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Department of Internal Medicine III (S.M.B.-P., R.L., L.W., W.W., H.V.), Division of Endocrinology and Metabolism, Core Unit of Medical Statistics and Informatics (G.H.), Department of Surgery (B.N.), and Clinical Institute of Pathology (K.K.), Medical University of Vienna, A-1090 Vienna, Austria
Address all correspondence and requests for reprints to: Sabina M. Baumgartner-Parzer, Ph.D., Department of Internal Medicine III, Division of Endocrinology and Metabolism, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: sabina.baumgartner-parzer{at}meduniwien.ac.at.
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Abstract |
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Objective/Design of the Study: Because frequencies of RET SNPs have not yet been evaluated in patients with elevated serum concentrations of calcitonin (hCt), a biochemical marker for medullary thyroid carcinoma (MTC), we studied RET SNPs in patients with FMTC (n = 22), patients with sporadic MTC (n = 45), and 71 subjects presenting with moderately elevated hCt concentrations (basal, >10 pg/ml; pentagastrin stimulated, > 50 < 100 pg/ml) in comparison with an age- and gender-matched control group (n = 79) with basal hCt concentrations in the normal range (<5 pg/ml).
Methods: After DNA extraction from citrated whole blood, RET exons 10, 11, 13, 14, 15, and 16 and exon/intron boundaries were analyzed by PCR-based cycle sequencing for RET germ line mutations, exonic (G691S, L769L, S836S, S904S) and intronic (IVS13+158; NCBI rs2472737 = IVS14–24) SNPs.
Results: In FMTC patients, the F791Y mutation was found to be associated (P = 0.001) with the L769L SNP. The exonic SNPs (G691S, L769L, S836S, and S904S) were not different among the four groups. The intron 14 SNP (IVS14–24), however, was more frequent in individuals with elevated hCt serum concentrations (P = 0.016) and patients with sporadic MTC (P < 0.001) when compared with the control group.
Conclusions: These data suggest that the exon 13 (L769L) and the intron 14 (IVS14–24) SNPs could act as genetic modifiers in the development of some forms of hereditary and sporadic MTC, respectively.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Several single-nucleotide polymorphisms (SNPs) of the RET protooncogene have been described in the general population as well as in patients with endocrine tumors (8), papillary thyroid carcinoma (9), familial and sporadic medullary thyroid carcinoma (MTC) (10, 11), and HSCR (12, 13). It still is a matter of debate to which extent neutral sequence variants (polymorphisms) could have interacting, predisposing, or modifying roles in the pathogenesis of MEN2 or sporadic MEN2-related tumors. Whereas exonic RET SNPs (G691S/exon 11, L769L/exon 13, S836S/exon 14, and S904S/exon 15) have been evaluated in a panel of different studies (10, 11, 14, 15, 16), although with controversial results, intronic SNPs were only rarely studied (17).
In that context, it is of note that the germ line sequence variant in intron 14 (IVS14–24; G 
A) was originally described in patients with HSCR (18). Because this sequence variant was exclusively found in HSCR patients but not in 300 alleles from unaffected control individuals, IVS14–24 was interpreted as a disease-causing mutation for HSCR (18). In contrast, Fitze et al. (17) reported this SNP to occur in 114 German blood donors with an allele frequency of 23%, which did not differ significantly from the frequency observed in patients with HSCR (26%) or that in patients with sporadic MTC (24%).
So far, however, frequencies of both exonic and intronic RET SNPs have not yet been evaluated in patients with elevated serum concentrations of calcitonin (hCt), a biochemical marker for MTC (19, 20). hCt is routinely determined in every individual referred to our thyroid outpatient department to detect MTC at an early potentially curable stage (21).
The present study evaluated frequencies of an intron 14 (IVS14–24) (17, 18), a recently reported (22) intron 13 (IVS13+158), and four known exonic SNPs (G691S, L769L, S836S, S904S) in patients with FMTC, sporadic MTC, and moderately elevated hCt serum concentrations (>50 < 100 pg/ml after pentagastrin stimulation test) in comparison with control subjects with hCt levels within the normal range (<5 pg/ml).
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Patients with familial or sporadic MTC. A hCt-screening program has been set up in our department in August 1994 (21). Since then, hCt serum concentrations have been determined in more than 15,000 patients referred to our outpatient clinic. This hCt screening program resulted in the detection of the patients with MTC included in the present study. The diagnosis of MTC was established histologically in 67 cases. FMTC was diagnosed when specific germ line mutations in the RET protooncogene (exons 10, 11,13, 14, 15, and 16) were detectable (n = 22; nine men aged 40–73 yr and 13 women aged 26–84 yr). In the absence of these mutations, the patients’ MTC was considered to be sporadic in nature (n = 45; 22 men and 23 women). Furthermore, these patients had neither a family history of MTC nor did they present symptoms of potentially related disorders such as phaeochromocytoma or parathyroid disease.
Patients with elevated hCt serum concentrations. This group included 71 individuals [54 men aged 19–75 yr (mean 55 ± 16 yr) and 17 women aged 24–79 yr (mean: 46 ± 17 yr)] with nodular thyroid disease, who had undergone a pentagastrin stimulation test because of a previously detected elevated (>10 pg/ml) basal hCt (23). Patients with positive family history for MTC were excluded. Blood samples for the determination of hCt were drawn through an indwelling catheter before as well as 2, 5, and 10 min after an iv bolus of 0.5 µg/kg pentagastrin (Cambridge Laboratories, Wallsend, UK). Basal concentrations of hCt in this group were ranged from 10.0 to 39.0 pg/ml and maximum hCt concentrations after PG stimulation ranged from 50.0 to 100.0 pg/ml.
Controls. RET protooncogene analysis was performed in samples obtained from 79 age- and sex-matched individuals (58 men and 21 women) seen in the endocrine and thyroid outpatient departments. Individuals with positive family history for FMTC or MEN2 as well as patients known to suffer from MTC, primary hyperparathyroidism, and/or phaeochromocytoma were excluded. Otherwise the only criterion for inclusion was hCt serum concentrations within the normal range (<5.0 pg/ml). No pentagastrin stimulation test was performed in these patients.
All patients were informed about the purpose of this investigation, and written consent was obtained in each case.
Determination of serum calcitonin
Serum calcitonin was determined with an acridinium ester-labeled chemiluminescent immunoassay running on the Advantage autoanalyzer (Nichols Institute Diagnostics, San Clemente, CA). Interassay coefficients of variation (at 10, 80, and 512 pg/ml) were 5% each (24).
RET analysis
Genomic DNA was extracted from peripheral blood leukocytes according to standard procedures (23, 25). Five fragments covering the exons 10, 11, 13, 14, 15, and 16 of the RET protooncogene were specifically amplified by selective PCR primers as described previously in more detail (23). After purification using the High Pure PCR purification kit (Roche Diagnostics, Mannheim, Germany) the respective PCR products were sequenced on a LI-COR model 4200 (LI-COR, Bad Homburg, Germany) using 5'-IRD-800-labeled primers (22) and the Thermo Sequenase cycle sequencing kit from United States Biochemical Corp. (Cleveland, OH).
Statistical analysis
Sequence data were compared among groups by logistic regression analysis of group indicator on allele dose (0, 1, 2). Because multiple SNPs were compared in multiple groups, P values were corrected using the Bonferroni-Holm method (26). Further group comparisons were done using Fisher’s exact test. P < 0.05 was considered as indicating statistical significance. The SAS System V9.1 (2003; SAS Institute Inc., Cary, NC) was used for statistical analysis.
Cross-validation
To assess the ability of significant associations to classify future observations into one group or another, an independent validation sample would be ideal. As outlined above, the MTC patients included in the present study are the result of a hCt screening program performed in more than 15,000 patients over the last 10 yr. Therefore, reevaluation of the data obtained in the present study in independent series of such patients is impossible in a reasonable period of time. Therefore, we addressed this issue using internal cross-validation. First, the original sample was randomly split into equal-sized training and validation sets. The training set was used to estimate probabilities of belonging to the control group, conditional on the number of mutant alleles, using logistic regression. The individuals of the validation set were then classified into either the control or the case group if the estimated probabilities of belonging to the control group were greater or smaller, respectively, than the proportion of controls among the original data set. The cross-validation classification error (CVCE) was then defined as the proportion of misclassified individuals of the validation set. This procedure was repeated 1000 times, and CVCE was averaged to avoid error due to random selection. Next, we estimated the distribution of the CVCE under the null hypothesis of no association by generating 10,000 data sets with randomly permuted group label. These permuted data sets were again randomly divided into training and validation sets, and CVCE was computed for each of the 10,000 permuted validation sets accordingly. The cross-validation P value was defined as the proportion of permuted validation sets that yield CVCEs smaller than the CVCE of the original sample.
In silico analysis was performed using prediction programs for splice sites (NNSPLICE: http://www.fruitfly.org/seq_tools/splice.html) for exonic-splicing enhancer (ESE) (ESEFinder: http:/exon.cshl.edu/ESE/) and branch sites (branch-site analyzer: http://ast.bioinfo.tau.ac.il/BranchSite.htm), as previously described by others (27, 28, 29). In brief, the complete RET-intron 14 sequence was extracted from the Ensembl Genome Browser (http:www.ensembl.org) and pasted into the respective program’s query field. For NNSPLICE the respective sequences were analyzed using a cut-off value of 0.01, for ESEFinder and the branch site analyzer, the default threshold values were used. In each case both the wild-type and mutant sequence were submitted.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Exonic RET SNPs
Statistical evaluation of the sequence data did not reveal significant differences between the control group (hCt < 5 pg/ml) and the patient groups (with elevated hCt concentrations > 50 < 100 pg/ml, with FMTC or sporadic MTC) with respect to frequency of the SNPs G691S (exon 11), L769L (exon 13), S836S (exon 14), and S904S (exon 15), as given in Table 1
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), the latter being present in heterozygous or homozygous form in all subjects (n = 12: nine unrelated index patients, three descendants) exhibiting the F791Y mutation. RET analysis in family members revealed three descendants to have inherited both F791Y and L769L, whereas four descendants and one mother exhibited neither the F791Y mutation nor the L769L SNP (Table 3). Such data indicate the F791Y mutation and the L769L SNP to be located on the identical allele in the studied individuals (Fisher’s exact test, P = 0.029).
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) there was not enough material available, for analysis of exonic and intronic SNPs in addition to the L769L SNP in exon 13. Therefore, this patient was included in Table 3 (index patient 4) but not into statistical evaluation with respect to other exonic and intronic SNPs (compare Tables 1 and 4).
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The intron 13 SNP (IVS13+158) was only recently reported (22) to occur in a patient with primary hyperparathyroidism harboring the F791Y germ line mutation. In the present study, this IVS13+158 SNP was only rarely detected and only in heterozygous form, e.g. in one control subject and one patient with sporadic MTC (Table 1).
The intron 14 (IVS14–24) SNP, however, was found with significantly higher frequency in the group with moderately elevated serum hCt concentrations (P = 0.016) and in patients with sporadic MTC (P < 0.001), when compared with the control group with normal hCt levels.
Mean (SD) age at diagnosis of MTC was 58 (11) yr in patients exhibiting homozygosity for the wild type as well as in patients being hetero- or homozygous for the IVS14–24 SNP.
Cross-validation
Cross-validation excluded chance as an explanation for the observed associations of the IVS 14–24 data with the elevated hCt group (CVCE = 38.6%, P = 0.013) and sporadic MTC patients’ group (CVCE = 27.4%, P = 0.001) in comparison with the control group.
In silico analysis
We used three sequence analysis packages as predictors of potential effects of the IVS14–24 sequence variant on RNA splicing. According to the splice site prediction program (NNSPLICE), the IVS14–24 variant created neither additional donor nor acceptor splice sites (an additional splice acceptor site was predicted only for the reverse strand); according to the ESE prediction program (ESEFinder), two ESEs are destroyed and one created by the IVS14–24 sequence variant. The branch-site analyzer identifies the GCCTGAC-motif (representing the nucleotides IVS14–17 to IVS14–23) as the putative branch site, the altered nucleotide (IVS14–24) being the first nucleotide beneath this motif.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Of note, controversial data exist with respect to all exonic RET SNPs studied so far. Although we cannot rule out that such differences depend on ethnic background or selection/inclusion criteria for the respective patients, we hypothesize that the studies’ results mainly depend on selection of the control group. Such an assumption results from the observation that in a considerable amount of studies either anonymous samples from healthy blood donors or respective DNA pools were used, excluding that these individuals have been checked for thyroid diseases. Even when healthy individuals were recruited as controls, in the majority of studies MTC and MEN2A-related tumors have not been excluded by inspection of the thyroid or determination of hCt serum concentrations. Thus, as far as we know, the present study is the only one restricting the control group to clinically healthy subjects with unremarkable family history with regard to MEN 2 and normal hCt serum concentrations.
As outlined above, the present study did not detect a significant difference of the frequency of the L769L SNP in the FMTC group, compared with the control group. Evaluation of a subgroup of patients with the F791Y germ line mutation, however, revealed that all these individuals (n = 12; nine index patients and three descendants) were heterozygous or homozygous for the nearby located L769L SNP (Tables 2 and 3), resulting in a significant association of F791Y and L769L. As far as can be deduced from pedigree analyses (Table 3), in all studied individuals, the F791Y mutation and L769L SNP are located on the same allele. Although we found this association in nine unrelated FMTC index patients, it remains speculative whether the presence of this SNP could predispose the respective allele for occurrence of a F791Y de novo mutation or could modulate the disease phenotype. That such association has not been reported so far may also relate to the circumstance that the F791Y mutation was described as a disease causing mutation for MEN2 and FMTC only later, when RET analysis was extended to individuals with so called sporadic MTC (31). Therefore, F791 mutations were previously categorized as rare mutations, whereas recent data (23) suggest that F791 mutations occur with a higher frequency than expected.
In contrast to exonic, a disease-related role of intronic RET SNPs has only rarely been evaluated in MTC patients. The IVS13+158 SNP was so far reported only in heterozygous form in one of our patients, who harbored the F791Y germ line mutation and suffered from primary hyperparathyroidism (22). A similar allele frequency for IVS13+158 (0.63% in the sporadic MTC and control group) was observed in the present study. Because IVS13+158 obviously is a rare SNP, a potential role of this SNP in MEN2, FMTC, or sporadic MTC cannot be evaluated.
Of note, the IVS14–24 SNP has originally been described in patients with HSCR (18). Because this sequence variant was not detectable in the respective study in 300 alleles from 150 unaffected control individuals (blood samples were obtained from a pool of anonymous donors), IVS14–24 was interpreted as a disease-causing mutation for HSCR (18). In contrast, Fitze et al. (17) reported 37.7% of control subjects to be heterozygous and 4.4% to be homozygous for this SNP, exhibiting no difference in comparison with patients with HSCR or sporadic MTC.
Although the IVS14–24 SNP obviously does not modulate age of onset of sporadic MTC, we found this sequence variant in a significant higher frequency in patients with sporadic MTC and subjects with moderately elevated hCt serum concentrations after pentagastrin stimulation when related to the control group. In our study IVS14–24 allele frequencies in elevated hCt and sporadic MTC patients were 18.3 and 27.7%, respectively. Because in Fitze’s study (17) IVS4–24 allele frequencies were at about 23% (MTC patients: 23.8%; controls: 23.2%) and thus similar to that in our MTC group, the controversial results of these two studies obviously relate to the significantly lower IVS14–24 allele frequency (e.g. 6.3%) found in our control group. Such findings could relate to the circumstance that our control group comprised only individuals with hCt serum concentrations within the normal range (<5 pg/ml), whereas in the majority of other studies, controls were not routinely checked for hCt serum concentrations, MEN2, and MTC-related diseases and respective family histories.
Although the differences of the IVS14–24 frequencies in the elevated hCt group and particularly in the sporadic MTC group are highly significant in comparison with the control group with normal hCt levels, we performed internal cross-validation, which excluded chance as an explanation for the observed associations.
The studied intron 14 RET sequence variant lies 24 nucleotides upstream of exon 15. Because bioinformatic studies show that transcriptional regulatory elements in addition to conserved splicing motifs are clustered toward intron/exon boundaries (32, 33), it is tempting to speculate that the observed nucleotide substitution (IVS14–24 GA) could result in aberrant splicing. Due to in silico analysis, the intron 14 SNP changes ESEs and is located next to the putative branch site. Whether the observed sequence variant could result in a cryptic branch site and whether the predicted changes of ESEs are of relevance in vivo remains speculative. Even if functional alterations of this RET variant could be demonstrated, its potential causative role in sporadic MTC remains to be elucidated because we and others (8, 34, 35) have demonstrated that RET splice variants are expressed at least partly in a tissue-specific manner. Moreover, it remains to be evaluated in the respective tumor tissue whether patients who are wild type or heterozygous for IVS14–24 in the germline exhibit as a putative second-hit loss of heterozygosity at the somatic level, as previously discussed for other RET-SNPs in patients with endocrine tumors (8).
In conclusion, the association of the IVS14–24 SNP with elevated hCt levels and sporadic MTC is of major interest; the molecular background, however, remains to be elucidated. Therefore, as outlined above, further in-depth studies are necessary to characterize a potential role of this RET sequence variant in the development of sporadic MTC.
Although such a role has not been proven so far for IVS14–24, patients exhibiting hetero- or homozygosity for this sequence variant will have to be carefully monitored clinically and biochemically for symptoms of MTC, even in the absence of elevated hCt serum concentrations.
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Acknowledgments |
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Footnotes |
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Abbreviations: CVCE, Cross-validation classification error; ESE, exonic-splicing enhancer; FMTC, familial form of medullary thyroid carcinoma; hCt, elevated serum concentrations of calcitonin; HSCR, Hirschsprung disease; MEN2, multiple endocrine neoplasia 2; MTC, medullary thyroid carcinoma; SNP, single-nucleotide polymorphism.
Received June 7, 2005.
Accepted August 8, 2005.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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