Thyroid Cancer Susceptibility and THRA1 and BAT-40 Repeats Polymorphisms

Introduction

The majority of cancers occur in the sporadic form, and it is currently accepted that both genetic and environmental factors are implicated in its origin (1). In addition, low-penetrance genes or rather polymorphisms in such genes could be of great significance to understand the tumorigenic processes of these common cancers (2). By contrast, familial cancers are less frequent in the population and are associated with rare high-penetrance genes (2). Therefore, at present there is an emerging interest to find gene variants and thereby to find associations between genetic polymorphisms and cancer risk factors (3).

Thyroid cancer is the most frequent endocrine cancer with geographic variation in its manifestation (4). Both familial and sporadic forms of this type of cancer exist and, as other types of cancer, the sporadic form is the most common type of thyroid cancer (5). The role of environmental factors in the etiology of thyroid cancer is well recognized (6, 7); moreover, genetic factors involved in the tumorigenic pathway have been characterized (7, 8). Thyroid tumors range from benign to malignant manifestations and relevant genetic alterations identified in the different progression stages, include ras mutations in follicullary tumors (9), RET gene rearrangements and BRAF mutations in papillary (10-12), p53 mutations in anaplastic carcinoma (13), and RET point mutations in medullary tumors (14). Although the association between these mutations and thyroid tumorigenesis is well accepted, the specific genetic pathways of such processes remain to be established (15).

One factor related with thyroid cancer is the expression of thyroid hormone receptors (16, 17). Thus, a correlation between increased expression of the thyroid hormone receptor-α1 gene (NR1A1a gene) and less aggressive thyroid cancers has been recently reported (17). Moreover, these authors also showed that the size of the THRA1 microsatellite, which resides in a noncoding region of exon 9 of the NR1A1a gene (18), correlates with the level of expression of this gene. In addition, several chromosome regions have been implicated in thyroid cancer, including both chromosome 1 arms (19-25). However, the putative genes relevant to thyroid carcinogenesis that reside in these chromosome regions have not yet been identified. The 3-β-hydroxysteroid dehydrogenase gene (HSD3B1 gene) maps on 1p13.1 and it has been reported to be involved in prostate cancer (26); therefore, the study of this gene on thyroid cancer risk would be of general interest, either to explore the possible implication of the HSD3B1 gene on thyroid tumorigenesis, or to use it as a marker of other genetic risk factors involved in thyroid cancer development. The HSD3B1 gene contains the commonly used intronic polymorphism BAT-40 (27, 28), which has been used in population-based studies.

The role of genetic variants in determining individuals susceptibility to cancer is an increasingly prominent research area. Association studies to identify genetic risk factors to cancer are just emerging (29-32). Related to thyroid cancer risk, few studies have been reported, mostly referring to environmental factors (33-37). Consequently, although specific genotypes associated with thyroid cancer risk have not yet been described; as in other common cancers, the importance of genetic variants to the risk of sporadic thyroid cancer might be expected. Here we have done a case-control study to investigate whether there is an association between the THRA1 repeat polymorphisms and thyroid cancer risk. In addition, we extended the analysis to the BAT-40 microsatellite marker to find possible associations of the HSD3B1 gene or its chromosome 1 containing region with thyroid cancer susceptibility.

Materials and Methods

Patient Groups and DNA Isolation

Control and patient groups in this study were unrelated subjects from Spain. Blood samples were collected from 141 healthy individuals (67 women and 74 men), mean age of 37.7 ± 10.8 years and 212 thyroid cancer patients (157 women, 55 men), mean age of 44.4 ± 14.6 years. The cancer patients were from the Nuclear Medicine Service of the Hospital Vall d'Hebron (Barcelona, Spain). Tumors of these patients were classified as papillary (n = 161) and follicular carcinomas (36), or Hürthle cell carcinomas (2). This information was not available for 13 individuals at the time of the study and they were considered as unclassified.

DNA was isolated from blood samples using a standard phenol-chloroform method and dissolved in 30 to 100 μL of TE [10 mmol/L Tris, 0.2 mmol/L EDTA (pH 7.5)].

Genotype Analysis

THRA1 and BAT-40 genotypes from both control and patient individuals were analyzed by separate PCR reactions. The primers used to amplify the THRA1 sequence were 5′-CTTAAGCAGTGGGGAACCTG-3′ and 5′-ATAGCATTGCCTTCCCATGT-3′ (38). Thus, the PCR product of 128 bp was considered to contain 18 CA repeats according to the sequence of the NR1A1a gene (Genbank accession no. X55068). The amplified products were labeled using 2.4 μCi of [α³³-P]dCTP (Amersham Biosciences, Buckinghamshire, England) and 2.5 units of Taq polymerase were used in the PCR reaction. The BAT-40 primers were 5′-AATAACTTCCTACACCACAAC-3′ (4,7,2′,4′,5′,7′-hexachloro-6-carboxyfluorescein-labeled) and 5′-GTAGAGCAAGACCACCTT-3′ (39), which amplify a 126-bp product containing the standard 40 A repeat, according to the sequence of the HSD3B1 gene (Genbank accession no. M38180). To amplify this sequence, 0.75 unit of Taq polymerase was used.

THRA1 and BAT-40 PCR reactions were done in a final volume of 30 μL using 100 ng of DNA in 1× PCR buffer [10 mmol/L Tris-HCl (pH 8.4), 50 mmol/L KCl, 0.01% gelatin], 2.5 mmol/L MgCl₂, 0.2 mmol/L of each deoxynucleotide triphosphates, and 0.1 μmol/L of each primer. Amplifications were done using a PT-100 thermocycler (MJ Research, Waltham, MA) as follows: for BAT-40, 35 cycles of 95°C for 30 seconds, 55°C for 45 seconds, and 72°C for 1 minute, with an initial denaturing step of 95°C for 5 minutes and a final extension step of 72°C for 5 minutes. For THRA1: 30 cycles of 94°C for 40 seconds, 54°C for 40 seconds, and 72°C for 40 seconds, with a denaturing step of 94°C for 3 minutes and a final extension step of 72°C for 5 minutes.

BAT-40 PCR products were analyzed using Applied Biosystems Automated Genetic Analysers (ABI310 or the ABI3100) with Genescan software. THRA1 PCR products were analyzed on 6% denaturing polyacrilamide gels (2.5 hours at 100 W) and visualized by autoradiography. The 30- to 330-bp ladder (Invitrogen, Carlsbad, CA) labeled with [γ³³-P]dATP (Amersham Biosciences) was used as molecular weight marker.

Statistical Analysis

Allele distributions of control and cancer patient groups were compared using the Mann Whitney test and the χ² test on the Minitab (v. 13) statistical package, with a 5% of level significance. Allele ranges were analyzed separately using the Fisher's exact test. Odds ratios of the genotype sets of patients with respect to the control were calculated at 95% confidence intervals. The last two analyses were done by using the SPSS version 11.5 statistical software (SPSS, Inc., Chicago, IL).

Results and Discussion

THRA1 Repeat Polymorphisms and Thyroid Cancer Risk

The THRA1 poly(CA)_n microsatellite, located in the NR1A1a gene, was examined in 141 healthy unrelated individuals and in 212 thyroid cancer individuals, who originated from the same geographic area. The pattern of PCR products of the THRA1 microsatellite obtained by autoradiography detection is shown in Fig. 1A. The genotype frequencies in control and patient cohorts are summarized in Table 1, and thereby the level of heterozygosity in control and patient groups was estimated as 70.9% and 71.2%, respectively. The previously reported characteristics of the THRA1 microsatellite also indicate a 78% of heterozygosity for this microsatellite (40), which is in concordance with our results. In addition, we have found that the THRA1 allelic variants range from 14 to 26 CA repeats (Fig. 2), and a similar allele range has also been reported in a group of thyroid cancer patients and cell lines (17). Both control and patient groups showed two most common alleles corresponding to PCR products of 132 bp (average frequency, 37.7%) and 128 bp (average frequency, 27.7%) with 20 and 18 CA repeats, respectively. When comparing the allele frequencies between patients and control, no significant differences were observed (P = 0.577, Mann-Whitney test). However, because the THRA1 repeat length has been directly related to the expression of the NR1A1a gene and because such expression has also been suggested to affect cellular differentiation in thyroid cancers (17), we have looked at the genotypes involving alleles at each end of the THRA1 CA repeat range (i.e., carriers of one or two alleles shorter than the 128-bp modal allele, or longer than the 132-bp modal allele). The results are presented in Table 2. Whereas carriers of the >132-bp alleles showed no association with thyroid cancer risk, carriers of at least one allele <128 bp could have some protective effect in thyroid cancer risk (odds ratio, 0.50; 95% confidence interval, 0.22-1.13), although the probability is on the border line and not statistically significant (P = 0.09). Authors of a recent study analyzing 30 thyroid tumors for NR1A1a expression postulate that lower than average expression of these gene is associated with shorter than average THRA1 microsatellite (<18 CA repeats) and with aggressive thyroid tumors (17). This assumption apparently disagrees with our results comparing normal and thyroid cancer cohorts for THRA1 variants. However, based on the known implication of the NR1A1a gene in gene regulation affecting different pathways important for cell differentiation (41-43), we propose that short THRA1 repeats and thereby a decreased expression of NR1A1a could provide a protective effect to thyroid cancer risk. On the other hand, a reduced presence of thyroid hormone receptor-α1 would possibly lead to cell invasion, as previously suggested by Onda et al. (17). Because papillary and follicular thyroid carcinoma are genetically and clinically different, a possible association of the type of thyroid cancer with genotypes involving short or long THRA1 alleles was investigated and no significant differences were observed (Fisher's exact test).

BAT-40 Genetic Marker and Thyroid Cancer Risk

Studies done to determine the utility of the BAT-40 poly(A)_n microsatellite in microsatellite instability studies have highlighted the polymorphic nature of this microsatellite (44-48). This characteristic of the BAT-40 repeat has been exploited in the present study to analyze whether there is an association between the BAT-40 containing region of the chromosome 1 with thyroid cancer susceptibility. Herein, low-penetrance genes involved in thyroid cancer risk could be identified.

The study was done with the control and patient subjects used in the THRA1 analysis. Specifically, 138 control and 207 patient individuals were genotyped for BAT-40 allele variants. Representative electropherograms of BAT-40 PCR products are shown in Fig. 1B. Each allele is displayed as a single peak complex, and to determine the allele size, we followed the same criteria used by others and us previously (45, 47). Thus, the predominant peak in each peak complex was considered as the allele size, given that the rest of the peaks are due to DNA polymerase slippage.

The BAT-40 genotype frequencies of control and patient subjects are presented in Table 1. Both groups showed a similar heterozygosity, 39.9% and 33.8% for controls and patients, respectively. Different levels of BAT-40 heterozygosity have been reported in different populations, ranging from 14.6% in a Japanese population (46) to 72% in American studies (44). The average heterozygosity found in the Spanish population of the present study was 36.8%, which also differs of the 59.7% of heterozygozity observed in a Scottish group (47).

In Fig. 3, the BAT-40 allele frequencies in control and patient groups are compared. A bimodal distribution of BAT-40 allele variants was found in both groups, with 120 and 123 bp as modal alleles. Previous studies also describe a bimodal distribution for BAT-40 alleles in different analyzed groups (44, 45, 47). In our study, differences in the BAT-40 allele between control and patient individuals were not statistically significant (P = 0.074, Mann Whitney test), but a wider allele range was observed in the patient cohort than in the control (96-130 and 110-130 bp, respectively; Fig. 3). Hence, the data was analyzed further and when the allele distributions were grouped in sets of equal intervals differing in 5 bp, significant differences between control and patient groups were observed (P = 0.035, likelihood ratio). Moreover, the 111- to 115-bp allele range was identified to be the cause for the differences (P = 0.008, Fisher's exact test). BAT-40 genotypes of patient and control cohorts also reflect the difference in the allele distributions (see Table 1). Thus, genotypes involving alleles in the 111 to 115 bp range are present in seven individuals of the control group (5.1%), but only one patient (0.05%), and the odds ratio indicate that these genotypes have a protective effect to thyroid cancer susceptibility in the studied population (odds ratio, 0.18; 95% confidence interval, 0.01-0.57; P = 0.02; Table 3), although showed no association with the type of thyroid cancer, papillary or follicular (Fisher's exact test). Moreover, genotypes involving alleles at both boundaries of the BAT-40 variant distributions (i.e., <111 and >125 bp), were found to be >2-fold more frequent in patient group than in the control group (4.8% and 2.1%, for genotypes with <111-bp alleles; 3.9% and 1.5%, for genotypes with >125-bp alleles; respectively). However, no association of either genotype group with thyroid cancer susceptibility was observed (see Table 3). The power of this analysis could be reduced by the low frequency of short allele carriers found in our population and the sample size of the group studied. In addition, taking into account that it has been reported that mutations at mononucleotide repeats take place with a bias to expansion (49, 50), it is unlikely that the higher frequency of the <111-bp alleles found in the patient group with respect to the control would have taken place by chance. Therefore, based on the present results, the hypothesis that short BAT-40 alleles would comprise a genetic marker for thyroid cancer risk can not be discarded; however, more extensive analyses are needed to validate this assumption, including haplotype analysis of the BAT-40 containing region. At the clinical level, analysis of the clinopathologic characteristics of individuals bearing <111-bp alleles, or >125-bp alleles, or individuals with <111-bp alleles together with individuals of >125-bp alleles, showed no differences with respect to sex, age of cancer diagnosis, and type thyroid cancer.

The HSD3B1 gene containing the BAT-40 microsatellite has been suggested to be related to prostate cancer susceptibility; thus, several gene variants have been described to be associated with risk of this type of cancer (26), but not the BAT-40 polymorphism. Because the BAT-40 A repeat resides in the intron 2 of the HSD3B1 gene, and no evidences of biological consequences of BAT-40 polymorphisms exist, any direct implication of BAT-40 alleles in cancer susceptibility is unlikely. However, our findings indicate that the BAT-40 region of chromosome 1 contains gene/s related to thyroid cancer susceptibility, which would suggest that genetic variants of these genes could have either a protective or a risk effect on thyroid cancer. Further studies are needed to identify these important genes for sporadic thyroid cancer.