Evidence for the founder effect of RET533 as the common Greek and Brazilian ancestor spreading multiple endocrine neoplasia 2A

in European Journal of Endocrinology

Objectives

About one-quarter of patients with medullary thyroid cancer (MTC) have inherited disease due to mutations in the RET gene. A rare mutation in exon 8 (G533C) of RET, previously described in a large Brazilian family with MEN2A, also appeared to be clustering in Greece, whereas it was rarely reported in other ethnic groups. The aim of this study was to identify a possible common ancestry between these carriers.

Patients and methods

Twelve RET G533C mutation carriers, four randomly selected from the Brazilian cohort and eight from apparently unrelated Greek families, were studied for a possible common ancestral origin. RET flanking microsatellite markers at chromosome 10q (D10S197, D10S196, D10S1652 and D10S537) were used.

Results

Genomic DNA analysis using these markers showed that many of these apparently unrelated individuals shared a common haplotype indicating a common ancestral origin.

Conclusion

Our data suggest that Brazilian and Greek patients with MTC carrying the G533C mutation in exon 8 of RET gene originate from a common ancestor. Due to historical reasons, we speculate that the more plausible explanation for the origin of this mutation is in Greece.

Abstract

Objectives

About one-quarter of patients with medullary thyroid cancer (MTC) have inherited disease due to mutations in the RET gene. A rare mutation in exon 8 (G533C) of RET, previously described in a large Brazilian family with MEN2A, also appeared to be clustering in Greece, whereas it was rarely reported in other ethnic groups. The aim of this study was to identify a possible common ancestry between these carriers.

Patients and methods

Twelve RET G533C mutation carriers, four randomly selected from the Brazilian cohort and eight from apparently unrelated Greek families, were studied for a possible common ancestral origin. RET flanking microsatellite markers at chromosome 10q (D10S197, D10S196, D10S1652 and D10S537) were used.

Results

Genomic DNA analysis using these markers showed that many of these apparently unrelated individuals shared a common haplotype indicating a common ancestral origin.

Conclusion

Our data suggest that Brazilian and Greek patients with MTC carrying the G533C mutation in exon 8 of RET gene originate from a common ancestor. Due to historical reasons, we speculate that the more plausible explanation for the origin of this mutation is in Greece.

Introduction

More than 25% of medullary carcinoma patients (MTC) have familial disease, which is due to germ line mutations of the RET gene. The clinical presentation varies according to the type of RET mutation. After the routine application of genetic analysis in all MTC cases (1), it became apparent that some apparently sporadic cases were in fact familial as they carried a RET mutation. Furthermore, a wider distribution of mutations across the RET gene was identified, which were different from those originally described. In this context, we recently described a large family with medullary thyroid carcinoma (MTC) carrying a missense mutation in exon 8 of RET, which corresponds to a glycine-to-cysteine substitution at codon 533 (2); this was later confirmed to be MEN2A. This family comprises 728 members of Caucasian origin with ancestors from Catalunia, Spain, who immigrated to Brazil at the end of the 19th century. Most of the family members live in Southeastern Brazil and have been followed at an outpatient Endocrine Unit either in Vitoria or São Paulo by the same team of physicians for over fifteen years. During this long-term follow-up, molecular and clinical characteristics of these patients have been described in detail (3, 4). However, the ancestry related to the origin of G533C RET mutation remains to be elucidated.

In 2007, Bethanis et al. reported a Greek patient with MEN 2A harboring the same RET G533C mutation (5). A few years later, Sarika et al. identified this mutation in 10 out of 129 patients with apparently sporadic medullary thyroid carcinoma in Greece (6). Gathering these results, we hypothesized whether Brazilian and Greek patients carrying the same RET mutation could share a common ancestor.

We proceeded with the investigation of putative founder effect as a point of coalescence between Brazilian and Greek affected families.

Patients and methods

Twelve patients with the G533C RET mutation were enrolled in the study: 4 randomly selected from the Brazilian cohort and 8 randomly selected from apparently unrelated Greek families and followed at the Endocrine Unit of Athens University Medical School (Athens, Greece). A control group comprising 29 unrelated thyroid-healthy Brazilian individuals was also studied. Blood samples were obtained from all participants, and all laboratory procedures were performed at the Laboratory of Molecular and Translational Endocrinology at the Federal University of Sao Paulo – UNIFESP, Brazil. The study protocol was approved by the internal review board of UNIFESP (São Paulo, Brazil) and University of Athens (Athens, Greece). All patients received genetic counseling before and after RET testing.

Genomic DNA was extracted from peripheral-blood leucocytes as previously reported (7). Haplotype analysis using four RET flanking microsatellite markers (D10S197, D10S196, D10S1652 and D10S537) was performed, as described by Qi et al. (8), to track a founder effect. The first marker was placed at position 50.04 cm and the other three at positions 70.07, 80.61 and 89.16 cm (Fig. 1A). The chromosome map distances were derived from the deCODE map (URL:http://www.ncbi.nlm.nih.gov/probe).

Figure 1

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Figure 1

(A) Schematic distribution of RET microsatellite markers in representative family members of apparently unrelated kindreds from Brazil and Greece. Microsatellite markers D10S197, D10S196, D10S1652 and D10S537, and their distance in cm, distributed throughout RET genomic region as haplotype analysis. (B) Phylogenetic tree of Brazilian and Greek families and controls.

Citation: European Journal of Endocrinology 176, 5; 10.1530/EJE-16-1021

Haplotypes of patients and controls were reconstructed using the statistical software package PHASE, version 2.1 (http://stephenslab.uchicago.edu/software.html) (9). We constructed the phylogenetic tree of the studied families with POPTREE2 software by estimating the genetic distance between them (10). Chi-square test was used to yield differences in allele distribution among Greek patients, Brazilian patients and Brazilian controls. GenePop was used to perform Hardy–Weinberg exact test (11), and all loci were in Hardy–Weinberg equilibrium.

Results

This study included 8 Greek patients (2 men and 6 women) and 4 Brazilian patients (2 men and 2 women). Clinical and biochemical data of these patients are summarized in Table 1. As can be seen from the table, all the examined cases had excellent long-term follow-up compatible with the relatively mild phenotype that has already been reported for carriers of this mutation (12). Furthermore, the age at presentation was in all cases relatively late.

Table 1

Clinical characteristics of the cohort of G533C patients.

Patient numberOriginAge at diagnosis (years)GenderpTNM stagingStatus at the last follow-up visit
1Brazil57FT1N0MxNED
2Brazil44FT2N1aMxNED
3Brazil33MNo MTCNED
4Brazil53MT3N1aM0NED
5Greece36FT1N0M0NED
6Greece34FT1bN0M0NED
7Greece55MT1N0M0NED
8Greece37MT1N0M0NED
9Greece35FT1N0M0NED
10Greece35FT1N0M0NED
11Greece24FT3N1aM0NED
12Greece50FT1N0M0NED

NED, no evidence of disease.

Four MTC patients (3 from Greece and one from Brazil) shared a common haplotype (D10S197-RET-D10S196-D10S1652: 168-RETG533C-100-173-148, Fig. 1A and Table 2), which was not seen in chromosomes bearing wild-type alleles (P < 0.005). In fact, Brazilian patients presented similar distribution of RET polymorphic microsatellite markers compared to Greek patients, suggesting that patients from both nationalities may share a unique genetic signature (Table 2). When comparing Brazilian and Greek affected patients to the control group, we observed a different distribution of D10S197 (P = 0.016), D10S196 (P = 0.042), D10S1652 (P < 0.001) and D10S537 alleles (P = 0.003; Table 3). These results reveal a genetic progenitor similarity between Brazilian and Greek RET G533C patients regarding RET flanking regions that was not observed in unrelated thyroid-healthy controls.

Table 2

Polymorphic microsatellite markers and allele frequency in Brazilian and Greek patients with medullary thyroid carcinoma bearing the RET G533C mutation.

MarkerAlleleRET G533C Brazilian patientsRET G533C Greek patientsP value
n%n%
D10S19716200337.50.223
16600112.5
1684100450
D10S196963754500.634
9800112.5
100125337.5
D10S1652157125000.312
16600112.5
17000225
173375562.5
D10S537139250112.50.262
14412500
148125562.5
15200112.5
15400112.5
Table 3

Comparison of RET haplotype frequency among Brazilian and Greek RET533 patients with thyroid-healthy controls.

MarkerAlleleRET G533C Brazilian patientsRET G533C Greek patientsThyroid-healthy controlsP valueaP valuebP valuec
D10S19716844180.0130.4240.016
Other alleles0840
D10S1969634140.0590.1990.042
Other alleles1444
D10S1652173351<0.001<0.001<0.001
Other alleles1357
D10S5371481580.4720.0040.003
Other alleles3350

Brazilian patients vs controls

Greek patients vs controls

Brazilian patients vs Greek patients vs controls.

According to the analysis of the phylogenetic tree of the MTC patients and controls, Brazilian and Greek patients pertained to a common root, corresponding to a putative most recent common ancestor (Fig. 1B). As a matter of fact, the control group shared reduced genetic similarity with the MTC patients.

Discussion

After the original identification that the exon 8 G533C RET mutation is associated with inherited MTC in our large Brazilian pedigree, there followed very few relative reports from the Mediterranean region and especially from Greece. Interestingly, the one family harboring this mutation in the USA proved to be of Greek ethnic origin (13). Our data suggest that Brazilian patients with MTC caused by the G533C RET mutation have an ancestor common to the Greek patients that share the same RET genotype. Indeed, by tracking the founder effect, we brought scientific shreds of evidence to this sense. Probably, the G533C mutation observed in Brazilian patients, originally from Catalunia, Spain, was settled by a migratory movement in Europe, either from Greece or from those same who established the Greek population.

Since ancient times, more than thirty Greek city-states had several colonies over the Mediterranean Sea, including in the Iberian Peninsula, where Catalunia is located (14). In our first description of the G533C RET mutation, we speculated that ‘this large family has a Caucasian background from Catalunia, and it is conceivable that relatives living in Europe might also bear the same mutation; hence, it is likely to address a founder effect from Spain, and it is feasible that other relatives also migrated to other countries by the time the ancestor moved to Brazil at the end of the 19th century’ (2).

The fact that Brazilian (from Catalunia, Spain) and Greek patients have a common ancestor is fascinating and suggests that, historically, the more plausible explanation for the origin of this mutation is in Greece as Greek migratory currents have occurred in the Mediterranean Sea for many centuries; furthermore, the substantial proportion of the G533C RET mutation among the hereditary MTC patients in Greece reinforces this hypothesis (6).

It is worth noting that the world is a melting pot, in which our genetically heterogeneous population can be described as a complex fusion of ethnicities, nationalities and cultures. In this scenario, cultural assimilation in the setting of immigration is important not only for the maintenance of national unity but also for the understanding of the genetic distribution of deleterious alleles. This ethnographic consideration highlights the impact of the present study on public health.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

The authors’ research was supported by grants from the São Paulo State Research Foundation/FAPESP (2006/60402-1 and 2010/51547-1 to R M B M and M R D S and 2014/06570-6 to J M C) and from the Fleury Group (12518). S C L received a research fellowship grant from FAPESP (2009/50575-4). R M B M is an investigator of the Fleury Group. J M C is an investigator of the Brazilian National Research Council (CNPq).

Author contribution statement

Lucas L Cunha: Collection and assembly of data, data analysis and interpretation and manuscript writing; Susan C Lindsey: Clinical data analysis, interpretation and manuscript writing; Maria Inez C França: Provision of study patients; Leda Sarika: Provision of study patients, clinical data analysis and interpretation; Alexandra Papathoma: Technical support; Ilda S Kunii: Technical support; Janete M Cerutti: Data analysis and interpretation and manuscript writing; Magnus R Dias-da-Silva: Design, data analysis and interpretation and manuscript writing; Maria Alevizaki: Conception and design and manuscript writing; Rui M B Maciel: Conception and design and manuscript writing.

Acknowledgements

The authors thank the team from the Laboratory of Molecular and Translational Endocrinology, especially Dr Cléber Camacho.

References

  • 1

    EliseiRAlevizakiMConte-DevolxBFrank-RaueKLeiteVWilliamsGR.2012European thyroid association guidelines for genetic testing and its clinical consequences in medullary thyroid cancer. European Thyroid Journal20131216231. (doi:10.1159/000346174)

  • 2

    Da SilvaAMMacielRMDa SilvaMRToledoSRDe CarvalhoMBCeruttiJM.A novel germ-line point mutation in RET exon 8 (Gly(533)Cys) in a large kindred with familial medullary thyroid carcinoma. Journal of Clinical Endocrinology and Metabolism20038854385443. (doi:10.1210/jc.2003-030997)

  • 3

    OliveiraMNHemerlyJPBastosAUTamanahaRLatiniFRCamachoCPImpellizzeriAMacielRMCeruttiJM.The RET p.G533C mutation confers predisposition to multiple endocrine neoplasia type 2A in a Brazilian kindred and is able to induce a malignant phenotype in vitro and in vivo. Thyroid201121975985. (doi:10.1089/thy.2010.0190)

  • 4

    SignoriniPSFrancaMICamachoCPLindseySCValenteFOKasamatsuTSMachadoALSalimCPDelceloRHoffAOA ten-year clinical update of a large RET p.Gly533Cys kindred with medullary thyroid carcinoma emphasizes the need for an individualized assessment of affected relatives. Clinical Endocrinology201480235245. (doi:10.1111/cen.12264)

  • 5

    BethanisSKoutsodontisGPaloukaTAvgoustisCYannoukakosDBeiTPapadopoulosSLinosDTsagarakisS.A newly detected mutation of the RET protooncogene in exon 8 as a cause of multiple endocrine neoplasia type 2A. Hormones20076152156. (doi:10.14310/horm.2002.1111011)

  • 6

    SarikaHLPapathomaAGarofalakiMVasileiouVVlassopoulouBAnastasiouEAlevizakiM.High prevalence of exon 8 G533C mutation in apparently sporadic medullary thyroid carcinoma in Greece. Clinical Endocrinology201277857862. (doi:10.1111/j.1365-2265.2012.04462.x)

  • 7

    KizysMMCardosoMGLindseySCHaradaMYSoaresFAMeloMCMontoyaMZKasamatsuTSKuniiISGiannoccoGOptimizing nucleic acid extraction from thyroid fine-needle aspiration cells in stained slides, formalin-fixed/paraffin-embedded tissues, and long-term stored blood samples. Arquivos Brasileiros de Endocrinologia e Metabologia201256618626. (doi:10.1590/S0004-27302012000900004)

  • 8

    QiXPMaJMDuZFYingRBFeiJJinHYHanJSWangJQChenXLChenCYRET germline mutations identified by exome sequencing in a Chinese multiple endocrine neoplasia type 2A/familial medullary thyroid carcinoma family. PLoS ONE20116e20353. (doi:10.1371/journal.pone.0020353)

  • 9

    StephensMScheetP.Accounting for decay of linkage disequilibrium in haplotype inference and missing-data imputation. American Journal of Human Genetics200576449462. (doi:10.1086/428594)

  • 10

    LatterBD.Selection in finite populations with multiple alleles. 3. Genetic divergence with centripetal selection and mutation. Genetics197270475490.

  • 11

    RoussetF.Genepop’007: a complete re-implementation of the genepop software for Windows and Linux. Molecular Ecology Resources20088103106. (doi:10.1111/j.1471-8286.2007.01931.x)

  • 12

    WellsSAJrAsaSLDralleHEliseiREvansDBGagelRFLeeNMachensAMoleyJFPaciniFRevised American Thyroid Association guidelines for the management of medullary thyroid carcinoma. Thyroid201525567610. (doi:10.1089/thy.2014.0335)

  • 13

    CastroMRThomasBCRichardsMLZhangJMorrisJC.Multiple endocrine neoplasia type 2A due to an exon 8 (G533C) mutation in a large North American kindred. Thyroid20132315471552. (doi:10.1089/thy.2012.0599)

  • 14

    BraudelF.The Mediterranean and the Mediterranean World in the Age of Philip II. Berkeley, California, USA: University of California Press1995.

 

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Figures

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    (A) Schematic distribution of RET microsatellite markers in representative family members of apparently unrelated kindreds from Brazil and Greece. Microsatellite markers D10S197, D10S196, D10S1652 and D10S537, and their distance in cm, distributed throughout RET genomic region as haplotype analysis. (B) Phylogenetic tree of Brazilian and Greek families and controls.

References

1

EliseiRAlevizakiMConte-DevolxBFrank-RaueKLeiteVWilliamsGR.2012European thyroid association guidelines for genetic testing and its clinical consequences in medullary thyroid cancer. European Thyroid Journal20131216231. (doi:10.1159/000346174)

2

Da SilvaAMMacielRMDa SilvaMRToledoSRDe CarvalhoMBCeruttiJM.A novel germ-line point mutation in RET exon 8 (Gly(533)Cys) in a large kindred with familial medullary thyroid carcinoma. Journal of Clinical Endocrinology and Metabolism20038854385443. (doi:10.1210/jc.2003-030997)

3

OliveiraMNHemerlyJPBastosAUTamanahaRLatiniFRCamachoCPImpellizzeriAMacielRMCeruttiJM.The RET p.G533C mutation confers predisposition to multiple endocrine neoplasia type 2A in a Brazilian kindred and is able to induce a malignant phenotype in vitro and in vivo. Thyroid201121975985. (doi:10.1089/thy.2010.0190)

4

SignoriniPSFrancaMICamachoCPLindseySCValenteFOKasamatsuTSMachadoALSalimCPDelceloRHoffAOA ten-year clinical update of a large RET p.Gly533Cys kindred with medullary thyroid carcinoma emphasizes the need for an individualized assessment of affected relatives. Clinical Endocrinology201480235245. (doi:10.1111/cen.12264)

5

BethanisSKoutsodontisGPaloukaTAvgoustisCYannoukakosDBeiTPapadopoulosSLinosDTsagarakisS.A newly detected mutation of the RET protooncogene in exon 8 as a cause of multiple endocrine neoplasia type 2A. Hormones20076152156. (doi:10.14310/horm.2002.1111011)

6

SarikaHLPapathomaAGarofalakiMVasileiouVVlassopoulouBAnastasiouEAlevizakiM.High prevalence of exon 8 G533C mutation in apparently sporadic medullary thyroid carcinoma in Greece. Clinical Endocrinology201277857862. (doi:10.1111/j.1365-2265.2012.04462.x)

7

KizysMMCardosoMGLindseySCHaradaMYSoaresFAMeloMCMontoyaMZKasamatsuTSKuniiISGiannoccoGOptimizing nucleic acid extraction from thyroid fine-needle aspiration cells in stained slides, formalin-fixed/paraffin-embedded tissues, and long-term stored blood samples. Arquivos Brasileiros de Endocrinologia e Metabologia201256618626. (doi:10.1590/S0004-27302012000900004)

8

QiXPMaJMDuZFYingRBFeiJJinHYHanJSWangJQChenXLChenCYRET germline mutations identified by exome sequencing in a Chinese multiple endocrine neoplasia type 2A/familial medullary thyroid carcinoma family. PLoS ONE20116e20353. (doi:10.1371/journal.pone.0020353)

9

StephensMScheetP.Accounting for decay of linkage disequilibrium in haplotype inference and missing-data imputation. American Journal of Human Genetics200576449462. (doi:10.1086/428594)

10

LatterBD.Selection in finite populations with multiple alleles. 3. Genetic divergence with centripetal selection and mutation. Genetics197270475490.

11

RoussetF.Genepop’007: a complete re-implementation of the genepop software for Windows and Linux. Molecular Ecology Resources20088103106. (doi:10.1111/j.1471-8286.2007.01931.x)

12

WellsSAJrAsaSLDralleHEliseiREvansDBGagelRFLeeNMachensAMoleyJFPaciniFRevised American Thyroid Association guidelines for the management of medullary thyroid carcinoma. Thyroid201525567610. (doi:10.1089/thy.2014.0335)

13

CastroMRThomasBCRichardsMLZhangJMorrisJC.Multiple endocrine neoplasia type 2A due to an exon 8 (G533C) mutation in a large North American kindred. Thyroid20132315471552. (doi:10.1089/thy.2012.0599)

14

BraudelF.The Mediterranean and the Mediterranean World in the Age of Philip II. Berkeley, California, USA: University of California Press1995.

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