Common Variants of the Thyroglobulin Gene Are Associated with Differentiated Thyroid Cancer Risk

Abstract

Background:

Genetic factors are important in thyroid cancer susceptibility. Recently, it has been reported that there are associations of certain chromosome regions with thyroid cancer. In this case–control study, we sought to determine whether there is an association between differentiated thyroid cancer (DTC) and variants in regions of chromosome 8q.

Methods:

We used a case–control association design in a population of 877 individuals (398 patients with sporadic DTC and 479 healthy controls). The iPLEX technology was applied to analyze seven single-nucleotide polymorphisms (SNPs) in chromosome 8q: two SNPs that map at 8q24, previously reported as risk markers in different types of cancer, two SNPs in the thyrotropin-releasing hormone receptor gene (TRHR), and three SNPs in the thyroglobulin gene (TG). Risk assessment was done by unconditional regression analysis.

Results:

The two SNPs that map at 8q24, rs6983267 and rs1447295, and the two TRHR polymorphisms showed no association with DTC. No association was also found for the exon 33 TG polymorphism. The two TG polymorphisms in the exon 10–12 cluster, however, were associated with an increased risk of DTC (dominant model odds ratio = 1.80, 95% confidence interval = 1.30–2.50, p < 0.001).

Conclusions:

In this study, we show for the first time that the TG gene is a susceptibility factor for thyroid cancer. Although these conclusions are based on a large population, additional studies are warranted to support these data.

Introduction

T hyroid cancer is the most common endocrine malignancy (1). Papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC) are subtypes of differentiated thyroid carcinoma (DTC), representing more than 90% of thyroid neoplasms (2). Environmental factors and common genetic variants are relevant for thyroid cancer susceptibility (2,3). As a result, several recent studies identified these risk factors and their interactions, using both genome-wide and candidate gene approaches. These studies have shown genetic variants on chromosome 9q22.33 and 14q13.3 to be related to thyroid cancer. Interestingly, these regions contain thyroid differentiation genes FOXE1 and NKX2-1, respectively (4,5). Additional polymorphisms at 5q33 and 1p12 are also susceptibility loci for thyroid cancer (6,7), and more recently, the WDR3 gene that maps at 1p12 was found to be associated with the disease (8). In chromosome 8q24, three independent regions have been related to risk of different types of cancers, including thyroid cancer, but no known genes reside in that susceptibility region (9,10). Using a linkage analysis approach, He et al. identified a susceptibility locus (AK023948) for thyroid cancer that resides in the thyroglobulin (TG) gene on 8q24 (11). It is relevant to mention that amplification of 8q24 in several types of cancer was already established before the susceptibility studies emerged (12,13).

Because the 8q23 and 8q24 regions contain genes involved in thyroid function and based on the indication that genetic variation in 8q24 is associated with cancer, we postulated that chromosome 8q23 and 8q24 may be associated with thyroid cancer. In the present study, we carried out a case–control association analysis using seven single-nucleotide polymorphisms (SNPs) of these chromosome regions: the rs4129682 and rs7823804 SNPs that are located in the thyrotropin-releasing hormone receptor (TRHR) gene that maps at 8q23, the rs180223, rs853326, and rs2076740 SNPs that are in the TG gene that maps at 8q24, and two additional SNPs, rs6983267 and rs1447295, at 8q24 that have been reported as risk factors for different types of cancer.

Materials and Methods

Subjects

The study was carried out in a Spanish population of 881 unrelated subjects who were recruited during a 5-year period (2004–2008). All subjects participating in this study reported no previous whole-body exposure to radiation, were of Caucasian origin with Spanish ancestors, and were from the same geographic area.

The patients consisted of 398 individuals who were newly diagnosed with differentiated thyroid cancer (DTC) at two Spanish hospitals (Hospital Vall d'Hebron, Barcelona; and Hospital Josep Trueta, Girona). There were 309 women and 89 men (mean age ± standard deviation: 47.06 ± 15.48). Clinical information of patients was obtained from the medical records. There were 339 patients with PTC (84.3%) and 59 patients with FTC (14.7%). The control individuals were 479 volunteers without a diagnosis of cancer. There were 283 women and 196 men. There mean age of 45.99 ± 17.24 years was comparable to the mean age of the patients (p = 0.09). All control subjects were from the same geographic area as the patients. Informed consent from all participants and clearances from the ethical committees of our institutions were obtained.

Selection of SNPs

The selection of SNPs was based on the information available in literature and public databases. The rs6983267 and rs1447295 SNPs have been extensively analyzed in different cancer risk association studies. The rs4129682 and rs7823804 polymorphisms are located in the only intron of the TRHR gene. Finally, the TG polymorphisms rs180223 (exon 10, Ser734Ala), rs853326 (exon 12, Met1028Val), and rs2076740 (exon 33, Arg1999Trp) are common genetic variants and were previously used in association studies of autoimmune thyroid disease (14). More information of the selected SNPs used in this study is listed in Table 1.

Table 1.

Single-Nucleotide Polymorphisms Studied on Chromosome 8q in a Case–Control Study of Thyroid Cancer in a Spanish Population

SNP	Genomic position ^a	Locus	Allele variant	Gene polymorphism
rs4129682	110191315	TRHR	C/T	Intron 1
rs7823804	110198554	TRHR	G/T	Intron 1
rs6983267	128482487	8q24	G/T	Intergenic
rs1447295	128554220	8q24	A/C	Intergenic
rs180223	133969434	TG	G/T	Ser734Ala
rs853326	133979156	TG	C/T	Met1028Val
rs2076740	134053240	TG	C/T	Arg1999Trp

Position according to NCBI reference sequence.

SNP, single-nucleotide polymorphism; TRHR, thyrotropin-releasing hormone receptor; TG, thyroglobulin.

DNA isolation and genotyping

DNA for both cases and controls was isolated from peripheral blood, using the standard phenol–chloroform method. For some controls (<20% of control subjects), DNA was obtained from saliva samples using the Oragene DNA Self-Collection kit (DNA Genotek).

All genotype analyses were performed at the Spanish National Genotyping Center (CeGen) using the iPLEX technology (Sequenom, Inc.). To guarantee genotyping reliability, double genotyping was performed of 10% of randomly selected samples in multiple 96-well plates. In addition, two HapMAp reference trios were incorporated in plates, and the genotype concordance and correct Mendelian inheritance were verified.

Statistical analysis

Comparison of sex proportion between cases and control groups and the Hardy–Weinberg equilibrium investigation of the genotype distribution in the control population were examined using the chi-square test with a 5% level of significance. Mean ages of cases and controls were compared by the Mann–Whitney test. Thyroid cancer risk was assessed using unconditional logistic regression analyses, adjusted for sex and age, to determine the odds ratios (OR) and 95% confidence intervals (CI).

The Haploview software (15) was used to examine the linkage disequilibrium (LD) between SNPs. A logistic regression analyses was used to obtain the adjusted OR and 95% CI of haplotypes.

The SNPStats software (16) with a 5% level of significance was used to perform all statistical analyses.

The power of the study was calculated using the CaTS Power Calculator software (17). For the sample size of the study and type error 1 of 0.05, the power was 95% to detect an OR of 1.41, considering a minor allele frequency of 0.40, and 1.95, considering a minor allele frequency of 0.05.

Results

In this case–control association study, we genotyped seven SNPs of chromosome 8q in 398 patients with DTC and 479 control individuals. The group of patients consisted of 309 women and 89 men with a mean age of 47.06 ± 15.48 years. The control subjects were 283 women and 196 men with a mean age of 45.99 ± 17.24 years. In our studied population, case and control groups had a different distribution of sexes (77.6% and 59.1% of females in cases and controls, respectively; p < 0.001). In the control group, within gender there was no difference in genotype distribution (data not shown). Thus, the different sex proportion in cases and control groups in our population did not influence successive association studies.

The allele frequencies and genotype distribution of the SNPs are shown in Table 2. The data are referred to the analysis of the total group of patients and after subgrouping for type of thyroid cancer, PTC and FTC. Significant differences in allele frequencies between cases and control were found for two of the three TG nonsynonymous SNPs, rs180223 and rs853326 (p < 0.01). However, no difference of allele frequency was observed for the rest of the SNPs studied.

Table 2.

Allele Frequencies, Genotype Distribution, and Risk Analysis of Seven Single-Nucleotide Polymorphisms on Chromosome 8q for Their Relationship with Thyroid Cancer in a Case–Control Study in a Spanish Population

	MAF
SNP	Controls N = 479	Patients N = 398	p-Value ^a	SNP genotype	Controls N (%) N = 479	Patients N (%)N = 398	Odds ratio ^b (95% CI)	p-Value ^c	PTC N (%)N = 339	Odds ratio ^b (95% CI)	p-Value ^c	FTC N (%) N = 59	Odds ratio ^b (95% CI)	p-Value ^c
TRHR
rs4129682	0.48	0.47	0.91	C/C	127 (26.9)	100 (25.2)	1.00		80 (23.7)	1.00		20 (33.9)	1.00
				C/T	240 (50.9)	217 (54.8)	1.04 (0.73–1.49)		184 (54.6)	1.14 (0.78–1.66)		33 (55.9)	0.82 (0.41–1.66)
				T/T	105 (22.2)	79 (19.9)	0.85 (0.55–1.33)	0.59	73 (21.7)	1.01 (0.64–1.61)	0.75	6 (10.2)	0.25 (0.08–0.75)	0.02
rs7823804	0.36	0.34	0.55	T/T	186 (39.4)	165 (41.7)	1.00		135 (40.1)	1.00		30 (50.9)	1.00
				G/T	233 (49.4)	189 (47.7)	0.83 (0.61–1.15)		163 (48.4)	0.86 (0.61–1.21)		26 (44.1)	0.71 (0.36–1.38)
				G/G	53 (11.2)	42 (10.6)	0.85 (0.51–1.42)	0.52	39 (11.6)	0.96 (0.56–1.64)	0.68	3 (5.1)	0.27 (0.07–1.09)	0.12
8q24
rs6983267	0.47	0.47	0.81^*	G/G	142 (30.7)	114 (29.3)	1.00		91 (27.6)	1.00		23 (39.0)	1.00
				G/T	204 (44.1)	187 (48.1)	1.11 (0.78–1.58)		161 (48.8)	1.22 (0.83–1.78)		26 (44.1)	0.82 (0.40–1.66)
				T/T	117 (25.3)	88 (22.6)	0.98 (0.64–1.48)	0.76	78 (23.6)	1.12 (0.72–1.73)	0.6	10 (16.9)	0.40 (0.15–1.05)	0.15
rs1447295	0.07	0.08	0.33	C/C	414 (86.8)	334 (85)	1.00		285 (84.8)	1.00		49 (86.0)	1.00
				A/C	60 (12.6)	54 (13.7)	1.00 (0.64–1.56)		47 (14.0)	1.00 (0.63–1.59)		7 (12.3)	0.85 (0.32–2.25)
				A/A	3 (0.6)	5 (1.3)	2.43 (0.46–12.80)	0.56	4 (1.2)	1.98 (0.37–10.67)	0.73	1 (1.8)	2.21 (0.14–35.11)	0.81
TG
rs180223	0.40	0.47	<0.01	G/G	177 (37.3)	109 (27.7)	1.00		94 (28.1)	1.00		15 (25.4)	1.00
(Ser734Ala)				G/T	214 (45.1)	197 (50.1)	1.78 (1.26–2.52)		166 (49.7)	1.69 (1.17–2.43)		31 (52.5)	2.79 (1.28–6.08)
				T/T	83 (17.5)	87 (22.1)	1.86 (1.20–2.87)	0.001	74 (22.2)	1.86 (1.18–2.93)	<0.01	13 (22.0)	1.78 (0.67–4.72)	0.02
rs853326	0.40	0.47	<0.01	C/C	178 (37.5)	110 (27.8)	1.00		95 (28.2)	1.00		15 (25.4)	1.00
(Met1028Val)				C/T	213 (44.9)	199 (50.2)	1.78 (1.26–2.52)		168 (49.9)	1.69 (1.18–2.43)		31 (52.5)	2.85 (1.31–6.20)
				T/T	83 (17.5)	87 (22.0)	1.85 (1.19–2.85)	0.001	74 (22.0)	1.84 (1.17–2.90)	<0.01	13 (22.0)	1.82 (0.69–4.81)	0.02
rs2076740	0.39	0.36	0.35	C/C	172 (38.0)	153 (41.1)	1.00		129 (40.7)	1.00		24 (43.6)	1.00
(Arg1999Trp)				C/T	210 (46.5)	166 (44.6)	0.86 (0.61–1.21)		142 (44.8)	0.85 (0.60–1.22)		24 (43.6)	0.98 (0.48–2.01)
				T/T	70 (15.5)	53 (14.2)	0.90 (0.56–1.44)	0.68	46 (14.5)	0.90 (0.54–1.47)	0.67	7 (12.7)	1.01 (0.34–3.01)	1.0

Significant values are indicated in bold.

Two-sided chi-square test for distribution of allelic frequencies: ^*Compared by the Armitage's trend test.

Adjusted for age and sex.

p-Value corresponding to codominant model.

N, number of subjects; MAF, minor allele frequency; PTC, papillary thyroid carcinoma; FTC, follicular thyroid carcinoma; CI, confidence interval.

In controls, the genotype distributions were according to the Hardy–Weinberg equilibrium for six of the seven polymorphisms (p = 0.13–0.71), except for rs6983267 (p = 0.01). In this case, genotyping errors were not considered to be the cause of the Hardy–Weinberg deviation (see Materials and Methods section). A correction for this deviation was used when comparing allele frequencies between cases and controls for this SNP (Armitage's trend test).

According to the different allele frequency found between cases and controls for two of the three TG polymorphisms studied, rs180223 and rs853326, we also found a significant association of these TG SNPs with thyroid cancer (see Table 2, codominant model). These two polymorphisms are in strong LD (r ² = 0.9956) and they displayed the same genotype distribution in our population. Consequently, the risk associated with the variant allele of both loci was very similar. For example, as shown in Table 2, in the total of cases the significant OR for the Ser734Ala polymorphism (rs180223) was 1.78 (95% CI = 1.26–2.52) for the heterozygous and 1.86 (95% CI = 1.20–2.87) for the homozygous variant Ala (p = 0.001), which indicates that one copy of the variant allele is sufficient to confer thyroid cancer susceptibility. The same was observed for the Met1028Val polymorphism (rs853326). In addition, both polymorphisms showed significant ORs in PTC and FTC. In contrast, no risk association was found for the third TG polymorphism studied, Arg1999Trp (rs2076740). Moreover, this polymorphism showed no linkage with the TG SNPs rs180223 and rs853326 (r ² < 0.0004). The four additional polymorphisms investigated also showed no association with thyroid cancer susceptibility (Table 2).

For the TG Ser734Ala (rs180223) and Met1028Val (rs853326) polymorphisms, the risk estimates are the same for homozygous variant and heterozygous (see above), suggesting a dominant model of inherence. Therefore, we evaluated risk assessment according to a dominant model for these polymorphisms and after stratification of the patients group for PTC and FTC. The results are presented in Table 3. For both polymorphisms, a statistically significant association of the variant allele carriers was observed with thyroid cancer and when the two types of thyroid cancer, PTC and FTC, were examined separately. Further, the risk estimates for all the patients (Ser734Ala: OR = 1.82, 95% CI = 1.31–2.52, p < 0.001) and for the patient subgroups separately, PTC (Ser734Ala: OR = 1.73, 95% CI = 1.23–2.44, p = 0.001) and FTC (Ser734Ala: OR = 2.45, 95% CI = 1.17–5.13, p = 0.01), were similar. Further investigation of haplotype combining the two significant SNPs of TG, rs180223 (Ser734Ala) and rs853326 (Met1028Val), inferred only two haplotypes with a frequency of 99.9%. As shown in Table 3, the haplotype composed by the two variant alleles, TT, had a different distribution between cases and controls, with a statistically significant OR of 1.41 (95% CI = 1.14–1.75, p = 0.001) for the total group of patients.

Table 3.

Genotype Distribution and Risk Estimates According to the Dominant Model, and Haplotype Analysis of Thyroglobulin Single-Nucleotide Polymorphisms, rs180223 and rs853326, in a Case–Control Study of Thyroid Cancer in a Spanish Population

TG genotype SNP	SNP genotype	Controls N (%)N = 479	Patients N (%)N = 398	Odds ratio ^a (95% CI)	p-Value ^b	PTC N = 339	Odds ratio ^a (95% CI)	p-Value ^b	FTC N = 59	Odds ratio ^a (95% CI)	p-Value ^b
rs180223	G/G	177 (37.3)	109 (27.7)	1.00		94 (28.1)	1.00		15 (25.4)	1.00
(Ser734Ala)	G/T-T/T	297 (62.7)	284 (72.3)	1.80 (1.30–2.50)	<0.001	240 (71.9)	1.73 (1.23–2.44)	0.001	44 (74.6)	2.45 (1.17–5.13)	0.01
rs853326	C/C	178 (37.5)	110 (27.8)	1.00		95 (28.2)	1.00		15 (25.4)	1.00
(Met1028Val)	C/T-T/T	296 (62.5)	286 (72.2)	1.80 (1.30–2.49)	<0.001	242 (71.8)	1.73 (1.23–2.44)	0.001	44 (74.6)	2.50 (1.19–5.22)	0.01

TG haplotype rs180223	rs853326	Controls	Patients	Odds ratio ^a (95% CI)	p-Value ^c	PTC	Odds ratio ^a (95% CI)	p-Value ^c	FTC	Odds ratio ^a (95% CI)	p-Value ^c
G	C	0.5998	0.5289	1.00	—	0.5310	1.00	—	0.5169	1.00	—
T	T	0.4002	0.4711	1.40 (1.13–1.74)	<0.01	0.4690	1.39 (1.11–1.74)	<0.01	0.4831	1.43 (0.92–2.24)	0.11

Significant values are indicated in bold.

Adjusted for age and sex.

p-Value corresponding to dominant model.

p-Value corresponding to haplotype association.

Subsequently, we analyzed the possible interaction between either of the two TG SNPs that were associated with DTC (rs180223 or rs853326) in our population and the TG SNPs showing lack of association (rs2076740). No interaction was found (interaction p-value = 0.39).

Finally, because of the higher incidence of thyroid cancer in women and the prognostic indicator of age at diagnosis in thyroid cancer, the risk for the TG Ser734Ala (rs180223) and Met1028Val (rs853326) polymorphisms in women and men subgroups according to age was estimated. The results are shown in Table 4, where it can be observed that in women, regardless of age at diagnosis, the ORs were significant according the dominant model for both TG polymorphisms. In this case, the risk estimates were similar to the ones found in the total population (Table 3). Similar trends were observed for <45-year-old men. However, the TG SNPs, rs180223 and rs853326, did not show association with DTC in ≥45-year-old men.

Table 4.

rs180223 and rs853326 Thyroglobulin Genotypes and Thyroid Cancer Risk in Women and Men According to Age at Diagnosis

		<45 years of age				≥45 years of age
TG genotype SNP	SNP genotype	Controls	Patients	Odds ratio (95% CI)	p-Value ^a	Controls	Patients	Odds ratio (95% CI)	p-Value ^a
Women		N = 164	N = 153			N = 119	N = 154
rs180223	G/G	74 (45.7)	49 (32.2)	1.00		42 (35.6)	38 (24.8)	1.00
(Ser734Ala)	G/T-T/T	88 (54.3)	103 (67.8)	1.77 (1.12–2.80)	0.01	76 (64.4)	115 (75.2)	1.67 (0.99–2.83)	0.05
rs853326	C/C	74 (45.7)	49 (32.0)	1.00		43 (36.4)	38 (24.7)	1.00
(Met1028Val)	C/T-T/T	88 (54.3)	104 (68.0)	1.78 (1.13–2.83)	0.01	75 (63.6)	116 (75.3)	1.75 (1.04–2.96)	0.04
Men		N = 89	N = 39			N = 107	N = 50
rs180223	G/G	31 (35.2)	8 (20.5)	1.00		30 (28.3)	14 (28.6)	1.00
(Ser734Ala)	G/T-T/T	57 (64.8)	31 (79.5)	2.11 (0.86–5.14)	0.09	76 (71.7)	35 (71.4)	0.99 (0.47–2.09)	0.97
rs853326	C/C	31 (35.6)	8 (20.5)	1.00		30 (28.0)	15 (30.0)	1.00
(Met1028Val)	C/T-T/T	56 (64.4)	31 (79.5)	2.15 (0.88–5.24)	0.08	77 (72.0)	35 (70.0)	0.91 (0.43–1.90)	0.80

Significant values are indicated in bold.

p-Value corresponding to dominant model.

Discussion

In this study, we have evaluated the association of genetic variants at different loci on chromosome 8q23 and 8q24 with DTC, including two SNPs markers previously reported to be associated with different types of cancer, and polymorphisms in two genes involved in thyroid function, TRHR and TG.

Several studies have shown that common genetic variants at rs6983267 and rs1447295 are related to colorectal, breast, and prostate cancers (9,18 –22). In our study, neither of these polymorphisms was significantly associated with DTC. This is the first analysis of the rs1447295 in thyroid cancer. Therefore, these results showing lack of association suggest that this SNP is not a risk marker for DTC. On the other hand, a previous study showed that the rs6983267 SNP was associated with thyroid cancer (10), which is in contradiction with our results in a Spanish population. We have analyzed a total of 402 patients and 479 controls. Therefore, the size of our study had sufficient power to detect an association if it was present. Nevertheless, genetic differences between the populations studied could explain the discrepancies between our study and the previous study (10), although random chance events are possible.

We found no evidence for an association of variants of the TRHR gene with DTC in our Spanish population. Thyrotropin-releasing hormone has a central role in regulating the secretion of TSH via the hypothalamic–pituitary–thyroid axis (23). This role makes TRHR a good candidate for thyroid cancer association studies. Mutations in TRHR can cause hypothyroidism (24), and recently, a genome-wide association study revealed that TRHR is an important gene for regulation of lean body mass (25). Therefore, as our study is the first to look for an association of TRHR variants with DTC, it would be important to confirm that variants of TRHR are not thyroid cancer risk factors in other populations. In addition, variants of the TRHR other than those studied here should be analyzed to determine whether they are risk factors for thyroid cancer.

There is little information regarding an association of TG variants with thyroid cancer. Thyroglobulin is the most highly expressed protein in the thyroid gland and plays important roles in thyroid function. Loss-of-function germline mutations in TG have been identified and linked to thyroid dyshormonogenesis (26,27). However, to our knowledge, only one report exists describing the influence of a genetic variant of TG in the probability of developing thyroid cancer (28). Here, we have analyzed three common TG SNPs that introduce missense amino acid changes in the protein and that were previously identified to be associated with autoimmune thyroid disease (14).

Two of the three TG SNPs studied here are in the exon 10–12 cluster and the other SNPs in exon 33. Both the exon 10–12 cluster and exon 33 are in different LD blocks. The exon 33 SNP was not associated with DTC. We found, however, that allele frequencies in the exon 10–12 cluster were statistically different in DTC compared with control subjects (p < 0.01), indicating that TG is a susceptibility gene for thyroid cancer. Except for men over the age of 44, these TG variants were associated with DTC in men and women. We do not have a clear explanation for the data in men over the age of 44 being different from younger men and women, but it may be related to decreased statistical power. Haplotype analysis combining the two TG risk SNPs, rs180223 and rs853326, showed that the risk estimate for the haplotype generated with the variant alleles of the two polymorphisms was similar to the OR found individually for each polymorphism. An explanation for these results is that any of these nonsynonymous SNPs could be responsible for the thyroid cancer susceptibility, but there are not sufficient data to resolve what the susceptibility locus is. Alternatively, neither of these polymorphisms may be directly responsible for the TG-associated susceptibility, but they define a risk haplotype in the TG gene, where rs180223 and rs853326 act as TG risk markers for thyroid cancer. In a previous study, linkage analysis on 26 families with PTC identified one susceptibility locus where both TG and Src-like adaptor (SLA) genes are located (11). In fact, the SLA gene is encoded by an intronic sequence on the antisense strand of the TG gene.

The size of our study population is sufficient to conduct association analysis using common genetic variants. On the other hand, as the majority of polymorphisms in the present study have not been previously analyzed in thyroid cancer association studies, other populations should be studied to confirm these results. In the case of the genetic variants of the TG gene associated with thyroid cancer in a Spanish population, replicated results will define the TG gene as a susceptibility factor for thyroid cancer. In addition, further studies will be required to determine the role of genetic variants in the TG gene in thyroid cancer susceptibility.

Footnotes

Acknowledgments

The authors thank all the subjects who participated in this study as well as the members of the Nuclear Medicine Service, Hospital Vall d'Hebron (Barcelona), and the Endocrinology Unit of the Hospital Josep Trueta (Girona) for providing patient blood samples. Eddy González-Flores, Esteban-Mariano Giménez, Wilser García-Quispes, and Cristian Valiente actively participated in collecting and preparing the samples for genotype analysis. This work was partially funded by the Spanish Ministry of Education and Science (project SAF2007-6338) and the Generalitat de Catalunya (CIRIT; 2009SGR-725). A. Akdi was supported by a predoctoral fellowship from the Universitat Autònoma de Barcelona.

Disclosure Statement

The authors declare that no competing financial interests exist.

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