Group-specific component and 25-hydroxylase gene polymorphisms in nasopharyngeal carcinoma: Associations with susceptibility and radiotherapy response

Abstract

BACKGROUND

Nasopharyngeal carcinoma (NPC), exhibiting high incidence in southern China, is linked to genetic and environmental factors. Vitamin D metabolism, involving transport [group-specific component (GC) protein] and activation [25-hydroxylase (CYP2R1) enzyme], may influence NPC susceptibility and radiotherapy response. Polymorphisms in GC and CYP2R1 genes affect protein function and serum 25-hydroxyvitamin D [25(OH)D] levels, and are implicated in other cancers. However, their role in NPC – particularly in high-risk Han Chinese populations – and interaction with vitamin D status remains unclear. This case control study (360 NPC patients, 550 controls) investigates these relationships to inform prevention and personalized therapy.

AIM

To investigate the association between vitamin D binding protein (GC) and CYP2R1 gene polymorphisms with susceptibility to NPC and radiotherapy response.

METHODS

A case control study design was adopted, and 360 patients with NPC and 550 healthy controls were included. TaqMan method was used to perform genotyping on GC gene loci rs4588, rs7041, and CYP2R1 gene loci rs10741657, rs12794714. Serum 25(OH)D levels were detected, and the relationship between gene polymorphisms and NPC risk and radiotherapy response was analyzed.

RESULTS

The GC gene rs4588 TT genotype was significantly associated with the risk of NPC in both the codominant model [odds ratio (OR) = 1.68, 95%CI: 1.15-2.45, P = 0.007] and the recessive model (OR = 1.56, 95%CI: 1.02-2.38, P = 0.039). The association between the rs4588 TT genotype and the risk of NPC was more significant in the male subgroup (OR = 1.87, 95%CI: 1.11-3.15, P = 0.019) and the squamous cell carcinoma subgroup (OR = 1.89, 95%CI: 1.19-3.00, P = 0.007). The serum 25(OH)D level of the rs7041 AA genotype carriers was significantly lower than that of the CC genotype (P < 0.001). The CYP2R1 gene rs10741657 AA genotype was associated with higher serum 25(OH)D levels (P = 0.003). The rs12794714 AA genotype was associated with radiotherapy resistance (OR = 1.76, 95%CI: 1.18-2.63, P = 0.005). Stratified analysis showed that the association between rs4588 and rs12794714 was significant only in the subgroup with higher 25(OH)D levels.

CONCLUSION

GC and CYP2R1 genes polymorphisms are associated with NPC susceptibility and radiotherapy response, and this association may be affected by serum 25(OH)D levels. This study provides a new idea for the prevention and individualized treatment in NPC.

Key Words: Group-specific component protein; 25-hydroxylase; Single nucleotide polymorphism; Nasopharyngeal carcinoma; Susceptibility; Radiotherapy response

Core Tip: This study reveals that polymorphisms in the vitamin D-binding protein gene [group-specific component (GC), rs4588] and 25-hydroxylase gene (CYP2R1) (rs12794714) are associated with nasopharyngeal carcinoma (NPC) susceptibility and radiotherapy response, particularly in individuals with sufficient serum 25-hydroxyvitamin D levels. The GC rs4588 TT genotype increases NPC risk, while the CYP2R1 rs12794714 AA genotype predicts radiotherapy resistance. These findings highlight the interplay between genetic predisposition and vitamin D status, offering new strategies for precision prevention and personalized treatment in NPC.

INTRODUCTION

Nasopharyngeal carcinoma (NPC) is a malignant tumor that occurs in the nasopharynx and has a high incidence rate in southern China[1]. Despite significant progress being made in the diagnosis and treatment of NPC in recent years, some patients still do not respond well to radiotherapy and have a poor prognosis[2]. Therefore, it is of great significance to explore the factors related to susceptibility and radiotherapy response of NPC. NPC exhibits significant regional differences in China, with incidence rates in southern provinces (such as Guangdong Province) reaching 20-30 per 100000, which is more than 10 times higher than in northern provinces[3], and is closely related to several gene polymorphism and Epstein-Barr virus infection[4,5]. This study focuses on the high-risk population of southern Han Chinese and aims to explore the regulatory role of vitamin D metabolism genes in NPC risk.

Vitamin D is vital in bodily functions, with anti-cancer effects drawing attention[5]. Vitamin D binding protein [group-specific component (GC) protein] is key in vitamin D metabolism/transport; CYP2R1 is vital for its activation[6,7]. The GC gene encodes GC protein, which plays an important role in the transport and metabolism of vitamin D[7]. Besides transporting vitamin D, GC protein also engages in immune functions like macrophage activation and complement regulation[8]. In addition, GC protein also has the ability to bind fatty acids and participates in the regulation of lipid metabolism[9]. In the metabolism of vitamin D, vitamin D-binding protein (VDBP) (encoded by the GC gene) and 25-hydroxyvitamin D [25(OH)D]-1α-hydroxylase (a key enzyme encoded by genes such as CYP2R1) play crucial roles. VDBP is responsible for the transport of vitamin D and its metabolites to the target tissues, whereas CYP2R1 is involved in the conversion of vitamin D to the active form 25(OH)D[10]. An association between vitamin D levels and the risk of nasopharyngeal cancer has been shown. However, vitamin D levels are not only influenced by environmental factors such as dietary intake and sun exposure, but may also be regulated by genetic factors[11]. Vitamin D may exert anti-cancer effects by modulating the activity of immune cells, inhibiting the production of inflammatory factors, and promoting apoptosis[12]. Vitamin D plays an important role in central nervous system development and function, and deficiency is associated with a variety of neurodevelopmental disorders and neurodegenerative diseases, such as autism, schizophrenia, and Parkinson's disease[13].

The 25-hydroxylase gene encode CYP2R1, which is a key enzyme for vitamin D activation[14]. While CYP2R1 is the primary enzyme responsible for 25-hydroxylation of vitamin D, other enzymes such as CYP27A1 and CYP3A4 also contribute to this activity[15]. Previous studies have shown that vitamin D enhances the anti-tumor effect of radiotherapy[16]. Vitamin D boosts radiotherapy efficacy via promoting apoptosis, inhibiting DNA repair, and enhancing immunity[17].

Previous studies have shown that GC and CYP2R1 genes polymorphisms are associated with the risk of various cancers[18,19]. Previous studies have shown that the GC gene rs4588 can cause structural changes in the GC protein, affecting its affinity for vitamin D and thereby regulating free vitamin D levels[20]. The GC gene rs7041 can alter the charge distribution of the GC protein, reducing its binding capacity with 25(OH)D and leading to differences in serum levels[21]. The CYP2R1 gene rs10741657 (promoter region) regulates CYP2R1 expression, affecting CYP2R1 activity and thereby altering serum 25(OH)D levels[22]. Although the CYP2R1 gene rs12794714 does not alter the amino acid sequence, it may influence enzyme activity through mRNA stability or translation efficiency[23]. However, the relationship between these gene polymorphisms and NPC remains unclear. In addition, although GC and CYP2R1 genes polymorphisms have been shown to be associated with NPC or vitamin D metabolism in populations such as Malaysia[24] and Tunisia[25], their role in the Han Chinese population in high-incidence areas remains unclear. This study aimed to explore the association between the polymorphisms of GC gene rs4588, rs7041 and CYP2R1 gene rs10741657, rs12794714 and NPC susceptibility and radiotherapy response, and to analyze the role of serum 25(OH)D levels in this association, so as to provide new ideas for the prevention and individualized treatment of NPC.

MATERIALS AND METHODS

Study subjects

This study adopted a case control design. From January 2018 to December 2021, 360 patients with newly diagnosed NPC were recruited from the Department of Otolaryngology-Head and Neck Surgery of a tertiary hospital as the case group. During the same period, 550 healthy subjects were recruited from the physical examination center as the control group (both NPC patients and healthy individuals were recruited from Shenzhen and its surrounding areas in China, sharing similar geographical and living environments. All participants were Han Chinese, excluding individuals of other ethnicities or mixed heritage to minimise the impact of genetic heterogeneity on the results). Inclusion criteria for the case group were as follows: (1) Pathologically confirmed NPC; (2) Aged 18-75 years; (3) Han Chinese; and (4) No prior anti-tumor treatment. Exclusion criteria included: (1) Combined with other malignant tumors; (2) Severe heart, liver, or kidney dysfunction; and (3) Autoimmune diseases. The inclusion criteria for the control group were: (1) No abnormal physical examination results; (2) Age and gender matched with the case group; and (3) No family history of malignant tumors. All subjects signed informed consent forms. This study was approved by the hospital ethics committee. The diagnosis and pathological classification of NPC were independently confirmed by two senior pathologists and standardised according to the International Classification of Diseases-10 code and the World Health Organization classification of head and neck tumours (2017). The sample size was calculated using PASS 15.0 software based on the following assumptions: Association with detection of the GC gene rs4588 polymorphism [odds ratio (OR) = 1.5, TT frequency in the control group = 8.7%], α = 0.05, and power = 80%. A total of 360 cases and 550 controls were ultimately included, meeting the minimum requirement.

Clinical data collection

A standardized questionnaire was used to collect the general information of the subjects, including age, gender, smoking history, drinking history, etc. Clinical data such as tumor stage and pathological type were also collected for the case group [the research questionnaire and data collection were conducted in Chinese (Mandarin or local dialect) to ensure that participants could accurately understand and complete the survey]. Patients who received radical radiotherapy were followed up to evaluate the radiotherapy response. The radiotherapy regimen used intensity-modulated radiotherapy, with a total dose of 66-70 Gy and a fractionated dose of 2.0-2.12 Gy, 5 times a week for a duration of 6 weeks and 7 weeks. The initial efficacy evaluation was performed 6-8 weeks after radiotherapy completion. According to the Response Evaluation Criteria in Solid Tumors (RECIST 1.1)[26], complete response (CR) and partial response (PR) were classified as radiotherapy sensitivity, and stable disease (SD) and progressive disease (PD) were classified as radiotherapy resistance. Radiotherapy responses were classified as sensitive (CR/PR) or resistant (SD/PD) according to RECIST 1.1 criteria, and assessments were performed by two independent physicians. CR was defined as disappearance of all target lesions; PR was defined as a reduction in target lesions of ≥ 30%; SD was defined as the lesion had neither shown any significant progression nor improved noticeably; and PD was defined as an increase in lesions of ≥ 20% or the appearance of new lesions.

Serum 25(OH)D level measurement

A 5 mL fasting venous blood sample was collected from each participant EDTA anticoagulant tubes. The samples were centrifuged at 4 °C (3000 rpm, 10 minutes) to separate the serum. The 25(OH)D levels were detected using chemiluminescent immunoassay (CLIA). The LIAISON^® 25 OH Vitamin D TOTAL Assay Kit (DiaSorin Inc., Stillwater, MN, United States) was used for detection on the LIAISON^® XL automated CLIA analyzer. The detection range was 4-150 ng/mL, with an intra-assay coefficient of variation of < 5% and an inter-assay coefficient of variation of < 10%. According to the Endocrine Society guidelines[27], 25(OH)D levels < 50 nmol/L were defined as vitamin D deficiency, 50-75 nmol/L as insufficiency, and > 75 nmol/L as sufficiency.

DNA extraction and genotyping

The genomic DNA of peripheral blood leukocytes was extracted using a Whole Blood Genomic DNA Extraction Kit (Tiangen Biochemical, Beijing, China). The specific steps are as follows: (1) Take 200 μL of whole blood and add it to a 1.5 mL centrifuge tube; (2) Add 20 μL of proteinase K and 200 μL of lysis buffer, mix well, and incubate in a 56 °C water bath for 20 minutes; (3) Add 200 μL of anhydrous ethanol and mix well; (4) Transfer the mixture to the adsorption column and centrifuge at 12000 rpm for 1 minute; (5) Add 500 μL of wash buffer 1 and centrifuge at 12000 rpm for 30 seconds; (6) Add 500 μL of wash buffer 2 and centrifuge at 12000 rpm for 30 seconds, and repeat once; (7) Dry-spin at 12000 rpm for 2 minutes; and (8) Add 50 μL of elution buffer, stand at room temperature for 2 minutes, and centrifuge at 12000 rpm for 1 minute. The DNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States), with an acceptable OD₂₆₀/OD280 ratio of 1.8-2.0.

A TaqMan SNP Genotyping Kit (Applied Biosystems, United States) was used to perform genotyping of GC gene rs4588 and rs7041, and CYP2R1 gene rs10741657 and rs12794714 on ABI 7900HT real-time PCR system. The PCR reaction mixture (10 μL) included: (1) 5 μL of TaqMan Universal PCR Master Mix; (2) 0.25 μL of 40 × SNP Genotyping Assay Mix; (3) 1 μL of DNA template (10 ng/μL); and (4) 3.75 μL of nuclease-free water. The PCR reaction conditions were initial denaturation at 95 °C for 10 minutes; followed by denaturation at 95 °C for 15 seconds, then annealing/extension at 60 °C for 1 minute, for a total of 40 cycles. Three replicate wells were set for each sample, and negative and positive controls were set for each batch of reactions. Genotype interpretation was performed using SDS 2.4 software. To ensure the accuracy of the genotyping results, 10% of the samples were randomly selected for repeated testing, and the consistency of the results was 100%.

Statistical analysis

Statistical Package for the Social Sciences 25.0 software was used for statistical analysis. Continuous data were expressed as mean ± SD, and t-test or analysis of variance were used for comparison between groups; count data were expressed as frequency (percentage), and χ² test was used for comparison between groups. Hardy-Weinberg equilibrium test was used with χ² goodness-of-fit test. Logistic regression model was used to analyze the association between gene polymorphism and NPC risk and radiotherapy response, OR and 95%CI were calculated. Logistic regression can directly provide the degree of influence of the independent variable on the probability of occurrence of the dependent variable, which can be quantified by calculating the OR, and can intuitively explain the associations between genetic polymorphisms and the risk of NPC and response to radiotherapy. Generalized linear model was used to analyze the relationship between genotype and serum 25(OH)D level. P < 0.05 was considered statistically significant.

RESULTS

General characteristics of the study subjects

The general characteristics of the case group and the control group are shown in Table 1. There were no statistical differences between the two groups in terms of age, gender, smoking history, and drinking history (P > 0.05). However, the serum 25(OH)D level in the case group was significantly lower than that in the control group (P < 0.001). In terms of common diseases, there was no significant difference in the prevalence of hypertension and diabetes between the case group and the control group. Among the routine blood test and biochemical indicators, only albumin levels differed significantly, with the case group showing lower levels (P = 0.008), other indicators such as hemoglobin, white blood cell count, platelet count, and total cholesterol level were not significantly different between the two groups. In the case group, squamous cell carcinoma was the most common pathological type (76.7%). The majority were diagnosed at stage III (46.7%), with T3 (40.0%) and N1-2 (73.4%) being the most common T and N stages, respectively. Additionally, 10% of the patients had distant metastasis at initial diagnosis, with an average tumor volume of 28.5 ± 15.7 cm³.

Table 1 Comparison of general characteristics of the subjects, n (%).

Characteristic	Case group (n = 360)	Control group (n = 550)	P value
Age (years) (mean ± SD)	52.6 ± 11.3	51.8 ± 10.9	0.312
Gender			0.726
Male	256 (71.1)	385 (70.0)
Female	104 (28.9)	165 (30.0)
Smoking history			0.583
Yes	168 (46.7)	246 (44.7)
No	192 (53.3)	304 (55.3)
Drinking history			0.671
Yes	132 (36.7)	210 (38.2)
No	228 (63.3)	340 (61.8)
25-hydroxyvitamin D (nmol/L) (mean ± SD)	46.8 ± 18.5	62.3 ± 20.7	< 0.001
Hypertension	72 (20.0)	116 (21.1)	0.698
Diabetes	36 (10.0)	61 (11.1)	0.612
Hemoglobin (g/L) (mean ± SD)	132.5 ± 15.7	134.2 ± 14.9	0.128
White blood cell count (× 109/L) (mean ± SD)	6.8 ± 2.1	6.6 ± 1.9	0.156
Platelet count (× 109/L) (mean ± SD)	235.6 ± 58.4	241.3 ± 55.7	0.142
Albumin (g/L) (mean ± SD)	42.3 ± 4.5	43.1 ± 4.2	0.008
Total cholesterol (mmol/L) (mean ± SD)	4.8 ± 1.0	4.9 ± 0.9	0.113
Pathological type
Squamous cell carcinoma	276 (76.7)
Undifferentiated carcinoma	72 (20.0)
Other	12 (3.3)
TNM stage
Stage I	24 (6.7)
Stage II	84 (23.3)
Stage III	168 (46.7)
Stage IV	84 (23.3)
T stage
T1	36 (10.0)
T2	108 (30.0)
T3	144 (40.0)
T4	72 (20.0)
N-stage
N0	48 (13.3)
N1	132 (36.7)
N2	132 (36.7)
N3	48 (13.3)
M-stage
M0	324 (90.0)
M1	36 (10.0)
Tumor volume (cm3) (mean ± SD)	28.5 ± 15.7

M-stage: Distant metastasis; N-stage: Regional lymph node involvement; T stage: Tumor size/extent.

Association between gene polymorphism and NPC risk

The genotype distribution of the four single nucleotide polymorphism (SNP) loci in both the case group and the control group conformed to the Hardy-Weinberg equilibrium (P > 0.05). The results showed that the rs4588 polymorphism of the GC gene was significantly associated with the risk of NPC in both the codominant and recessive models (Table 2). In the codominant model, the risk of NPC in carriers of the TT genotype was significantly increased (OR = 1.68, 95%CI: 1.15-2.45, P = 0.007). In the recessive model, the TT genotype also showed a significantly increased risk compared to the GT + GG genotype (OR = 1.56, 95%CI: 1.02-2.38, P = 0.039). For the rs4588 polymorphism that showed a significant association, subgroup analysis of gender and tumor typing was performed (Table 3). Subgroup analysis results showed that the association between rs4588 TT genotype and NPC risk was more pronounced in the male subgroup (OR = 1.87, 95%CI: 1.11-3.15, P = 0.019) and squamous cell carcinoma subgroup (OR = 1.89, 95%CI: 1.19-3.00, P = 0.007). Other SNP loci (rs7041, rs10741657, and rs12794714) did not show significant association with NPC risk under different types of inheritance (P > 0.05).

Table 2 Association between group-specific component and 25-hydroxylase gene polymorphisms and nasopharyngeal carcinoma risk, n (%).

Single nucleotide polymorphism	Types of inheritance	Genotype	Case group	Control group	Odds ratio (95%CI)	P value
GC rs4588	Codominant	GG	156 (43.3)	272 (49.5)	1.00 (reference)
		GT	152 (42.2)	230 (41.8)	1.15 (0.87-1.53)	0.325
		TT	52 (14.5)	48 (8.7)	1.68 (1.15-2.45)	0.007
	Dominant (TT + GT vs GG)	TT + GT	204 (56.7)	278 (50.5)	1.28 (0.98-1.67)	0.071
		GG	156 (43.3)	272 (49.5)	1.00 (reference)
	Recessive (TT vs GT + GG)	TT	52 (14.5)	48 (8.7)	1.56 (1.02-2.38)	0.039
		GT + GG	308 (85.5)	502 (91.3)	1.00 (reference)
GC rs7041	Codominant	CC	132 (36.7)	214 (38.9)	1.00 (reference)
		CA	168 (46.7)	258 (46.9)	1.06 (0.79-1.41)	0.713
		AA	60 (16.6)	78 (14.2)	1.24 (0.83-1.85)	0.287
	Dominant (AA + CA vs CC)	AA + CA	228 (63.3)	336 (61.1)	1.10 (0.84-1.44)	0.498
		CC	132 (36.7)	214 (38.9)	1.00 (reference)
	Recessive (AA vs CA + CC)	AA	60 (16.6)	78 (14.2)	1.21 (0.84-1.74)	0.309
		CA + CC	300 (83.4)	472 (85.8)	1.00 (reference)
CYP2R1 rs10741657	Codominant	GG	168 (46.7)	242 (44.0)	1.00 (reference)
		GA	148 (41.1)	246 (44.7)	0.87 (0.66-1.15)	0.326
		AA	44 (12.2)	62 (11.3)	1.02 (0.66-1.58)	0.924
	Dominant (AA + GA vs GG)	AA + GA	192 (53.3)	308 (56.0)	0.90 (0.69-1.17)	0.423
		GG	168 (46.7)	242 (44.0)	1.00 (reference)
	Recessive (AA vs GA + GG)	AA	44 (12.2)	62 (11.3)	1.09 (0.73-1.65)	0.673
		GA + GG	316 (87.8)	488 (88.7)	1.00 (reference)
CYP2R1 rs12794714	Codominant	GG	152 (42.2)	250 (45.5)	1.00 (reference)
		GA	160 (44.5)	238 (43.3)	1.10 (0.83-1.46)	0.492
		AA	48 (13.3)	62 (11.2)	1.27 (0.83-1.95)	0.273
	Dominant (AA + GA vs GG)	AA + GA	208 (57.8)	300 (54.5)	1.14 (0.87-1.49)	0.339
		GG	152 (42.2)	250 (45.5)	1.00 (reference)
	Recessive (AA vs GA + GG)	AA	48 (13.3)	62 (11.2)	1.21 (0.81-1.81)	0.356
		GA + GG	312 (86.7)	488 (88.8)	1.00 (reference)

CYP2R1: 25-hydroxylase; GC: Group-specific component.

Table 3 Subgroup analysis of group-specific component gene rs4588 polymorphism and nasopharyngeal carcinoma risk, n (%).

Subgroup	Genotype	Case group	Control group	Odds ratio (95%CI)	P value
Gender
Male	GG	112 (43.8)	192 (49.9)	1.00 (reference)
	GT	108 (42.2)	160 (41.6)	1.16 (0.83-1.61)	0.389
	TT	36 (14.0)	33 (8.5)	1.87 (1.11-3.15)	0.019
Female	GG	44 (42.3)	80 (48.5)	1.00 (reference)
	GT	44 (42.3)	70 (42.4)	1.14 (0.68-1.92)	0.615
	TT	16 (15.4)	15 (9.1)	1.94 (0.88-4.28)	0.099
Tumor type
Squamous cell carcinoma	GG	120 (43.5)	272 (49.5)	1.00 (reference)
	GT	116 (42.0)	230 (41.8)	1.14 (0.84-1.55)	0.391
	TT	40 (14.5)	48 (8.7)	1.89 (1.19-3.00)	0.007
Other types	GG	36 (42.9)	272 (49.5)	1.00 (reference)
	GT	36 (42.9)	230 (41.8)	1.18 (0.72-1.94)	0.506
	TT	12 (14.2)	48 (8.7)	1.89 (0.92-3.88)	0.084

Association between gene polymorphism and serum 25(OH)D level

The analysis showed that the GC gene rs7041 polymorphism was significantly associated with serum 25(OH)D levels. The AA genotype carriers had significantly lower 25(OH)D levels than the CC genotype (P < 0.001; Figure 1A). The CYP2R1 gene rs10741657 AA genotype was associated with higher serum 25(OH)D levels (P = 0.003; Figure 1B). However, the rs4588 and rs12794714 polymorphisms were not significantly associated with 25(OH)D levels.

Figure 1 Association between group-specific component gene rs7041 and 25-hydroxylase gene rs10741657 polymorphisms and serum 25-hydroxyvitamin D levels. A: Box plot showing serum 25-hydroxyvitamin D [25(OH)D] levels corresponding to the group-specific component rs7041 genotype (CC, CA, AA) in the study population. The 25(OH)D levels of AA genotype carriers were significantly lower than those of CC genotype carriers (^cP < 0.001); B: Box plots showing serum 25(OH)D levels stratified by 25-hydroxylase rs10741657 genotype (GG, GA, AA). Serum 25(OH)D levels were higher in the AA genotype compared to the GG genotype (^bP < 0.01). 25(OH)D: 25-hydroxyvitamin D.

Association between gene polymorphism and radiotherapy response

Among the 278 patients who received radical radiotherapy, 192 (69.1%) were sensitive to radiotherapy and 86 (30.9%) were resistant to radiotherapy. The CYP2R1 gene rs12794714 AA genotype was significantly associated with radiotherapy resistance (OR = 1.76, 95%CI: 1.18-2.63, P = 0.005; Table 4). No other SNPs were significantly associated with radiotherapy response.

Table 4 Association between 25-hydroxylase gene rs12794714 polymorphism and radiotherapy response, n (%).

Genotype	Radiotherapy sensitivity	Radiotherapy resistance	Odds ratio (95%CI)	P value
GG	88 (45.8)	28 (32.6)	1.00 (reference)
GA	84 (43.8)	40 (46.5)	1.49 (0.85-2.62)	0.162
AA	20 (10.4)	18 (20.9)	1.76 (1.18-2.63)	0.005

Stratified analysis

To further explore the role of serum 25(OH)D levels in the association between gene polymorphisms and NPC risk and radiotherapy response, a stratified analysis was performed. The results showed that the association between the GC gene rs4588 TT genotype and NPC risk was significant only in the subgroup with 25(OH)D levels > 50 nmol/L (OR = 2.15, 95%CI: 1.32-3.51, P = 0.002; Figure 2A). Similarly, the association between the CYP2R1 gene rs12794714 AA genotype and radiotherapy resistance was also significant only in the subgroup with 25(OH)D levels > 50 nmol/L (OR = 2.43, 95%CI: 1.45-4.08, P < 0.001; Figure 2B).

Figure 2 Stratified analysis of gene polymorphisms and nasopharyngeal carcinoma risk/radiotherapy response by serum 25-hydroxyvitamin D levels. A: The association between the group-specific component gene rs4588 TT genotype and nasopharyngeal carcinoma risk was significant only in the subgroup with serum 25-hydroxyvitamin D [25(OH)D] levels > 50 nmol/L [odds ratio (OR) = 2.15, 95%CI: 1.32-3.51, P = 0.002]; B: The association between the 25-hydroxylase gene rs12794714 AA genotype and radiotherapy resistance was significant only in the subgroup with serum 25(OH)D levels > 50 nmol/L (OR = 2.43, 95%CI: 1.45-4.08, P < 0.001). AUC: Area under the curve; CYP2R1: 25-hydroxylase; GC: Group-specific component; ROC: Receiver operating characteristic; 25(OH)D: 25-hydroxyvitamin D.

DISCUSSION

This investigation delved into the correlation between polymorphisms in the genes encoding GC protein and CYP2R1 with susceptibility to NPC and the response to radiotherapy. Furthermore, it analyzed the regulatory role of serum 25(OH)D levels in these associations. The findings revealed a significant association between the rs4588 polymorphism in the GC gene and the risk of NPC, as well as a link between the rs12794714 polymorphism in the CYP2R1 gene and radiotherapy response. Notably, these associations were influenced by serum 25(OH)D levels.

The rs4588 TT genotype is significantly linked to NPC risk, which is consistent with past studies. Previous studies reported that the rs4588 TT genotype was associated with an increased risk of colorectal cancer and prostate cancer[28,29]. The amino acid change (Thr436 Lys) caused by rs4588 affects the structure and function of GC protein, thereby affecting the transport and metabolism of vitamin D[30]. It is worth noting that subgroup analysis showed that the association between the rs4588 TT genotype and the risk of NPC was more significant in males and squamous cell carcinoma subgroups, suggesting that GC gene polymorphism affects the occurrence of NPC through gender-specific and tissue-specific mechanisms. The study also found that the rs7041 polymorphism was significantly associated with serum 25(OH)D levels, and AA genotype carriers had lower 25(OH)D levels. This result has also been confirmed in other studies[31]. The amino acid change (Asp432Glu) caused by rs7041 affects the binding affinity of GC protein to vitamin D, thereby affecting the level of 25(OH)D in the circulation[32]. Furthermore, the study found that the rs10741657 AA genotype is associated with higher serum 25(OH)D levels, which is consistent with previous research results[19]. The rs10741657 is located in the promoter region of the gene and regulates the activity of CYP2R1 by affecting gene expression[33]. Studies have reported the association between the rs12794714 polymorphism of the CYP2R1 gene and the radiotherapy response in NPC. The results showed that the AA genotype was associated with radiotherapy resistance. The rs12794714 polymorphism regulates radiotherapy sensitivity by affecting the metabolism of vitamin D. Although rs12794714 is a synonymous mutation (Ser75Ser) that does not change the amino acid sequence, it regulates the expression level of CYP2R1 by affecting mRNA stability, splicing efficiency or translation rate[34].

The results of stratified analysis showed that the association between rs4588 and rs12794714 polymorphisms and NPC risk and radiotherapy response was significant only in the subgroup with higher 25(OH)D levels. This finding highlights the importance of gene-environment interaction in the development of NPC. Vitamin D level, as an effect modifier, affects the phenotypic effect of gene polymorphisms. In the case of high vitamin D levels, the small differences caused by gene polymorphisms are amplified, thus showing significant differences in disease risk. In contrast, in the state of vitamin D deficiency, the effect of gene polymorphisms is masked. This finding emphasizes the importance of considering environmental factors when evaluating the association between gene polymorphisms and disease risk.

The potential mechanism of the above findings involves the threshold effect of the vitamin D signaling pathway. When vitamin D levels are high, the differences in vitamin D metabolism caused by GC and CYP2R1 genes polymorphisms are more likely to exceed a certain functional threshold, thereby showing significant biological effects. In the case of sufficient vitamin D, the rs4588 TT genotype causes more vitamin D to exist in free form, increasing its concentration in target tissues, thereby affecting processes such as cell proliferation, differentiation, and apoptosis[35]. In addition, vitamin D levels also regulate the effects of gene polymorphisms through epigenetic mechanisms. Studies have shown that vitamin D affects DNA methylation and histone modification[36]. Some studies have shown that vitamin D can play a role in cancer prevention and treatment by regulating DNA methylation to affect gene expression[37]. Vitamin D can also regulate gene expression by affecting histone modifications. It has been shown that vitamin D can regulate the activities of histone acetyltransferases and histone deacetylases, which in turn can alter the acetylation status of histones, affecting chromatin structure and gene accessibility[38]. Therefore, different vitamin D levels lead to differential regulation of GC and CYP2R1 genes expression, which in turn affects the phenotypic effects of their polymorphisms. This epigenetic regulation explains why the same genotype exhibits different disease risks at different vitamin D levels.

Diet is one of the most important sources of vitamin D. Sun exposure is the body's main natural way of obtaining vitamin D. Diet and sun exposure together affect serum 25(OH)D levels, which in turn are closely related to the risk of NPC and response to radiotherapy. Therefore, maintaining appropriate serum 25(OH)D levels through a sensible diet and moderate sun exposure may help reduce the risk of NPC and improve radiotherapy response. However, due to the complex effects of individual differences, genetic factors, and other environmental factors (e.g., physical activity), this relationship needs to be clarified by further studies.

GC and CYP2R1 genes polymorphisms can serve as potential biomarkers for early screening of NPC. By detecting polymorphisms in these genes, especially the rs4588 and rs12794714 loci, which are significantly associated with NPC risk, high-risk individuals can be identified for closer monitoring and early intervention. By dynamically monitoring changes in the expression levels or polymorphisms of the GC and CYP2R1 genes in patients, combined with clinical symptoms and imaging findings, a more comprehensive assessment of the patient's disease state and treatment response can be made, providing a scientific basis for the adjustment of the treatment plan.

While traditional disease risk assessments tend to consider only the role of genetic or environmental factors alone, studies of gene-environment interactions are able to synthesise the interactions between the two to provide a more accurate assessment of disease risk. By identifying genetic polymorphisms associated with nasopharyngeal cancer risk (e.g., rs4588 in the GC gene and rs12794714 in the CYP2R1 gene) and their interactions with environmental factors [e.g., serum 25(OH)D levels], we can more accurately assess an individual's risk of developing nasopharyngeal cancer.

This study also has limitations. First, the sample size is relatively small, which may affect the statistical power. Second, this study included only Han Chinese participants, and there are significant differences in genetic background, environmental factors, and living habits among different races and populations, which may have an impact on the relationship between genetic polymorphisms and susceptibility to nasopharyngeal cancer and response to radiotherapy. Therefore, the results of this study need to be applied with caution to other races and populations. Furthermore, environmental factors such as diet and sun exposure that may affect serum 25(OH)D levels were not considered. To more accurately assess the impact of genetic polymorphisms, we included albumin levels as a covariate in regression models in future studies to control for their potential confounding effects. There were no significant differences between the case group and the control group in terms of the prevalence of hypertension and diabetes. However, patients with severe organ dysfunction and autoimmune diseases were excluded, which may have limited the analysis of comorbidities on NPC risk or treatment response. This study also has limitations in terms of multiple comparison correction. In future analyses, we plan to use Bonferroni correction or false discovery rate correction methods to adjust P values to control for the risk of false positives associated with multiple comparisons. Only four SNPs in the GC and CYP2R1 genes were examined in this study, and future plans are to expand the detection of genetic polymorphisms in the GC and CYP2R1 genes to include more tag SNPs. Albumin levels in this study may have had a confounding effect as a confounder on the association between vitamin D levels, genotype, and NPC risk. Due to sample size limitations, this study did not perform haplotype analysis of SNP loci in the GC and CYP2R1 genes. Future studies should expand the cohort and perform linkage disequilibrium assessment to explore the potential synergistic effects of haplotypes on NPC risk and treatment response. The case group in this study did not systematically collect data on family history of NPC, which may have overlooked the potential influence of genetic susceptibility. Although the control group excluded individuals with a family history of malignant tumours, future studies should include family history information (especially in high-incidence areas) to distinguish the independent contributions of genetic and environmental factors. Although some confounding factors were controlled through strict matching and exclusion criteria, the exclusion of body mass index, Epstein-Barr virus infection status, treatment adherence, and tumour microenvironment data may affect the interpretation of the results. Finally, this study was a case control design, and causal relationships cannot be inferred. Future prospective cohort studies are needed to verify the findings of this study.

CONCLUSION

In conclusion, the study revealed the important role of GC and CYP2R1 genes polymorphisms in NPC susceptibility and radiotherapy response and emphasized the regulatory role of serum 25(OH)D levels in these associations. The above findings provide new insights into the role of vitamin D metabolic pathways in the development of NPC and also have direct guidance significance for high-incidence areas in southern China, such as precision screening and vitamin D intervention for populations with high-risk genotypes of GC/CYP2R1. Additionally, the association between CYP2R1 rs12794714 and radiotherapy resistance may have cross-racial applicability, providing new insights for the prevention and treatment of NPC globally.