the American Thyroid Association (ATA) Task Force has published management
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guidelines (8) on the timing and extension of prophylactic thyroidectomy establishing four
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different risk categories (from A, the lowest, to D, the highest). There is also a different
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prevalence of PHEO and HPT according to the type of specific codon mutation (7,9,10).
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The most common mutation, accounting for 34% of European MEN2A families, affects
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codon 634 (11). “In vitro” assays have demonstrated that the codon 634 mutation had a higher
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probability of transforming NIH3T3 cells than other RET codon mutations (12). This biological
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characteristic of the highest transforming activity correlates with phenotype because codon 634
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mutations are associated with the more severe forms of the MEN 2A phenotype and it is
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classified as class C in the ATA guidelines (8).
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There are six different amino acid substitutions for the same cysteine on codon 634,
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and all of them displayed comparable transforming activity in one “in vitro” assay (12),
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indicating that the degree of the activity depends on the position of cysteine mutations rather
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than the substituted amino acids. However, the assay was performed only in fibroblasts; thus the
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biological effects of the substituted amino acids are not known in thyroid C cells, adrenal
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medullary cells, and parathyroid cells. From a clinical viewpoint, few reports have indicated
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whether there is any difference in the clinical profiles based on amino acid substitutions on
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codon 634 (4,6,9,10,13).
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The aim of our study was to evaluate any potential differences in clinical risk profiles
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and outcomes in Spanish patients caused by different mutations on codon 634. This information
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might be useful for tailoring more accurate clinical care.
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Cys634Arg (C634R) is the most common RET mutation in European countries (14),
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except in Italy where Val804Met is more common (15). However, the most common RET
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mutation in MEN 2A Spanish patients is Cys634Tyr (C634Y) (16). Consequently, we examined
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the largest number of patients ever reported with this specific mutation on codon 634, which
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provided a firmer basis for statistical analyses.
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Subjects and methods
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The Spanish Group for the Study of MEN and Pheochromocytomas/Paragangliomas,
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which depends on the Spanish Endocrinology and Nutrition Society, designed an online
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national database to collect extensive clinical and genetic information about Spanish MEN 1,
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MEN 2A, MEN 2B, and pheochromocytomas/paragangliomas patients. Informed consent was
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obtained for all diagnostic and therapeutic procedures and for the inclusion of family and
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personal clinical data in the Register. The anonymity of patients was not breached; therefore,
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the study met the conditions required under Spanish law for making medical and or personal
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data available for scientific research.
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Data from patients diagnosed with MEN 2A, according to international clinical
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guidelines (1), were included in the database from January 2009 to December 2011 and were
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analysed in the course of 2012. All of these patients were diagnosed and clinically examined by
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the authors. The study was retrospective and multicentric.
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Briefly, the data includes gender, birth date, family history, and type of diagnosis (index
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cases and diagnosis by genetic or biochemical screening), the type of RET mutation, and the
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dates of diagnosis for the syndrome and for each lesion. The age at MEN2A syndrome
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diagnosis was considered the age at the second manifestation of any clinical features in index
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cases and the first manifestation or genetic diagnosis in familial cases. The age at diagnosis of
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clinical disease was considered the age at histological diagnosis for MTC and biochemical
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diagnosis for PHEO and HPT.
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For thyroid disease, the following characteristics were recorded: calcitonin (CT) basal
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and/or pentagastrin-stimulated levels (positive or negative), extent of surgery, and outcome after
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thyroidectomy. With respect to thyroid histopathology, we recorded the presence or absence of
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C-cell hyperplasia (CCH) and MTC. For lymph node status, we collected data on lymph node
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dissection as N0, N1, and Nx (non-conclusive data). For analysis, the N0 classification was used
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to describe N0 or Nx data. The same approach was applied for distant metastases, which were
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recorded at thyroid surgery. Tumour stage was assessed according to the 2002 UICC TNM
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classification (17). Patients were considered biochemically free of the disease at the last follow-
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up, according to international guidelines.
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For PHEO, we collected data about the presence or absence of symptoms at diagnosis, uni- or bilateral involvement, type of surgery, and clinical outcome. For HPT, we included the presence or absence of clinical symptoms, type of surgery, and clinical outcome.
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Statistical analysis
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Categorical variables were summarized as frequency counts and percentages.
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Continuous variables were summarized as the mean ± standard deviation unless otherwise
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specified, and range. For continuous variables, normality was assessed with the Kolmogorov-
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Smirnov test, and logarithmic transformations were applied as necessary to ensure normal
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distribution. Baseline characteristics were compared using the 2 test or Fisher’s exact test for
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qualitative variables, or the Student’s t test or Mann-Whitney’s U test for quantitative variables.
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The Kaplan−Meier estimate for age-related penetrance of MTC, PHEO, HPT, lymph nodes and
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distant metastases, and for biochemically free of disease were compared among type of amino
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acid substitution using the long rank test. Risk factors for disease-free survival were assessed
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via the chi-square test or Fisher´s exact test and we also performed a Cox regression model.
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Five candidate risk factors were assessed: gender, age at the time of surgery, basal and/or
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pentagastrin-stimulated calcitonin levels, lymph nodes metastases, and type of amino acid
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substitution. Associations were expressed as hazard ratios (HR) with their 95% confidence
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intervals (95% CI). Results were considered statistically significant at P
