Thyroid cancer is the most common endocrine cancer [1
]. The etiology of thyroid cancer remains largely unknown, but individual susceptibility to thyroid cancer is likely to be influenced by the combined effect of the environment and multiple low-penetrance to moderate-penetrance genes [2
]. Several studies have shown that serum levels of 25-hydroxyvitamin D are lower in patients with thyroid cancer [4
]. This is in agreement with the growing body of evidence for a role of vitamin D in regulating cell proliferation and differentiation [6
There is an inter-individual variability in serum levels of vitamin D that is not explained alone by dietary intake or sunlight exposure [8
]. Studies have shown that a significant proportion of the variability of vitamin D levels is due to genetic and epigenetic factors that affect several steps along the vitamin D pathway [9
]. In particular, two large genome-wide association studies (GWAS), performed on individuals of European descent, have associated serum 25-hydroxyvitamin D levels with Single Nucleotide Polymorphisms (SNPs) close to genes involved in the synthesis (7-dehydrocholesterol reductase, DHCR7
), hydroxylation (cytochrome P450 subfamily IIR polypeptide 1, CYP2R1
) and degradation (cytochrome P450 family 24 subfamily A polypeptide 1, CYP24A1
) of vitamin D [10
(on chromosome 11q13.4) encodes the enzyme 7-dehydrocholesterol reductase, which is the penultimate enzyme of sterol biosynthesis that converts 7-dehydrocholesterol (7-DHC) to cholesterol. Homozygous mutations in DHCR7
cause the Smith-Lemli-Opitz syndrome, which is associated with elevated serum 7-DHC levels, low serum cholesterol levels, and multiple congenital abnormalities, but heterozygous carriers have been proposed to be protected against rickets and osteomalacia from hypovitaminosis D [11
(on chromosome 11p15.2) encodes microsomal vitamin D 25-hydroxylase, which catalyzes the first hydroxylation reaction in the biosynthesis of vitamin D. Homozygous mutations in CYP2R1
have been identified in cases of vitamin D-dependent rickets type 1B [10
(on chromosome 20q13.2) encodes vitamin D 24-hydroxylase, which is involved in the inactivation of vitamin D metabolites [11
]. Previous GWAS demonstrated that the rs12785878, rs2060793, and rs6013897 SNPs, located near the DHCR7
, and CYP24A1
genes, respectively, were strongly associated with serum vitamin D levels in both discovery and replication cohorts [10
The aim of this study was to determine if the DHCR7 rs12785878, CYP2R1 rs2060793, and CYP24A1 rs6013897 SNPs are also associated with susceptibility to thyroid cancer.
rs2060793, and CYP24A1
rs6013897 genotype and allele frequencies observed in patients and controls are presented in Table 1
, Table 2
and Table 3
, respectively. Observed genotype frequencies did not deviate from the expected frequencies under the Hardy-Weinberg equilibrium.
rs12785878 minor allele (G allele) was associated with overall thyroid cancer under an additive (OR 1.38, 95% CI 1.15–1.65, p
= 0.0004) and codominant inheritance model, in which the homozygous GG genotype had the greatest risk (OR 1.88, 95% CI 1.30–2.74) and the heterozygous TG genotype an intermediate risk (OR 1.41, 95% CI 1.07–1.86) (p = 0.002) (Table 1
). This association was observed for both papillary (OR 1.35, 95% CI 1.12–1.62, p = 0.0016, additive model) and follicular (OR 1.64, 95% CI 1.11–2.42, p = 0.013, additive model) thyroid carcinoma. All associations remained significant after adjusting for sex and age.
No significant differences between patients and controls were observed for the CYP2R1
rs2060793 (Table 2
) and CYP24A1
rs6013897 (Table 3
The distribution of SNPs did not differ with age at diagnosis (<45 vs. ≥45 years), gender (male vs. female), tumor subtype (papillary vs. follicular), tumor size (≤1 vs. >1 cm), or lymph node or distant metastasis (presence vs. absence). The CYP2R1
rs2060793 minor allele (A allele) was less frequent in patients with a lymph node or distant metastasis (Table 2
). Individuals with at least one A allele had a decreased risk of metastasis, under a dominant inheritance model (OR 0.61, 95% CI 0.40–0.93) (p
= 0.022). However, this p
-value was above the threshold of statistical significance (p
> 0.017) determined by the Bonferroni correction for multiple comparisons.
This case-control study revealed an increased frequency of the DHCR7
rs12785878 minor allele (G allele) in patients with thyroid cancer. The homozygous GG genotype was associated with 1.9 times greater risk for thyroid cancer (OR 1.88, 95% CI 1.30–2.74), when compared to the homozygous TT genotype, whereas the heterozygous TG genotype conferred an intermediate risk (OR 1.41, 95% CI 1.07–1.86). These results suggest that this genetic variant, which previous GWAS have associated with lower circulating vitamin D levels [10
], is also a risk locus for thyroid cancer in the Portuguese population.
To date, there are no functional studies of the rs12785878 polymorphism, so it remains unclear by which mechanism this polymorphism influences the serum levels of vitamin D and cancer risk. The rs12785878 polymorphism is located 8 kilobases upstream from the transcription initiation site of DHCR7
and it is unknown if it has any effect on enzyme expression or if it has only an indirect effect through linkage disequilibrium with other functional variants in the region. In fact, rs12785878 is located in an intron of an entirely different gene (NADSYN1
, encoding nicotinamide adenine dinucleotide (NAD) synthetase). Hence, the location of this SNP is often referred to as the NADSYN1/DHCR7
]. However, it is DHCR7
that has been assumed to be the candidate gene for vitamin D levels, as it encodes the enzyme 7-dehydrocholesterol reductase, which converts 7-dehydrocholesterol to cholesterol. Therefore, this removes the substrate from the synthetic pathway of vitamin D3 in the skin [10
Our study is the first to report an association of DHCR7
rs12785878 with any form of cancer. Other studies performed on colorectal [18
], breast [20
], prostate [20
], ovarian [20
], lung [20
], pancreatic [20
], neuroblastoma [20
], and overall cancer risk [23
] did not find an association with DHCR7
rs12785878. However, the association of these cancers with vitamin D levels is more controversial than for thyroid cancer [4
], and this might explain the lack of an association with DHCR7
. No other study of this SNP in thyroid cancer has so far been reported.
rs2060793 (G > A) and CYP24A1
rs6013897 (T > A) variants were not found to be associated with thyroid cancer in our study, despite their reported association with lower vitamin D levels [10
]. This is in agreement with Penna-Martinez et al. [24
] who analyzed different SNPs within CYP2R1
(rs12794714 and rs10741657) and CYP24A1
(rs927650, rs2248137, rs2296241) and found no association of thyroid cancer with each individual SNP, even though certain haplotypes of CYP24A1
were associated with a higher risk.
Clinical parameters (age at diagnosis, gender, tumor subtype, tumor size, presence of lymph node, or distant metastasis) were analyzed to assess if genetic susceptibility was limited to subgroups of patients, which has been reported for other genetic variants [12
]. Our study revealed a decreased frequency of the CYP2R1
rs2060793 (G > A) minor allele in patients with metastatic disease (OR 0.61, 95% CI 0.40–0.93), which suggests a protective effect of this SNP against more advanced disease. However, this association failed to reach statistical significance after correction for multiple comparisons. It is noteworthy that other genetic variants associated with lower serum vitamin D have been associated with protection against aggressive (but not nonaggressive) prostate cancer [21
] and with improved survival after high-grade glioma [27
]. Thus, the significance of our findings needs to be clarified in further investigations.
GWAS on thyroid cancer have not identified DHCR7
or any other vitamin D-related gene, as risk loci for thyroid cancer [3
]. Susceptibility SNPs identified in case-control association studies have not always been confirmed by GWAS [2
]. This could indicate the absence of a real effect of some SNPs, but an alternative explanation is that the high stringent criteria applied in GWAS, designed to prevent false-positive findings, may exclude SNPs truly associated with the risk [2
Our study has some limitations. First, serum vitamin D levels are not routinely obtained in the preoperative work-up of patients and, therefore, it was not possible to correlate the vitamin D levels with the genetic profile and with the disease status. However, measurements of circulating vitamin D also have limitations, which include inter-laboratory differences, seasonal fluctuations, fasting status, and other confounding effects caused by the disease or its treatment [28
]. Second, there are no functional studies of the genetic variants to confirm their role in vitamin D homeostasis. Although the effect of the variants on cancer susceptibility may be through lower vitamin D levels, a vitamin D-independent mechanism of these variants on thyroid tumorigenesis has not been excluded. In particular, there is evidence that the DHCR7 enzyme regulates the switch between cholesterol and vitamin D production [29
] and that cholesterol metabolism may also be implicated in cancer development [30
]. Lastly, the associations found in this study may be population-specific and not replicated in other populations with different environmental exposures and genetic profiles. In contrast to the association of DHCR7
rs12785878 with vitamin D levels in individuals of European descent, a study with Arab and South Asian individuals did not identify such an association [31
]. Therefore, it would be of interest to replicate our study in other European and non-European populations.
In conclusion, our data suggest that DHCR7 rs12785878 is a novel risk locus for thyroid cancer and that CYP2R1 rs2060793 may confer a protective effect against disease progression. These findings may contribute to a better understanding of the role of vitamin D in the development and progression of thyroid cancer.