CYP24A1 Overexpression and Vitamin D Deficiency: A Potential Prognostic Axis in Thyroid Nodules Classified by the Bethesda System

Abstract

Introduction:

Vitamin D, crucial for calcium balance and bone health, also regulates the cell cycle and has anti-tumour properties. Given the rising incidence of thyroid cancer, this study investigates the association between preoperative vitamin D levels and CYP24A1 mRNA expression in thyroid nodules (TNs) classified by the Bethesda system.

Methods:

A prospective observational study included 40 adults with TNs. Exclusion criteria applied. Clinical evaluation involved blood tests, thyroid ultrasound, fine-needle aspiration cytology, and histopathology. Serum vitamin D, calcium, and CYP24A1 mRNA expression were analysed for correlations.

Results:

Participants had a mean age of 37.3 years; 75% were female. Thyroid Imaging Reporting and Data System (TIRADS) identified suspicious nodules. Most patients (95%) had normal thyroid-stimulating hormone levels, but 52.5% showed vitamin D deficiency, and 47.5% had low calcium levels. CYP24A1 mRNA expression was elevated, especially in papillary thyroid carcinoma (PTC). Positive correlations were found between vitamin D, calcium, and CYP24A1 expression.

Conclusion:

The study highlights the role of vitamin D deficiency and elevated CYP24A1 expression in TNs, particularly PTC. TIRADS and Bethesda classifications remain valuable diagnostic tools. CYP24A1 may serve as a prognostic marker or therapeutic target, suggesting the need for further research into vitamin D pathways in thyroid disease.

INTRODUCTION

Thyroid nodules (TNs) are increasingly detected worldwide, with a prevalence of 19–68% by high-resolution ultrasonography, and their frequency rises with age.[1,2,3] While most are benign, some progress to malignancy, highlighting the importance of accurate ultrasound-based classification.[4] Risk factors include age, sex, irradiation, iodine status, obesity, and metabolic syndrome. Vitamin D deficiency has recently been implicated in several cancers, including thyroid cancer.[5,6,7,8,9] Active vitamin D exerts anti-tumour effects via the vitamin D receptor (VDR), regulating target genes through vitamin D response elements. Impaired vitamin D–VDR signalling is associated with thyroid carcinogenesis.[10,11,12]

Beyond its classical role in calcium homeostasis, vitamin D influences cell cycle, apoptosis, angiogenesis, and invasion. Low serum vitamin D correlates with higher papillary thyroid carcinoma (PTC) risk, although its relationship with TN malignancy remains underexplored.[13] Vitamin D metabolism involves CYP24A1, a mitochondrial cytochrome P450 enzyme that inactivates 1,25-D3. Overexpression of CYP24A1 facilitates carcinogenesis in colorectal and thyroid cancers, while downregulation enhances vitamin D’s anti-tumour effects.[14] Genome-wide association studies also implicate CYP24A1 variants in vitamin D regulation. Experimental studies demonstrate that vitamin D treatment upregulates CYP24A1 in thyroid cancer cell lines, suggesting that tumour response depends on both vitamin D levels and CYP24A1 expression.[15,16,17,18]

This study examines the correlation between preoperative vitamin D and CYP24A1 mRNA expression in TNs classified by the Bethesda system, aiming to clarify vitamin D’s prognostic role and its potential as a therapeutic adjunct in TN patients.

MATERIALS AND METHODS

This prospective observational study was conducted at the Department of Otorhinolaryngology and Head and Neck Surgery in collaboration with the Departments of Endocrinology, Histopathology, and Cytology of the same institute, which spanned from January 2019 to December 2020. A total of 40 patients with TNs, including various thyroid conditions classified according to the Bethesda system, were recruited in this study. Patients underwent comprehensive clinical assessments, including blood tests, serum vitamin D and calcium measurements, body mass index (BMI) calculation, chest X-ray, electrocardiogram, thyroid function tests, ultrasound scans, and fine-needle aspiration cytology (FNA).

Cases were categorized into three groups based on the Bethesda classification:

Group I: Benign (Bethesda 2 cytology)

Group II: Indeterminate (Bethesda 3, 4, 5 cytology)

Group III: Malignant (Bethesda 6 cytology)

Serum vitamin D, calcium levels, FNA samples, and surgically resected TN specimens were collected for all recruited cases. Ultrasound scans were performed to calculate the Thyroid Imaging Reporting and Data System (TIRADS) score to assess the likelihood of malignancy. A single cytopathologist conducted all FNA cytological examinations. Patients who met the criteria for surgery based on ATA guidelines underwent surgery, and a single histopathologist performed the histopathological examination. The patients who were pregnant, aged less than 40 years, had a history of head and neck radiation, had an infection, and had a history of prior thyroid surgery were excluded from the study. The quantitative mRNA expression of the CYP24A1 gene was evaluated in surgically resected TN specimens. All the results were correlated with the cytology and histopathology of TNs. Statistical analysis was conducted to compare age, sex, serum vitamin D, serum calcium levels, BMI, thyroid-stimulating hormone (TSH) levels, TIRADS grading, and CYP24A1 mRNA expression among the three groups

Sample collection

Five ml of peripheral blood samples was collected from all patients to measure serum vitamin D, TSH, and serum calcium levels. The quantitative expression of CYP24A1 mRNA was assessed in resected TN specimens. Tissue samples were collected from 40 patients immediately after surgery, snap-frozen, and stored at –80°C.

RNA isolation and cDNA synthesis

Total RNA was extracted from the nodule and control tissues using TRI reagent (Sigma-Aldrich, USA). RNA purity (A260/280 = 1.9–2.0) was assessed with a Biophotometer Plus (Eppendorf, Germany) [Supplementary Figure 1 ^{(219.3KB, tif)} ], and integrity checked on 1.5% agarose gel. cDNA was synthesized from 4–6 μg RNA using a cDNA synthesis kit (Fermentas Life Sciences, USA).

Gene expression analysis

Specific CYP24A1 primers were designed for mRNA analysis using Primer3 software. The quantitative real-time polymerase chain reaction (qRT-PCR) was conducted with SYBR Green I dye on a Light Cycler 480 Real-Time PCR system. B-actin gene was used for the normalization of gene expression. The melting curve analysis confirmed the specificity of qRT-PCR products.

Statistical analysis

Data were collected using a predefined proforma and analysed with descriptive statistics. Mean ± standard deviation or median (interquartile range) were reported as appropriate, with 95% confidence intervals (CIs). Normality was tested by Kolmogorov–Smirnov. Proportions were compared using the Chi-square/Fisher’s exact test, and correlations by Pearson’s coefficient. Two-sided tests with α = 0.05 were performed in Statistical Package for the Social Sciences v22.0.

Ethical aspects

The study was conducted in accordance with the principles of the Declaration of Helsinki 1964 and subsequent amendments. The study protocol was reviewed and approved by the Institutional Ethics Committee of PGIMER Chandigarh, India, NK/S116/MS/417, dated 23.8.2025. However, the above-mentioned study was already discussed and approved in the meeting of the Institute Ethics Committee (Intramural) PGIMER, Chandigarh, held on 30 January 2020. A new copy of ethical approval was provided on 23 August 2025, on request from the corresponding author of the study, due to the unavailability of the previous one. Written informed consent was obtained from all individual participants. Participant confidentiality and data privacy were strictly maintained throughout the research.

RESULTS

Sociodemographic analysis

Over 16 months, 40 patients with TNs were included in the study. The mean age was 37.3 ± 13.38 years [Supplementary Figure 2 ^{(228.1KB, tif)} ]. The distribution of patients by age showed higher numbers in the 26–40 age group.

Gender distribution was skewed, with more females (75%) than males (25%) [Supplementary Figure 3a ^{(272.2KB, tif)} ]. Gender distribution significantly correlated with Bethesda classification [Supplementary Figure 3b ^{(272.2KB, tif)} ].

Cytopathological analysis

All 40 patients were classified according to the Bethesda classification for TNs. Categories were as follows: 0% in Category 1, 47.5% in Category 2, 2.5% in Category 3, 10% in Category 4, 0% in Category 5, and 40% in Category 6 [Supplementary Figure 4a ^{(249.1KB, tif)} ]. Further classification placed patients into three groups: Group I (47.5%), Group II (12.5%), and Group III (40%) [Supplementary Figure 4b ^{(249.1KB, tif)} ].

Endocrinological analysis

Most patients (95%) had normal serum TSH levels [Supplementary Figure 5a ^{(341.9KB, tif)} ]. The distribution of TSH levels was similar among the three groups [Supplementary Figure 5b ^{(341.9KB, tif)} ]. More than half of the patients (52.5%) had decreased serum vitamin D3 levels [Figure 1a]. Vitamin D3 levels varied across the three groups but without significant correlation [Figure 1b].

Figure 1.

Serum vitamin D3 level of patients (a) and Distribution in patient groups (b)

Nearly half of the patients (47.5%) had decreased serum calcium levels [Figure 2a]. Calcium levels varied among the three groups but not significantly [Figure 2b].

Figure 2.

Serum calcium level of patients (a) and Distribution in patient groups (b)

Radiological analysis

All patients were assessed using TIRADS grading on the USG neck [Figure 3]. No patients were categorized as TIRADS 1 or 2. TIRADS 3 had 20%, TIRADS 4A had 27.5%, TIRADS 4B had 12.5%, TIRADS 4C had 12.5%, and TIRADS 5 had 27.5%.

Figure 3.

TIRADS grading of patients

Histopathological analysis

Post-surgery, 57% of patients had benign pathology, including colloid goitre (40%) and follicular adenoma (17.5%). Forty-three per cent of patients had malignant pathology, including papillary carcinoma (35%) and follicular carcinoma (7.5%) [Figures 4a and 4b].

Figure 4.

Histopathology of patients (a) and Histopathological classification of patient groups (b)

CYP24A1 gene expression

The quantitative expression analysis of CYP24A1 in TNs subjects (n = 40) was studied in surgically resected TN tissue samples with taking adjacent normal thyroid tissue as control for individual subject and denoted as CYP24A1-D and CYP24A1-N, respectively. Individual fold change value was calculated by dividing both the values for CYP24A1-D and CYP24A1-N and analysed with respect to each other statistically to yield relative fold change of CYP24A1 expression. In our study, overall CYP24A1 gene expression was upregulated in TNs compared to adjacent normal tissue [Figure 5a]. However, CYP24A1 gene expression in both TNs and adjacent normal thyroid tissues of PTC significantly differed from that in follicular neoplasm and colloid goitre [Figures 5b and 5c].

Figure 5.

Relative gene expression of CYP24A1 (a), in TNs (CYP24A1-D) (b) and in adjacent normal thyroid tissues (CYP24A1-N) (c)

Correlation analysis

Correlation analysis was performed in between the endocrinological parameters and CYP24A1 gene expression. In our study, Spearman’s correlation between serum vitamin D3 and serum calcium was statistically significant with P = 0.008 and correlation coefficient as 0.416. However, the correlation between serum vitamin D3 and BMI was not statistically significant with P = 0.373 and a correlation coefficient as –1.45. Serum vitamin D3 was positively correlated with CYP24A1 gene expression in adjacent normal thyroid tissues (P = 0.044). However, positive correlation was not found in between serum vitamin D3 and CYP24A1 gene expression in TNs (P = 0.972). Additionally, we found statistically significant positive correlation in between serum calcium and CYP24A1 gene expression in both TNs (P = 0.003) and adjacent normal thyroid tissues (P = 0.031).

DISCUSSION

The age distribution in this study demonstrates a broad spectrum of individuals affected by TNs, with a mean age of 37.3 years. Traditionally, advanced age has been considered a negative prognostic factor in thyroid cancer, a notion first proposed by Crile and Hazard in 1953.[19] However, more recent research, such as that by Hyeyeun Lim et al.,[20] has shown that both incidence and mortality of thyroid cancer are rising across all age groups. This emerging trend challenges the use of a fixed age threshold in prognostic models and underscores the need for more nuanced approaches to thyroid cancer risk stratification.

A marked gender disparity was also observed, with females accounting for 75% of the patient population. This aligns with findings from Hyeyeun Lim et al.,[20] who also reported a higher prevalence of thyroid cancer among women. The underlying reasons for this gender predominance remain unclear but may involve hormonal influences, autoimmunity, or genetic susceptibility. Further research is warranted to elucidate the mechanisms contributing to this disparity.

The analysis of BMI revealed that 55% of the patients were overweight, while none were underweighted. Notably, BMI distribution varied across Bethesda categories. This observation reinforces the relevance of metabolic and nutritional factors in TN pathophysiology. Previous studies by Jing et al.[21] and Junya Sado et al.[22] support this finding, reporting that elevated BMI is associated with an increased risk of developing thyroid malignancies, particularly PTC.

Imaging assessments using the TIRADS identified a significant proportion of suspicious TNs, reinforcing the utility of this system in guiding clinical management and the decision for surgical intervention. When used in conjunction with FNAC, imaging enhances diagnostic accuracy and helps in the stratification of malignancy risk.

Cytological classification through the Bethesda system further contributed to diagnostic clarity. Patients in this study were distributed across six Bethesda categories, ranging from benign to malignant. Importantly, no patients were classified in Bethesda Category I (non-diagnostic), reflecting the effectiveness of FNAC in obtaining sufficient cellular material for reliable interpretation. The combined use of TIRADS and Bethesda systems provides a robust framework for clinical decision-making and risk assessment.

Thyroid function analysis showed that the majority of patients (95%) had normal serum TSH levels, suggesting an absence of overt thyroid dysfunction in most cases. However, 5% of patients exhibited decreased TSH levels. Previous findings by Huang et al.[23] have linked low TSH levels with an increased risk of PTC in women (odds ratio = 3.74; 95% CI: 1.53–9.19), indicating that even subclinical alterations in thyroid function may have prognostic implications. This underscores the importance of routine thyroid function testing in patients with TNs, as subtle hormonal imbalances may reflect or contribute to underlying pathological changes.

The study found that 52.5% of patients had normal serum calcium levels, while 47.5% exhibited decreased levels. In Group I, 37.5% had normal calcium levels and 10% had decreased levels. Group II included 2.5% with normal calcium and 10% with decreased levels, whereas Group III had 12.5% with normal calcium and 27.5% with decreased levels. These findings are consistent with the 2016 study by Ying Xue et al.,[24] which demonstrated that patients with non-medullary thyroid cancer (NMTC) typically exhibit lower preoperative serum calcium levels compared to those with benign TNs. Their study also reported a negative association between calcium levels and NMTC, particularly in the context of primary hyperparathyroidism. Furthermore, serum calcium levels below 2.67 mmol/L were effective in distinguishing NMTC from benign TNs. These results suggest that low serum calcium may serve as a predictive marker for NMTC, especially in patients with primary hyperparathyroidism; however, further research incorporating parathyroid hormone levels is warranted.

A significant finding of the present study was that over half of the patients had decreased serum vitamin D3 levels, and nearly half exhibited reduced calcium levels. These observations imply a potential association between vitamin D3, calcium, and thyroid health, highlighting the need for further investigation into their roles in thyroid disease pathogenesis.

The study identified notable correlations among several clinical factors. Serum vitamin D3 levels correlated positively with serum calcium levels, indicating a biological relationship between these two biomarkers. Additionally, serum vitamin D3 levels were positively associated with CYP24A1 expression in adjacent normal thyroid tissue, suggesting complex regulatory interactions within the thyroid microenvironment. These findings are in agreement with previous research by Zahra Heidari et al.[25] and a meta-analysis by Zhao et al.,[13] which concluded that lower serum 25-hydroxyvitamin D levels are associated with an increased risk of thyroid cancer, and that vitamin D deficiency may contribute to its development. Similarly, low serum 25(OH) D level was also correlated with aggressiveness of thyroid cancer by a study of Kim et al.[26] However, studies in other cancers like human oral squamous cell carcinoma revealed that the expression of CYP24A1 was associated with tumour differentiation and further significantly correlated with patients’ prognosis.[27] Moreover, the study observed upregulated CYP24A1 expression in TNs compared to normal tissue, supporting its potential role in thyroid pathology. This aligns with previous studies by Hu and Zhang et al.[28] and Zou et al.,[29] which provided evidence that CYP24A1 functions as an oncogene in PTC. These studies demonstrated that CYP24A1 overexpression activates multiple signalling pathways that contribute to malignant progression and therapy resistance. The significantly elevated CYP24A1 expression observed in our study provides additional evidence supporting its involvement in thyroid tumorigenesis and supports the consideration of CYP24A1 as a novel therapeutic target in the treatment of thyroid cancer.

Collectively, these findings underscore the multifactorial nature of TNs and emphasize the importance of integrating demographic variables (age, gender, BMI), serum biomarkers (vitamin D3, calcium, TSH), and imaging modalities in the comprehensive assessment and management of thyroid disease. The observed correlation between CYP24A1 expression and thyroid pathology offers promising insights into potential therapeutic strategies and enhances our understanding of the molecular mechanisms involved in thyroid cancer. This study contributes meaningfully to the growing body of the literature on thyroid disorders and highlights important avenues for future research.

CONCLUSION

This study highlights improved diagnostic accuracy in TNs by combining the Bethesda system with TIRADS. CYP24A1 mRNA was significantly upregulated in malignant nodules, indicating potential as a therapeutic target. Its correlation with serum calcium suggests a role in calcium homeostasis and thyroid tumorigenesis. The findings emphasize the interplay of vitamin D, calcium metabolism, and gene regulation, supporting further research on CYP24A1 and novel strategies in thyroid cancer management.

Authors’ contributions

All authors contributed to the study conception and design. Drafting of the original article was conducted by AB, SB, and NR and further critically revised for intellectual content by RG and all the other authors. The manuscript has been read and approved by all the authors.

Conflicts of interest

There are no conflicts of interest.

Use of artificial intelligence

NA.

Data availability statement

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Supplementary Figure 1

The spectrometric reading of RNA isolated from tissues

Supplementary Figure 2

Distribution of age

Supplementary Figure 3

Distribution of gender (a) and Correlation of gender with Bethesda classification (b)

Supplementary Figure 4

Bethesda classification of patients (a) and Categorization of patients in groups (b)

Supplementary Figure 5

Serum TSH levels of patients (a) and Distribution in patient groups (b)

Funding Statement

Nil.