Interaction of chronic daily headache, dietary intake of iron, and FTO genotype

Abstract

Background:

Chronic daily headache (CDH) is known to be a common and debilitating disease that is influenced by genetic, dietary, and lifestyle factors. Iron (Fe) plays a key role in energy metabolism and neurotransmitter synthesis, while the FTO gene polymorphism (rs9939609) may play a role in modulating nutritional responses and nutrient metabolism. This study aimed to determine the interaction between CDH odds, dietary iron intake, and FTO genotype.

Methods:

This case-control study was conducted on 150 CDH patients and 150 healthy individuals aged 40–80 years. Participants were assessed for anthropometric measurements, assessment of food intake using a validated food frequency questionnaire, and FTO genotype. The association between dietary iron intake and CDH stratified by FTO genotype was assessed using logistic regression analysis after adjusting for age, gender, body mass index (BMI), physical activity, energy intake, and hypertension.

Results:

Among individuals with AT/AA genotypes, those with CDH had lower dietary iron intake than controls (17.10 ± 4.02 vs 18.63 ± 4.62 mg/day, p = 0.023). Logistic regression after full adjustment found an inverse association between dietary iron and CDH in AT/AA genotype carriers (p = 0.042). No significant association was observed in TT genotype carriers.

Conclusion:

Dietary iron intake may be associated with a reduced odds of CDH in FTO A allele carriers, but no association was observed in TT genotype carriers, suggesting a gene-nutrient interaction in the prevention and management of CDH. Further studies are necessary to substantiate these findings.

Introduction

Chronic daily headache (CDH) has historically been defined as the presence of headache on 15 or more days per month for at least 3 months, as described in earlier clinical studies. ¹ However, CDH is not recognized as a formal diagnostic entity in the International Classification of Headache Disorders (ICHD-3), ² and is currently used as a descriptive umbrella term encompassing several chronic headache disorders. It is estimated to affect approximately 3%–5% of the general population, making it a significant public health concern. ³ CDH may have a profound impact on an individual’s quality of life, leading to decreased productivity, increased healthcare utilization, and psychological distress. ⁴ The etiology of CDH is multifactorial, involving a complex interplay of genetic, environmental, and lifestyle factors. ⁵ One such factor that has gained attention in recent years is dietary intake, specifically the intake of iron (Fe). Iron is an essential mineral involved in various physiological processes, including oxygen transport, energy metabolism, and neurotransmitter synthesis. ⁶ Fe also serves as an essential cofactor for the enzyme tryptophan hydroxylase, which is involved in the synthesis of serotonin, a neurotransmitter implicated in the pathophysiology of migraines. ⁷

On the other hand, genetic factors may modify the relationship between dietary iron intake and chronic headache susceptibility. The FTO gene, also known as fat mass and obesity-associated gene, has been extensively studied in the context of obesity and body weight regulation. ⁸ However, emerging evidence suggests that the FTO gene may also play a role in the regulation of food intake, energy expenditure, and nutrients’ metabolism. ⁹ Several single nucleotide polymorphisms (SNPs) within the FTO gene have been associated with obesity and related metabolic disorders. ¹⁰ Given the potential interplay between FTO genotype, dietary intake, and CDH, investigating the association between Fe intake and CDH in carriers of different FTO genotypes is of great interest.

Previous studies examining the association between Fe intake and CDH have yielded conflicting results. Some studies have reported a positive association between low Fe intake and CDH, suggesting that Fe deficiency may contribute to the development and exacerbation of headaches. ¹¹ Other studies have found no significant association between Fe intake and CDH. ¹² Differences between the findings of these studies may stem from variations in the sequence or functional polymorphisms of iron‑related genes, which can influence iron metabolism and downstream biological pathways. ¹³ Understanding the role of FTO genotype in this context may help elucidate the underlying genetic mechanisms that influence Fe metabolism and CDH susceptibility. It is plausible that carriers of certain FTO genotypes may be more susceptible to the effects of Fe deficiency or excess on CDH, potentially due to alterations in Fe transport, absorption, or utilization. ¹⁴

To date, limited research has investigated the association between Fe intake and CDH in carriers of different gene variations. Therefore, the aim of this study is to examine the relationship between dietary intake of Fe and CDH in a sample of individuals with different FTO genotypes.

Methods

This case-control study was conducted in 2023 at Shohadaye Tajrish Hospital in Tehran, Iran. The case group consisted of patients with chronic headache disorders who were diagnosed by a physician. The control group was selected from individuals visiting the hospital for other reasons or hospital staff who did not have persistent headaches. At the beginning of the study, a verbal explanation regarding the study’s objectives, methodology, and confidentiality of information was provided, followed by obtaining written informed consent. Participants then completed the informed consent form. The sample size was calculated using the OpenEpi platform, assuming α = 0.05, 80% statistical power, a 1:1 case–control ratio, and the odds ratio reported in a previous comparable study. ¹⁵ This process yielded a required sample of 150 participants per group.

Trained interviewers collected data on general characteristics, medical history, anthropometric measurements, physical activity levels, and dietary intake. Inclusion criteria for the case group included patients aged 40–80 years with a diagnosis of chronic headache, defined as experiencing headaches on more than 15 days or more per month for at least 3 months. ³ The inclusion criteria for the control group included individuals aged 40–80 years without a history of recurrent or chronic headache disorders and were frequency-matched to cases by age. The age range of 40–80 years was chosen to limit heterogeneity due to dietary patterns, hormonal status, and characteristics of headaches. Exclusion criteria included pregnancy or lactation, iron supplementation, chronic kidney or inflammatory diseases known to affect iron metabolism, malignancy, psychiatric disorders, and regular use of alcohol or analgesic medications suggestive of medication-overuse headache. Participants were also excluded from the study if they failed to complete the questionnaires or if it was not possible to collect the required information.

To minimize selection bias, cases and controls were recruited consecutively from the same center during the same study period. Valid and reliable tools previously validated in other studies were employed to reduce information bias. Iron intake as part of the diet reflected through the FFQ shows average intake over time rather than the momentary biochemical status of the individual, thus, markers such as serum, ferritin, or hemoglobin were not used or analyzed in this current study.

A total of 353 individuals were screened for eligibility, of whom 53 were excluded due to not meeting inclusion criteria. In the non–chronic headache group, 76 participants carried the TT genotype (51%) and 74 carried the AT/AA genotypes (49%), with additional exclusions applied for alcohol or substance abuse (n = 1), iron supplementation (n = 2), and incomplete data (n = 2). In the chronic headache group, 85 participants exhibited the TT genotype (57%) and 65 the AT/AA genotypes (43%), with exclusions for alcohol or substance abuse (n = 3), iron supplementation (n = 1), and incomplete information (n = 1). After applying all exclusion criteria, 150 participants from each group were included in the final analysis, as illustrated in Figure 1.

Figure 1.

Flowchart of the study participants.

Data collection methods

Social and demographic information was collected through face-to-face interviews and phone calls. Furthermore, all participants were assessed for anthropometric status, dietary intake, and physical activity. Height and weight were measured using a calibrated Seca stadiometer and scale with an accuracy of 0.5 cm and 100 g, respectively. Body Mass Index (BMI) was then calculated using the standard formula, weight in kilograms divided by the square of height in meters. Physical activity was assessed using a validated form of the International Physical Activity Questionnaire (IPAQ), and MET values were calculated accordingly as standardized metabolic‑equivalent measures. ¹⁶ To ensure that headache status was not influenced by underlying causes, data on lipid profile parameters (i.e., LDL, HDL, and total cholesterol) were obtained from the participants’ existing medical records, as recorded in their files. Also, blood pressure was measured by trained personnel after at least 5 min of seated rest using a calibrated sphygmomanometer. Two measurements were taken from the right arm and averaged. Hypertension was defined as SBP ≥140 mmHg and/or DBP ≥90 mmHg, self-reported physician-diagnosed hypertension, or current use of antihypertensive medication.

Genotyping of the FTO gene

For FTO genotyping, 5 mm of blood were collected from participants at the start of the study and were placed into EDTA tubes for subsequent DNA extraction and analysis. Genomic deoxyribonucleic acid (DNA) was extracted using the GeneAll DNA extraction kit (Incheon, South Korea) following the manufacturer’s protocol. Extracted DNA samples were amplified using PCR with Ampliqon DNA polymerase master mix (Batch No. A180301; Ampliqon, Denmark). The FTO gene polymorphism rs9939609 was evaluated using the Tetra‑primer Amplification Refractory Mutation System Polymerase Chain Reaction (Tetra‑ARMS PCR) technique on the PCR products.

Dietary intake assessment

To assess dietary iron intake, a validated Food Frequency Questionnaire (FFQ) was used. ¹⁷ Food consumption over the past year was assessed through face-to-face interviews. Intake amounts were converted to grams using the USDA food composition tables. For local Iranian foods not listed in the FCT, the Iranian food composition table was employed. The data collected from the FFQ were processed using the Nutritionist IV software, modified for Iranian foods, to calculate the nutrient intake in grams (First Data Bank, San Bruno, CA; version 3.5.2). Macronutrient and dietary iron intake were then evaluated.

Statistical analysis

Normality of continuous variables was evaluated using the Kolmogorov–Smirnov test, and all variables met the assumptions for parametric testing. Social-demographic characteristics and dietary intake between the case and control groups were compared using the chi-square test (for qualitative variables) and independent t-test (for quantitative variables).

Daily intake of macronutrients and dietary iron was compared between the case and control groups using independent t-tests. To examine the association between chronic headache and dietary iron intake among carriers of different FTO genotypes (TT vs AT/AA), binary logistic regression analysis was performed. Regression models were adjusted incrementally as follows: Model 1 was adjusted for age, sex, hypertension status, and energy intake; Model 2 was further adjusted for BMI and physical activity. Statistical analysis was conducted using SPSS version 20 (SPSS Inc., Chicago, IL, USA). A p-value of <0.05 was considered statistically significant in all analyses.

Results

Baseline comparisons between the 150 chronic daily headache cases and 150 controls showed that the groups were similar in age, while a higher proportion of females was observed among cases (79.9% vs 52.2%, p = 0.001). Cases also had lower MET levels (37.53 ± 7.20 vs 38.78 ± 7.85, p = 0.001), shorter height (159.09 ± 8.31 cm vs 162.6 ± 9.25 cm, p = 0.001), and slightly higher BMI (28.58 ± 4.46 vs 28.14 ± 4.75 kg/m², p = 0.04). Diastolic blood pressure was marginally lower in cases (70.79 ± 11.34 vs 72.11 ± 10.42 mmHg, p = 0.01). The distribution of FTO genotypes was comparable between groups, with TT vs AT/AA frequencies of 51% and 49% in controls and 57% and 43% in cases, respectively, indicating no evidence of genotype distribution bias.

Table 1 presents the general characteristics of the participants categorized by FTO genotype. Among individuals with the TT genotype, no significant differences were observed between participants with CDH and participants without CDH in any demographic, anthropometric, or clinical variables. Among individuals with the AT/AA genotypes, participants with CDH showed a significantly lower physical activity level compared to individuals without CDH (0.98 ± 1.20 vs 1.55 ± 1.57 MET-h/day, p = 0.008),While other characteristics were similar between groups.

Table 1.

General characteristics of the participants.

Variables	TT genotype			AT/AA genotypes
	Controls (n = 76)	Cases (n = 85)	p	Controls (n = 74)	Cases (n = 65)	p
Age (years)	49.53 ± 8.421	50.15 ± 8.468	0.727	49.52 ± 8.469	50.18 ± 8.678	0.349
Height (cm)	155.893 ± 5.184	156.485 ± 4.739	0.564	155.695 ± 5.710	156.580 ± 6.015	0.203
Weight (kg)	72.357 ± 12.517	70.765 ± 13.067	0.557	70.360 ± 11.887	72.634 ± 10.868	0.329
BMI (kg/m2)	29.671 ± 4.350	28.889 ± 5.150	0.449	28.976 ± 4.350	29.615 ± 3.999	0.329
Physical activity (MET-h/day)	1.742 ± 1.629	1.411 ± 1.519	0.318	1.548 ± 1.568	0.982 ± 1.198	0.008
Right DBP (mmHg)	70.29 ± 8.017	68.59 ± 7.978	0.313	70.90 ± 10.056	71.21 ± 9.676	0.842
Right SBP (mmHg)	109.37 ± 13.728	105.59 ± 11.073	0.136	109.56 ± 14.92	110.14 ± 15.145	0.809
TG (mg/dL)	117.761 ± 71.951	130.641 ± 64.832	0.363	125.200 ± 67.053	130.654 ± 56.351	0.566
Total Cholesterol (mg/dL)	199.517 ± 38.352	204.126 ± 41.153	0.586	194.482 ± 38.900	186.504 ± 36.781	0.182
HDL (mg/dL)	57.943 ± 8.784	56.706 ± 10.931	0.567	55.546 ± 12.236	52.964 ± 10.760	0.150
LDL (mg/dL)	118.022 ± 33.144	121.292 ± 32.426	0.634	113.896 ± 33.049	107.408 ± 32.349	0.212

ALP: alkaline phosphatase; BMI: body mass index; DBP: diastolic blood pressure; HDL: high-density lipoprotein; LDL: low-density lipoprotein; RBC: red blood cell count; SBP: systolic blood pressure; SGOT: serum glutamate oxaloacetate transaminase; SGPT: serum glutamate pyruvate transaminase; TG: triglyceride; WBC: white blood cell count.

Table 2 presents participants’ dietary intake. Among individuals with the TT genotype, no significant differences were observed in energy (2609.04 ± 415.75 vs 2580.12 ± 460.36 kcal/day, p = 0.749), protein (85.88 ± 22.80 vs 84.25 ± 19.63 g/day, p = 0.723), carbohydrate (369.39 ± 50.32 vs 364.93 ± 79.28 g/day, p = 0.729), total fat (95.02 ± 22.59 vs 94.75 ± 19.03 g/day, p = 0.953), or dietary iron intake (18.19 ± 3.76 vs 18.86 ± 5.12 mg/day, p = 0.451) between participants with CDH and participants without CDH. In contrast, among individuals with the AT/AA genotypes, cases had a significantly lower daily intake of dietary iron compared to controls (17.10 ± 4.02 vs 18.63 ± 4.62 mg/day, p = 0.023). No significant differences were observed between groups for energy (2534.90 ± 319.64 vs 2613.57 ± 566.99 kcal/day, p = 0.226), protein (81.24 ± 14.91 vs 84.27 ± 30.96 g/day, p = 0.364), or total fat (91.32 ± 20.89 vs 94.18 ± 25.29 g/day, p = 0.420) intake. Carbohydrate intake was slightly lower in individuals with CDH compared with controls (362.84 ± 41.11 vs 377.02 ± 70.09 g/day), but this difference did not reach statistical significance (p = 0.083).

Table 2.

Dietary intake of the participants.

Nutrients	TT genotype			AT/AA genotypes
	Controls (n = 76)	Cases (n = 85)	p	Controls (n = 74)	Cases (n = 65)	p
Energy (kcal/day)	2580.121 ± 460.363	2609.0375 ± 415.752	0.749	2613.574 ± 566.992	2534.899 ± 319.636	0.226
Protein (g/day)	84.253 ± 19.625	85.875 ± 22.797	0.723	84.267 ± 30.957	81.244 ± 14.907	0.364
Carbohydrate (g/day)	364.927 ± 79.279	369.387 ± 50.320	0.729	377.019 ± 70.089	362.839 ± 41.106	0.083
Total fat (g/day)	94.753 ± 19.028	95.021 ± 22.593	0.953	94.180 ± 25.294	91.320 ± 20.889	0.420
Dietary Iron (mg/day)	18.859 ± 5.117	18.186 ± 3.756	0.451	18.631 ± 4.616	17.098 ± 4.024	0.023

To examine the association between dietary iron intake and chronic daily headache, two regression models were applied. As shown in Table 3, among individuals with the TT genotype, dietary iron intake was not significantly associated with CDH in either Model 1 (OR = 0.933, 95% CI: 0.829–1.051, p = 0.254) or Model 2 (OR = 0.929, 95% CI: 0.822–1.051, p = 0.243). In contrast, among individuals with the AT/AA genotypes, iron intake exhibited a protective association in Model 1 (OR = 0.924, 95% CI: 0.851–1.004, p = 0.062), which became statistically significant after further adjustments in Model 2 (OR = 0.913, 95% CI: 0.836–0.997, p = 0.042).

Table 3.

Chronic daily headache and dietary intake of Fe in different FTO genotypes.

Variable	TT genotype				AT/AA genotypes
	Model 1		Model 2		Model 1		Model 2
	OR (CI 95%)	p	OR (CI 95%)	p	OR (CI 95%)	p	OR (CI 95%)	p
Dietary iron	0.933 (0.829–1.051)	0.254	0.929 (0.822–1.051)	0.243	0.924 (0.851–1.004)	0.062	0.913 (0.836–0.997)	0.042

Model 1. Adjusted for age, sex, having hypertension, and energy intake, Model 2. Further adjusted for BMI and physical activity.

Discussion

The present study adds new evidence on the interplay between dietary iron intake and susceptibility to CDH. Higher iron intake was significantly associated with lower odds of CDH among individuals carrying the FTO A allele (AT/AA genotypes), while no such association was observed in participants with the TT genotype. These results highlight the importance of gene-nutrient interactions in headache pathophysiology and suggest that genetic background may shape individual responses to dietary factors.

Previous studies investigating iron and headache have reported mixed results. Singh et al. recently demonstrated a strong association between iron deficiency anemia and CDH, suggesting that inadequate iron availability may contribute to chronic headache through impaired oxygen delivery and altered neurotransmitter synthesis. ¹¹

In contrast, Dominguez et al. reported iron accumulation in the periaqueductal gray matter of chronic migraine patients, proposing a potential neurotoxic effect of iron overload. ¹² Such discrepancies may stem from differences in the level of iron intake, participant characteristics, or methods used to assess iron status. Our study adds a new layer to this debate by showing that genetic variation–in this case the FTO rs9939609 polymorphism- may partially explain why iron intake influences headache odds in some individuals but not in others.

The biological mechanisms underlying this interaction remain speculative but plausible. Iron plays a central role in serotonin synthesis, mitochondrial function, and oxidative balance, all of which have been implicated in headache disorders. ¹⁸ Carriers of the FTO A allele may have altered energy metabolism, inflammatory responses, or nutrient utilization, potentially increasing their vulnerability to iron deficiency. ¹⁹ In this case, A allele carriers might be more sensitive to changes in dietary iron intake, in such a way that adequate iron intake contributes to stable neurotransmitter levels, stable mitochondria, and stable neurons, which may decrease the risk for headaches. FTO was reported to have a role in cellular nutrient sensing. ²⁰ Furthermore, FTO genotype may influence the association between dietary components and the expression level of several genes. ²¹ Given these functions, FTO variants may also affect iron‑related cellular processes and the synthesis of iron‑containing proteins implicated in headache pathophysiology.

The lack of considerable correlation between dietary iron intake and CDH among TT genotype carriers can be attributed to many different things. First, TT genotype people may have less variable metabolic or inflammatory profiles caused by a dietary iron influenced neurological functions, hence, the stabilizing effect of the iron on the diets. Second, it is plausible that TT genotype carriers have more efficient baseline iron utilization or storage, making dietary intake a less critical determinant of headache risk. Other gene-nutrient interaction studies have documented similar attenuation of diet-disease associations generated by a specific genotype which adds credence to such possible explanations. ²²

Clinically, these findings highlight the potential relevance of precision nutrition approaches in the prevention and management of chronic headache. For FTO A allele carriers, a low-cost, low-risk, and dietary approach to headache management may be optimizing iron intake. Nevertheless, due to iron’s intricate and dual-sided relationship with neurologic health, and the deficiency and excess of iron being harmful, individualized nutrition approach is crucial. This study has several strengths, including its focus on gene-diet interaction, the use of validated dietary assessment tools, and adjustment for important confounders such as BMI and physical activity. However, limitations must also be acknowledged. The case-control design precludes causal inference, dietary intake was self-reported and thus as prone to recall bias, and iron statues was not confirmed with biomarkers such as serum ferritin or transferrin saturation. Furthermore, the sample was restricted to middle-aged and older adults from a single clinical setting, limiting generalizability. Future research should employ longitudinal or interventional designs to validate these finding and clarify the causal role of iron in CDH.

Conclusion

In conclusion, our results suggest that dietary iron intake exerts an inverse association with chronic daily headache in carriers of the FTO A allele but not in individuals with the TT genotype. These findings highlight the potential of precision nutrition approaches in headache prevention, where tailoring dietary recommendations to genetic background may improve effectiveness and reduce disease burden. Studies integrating genetic profiling with objective measures of iron status, as well as exploring potential thresholds for deficiency or overload, would be especially valuable. Expanding the scope to include diverse populations and other candidate genes could further enhance our understanding of nutrient-gene interactions in headache disorders.