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Original Studies |
Unit of Genetic Cancer Susceptibility (F.L., M.S., T.T., G.R., F.C.) and Unit of Genetic Cancer Epidemiology (D.E.G.), International Agency for Research on Cancer, 69372 Lyon, France; Faculté de Médecine, Université de Sfax (H.A.), 3028 Sfax, Tunisie; Institut Jean Godinot (M.J.D.), 51056 Reims, France; and Service de Médecine Nucléaire, Institut Gustave Roussy (M.S.), 94805 Villejuif, France
Address all correspondence and requests for reprints to: Prof. Giovanni Romeo, Unit of Genetic Cancer Susceptibility, International Agency for Research on Cancer, 150 cours Albert Thomas, 69372 Lyon Cedex 08, France. E-mail: romeo{at}iarc.fr
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Abstract |
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Introduction |
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Genetic risk factors are poorly known. Families with recurrence of NMTC have been repeatedly reported in the literature (14, 15, 16, 17, 18). Familial NMTC (FNMTC) accounts for about 3–7% of all thyroid tumors and is a clinical entity characterized by a more aggressive phenotype than its sporadic counterparts. FNMTC is often multifocal, relapses more frequently, and occurs at an earlier age than the sporadic forms (19).
Familial clustering of NMTC and therefore the possible existence of genetic risk factors are demonstrated also by epidemiological studies. The Utah Population Database resource was used to systematically investigate familial clustering of 28 distinct cancer site definitions among first degree relatives of cancer probands. All sites showed an excess of cancers of the same site among relatives, with thyroid cancer having the highest relative risk for first degree relatives of probands with thyroid cancer (familial relative risk = 8.60) (20). These results are supported by a second population-based study carried out in Sweden, suggesting an increased thyroid cancer risk of offspring when one parent has thyroid cancer. This risk is higher when the second parent is also affected (21).
Many published pedigrees suggest an autosomal dominant mode of inheritance with reduced penetrance (14, 15, 16, 17, 18), but polygenic inheritance cannot be excluded, which could also explain the rarity of large families with many cases of thyroid cancer.
To map the gene(s) affecting susceptibility to NMTC, an international consortium for the recruitment of families with recurrence of NMTC has been organized at International Agency for Research on Cancer (IARC) in collaboration with clinicians from several countries. As the mode of inheritance of FNMTC is not clear, a very simple selection criterium was used, i.e. families were selected for the study when at least two cases of NMTC were present, either papillary thyroid carcinoma (PTC) or follicular thyroid carcinoma (FTC). Family members diagnosed with thyroid pathologies, such as multinodular goiter (MNG) or adenoma were also included in the study and were considered as affected, as such benign diseases are risk factors for thyroid cancer (22, 23). One hundred and sixty-two NMTC families have been collected to date. Among them, several kindreds also show recurrence for MNG. There could therefore be genes predisposing to both benign and malignant thyroid tumors.
We have previously studied several large pedigrees with recurrence of NMTC and MNG by a genome-wide search approach. This suggested genetic heterogeneity in susceptibility to NMTC. Particularly, three interesting genes have been shown to be involved in some thyroid cancers.
MNG1, mapped to chromosome 14q31, is involved in susceptibility to multinodular goiter in a large Canadian family with 18 cases of MNG in which 2 individuals also had PTC. The same gene did not play a major role in the 37 small FNMTC pedigrees under study (24). To reevaluate its involvement in FNMTC, we investigated MNG1 in our larger sample of NMTC families. Moreover, this region of chromosome 14 has also been suggested to harbor a major susceptibility locus for Graves’ disease (25).
TCO, mapped to chromosome 19p13.2, is responsible for predisposition to thyroid tumors in a French family with six individuals showing MNG and three PTC cases (26).
Germline and somatic mutations of the protooncogene RET are frequently found in both isolated and familial forms of medullary thyroid cancer (27). Moreover, rearrangements in this protooncogene have been found frequently in sporadic cases of papillary thyroid carcinoma (27). However, the involvement of RET in predisposition to familial NMTC has not been the subject of detailed investigation.
In this study, we have focused on 56 informative families from our collection in these three candidate regions. In addition, a large kindred with recurrence of Graves’ disease and Hashimoto’s disease (28) was analyzed at 14q31, where a susceptibility locus for Graves’ disease has been localized (25) in the region of MNG1.
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Subjects and Methods |
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An international consortium has been established for the collection of biological samples from NMTC families. The collaborating centers are either clinical or epidemiological institutions. The search for families is therefore carried out with a variety of methods, from self-referral of the patients to automated cross-checking of cancer and population registries (29). This study was approved by the committee for protection of persons in biomedical research of Lyon.
The criterium for eligibility of the families is that at least 2 family members must be affected with NMTC. Family members having benign thyroid diseases such as goiters, adenomas, or thyroiditis were also sampled. Informed consent was obtained by a clinician belonging to 1 of the collaborating centers, and a blood sample (10–20 mL) was drawn. A clinical questionnaire was also proposed to the patients, and their pedigrees were constructed. All of the material was sent to the IARC, where DNA was extracted from blood samples using the Puregene Kit (Gentra Systems, Minneapolis, MN), and lymphoblastoid cell lines were established. Thus, a panel of 56 informative families with at least 2 relatives affected with NMTC was selected for genotyping. When available, samples of tumor tissues were also collected for future studies.
One hundred and fifty individuals from the 56 NMTC families were used for the genotyping. Among the 56 families, we observed 37 families with 2 cases of NMTC: 14 families with 3 cases of NMTC, 3 families with 4 cases of NMTC, and 2 families with 5 cases of NMTC. Most families also showed cases of other thyroid diseases, such as goiters, adenomas, or thyroiditis. In addition, 37 of 49 affected members of a large kindred (28) with recurrence of Graves’ disease or Hashimoto’s thyroiditis were genotyped for a susceptibility locus to Graves’s disease with selected markers (25).
Genotyping
The DNA samples were genotyped with microsatellite markers from chromosome 10 (D10S141, sTCL2-RET, RET-INT5), chromosome14 (D14S617, D14S749, D14S1030, D14S1054, D14S611), and chromosome 19 (D19S391, D19S916, D19S413, D19S586, D19S535, D19S221). Primer sequences were obtained from public databases (markers of chromosomes 14 and 19) or from the literature for markers of chromosome 10 [RET-INT5 (30), D10S141 (31), sTCL2-RET (32)]. Primers were labeled with fluorescent dyes, or alternatively, unlabeled primers were used, and the PCR reactions were supplemented with fluorescently labeled dCTP (PE Applied Biosystems, Foster City, CA). The PCR reactions were carried out in a volume of 8 µL and included 1 x buffer PCR, 1.5 mmol/L MgCl2, 200 µmol/L of each deoxy-NTP, 50 ng DNA, 0.2 U Red Hot DNA polymerase (Advanced Biotechnologies, Epsom, UK), and 5 pmol of each primer. The reactions were carried out in a 9600 GeneAmp PCR System with the following thermal profile: denaturation at 96 C for 5 min; 30 cycles of 94 C for 30 s, 53 C (or 55 C) for 30 s, and 72 C for 30 s; with a final extension at 72 C for 5 min. The fluorescent products were pooled, and an aliquot was run in a 377 Automated Sequencer (PE Applied Biosystems). The data were automatically collected and analyzed by the GeneScan and Genotyper softwares (PE Applied Biosystems).
Genetic model of thyroid cancer susceptibility
As many genetic analyses require the specification of a model relating the phenotype (in this case, NMTC, or NMTC+MNG) to the genotype at a hypothesized susceptibility locus, we derived two models that were chosen to fit the epidemiological data in terms of population prevalence (0.002) (33) and the observed familial risk (8.6) (20). Because the mode of inheritance is unknown for familial NMTC, both a dominant and a recessive model were analyzed. Under the dominant model we assumed a disease allele frequency of 0.001, a penetrance of 0.18 (probability of having a given phenotype given a certain genotype), and a phenocopy rate of 0.0016 (probability of having the disease without having the disease allele). Under the recessive model these parameters were fixed at 0.05, 0.21, and 0.0015, respectively.
Linkage analysis
To examine coinheritance of the postulated thyroid cancer susceptibility locus and the three candidate loci examined, a series of genetic linkage analyses was performed. To avoid overreliance on the assumed genetic models as defined above, nonparametric linkage analyses were performed in addition to those based on the assumed genetic models. Both sets of analyses were carried out using the program GeneHunter (34). For the parametric analyses, the evidence for or against such coinheritance is quantified by the LOD score which is the decimal logarithm of the ratio of the probabilities of the observed phenotype and marker data under the hypotheses of linkage to the chromosomal region of interest defined by the marker loci to that under the null hypotheses that the disease locus and marker loci are independent. A LOD score of 3, for example, indicates odds of 1000:1 in favor of linkage to a given genetic marker or set of markers, whereas a value of -2 would indicate odds of 100:1 against such linkage. Because there is likely to be genetic heterogeneity (more than one disease-causing gene) present in our sample, we also estimated the maximum LOD score as a function of the proportion of families linked to the candidate regions.
For the nonparametric approach, the analysis is based on nonrandom sharing of alleles at the genetic marker loci among affected individuals within each family. The test statistic of interest has a standard normal distribution reflecting the deviation from the null hypothesis of haplotype sharing according to expectations based solely on the genetic relationship between the affected individuals.
Allele frequencies for the microsatellite markers were obtained from the Genome Database. All families in our dataset are of Caucasian European origin. All European populations belong to a very homogeneous group, if compared with other world populations (35); therefore, we assumed that allele frequencies are the same for all of them. Only two of the families are from Finland, i.e. from a population slightly more distant from the rest of European ethnic groups, but we used the same allele frequencies for these families, as allele frequencies of Finnish samples usually do not differ from those from the rest of Europe in anonymous DNA markers. When allele frequencies for Caucasians were unavailable, allele frequencies were calculated from the genotyping of our samples, choosing one individual per family and assuming that the families are unrelated (102 alleles).
The markers used and assumed genetic maps for the three candidate regions were: for chromosome 10, D10S141 - 0.1cM - sTCL2-RET - 0.1cM - RET-INT5; for chromosome 14, D14S617 - 2.7cM - D14S749 - 5.0cM - D14S1030 - 0.1cM - D14S1054 - 2.7cM - D14S611; and for chromosome 19, D19S391 - 2.5cM - D19S916 - 1.1cM - D19S413 - 0.6cM - D19S586 - 0.7cM - D19S535 - 2.6cM - D19S221.
To assess the utility of our family resource for detecting linkage to a candidate susceptibility locus, we carried out a series of simulations using the SLINK program of LINKAGE (36). The simulations were performed assuming first that all families were due to the candidate locus, and then under varying levels of genetic heterogeneity. In all simulations reported, the assumed marker locus was D19S413 (chosen randomly from our set of markers), no recombination was allowed between the disease and marker loci, and the dominant and recessive models specified above were used.
Genetic heterogeneity was tested using the HET function of GeneHunter and the HOMOG program (37).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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). In these families, samples from 115
cases of other thyroid pathologies were also collected. In all
families, biological samples have been collected from at least 1 person
affected with NMTC. In 38 families, the collection of samples from NMTC
cases is not yet completed; therefore, these families were classified
as informative if completed. In 34 families with only 2 cases of NMTC,
the affected individuals are a pair of parents and offspring;
therefore, these families are not suitable for the linkage analysis and
were classified in the low informativity category. These families will
be useful for mutation testing when a major gene of predisposition to
NMTC is cloned. For 28 other families, detailed information on the
pedigree structure or the clinical history of the patients has not yet
been collected, and these families have been classified as low
informativity as well.
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sums up the number of cases of thyroid
cancer and other thyroid pathologies diagnosed in this set of families.
Among the remaining 6 informative families, 2 large pedigrees have
already been studied (26) (Stark, M., et al., unpublished
results), and detailed clinicopathological data of the last 4 families
were not available at the time of this study. Figure 1 shows some examples of typical
pedigrees used in this study.
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).
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A single point linkage analysis (i.e. analyzing 1 marker at
a time) was performed with GeneHunter on the 56 pedigrees with the same
parameters we used for the simulation; the LOD scores we obtained were
negative with both dominant and recessive models for the 3 candidate
regions. The multipoint analysis performed with GeneHunter gave similar
results using the 2 models of inheritance and nonparametric analysis
(summarized in Table 3).
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We also performed additional analysis with subsets of the 56 families to address various alternative hypothesis. All of these analyses did not change the results obtained with the entire dataset.
We speculated that the criterion for inclusion of a family in the study could be too loose, and that a few families with only 2 cases of NMTC might not be due to genetic predisposition. Although this is already taken into account in the general analysis, because genetic heterogeneity is tested for, we repeated an analysis including only the 19 families with 3 or more cases of NMTC. The results of this analysis did not significantly differ from those obtained with all 56 families (data not shown).
Inclusion of adenoma, MNG, and thyroiditis cases as affected
individuals could dilute the analysis of linkage for NMTC; therefore,
we performed a separate analysis using NMTC alone. Again, the results
were largely negative with the three loci (Table 4).
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In addition, a large Tunisian pedigree with recurrence of Graves’ disease and Hashimoto’s thyroiditis (28) was also studied for linkage on chromosome 14 because of the reported linkage of susceptibility to Graves’ disease to chromosome 14q31 in a recent study (25). This pedigree is very large and, due to its complexity, cannot be analyzed by ordinary methods for linkage analysis. Nevertheless, examination of the genotypes of chromosome 14 showed an absence of haplotype sharing among the affected individuals (data not shown) and therefore rules out linkage of this kindred to MNG1.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Although genetic risk factors for NMTC are not fully elucidated, three chromosomal regions stand out as candidates for genetic susceptibility to NMTC. Two of them have been directly related to susceptibility to NMTC by studies in large kindreds performed by us and others. MNG1, mapped to chromosome 14q31, is involved in susceptibility to multinodular goiter in a large Canadian family showing 18 cases of MNG and 2 cases of PTC (24). We have recently found evidence for linkage between the gene TCO, mapped to chromosome 19p13.2, and susceptibility to FNMTC and goiter in a French family with six cases of multinodular goiter and three cases of PTC (26). The French and Canadian pedigrees have a similar structure. Despite this, they are linked to different genes of predisposition. The third region, harboring the protooncogene RET, has never been directly linked to FNMTC, but it is a strong candidate because rearrangements in RET have been found frequently in sporadic cases of NMTC (27). It is also known that the great majority of familial MTC as well as some sporadic MTC show missense mutations in the RET protooncogene (27).
In this study, we have tested kindreds of small and medium size from our collection by linkage analysis to assess which proportion of NMTC cases could be linked to RET, TCO, or MNG1. Results using both dominant and recessive hypothesis and also following a nonparametric method are reported. All three models show that there is no evidence for linkage; the proportion of linked families is not statistically significant for any of the 3 loci. Multipoint linkage analysis in 56 kindreds reinforces the hypothesis of the existence of genetic heterogeneity in FNMTC. Furthermore, this raises the possibility that large families with NMTC and MNG are in linkage, each with a different gene, and that such genes do not have a role in the susceptibility observed in smaller pedigrees.
The study of the Tunisian family with recurrence of Graves’ disease did not support the results of Tomer et al. (25), as no linkage has been found to the 14q31 locus in this family. This suggests that a new susceptibility gene to a disease phenotypically indistinguishable from Graves’ disease could be involved. Further investigations on diagnosis of the patients should be performed.
Further studies will include mapping of new NMTC susceptibility genes by whole genome scan of new large pedigrees and on the whole of our collection of small and medium-sized families. As susceptibility to NMTC could be linked to a number of genes, each with a small individual effect, more informative families will have to be collected to have sufficient analytical power.
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Acknowledgments |
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Footnotes |
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2 Recipient of a Special Training Award from the International Agency
for Research on Cancer.
3 The members of the NMTC Consortium who were involved in this study
are Dr. A. M. Bernard, Centre Eugène Marquis (Rennes,
France); Dr. A. Boneu, Centre Claudius Régaud (Toulouse, France);
Dr. M. J. Bugalho, Instituto Portugues de Oncologia (Lisboa,
Portugal); Dr. M. Cavarec, CHU Morvan (Brest, France); Dr. J. P.
Fricker, Centre Paul Strauss (Strasbourg, France); Drs. O. Haas and A.
Weinhausl, St. Anna Children’s Hospital (Wien, Austria); Prof. J.
Leclère, CHU de Nancy, Hôpitaux de Brabois
(Vandoeuvre-lès-Nancy, France); Dr. F. Leprat, CHU de Bordeaux,
Hôpital Haut-Lévèque (Bordeaux, France); Dr. A.
Murat, CHU de Nantes, Hôtel Dieu (Nantes, France); Dr. F. Pacini,
Universita’ di Pisa (Pisa, Italy); Dr. B. Pasini, Istituto Nazionale
Tumori (Milan, Italy); Prof. C. Reiners and Dr. J. Farahati,
Universität Würzburg (Würzburg, Germany); Prof. G.
Riccabona, Universität Innsbruck (Innsbruck, Austria); Dr. M.
Rodier, CHU de Nîmes, Hôpital Caremeau (Nîmes,
France); Dr. R. Sankila, Finnish Cancer Registry (Helsinki,
Finland); Dr. H. Sobol, Institut J. Paoli-I. Calmettes (Marseille,
France); Dr. M. E. Toubert, Hôpital Saint-Louis (Paris,
France); Prof. J. Tourniaire and Dr. M. Bertholon-Grégoire,
Hôpital de l’Antiquaille (France); and Dr. M. Zini,
Arcispedale Santa Maria Nuova (Reggio Emilia, Italy).
Received November 4, 1998.
Revised February 23, 1999.
Accepted March 17, 1999.
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References |
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of cyclic AMP-dependent protein kinase A. Mol Cell Biol. 13:358–366.This article has been cited by other articles:
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  F. Weber and C. Eng Update on the Molecular Diagnosis of Endocrine Tumors: Toward -omics-Based Personalized Healthcare? J. Clin. Endocrinol. Metab., April 1, 2008; 93(4): 1097 - 1104. [Abstract] [Full Text] [PDF]
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 J. W. B. de Groot, T. P. Links, J. T. M. Plukker, C. J. M. Lips, and R. M. W. Hofstra RET as a Diagnostic and Therapeutic Target in Sporadic and Hereditary Endocrine Tumors Endocr. Rev., August 1, 2006; 27(5): 535 - 560. [Abstract] [Full Text] [PDF]
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 J D McKay, D Thompson, F Lesueur, K Stankov, A Pastore, C Watfah, S Strolz, G Riccabona, R Moncayo, G Romeo, et al. Evidence for interaction between the TCO and NMTC1 loci in familial non-medullary thyroid cancer J. Med. Genet., June 1, 2004; 41(6): 407 - 412. [Abstract] [Full Text] [PDF]
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 A. Matakidou, N. Hamel, S. Popat, K. Henderson, T. Kantemiroff, C. Harmer, S. E.M. Clarke, R. S. Houlston, and W. D. Foulkes Risk of non-medullary thyroid cancer influenced by polymorphic variation in the thyroglobulin gene Carcinogenesis, March 1, 2004; 25(3): 369 - 373. [Abstract] [Full Text] [PDF]
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F Lesueur, M Corbex, J D McKay, J Lima, P Soares, P Griseri, J Burgess, I Ceccherini, S Landolfi, M Papotti, et al. Specific haplotypes of the RET proto-oncogene are over-represented in patients with sporadic papillary thyroid carcinoma J. Med. Genet., April 1, 2002; 39(4): 260 - 265. [Abstract] [Full Text] [PDF]
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S. Bevan, T. Pal, C. R. Greenberg, H. Green, J. Wixey, G. Bignell, S. A. Narod, W. D. Foulkes, M. R. Stratton, and R. S. Houlston A Comprehensive Analysis of MNG1, TCO1, fPTC, PTEN, TSHR, and TRKA in Familial Nonmedullary Thyroid Cancer: Confirmation of Linkage to TCO1 J. Clin. Endocrinol. Metab., August 1, 2001; 86(8): 3701 - 3704. [Abstract] [Full Text] [PDF]
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C. Eng Editorial: Familial Papillary Thyroid Cancer--Many Syndromes, Too Many Genes? J. Clin. Endocrinol. Metab., May 1, 2000; 85(5): 1755 - 1757. [Full Text]
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C. D. Malchoff, M. Sarfarazi, B. Tendler, F. Forouhar, G. Whalen, V. Joshi, A. Arnold, and D. M. Malchoff Papillary Thyroid Carcinoma Associated with Papillary Renal Neoplasia: Genetic Linkage Analysis of a Distinct Heritable Tumor Syndrome J. Clin. Endocrinol. Metab., May 1, 2000; 85(5): 1758 - 1764. [Abstract] [Full Text]
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, Unaffected males; 
, unaffected females; • and
, cases of NMTC; 
and 
, cases of MNG; |KR and
, cases of
adenoma.