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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-2059
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 4 2127-2130
Copyright © 2005 by The Endocrine Society

BRIEF REPORT

Succinate Dehydrogenase D Variants Do Not Constitute a Risk Factor for Developing C Cell Hyperplasia or Sporadic Medullary Thyroid Carcinoma

Alberto Cascon, Arancha Cebrian, Marina Pollan, Sergio Ruiz-Llorente, Cristina Montero-Conde, Rocio Leton, Ruth Gutierrez, Fabienne Lesueur, Roger L. Milne, Olga Gonzalez-Albarran, Tomas Lucas-Morante, Javier Benitez, Bruce A. J. Ponder and Mercedes Robledo

Hereditary Endocrine Cancer Group (A.Ca., S.R.-L., C.M.-C., R.L., R.G., M.R.), Centro Nacional de Investigaciones Oncológicas, Madrid, Spain; Cancer Research United Kingdom (A.Ce., F.L., B.A.J.P.), Department of Oncology, University of Cambridge, Cambridge, United Kingdom; Cancer Epidemiology Service (M.P.), National Center for Epidemiology, Carlos III Institute of Health, Madrid, Spain; Department of Human Genetics (R.L.M., J.B.), Centro Nacional de Investigaciones Oncológicas, Madrid, Spain; Endocrinology Service (O.G.-A.), Hospital Ramon y Cajal, E-28029 Madrid, Spain; and Endocrinology Service (T.L.-M.), Clinica Universitaria Puerta de Hierro, Madrid, Spain

Address all correspondence and requests for reprints to: Mercedes Robledo, Hereditary Endocrine Cancer Group, Department of Human Genetics, Centro Nacional de Investigaciones Oncológicas, Melchor Fernández Almagro 3, E-28029 Madrid, Spain. E-mail: mrobledo{at}cnio.es.


   Abstract
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 Abstract
Introduction
 Subjects and Methods
 Results and Discussion
 References
 
Medullary thyroid carcinoma (MTC) is a tumor that arises from parafollicular cells of the thyroid gland. MTC can occur sporadically (75%) or as part of inherited cancer syndromes (25%). In most cases, hereditary MTC evolves from preneoplastic C cell hyperplasia (CCH), so early detection of this pathology would evidently be critical. A recent study reports that alterations in succinate dehydrogenase (SDH) D are responsible for familial non-RET CCH. First, we studied SDHD in two families with hereditary non-RET CCH and found no alterations related to the inheritance of this disease. Then, we investigated whether the H50R variant could be a risk factor in the sporadic development of MTC in both Spanish and English patients. We found no evidence that the presence of the H50R is strongly associated with the risk of sporadic MTC, although we did observe an association with age at diagnosis of MTC in Spanish H50R carriers that we did not find in English patients. Finally, we looked for evidence of CCH or any other thyroid disease in a panel of germ-line SDH (B or D) mutation carriers and found none. We conclude that SDHD variants do not constitute a risk factor for developing CCH or sporadic MTC.


   Introduction
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 Abstract
 Introduction
Subjects and Methods
 Results and Discussion
 References
 
MEDULLARY THYROID CARCINOMA (MTC) is a calcitonin-secreting tumor originating in the parafollicular cells (C cells) of the thyroid gland, and it represents about 5–10% of all thyroid malignancies. MTC occurs in both hereditary (25%) and sporadic (75%) clinical settings (1). In familial cases, it only occurs in familial MTC or as a component of multiple endocrine neoplasia type 2A or multiple endocrine neoplasia type 2B (2). Although C cell hyperplasia (CCH) is a relatively common abnormality in middle-aged adults, CCH usually precedes as precursor lesion the development of MTC (3, 4). In fact, most patients with hereditary MTC first develop CCH (5); hence, early detection of this pathology is vital in the clinical outcome of these patients.

A recent study (6) described a family with CCH as being attributable to a change in succinate dehydrogenase (SDH) D. This gene codes for one of the mitochondrial SDH subunits and has been found altered (as indeed have both the SDHB and SDHC genes) in both paraganglioma and pheochromocytoma (PCC) families (7, 8, 9). The authors found that the three affected members of the family had the variant c.149A>G (H50R), an alteration with controversial significance that has been related to both Merkel cell carcinomas and midgut carcinoids (10). Contrary to its apparently pathogenic character, the H50R variant has been found in 3% of two distinct control populations (11, 12). Moreover, it has been recently demonstrated that this alteration does not affect the activity of the SDHD protein (13).

The goal of this analysis was to study the involvement of SDHD and related genes in non-RET CCH using three approaches: 1) screening these genes for abnormalities in non-RET CCH families; 2) performing an association study in MTC; and 3) investigating evidence of raised calcitonin levels in individuals carrying either the H50R variant or SDH mutations.


   Subjects and Methods
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 Abstract
 Introduction
 Subjects and Methods
Results and Discussion
 References
 
Subjects

Informed consent was obtained from all patients.

CCH families. We studied the presence of mutations in SDH genes in all affected and nonaffected members of two CCH families (F1 and F2) who tested negative for mutations in RET (exons 1–20). RET gene segregation with the disease was studied in both families by means of haplotype analysis, using intragenic single nucleotide polymorphisms (SNP) located in exons 2 (rs1800858), 7 (rs1800860), 11 (rs179939), and 15 (rs1800863). We also studied SDHD segregation by using three SNPs (rs10789859, rs3839946, and rs7944155) that were chosen using PupaSNP, available at http://pupasnp.bioinfo.cnio.es/, and HapMap, available at http://www.hapmap.org. The proband of F1 was surgically operated for MTC in 1984, and he has 12 relatives with elevated basal calcitonin (BC) or pentagastrin-provoked (PG) calcitonin levels. Total prophylactic thyroidectomy was performed on 10 of these relatives, and CCH has been confirmed in nine of these cases by means of pathological studies (Table 1Go). The proband’s mother in F2 was diagnosed and operated for MTC in 1982. Subsequently, four relatives showed elevated BC or PG calcitonin levels, and three of them underwent prophylactic thyroidectomy (Table 1Go).


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  		TABLE 1. Clinical data of members of F1 and F2

 
Sporadic MTC patients. We studied the H50R variant in both Spanish and English patients. All Spanish (n = 120) and English (n = 135) MTC patients previously tested negative for mutations in RET by means of sequencing of exons 10, 11, and 13–16. No patients had a family history.

SDH (B or D) mutation carriers. Thyroid pathology was excluded in all cases by evaluating the BC level and, in some cases, by ultrasound scan, although CCH is difficult to exclude using these methods.

Controls. A set of 319 controls were selected from a larger group (up to 382) of unrelated and nonaffected individuals representative of the Spanish population. The Spanish control population was selected to be frequency matched by age and sex strata to the population of cases, with a control to case ratio equal to 3:1. Among Spanish men aged 45–64, however, only one control per case was available. A group of 547 anonymous individuals were used as representative of the English population. These English individuals were between 47 and 76 yr old, and they were randomly selected from the European Prospective Investigation of Cancer study (14).

Methods

Amplification and sequencing analysis. Genomic DNA was extracted from both patients’ and controls’ blood samples following a standard method (15).

Denaturing HPLC (dHPLC). To analyze the prevalence of the H50R variant in sporadic MTC cases, DNA samples from all patients were prepared in PCR-96 plates for dHPLC analysis (Transgenomic, Cheshire, UK). We used DNA from unrelated and unaffected individuals as a control population. Exon 2 SDHD primers were used to genotype both control and sporadic MTC samples for H50R polymorphism using dHPLC (16). Representative samples of all defined dHPLC patterns were studied using direct sequencing to verify different genotypes.

Statistical analysis. In the Spanish population, odds ratios (OR) were calculated to estimate the association between H50R polymorphism and sporadic MTC, using unconditional regression analysis, adjusted for age and sex. Because the ages of diagnosis for cases with and without the studied polymorphism were substantially different (Wilcoxon text), OR were also computed separately for two age groups, considering a cutoff equal to 45 yr. Furthermore, the statistical significance of the interaction between H50R polymorphism and age was evaluated, including the corresponding interaction term in the logistic model. Because we did not have information on the ages of English controls, we were only able to establish the general effect of the variant using all controls, without adjusting by age.


   Results and Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results and Discussion
References
 
First, we studied SDH in families with hereditary non-RET CCH (with some cases affected with MTC), to determine whether the H50R variant, or some other SDH change, could be involved in the inheritance of this disease. We screened two CCH families (Table 1Go) that previously tested negative for RET alterations not only by a complete mutation screening but also by means of a haplotype study. We carried out the analysis of the H50R variant and found no change in the probands or in any of their relatives. Extended study of the remaining SDHD exons and of the other SDH subunits, SDHB and SDHC, similarly did not detect any alteration among these families. We also excluded both SDHD segregation with the disease and the presence of gross deletions in the gene in F1, by means of a haplotype study using three informative markers throughout the gene. After the molecular analysis of all SDH genes, we can conclude that the development of non-RET familial CCH was not attributable to the H50R variant or any other alteration of the SDHD subunit in these two families. We also suggest that the other SDH subunits are not related to the disease either.

Second, we adopted an approach to consider whether H50R is causally associated with CCH. Because CCH precedes MTC in most cases as a premalignant precursor, we carried out a case-control study of 120 Spanish MTC cases and 319 control individuals (Table 2Go). We found no evidence that the presence of H50R was associated with risk for sporadic MTC [P = 0.261; OR = 1.68; 95% confidence interval (CI) = 0.68–4.16]. Nevertheless, a formally significant interaction between age and the effect of the H50R polymorphism was found (P = 0.03). The mean age at onset of our sporadic MTC was 52 yr, and interestingly, among H50R patient carriers, five eighths were under 40 yr of age when disease became apparent. Considering these findings, we estimated the effect of the presence of this variant in individuals younger than 45 yr, a cutoff that corresponded to the first tertile of the age distribution among cases. A statistically significant association between the H50R variant and sporadic MTC was found (P = 0.025). The effect found in the group younger than 45 yr, for whom the influence of genetic risk factors could be stronger, suggested that this variant might play a role as a low penetrance gene. To examine this further, we extended the study to an English series of 135 MTC patients compared with 547 control individuals. We did not find any statistical association between the presence of this variant and the risk for sporadic MTC, studying the effect of H50R in all English cases vs. controls (Table 2Go). We could not perform unconditional regression analysis adjusted for age because we lacked these data, but no interaction between age and the effect of the H50R polymorphism was apparent in the English cases (observed frequencies of H50R were 1/61 and 1/71 in younger and older cases, respectively). Combining data from both studies gave a crude OR estimate of 1.22 (95% CI = 0.52–2.63). It is likely that the association found in Spanish patients occurred randomly due to the size of our series of patients. To exclude the possibility that this variant was in linkage disequilibrium with another alteration with potential phenotypic effect and present only is the Spanish population, we studied further one of the SNPs used in the haplotype analysis of SDHD (rs3839946). This SNP was located in the promoter region of SDHD and could affect the FOXD3 (transcription factor) recognition sequence. We checked for linkage disequilibrium between these two variants among the Spanish cases, and we did not find any association between the presence of H50R and this putative functional polymorphism (data not shown).


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  		TABLE 2. Association between H50R variant and sporadic MTC in Spanish and English populations

 
Finally, because the H50R SDHD variant had been described as causative of CCH, it seemed logical to assume that other SDH variants might have similar consequences. We used plasma calcitonin measurements to test for the presence of CCH (17). We collected clinical data from 10 patients with SDH (B or D) mutations and 3 H50R carriers. We did not find any abnormal BC levels in the H50R carriers or among our SDH mutation cases. The ultrasound scan data did not reveal any thyroid pathology either. In some RET mutation carriers, calcitonin levels are normal with C cell disease in early stages of development, so we cannot exclude thyroid pathology entirely. Although we had no PG data (using this test in our cases with BC levels less than 10 pg/ml was considered unjustified because of the adverse effects), it is interesting to emphasize that none of the H50R carriers had familial antecedents of PCC or thyroid pathologies. In addition, one of the H50R carriers had inherited the variant from his mother, and it is clear that the disease transmission associated to SDHD mutations exclusively occurs through paternal transmission (18). Penetrance and phenotypic expressiveness of mutations in the SDH genes have not been established yet, so we cannot exclude that some pathogenic mutations lead to disease at different ages and in different locations. A recent study suggested SDHB mutations predisposing to early onset kidney cancer with clinical implications for medical surveillance (19). Nevertheless, despite the fact that mutations of SDHD have been suggested as causing other neuroendocrine tumor development, to date there is no evidence of these genes being involved in diseases other than paraganglioma or PCC, and the present study further excludes its involvement in CCH and MTC development.


   Footnotes
 
This work was supported by the Fondo de Investigaciones Sanitarias projects CP03/00028 and PI02/0919. S.R.-L. and C.M.-C. are predoctoral fellows of Majadahonda City Council and Gobierno Vasco, respectively.

First Published Online December 28, 2004

Abbreviations: BC, Basal calcitonin; CCH, C cell hyperplasia; CI, confidence interval; dHPLC, denaturing HPLC; F1, family 1; F2, family 2; MTC, medullary thyroid carcinoma; OR, odds ratio(s); PCC, pheochromocytoma; PG, pentagastrin-provoked calcitonin; SDH, succinate dehydrogenase; SNP, single nucleotide polymorphism(s).

Received October 18, 2004.

Accepted December 14, 2004.


   References
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 Introduction
 Subjects and Methods
 Results and Discussion
 References
 

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