Screening of the RET Proto-oncogene in Families with Multiple Endocrine Neoplasia Type 2 in Algeria  

Soumeya N Fedala , Mahdi Ali Haddam , Farida Chentli , Lakhdar Griene , Mohamed Chikouche , Radhia Ridas
1 Bab El Oued Hospital, Algeria
2 Bologhine Hospital, Algeria
3 Center of Pierre Et Marie Curie, Algeria
4 Ain naadja Hospital, Algeria
Author    Correspondence author
International Journal of Clinical Case Reports, 2013, Vol. 3, No. 16   doi: 10.5376/ijccr.2013.03.0016
Received: 24 Oct., 2013    Accepted: 05 Nov., 2013    Published: 06 Nov., 2013
© 2013 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

The MEN 2 is a hereditary disorder of dominant autosomal inheritance with complete penetrance associated with germeline RET gene mutations a genetic marker of MTC. The systematic analysis of this gene in any MTC or suspiscion of MEN2A allows for the presymptomatic diagnosis of familial forms. Specific and early treatment, by a surgery cure, of related with genetic risk at an early clinicopathological stage allows a fit treatment, the only guarantee. Three index cases with endocrine neoplasia were hospitalized at the Department of Endocrinology: Two patients 36 years old and 44 years old for exploration and management of bilateral adrenal pheochromocytoma and the other patient aged 40 years old to support a medullary thyroid carcinoma. The three patients underwent endocrine and morphological exploration seeking MEN 2a. The three patients had primary hyperparathyroidism by adenoma, MTC and pheochromocytoma. Direct sequencing of exons in three index cases revealed a mutation at codon 634 in exon 11 of the RET gene with two types of mutations: A transition TgC in the heterozygous state transforming cysteine to arginine (C634R) in the first and third family (2/3 of the cases studied); A transition G gA in the heterozygous state changing the cysteine to tyrosine (C634Y) in the second group (1/3 of the cases studied). Phenotypic and genotypic correlation was found. The extended to members of the three families (siblings, parents and direct and collateral descendants) survey has been accepted by 11 subjects. 5 of them will carry the familial mutation (45.5% of subjects). The clinical and paraclinical found no manifestation of MEN 2 out of MTC. Total thyroidectomy and central neck dissection were indicated for them. The MEN2A syndromic disorders are serious multi-cancerous conditions. Their diagnosis is based on the recognition of their family character, and on exploration highlighting various endocrine abnormalities. Confirmation by the genetic study of RET gene enables a presymptomatic screening and early therapeutic care in related. 

Keywords
Multiple endocrine neoplasia typ2; RET gene; Medullary thyroid cancer; Genetic study

1 Introduction
Multiple endocrine neoplasia type 2 (MEN 2) is an inherited multiglandular disease whose prevalence is estimated at 1/5000(1). There are three variants of the phenotypic MEN2 with a constant presence of MeduLlary thyroid cancer (MTC): MEN2A (Sipple syndrome), the most common form (60% of MEN 2) associated with the MTC: pheochromocytoma in 20%~50% of cases and primary hyperparathyroidism (HPT) in 5%~20% cases, MEN2B (Gorlin syndrome), rarer (5% of MEN 2) associated with the MTC pheochromocytoma (50% of cases), dysmorphic type Marfan, digestive and submucosa ganglioneuromato-sis (lips, tongue, eyelids, conjunctival tissue) and MTC family isolated syndrome (Farndon: 35% of MEN 2) in which the other components of the disease are absent (Romei et al., 2012; Brandi et al., 2001).

The MEN 2 is a hereditary disorder of dominant autosomal inheritance with complete penetrance associated with germeline RET gene mutations a genetic marker of MTC (Hunt, 2002). The systematic analysis of this gene in any MTC or suspiscion of MEN2A allows for the diagnosis of familial forms and allow presymptomatic diagnosis. Specific and early treatment, by a surgery cure, of related with genetic risk at an early clinicopathological stage allows a fit treatment, the only guarantee (Romei et al., 2012; Brandi et al., 2001; Hunt, 2002).

2 Purpose
Search for a mutation in the RET gene in three  index cases with MEN 2a and their families, secondary to correlate genotype phenotype among them.

3 Population, methodology
Three index cases with endocrine neoplasia were hospitalized at the Department of Endocrinology:

The patient (II.8) 36 years old, mother of four children and the patient (II.3) , 44 years old for exploration and management of bilateral adrenal pheochromocytoma.

The patient (I.1) aged 40 years old, mother of 6 children to support a medullary thyroid carcinoma.

The three patients underwent endocrine and morphological exploration seeking MEN 2a: urinary methoxylated derivatives, magnetic resonance imaging, MIBG scintigraphy for the diagnosis of pheochromocytoma, TCT dosage, thyroid scintigraphy with I131, thyroid ultrasound and fine needle aspiration for the diagnosis of thyroid carcinoma, calcium, phosphate, parathyroid hormone assay and the MIBI parathyroid scintigraphy for the diagnosis of primary hyperparathyroidism with parathyroid adenoma. Medullary carcinoma of the thyroid were found in the three patients. the assessment was completed by the study of the RET gene and morphological staging looking for secondary locations. Front positivity of the RET gene mutation in three patients, genetic testing is undertaken in their families. If molecular abnormality, endocrine and morphological assessment was undertaken to find a  multiple endocrine neoplasia.

To achieve the genetic study, blood samples were collected in EDTA tubes after informed consent of all subjects participating in this study. Blood is initially subjected to a hypotonic solution to burst red cells. The lysate was centrifuged (15 min, 1 500 g) and, after removing the supernatant, the cell pellet containing the leukocytes (0.3% of circulating cells) is taken up in a saline solution (0.15 mol/L NaCl/EDTA 0.1 mol/L, pH 10.5) and is treated with a solution of the white blood cells lysis.
Nuclear DNA released into the medium is treated for one to two hours at 65? by an active proteinase, proteinase K, which is designed to digest proteins that were associated with him. The cell lysate was treated with equal volume of phenol, and after centrifugation, with a mixture of chloroform and isoamyl alcohol. Thereafter, an in vitro DNA amplification by Polymerase Chain Reaction (PCR) is performed. Amplification products were then analyzed by electrophoresis on 2% agarose gel in the presence of ethidium bromide for visualization under UV, using automatic sequencers and marking nucleotides with different fluorochromes. The search of mutation is performed by sequencing direct PCR products, in 7 of the 21 exons of the RET proto-oncogene known to be the seat of mutations: 8, 10, 11, 13, 14, 15 and 16 exons.

4 Results
Direct sequencing of exons in three patients (AII.8; BII.3 and CI.1) revealed a mutation at codon 634 in exon 11 of the RET gene.

Analysis of codon 634 of the three index cases revealed two types of mutations:

-A transition T C in the heterozygous state transforming cysteine to arginine (C634R) in the first and third family (2/3 of the cases studied).

-A transition G A in the heterozygous state changing the cysteine to tyrosine (C634Y) in the second group (1/3 of the cases studied).

5 Results of family screening
The extended to members of the three families (siblings, parents and direct and collateral descendants) survey has been accepted by 11 subjects. 5 of them will carry the familial mutation (45.5% of subjects) (Figure 1). The clinical and paraclinical found no manifestation of  MEN 2 out of MTC.
 


Figure 1 Results of family A/B/C screening


6 Phenotypic characteristics of subjects RET+ (Table 1)
The breakdown by age group shows that the disease has been diagnosed in adulthood with a peak incidence between 30 and 40 years (6/15).

The distribution of patients investigated showed no gender predominance.

Correlation phenotype/genotype:A correlation phenotype/genotype was observed within and inter families was found. All patients with the codon 634 mutation developed the three neoplasia of MEN 2a: MTC, CCP and HPP (Table 1). All patients with the RET mutation in groupA had a bilateral  pheochromocytoma, MTC and parathyroid adenoma at diagnosis.
 


Table 1 Phenotypic characteristics of subjects RET +


7 Discussion
The MEN 2 is the only known hereditary cancer linked to the dominant mutation of germeline proto-oncogene: the RET gene (Hunt, 2002). The RET gene (60 kilobases, 21 exons), located on the pericentromeric region of chromosome 10 (10q11.2) encodes a tyrosine kinase membrane receptor that relays signals of growth and differentiation in neural crest. The RET mutations associated to MEN2 lead to constitutive self-activation of the tyrosine kinase domain and oncogene transforming activity (Arighi, 2005). Specific RET mutations are directly related to the types of MEN2 and therefore the aggressiveness of  MTC and the presence of other endocrine tumors (Hansford and Mulligan, 2000).

The mutations associated with MEN2A phenotype sit mostly in exon 11 (codon 634). Several studies in different populations relate the prevalence found in our study (Table 2).
 


Table 2 The frequency of codon 634 mutations in different populations


Mutations in exon 10 (codons 609-611-618-620) is found in 15%~20% of cases. Other types of mutations located in exons 11 and 13~15 were linked to the MEN2A in a limited number of cases (Bethanis et al., 2007; Wagner et al., 2012; Santoro et al., 2010; Santoro et al., 1998).

There is a genotype/phenotype relationship in these conditions. Une corrélation génotype-phénotype dans les NEM 2A a été démontrée. La mutation au codon 634 est fortement associée au développement d‘un CMT, un PCC et à l‘ HPT (Romei et al., 2010; Mukherjee and Zakalik, 2010). 

The risk of pheochromocytoma is 50% (vs 100% our study) for patients with mutation 634 and is much lower for other mutations (codons 790, 791, 804 and 891) (Romei et al., 2010).

Similarly, 634 mutations are associated more often notalgia (36% vs. 0% this study) and hyperparathyroidism (Santoro et al., 2010; 1998). This correlation is observed for other mutations such mutations 609, 618 or 620 which are associated with Hirschsprung‘s syndrome (Romei et al., 2010).

The discovery of a MTC is the most common diagnostic circumstance of an index case of a new family, but MEN2 should be sought in pheochromocytoma and atypical HPT (young, multiglandular reached). Each syndrome has biomarkers for the diagnosis but also to ensure the post-treatment surveillance. Related reported as free RET (-) will be excluded permanently monitoring and support.

On the therapeutic level, the management of MEN2A involves the treatment of MTC, pheochromocytoma and hyperparathyroidism. MTC surgery in MEN 2 is identical to that of sporadic MTC. A total thyroidectomy and central neck dissection is systematic given the frequency of lymph node involvement (Willem de Groot et al., 2006). The identification of related with risk whose carrying the mutation of RET allows prophylactic or early management of CMT whose benefit it is well established (Willem de Groot et al., 2006; Murat and Nicolli-Sire, 2000; Niccoli-Sire, 2006). Given the risk of recurrent, laryngeal and parathyroid disease related to thyroid surgery, age of thyroidectomy must be decided depending on the genotype. Indeed, it is a cation of risk stratification based on the transfer of the results of functional studies and oncogenic potential.

Thus, mutations in codons 918, 922 and 883 are classified as high risk, mutations in codons 634, 611, 618 and 620 medium risk, mutations 609, 768, 790, 791, 804 and 891 considered less aggressive, low risk (Niccoli-Sire, 2006; Brandi et al., 2001; Niccoli-Sire and Conte-Devolx, 2005).

Whatever the genotype, annual biological screening of pheochromocytoma and HPT should be initiated in all subjects with the mutation or with the disease. The age at which to begin the screening of patients at risk is not yet consensus: from the age of 15 regardless of the genotype for some, at five and ten years for NEM2B and with the MEN2A mutation RET codon534, after 20 years for other genotypes (Raue and Raue, 2009; Plaza-Menacho et al., 2006).

8 Conclusion
The MEN2A syndromic disorders are serious multi-cancerous conditions. Their diagnosis is based on the recognition of their family character, and on exploration highlighting various endocrine abnormalities. Confirmation by the genetic study of RET gene enables a presymptomatic screening and early therapeutic care in related.

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