Evaluation of Vitamin D Receptor rs1544410 Gene Polymorphism in Children and Their Families Presenting With Headache and Low Serum 25-OH Vitamin D Levels to a Pediatric Neurology Outpatient Clinic

Evaluation of Vitamin D Receptor rs1544410 Gene Polymorphism in Children and Their Families Presenting With Headache and Low Serum 25-OH Vitamin D Levels to a Pediatric Neurology Outpatient Clinic | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Systematic Review Evaluation of Vitamin D Receptor rs1544410 Gene Polymorphism in Children and Their Families Presenting With Headache and Low Serum 25-OH Vitamin D Levels to a Pediatric Neurology Outpatient Clinic PINAR ÇAY, Fatma Müjgan Sönmez, Eyyüp Üçtepe, Esra Gündüz This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6641383/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background : Primary headaches are common in childhood and may be influenced by genetic and environmental factors. The vitamin D receptor (VDR) rs1544410 (BsmI) polymorphism has been implicated in neurological disorders, including headaches. Objective : To assess the association between VDR rs1544410 polymorphism and primary headaches in children and their families. Methods : A prospective case-control study was conducted at Turgut Özal University Faculty of Medicine. The study included 8 pediatric patients with primary headaches, 20 family members with vitamin D deficiency and headaches, and 36 age-matched healthy controls. Genotyping was performed by PCR-RFLP. Serum 25-OH vitamin D levels were measured. Results: Patients had significantly lower vitamin D levels compared to controls (p = 0.001). There were no significant differences in VDR rs1544410 genotype distributions between patients, family members, and controls (p > 0.05). Genotype frequencies were consistent with Hardy-Weinberg equilibrium. Conclusions : Although a potential association between VDR rs1544410 polymorphism and childhood primary headaches is suggested, no statistically significant results were observed. Further studies with larger cohorts are warranted. vitamin D receptor rs1544410 polymorphism primary headache children genetic association vitamin D deficiency Figures Figure 1 INTRODUCTION Headache is one of the most common neurological complaints in childhood, with migraine and tension-type headaches standing out as the primary headache types in this age group. These conditions can adversely affect the quality of life of children, reduce academic performance, and disrupt psychosocial development. The etiology of primary headaches is considered multifactorial, shaped by the interaction of genetic and environmental factors (1). In recent years, associations between vitamin D deficiency and various neurological disorders have been identified, with particular attention drawn to studies indicating lower vitamin D levels in patients with migraine-type headaches. Vitamin D plays a role not only in bone health but also in neuroprotective effects, regulation of inflammation, neurotransmitter synthesis, and calcium homeostasis. These effects are largely mediated by the intracellular Vitamin D receptor (VDR) located in the cell nucleus (2,3). The VDR gene is located at the q13.11 region of chromosome 12 and belongs to the nuclear hormone receptor family. It exerts its genomic effects by forming a complex with the active form of vitamin D (1,25(OH)₂D₃), heterodimerizing with the retinoid X receptor (RXR), and binding to vitamin D response elements (VDRE) on target genes, thereby regulating the transcription of genes involved in various cellular processes. Genes regulated by VDR include those related to calcium transport, immune regulation, and neurotransmitter metabolism (4). Many polymorphisms of the VDR gene have been identified; among them, the rs1544410 (BsmI) polymorphism, located in an intronic region near the 3' UTR, is one of the most extensively studied. This polymorphism is believed to affect VDR mRNA stability, translation efficiency, and receptor levels. Therefore, genetic variations such as rs1544410 may lead to interindividual differences in vitamin D signaling and potentially contribute to susceptibility to neurological disorders (5). The rs1544410 (BsmI) polymorphism of the VDR gene has been associated with genetic predisposition in various diseases. It is hypothesized that this polymorphism may influence VDR gene expression and intracellular signal transduction mediated by vitamin D. Accordingly, VDR rs1544410 variants may also play a role in the development of primary headaches in children (2,3). In this study, we aimed to investigate the relationship between headaches and VDR rs1544410 polymorphism in children using both a case-control design and a family-based genetic approach. Evaluating the genotypes of family members was important for revealing possible hereditary characteristics of headache and examining genetic susceptibility from a broader perspective. MATERIALS AND METHODS This prospective study was conducted at Turgut Özal University Faculty of Medicine with the approval of the Ethics Committee, between November 2013 and October 2014. The study included 8 pediatric patients diagnosed with primary headache (migraine or tension-type headache), 20 family members with identified vitamin D deficiency, and 36 healthy control individuals of similar age. Individuals with chronic diseases (such as diabetes mellitus, chronic renal failure, metabolic disorders), genetic syndromes, malabsorption, or immobilization were excluded from the study. Informed consent forms were obtained from all participants. Peripheral venous blood samples were collected in tubes containing ethylenediaminetetraacetic acid (EDTA), and DNA was isolated using standard protocols. The VDR rs1544410 (BsmI) polymorphism was analyzed using the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method. The amplified PCR products were digested with the BsmI enzyme and separated by agarose gel electrophoresis. For the VDR R = G/A (C/T) (rs1544410) polymorphism, the HpyCH4V enzyme (recognition site: TG’CA) was used to cut a 300 bp genomic fragment. Genotyping was determined as follows: GG : 189 bp + 111 bp = homozygous wild-type AA : 150 bp + 111 bp + 39 bp = homozygous polymorphic AG : 189 bp + 150 bp + 111 bp + 39 bp = heterozygous polymorphic (Fig. 1 ) Statistical Analysis Data were analyzed using SPSS Statistics 20.0 software (IBM Corp., Armonk, NY, USA). Genotype and allele frequencies were calculated as counts and percentages, and differences between groups were evaluated using the Pearson chi-square test. For cells with expected counts less than 5, Fisher’s exact test was applied. Allele frequencies were also analyzed for pairwise group comparisons. Hardy-Weinberg equilibrium (HWE) was tested separately for each group to assess population equilibrium. The relationship between vitamin D levels and genotypes was analyzed categorically, and a p-value of < 0.05 was considered statistically significant. Additionally, a secondary analysis was performed to evaluate the distribution of genotypes according to the presence or absence of headache history in family members. The chi-square test and odds ratio comparisons were also used for this analysis. RESULTS Between November 2013 and October 2014, a total of 64 participants were included in the study: 8 pediatric patients who presented with headaches to the Pediatric Neurology Department of Turgut Özal University Faculty of Medicine, 20 family members diagnosed with vitamin D deficiency, and 36 healthy children (control group) who attended routine visits to the general pediatrics outpatient clinics and were found to have vitamin D deficiency. The mean age of the patient group was 12.75 ± 3.49 years; the mean age of the control group was 8.14 ± 3.44 years; and the mean age of the family members was 32.30 ± 15.4 years. A statistically significant difference was observed between the patient and control groups in terms of age (p = 0.002, < 0.05). In the patient group, 3 (37.5%) were male and 5 (62.5%) were female, whereas the control group included 20 (55.6%) males and 16 (44.4%) females. There was no statistically significant difference in sex distribution between the patient and control groups (chi-square = 0.855, p = 0.448 > 0.05). Comparison of biochemical data between the patient group and family members showed higher levels of calcium, ALP, and phosphorus in the patients; however, only ALP levels were statistically significantly elevated (p < 0.05). There was no statistically significant difference in PTH levels between the two groups. The mean serum 25-OH vitamin D levels were 11.46 ± 6.91 ng/mL in family members, 9.90 ± 3.58 ng/mL in patients, and 18.98 ± 7.76 ng/mL in the control group. Patients had lower vitamin D levels compared to their family members, but this difference was not statistically significant. However, the control group had significantly higher vitamin D levels compared to the patient group (p = 0.001 < 0.05). Vitamin D levels were classified as: insufficiency (20–30 ng/mL), deficiency (10–19 ng/mL), and severe deficiency (0–9 ng/mL). No patients had vitamin D insufficiency, while 3 (37.5%) had deficiency and 5 (62.5%) had severe deficiency. In the control group, 17 (47.2%) had insufficiency, 14 (38.9%) had deficiency, and 5 (13.9%) had severe deficiency. Insufficiency was significantly more common in the control group, whereas severe deficiency was significantly more common in the patient group (chi-square = 9.833, p = 0.003 < 0.05). Among family members, 3 (15%) had insufficiency, 8 (40%) had deficiency, and 9 (45%) had severe deficiency. There were no statistically significant differences between the patient and control groups for VDR rs1544410 genotypes (GG wild-type homozygous, AA homozygous polymorphic, AG heterozygous polymorphic) (chi-square = 2.073, p = 0.453 > 0.05). Similarly, when the polymorphic genotypes (AG + AA) were grouped together, there was no significant difference (chi-square = 0.786, p = 0.434 > 0.05). When comparing the patient group and their family members, there was no statistically significant difference in VDR rs1544410 genotypes (chi-square = 0.952, p = 0.768 > 0.05). Combining the polymorphic genotypes (AG + AA) also showed no difference (chi-square = 0.233, p = 0.472 > 0.05). There was no statistically significant association between vitamin D groups and VDR rs1544410 genotypes, either individually or combined (GG vs. AG + AA) (chi-square = 1.808, p = 0.736 > 0.05 and chi-square = 0.034, p = 0.993 > 0.05). The Hardy-Weinberg equilibrium test indicated that the distribution of alleles in the study population was consistent with equilibrium. No statistically significant differences were found between the patient and control groups, or between the patient and family groups, in terms of allele frequencies (p = 0.453 and p = 0.768, respectively). DISCUSSION The most common primary headache syndromes in children are migraine, tension-type headache, and cluster headache. An accurate diagnosis of primary headaches is essential once secondary causes have been excluded. Since migraine and tension-type headaches can present with overlapping features, distinguishing between them can be challenging, which may impact treatment. According to the literature, the prevalence of headaches in children is approximately 50%, with genetic predisposition recognized as a major risk factor. Studies investigating the hereditary nature of migraine have shown that individuals with first-degree relatives suffering from migraine have a significantly increased risk of developing the condition themselves. Genome-wide association studies (GWAS) investigating the genetic basis of migraine have identified associations between various genomic regions and migraine. The vitamin D receptor (VDR) gene has emerged as one of the candidate genes, as vitamin D is thought to contribute to headache pathophysiology through its neuroprotective and anti-inflammatory properties. Several studies have also demonstrated associations between VDR polymorphisms and various neurological disorders, including headache, migraine, depression, and epilepsy [ 2 – 4 ]. Recent studies have focused on the relationship between the vitamin D receptor (VDR) BsmI polymorphism (rs1544410) and headache disorders, particularly migraine and cluster headaches, in adults. A case-control study conducted in Southeastern Europe investigated the association between three common VDR gene variants (FokI, TaqI, and BsmI) and migraine susceptibility. The study reported that individuals with the heterozygous GA genotype of the BsmI polymorphism had a higher risk of developing migraine, particularly migraine without aura, suggesting a possible role of this variant in migraine susceptibility in this population [ 6 ]. Another study examining VDR gene polymorphisms and cluster headache (CH) in a Southeastern European Caucasian population found no significant difference in genotype frequencies between CH patients and controls. However, haplotype analysis suggested that the TAC haplotype, which includes the BsmI polymorphism, may be associated with reduced susceptibility to CH. Additionally, the GG genotype of BsmI was associated with more frequent cluster headache attacks, indicating a potential role in disease severity [ 7 ]. In a study including 105 fibromyalgia patients and 105 controls, no significant associations were found between the VDR BsmI polymorphism and either genotype or allele frequencies, nor with the clinical symptoms of fibromyalgia (p > 0.05). The authors concluded that larger sample sizes are needed to explore this relationship further [ 8 ]. Schürks et al. (2009) investigated 77 polymorphisms in 52 candidate genes in 25,713 women over the age of 45 with and without migraine. Although some polymorphisms (TNF rs673, TGFB1 rs1800469, CCR2 rs1799864) were found to be associated with migraine, the VDR rs1544410 polymorphism showed no statistically significant association [ 9 ]. CONCLUSION These findings suggest a possible association between the VDR BsmI polymorphism and headache disorders in adults. However, further research is needed to fully understand the underlying mechanisms and clinical relevance of these associations. A strength of our study is the inclusion of family members, which allowed us to evaluate hereditary tendencies. Nevertheless, the limited sample size may have prevented the results from reaching statistical significance. Additionally, examining only one polymorphism may have excluded the potential effects of other variants within the VDR gene. In conclusion, our study suggests a potential relationship between primary headaches in childhood and the VDR rs1544410 polymorphism. However, further large-scale, multicenter studies examining multiple genetic polymorphisms are needed to confirm this association. Declarations Author Contribution Pınar Çay Writing – review and editingFatma Müjgan Sönmez - SupervisionEyyüp Üçtepe- MethodologyEsra Gündüz - Methodology References Alehan F (2003) Prospective evaluation of childhood headaches. J Pediatr Health Dis 46:38–42 Champe PC, Harvey RA, Ferrier DR (2007) Biochemistry. 3rd ed. Ulukaya E, editor. Lippincott's Illustrated Reviews. Istanbul: Nobel Medical Publishing Bringhurst FR, Demay MB, Krane SM, Kronenberg HM (2005) Bone and mineral metabolism in health and disease. In: Kasper DL, Braunwald E, Fauci AS, Hauser SL, Longo DL, Jameson JL (eds) Harrison’s Principles of Internal Medicine, 16th edn. McGraw-Hill Companies, New York, pp 2238–2286 Labuda M, Fujiwara TM, Ross MV et al (1992) Two hereditary defects related to vitamin D metabolism map to the same region of human chromosome 12q13-14. J Bone Min Res 7:1447–1453 Çakır Ö, Tunalı N (2010) The role of VDR polymorphisms in calcium oxalate kidney stone formation. J Cell Mol Biol 8(2):1–12 Papasavva M, Vikelis M, Siokas V, Katsarou MS, Dermitzakis EV, Raptis A et al (2023) Genetic variability in vitamin D receptor and migraine susceptibility: a Southeastern European case-control study. Neurol Int 15(3):1117–1128. 10.3390/neurolint15030069 Papasavva M, Vikelis M, Siokas V, Katsarou MS, Dermitzakis E, Raptis A et al (2022) VDR gene polymorphisms and cluster headache susceptibility: case-control study in a Southeastern European Caucasian population. J Mol Neurosci 72(2):382–392. 10.1007/s12031-021-01892-w Parvez S, Fatima G, Mehdi F, Hadi NR, Fedacko J (2022) Relationship between vitamin D receptor gene BsmI polymorphism and fibromyalgia syndrome. Cureus 14(7):e27113. 10.7759/cureus.27113 Schürks M, Kurth T, Buring JE, Zee RYL (2009) A candidate gene association study of 77 polymorphisms in migraine. J Pain 10(7):759–766 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6641383","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Systematic Review","associatedPublications":[],"authors":[{"id":455761977,"identity":"687c9e75-6895-4f05-9e01-7c36d2a8dd71","order_by":0,"name":"PINAR ÇAY","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYDCCA2AEAszHwBQbO/Fa2NIYGBKAFDMRWqCAxwyshYGQFr7bZw8eLqi5J88/I+fbg48/tsnzMTMwfviYg1uL5Lm8hMMzjhUbzriRu91wRsJtwzZmBmbJmdtwazE4w2NwmIctIYHhRu42aZ6E24xALWzMvAS1/EtIkL+R8wykxZ44LbxtCQkGN3LYQFoSCWqRPMOXcJi3L8Fw45lnZpIz0m4ntzEzNuP1C98Z3sOfeb4lyMsdT34m8cHmtu389uaDHz7i0QKMDigtkAATYWzApx5JC/8BAgpHwSgYBaNgxAIAgtZQ+4QdLeQAAAAASUVORK5CYII=","orcid":"","institution":"Muğla University","correspondingAuthor":true,"prefix":"","firstName":"PINAR","middleName":"","lastName":"ÇAY","suffix":""},{"id":455761978,"identity":"4d5a595a-61f8-4081-b6f4-7ccffada7438","order_by":1,"name":"Fatma Müjgan Sönmez","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Fatma","middleName":"Müjgan","lastName":"Sönmez","suffix":""},{"id":455761979,"identity":"0430fbdd-8dc8-4d30-b47c-26caa826562f","order_by":2,"name":"Eyyüp Üçtepe","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Eyyüp","middleName":"","lastName":"Üçtepe","suffix":""},{"id":455761980,"identity":"93f3c7a5-674c-459c-ae4d-35e29897a2f1","order_by":3,"name":"Esra Gündüz","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Esra","middleName":"","lastName":"Gündüz","suffix":""}],"badges":[],"createdAt":"2025-05-11 20:08:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6641383/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6641383/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82702524,"identity":"2f237f6d-4f63-47dc-8f0d-ff8175e49877","added_by":"auto","created_at":"2025-05-14 09:47:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":126929,"visible":true,"origin":"","legend":"\u003cp\u003eGenotyping pattern; lanes 1a and 3a = AA (homozygous polymorphic), lane 2a = GG (homozygous wild-type), lane 4a = AG (heterozygous polymorphic).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6641383/v1/4f637865cb105b840ecb1388.png"},{"id":82702525,"identity":"57e3f56e-5a1c-4a1c-a580-d058f3717eed","added_by":"auto","created_at":"2025-05-14 09:48:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":460857,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6641383/v1/b9f3580c-c667-431b-b027-5806c2b0324b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eEvaluation of Vitamin D Receptor rs1544410 Gene Polymorphism in Children and Their Families Presenting With Headache and Low Serum 25-OH Vitamin D Levels to a Pediatric Neurology Outpatient Clinic\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eHeadache is one of the most common neurological complaints in childhood, with migraine and tension-type headaches standing out as the primary headache types in this age group. These conditions can adversely affect the quality of life of children, reduce academic performance, and disrupt psychosocial development. The etiology of primary headaches is considered multifactorial, shaped by the interaction of genetic and environmental factors (1).\u003c/p\u003e \u003cp\u003eIn recent years, associations between vitamin D deficiency and various neurological disorders have been identified, with particular attention drawn to studies indicating lower vitamin D levels in patients with migraine-type headaches. Vitamin D plays a role not only in bone health but also in neuroprotective effects, regulation of inflammation, neurotransmitter synthesis, and calcium homeostasis. These effects are largely mediated by the intracellular Vitamin D receptor (VDR) located in the cell nucleus (2,3).\u003c/p\u003e \u003cp\u003eThe VDR gene is located at the q13.11 region of chromosome 12 and belongs to the nuclear hormone receptor family. It exerts its genomic effects by forming a complex with the active form of vitamin D (1,25(OH)₂D₃), heterodimerizing with the retinoid X receptor (RXR), and binding to vitamin D response elements (VDRE) on target genes, thereby regulating the transcription of genes involved in various cellular processes. Genes regulated by VDR include those related to calcium transport, immune regulation, and neurotransmitter metabolism (4).\u003c/p\u003e \u003cp\u003eMany polymorphisms of the VDR gene have been identified; among them, the rs1544410 (BsmI) polymorphism, located in an intronic region near the 3' UTR, is one of the most extensively studied. This polymorphism is believed to affect VDR mRNA stability, translation efficiency, and receptor levels. Therefore, genetic variations such as rs1544410 may lead to interindividual differences in vitamin D signaling and potentially contribute to susceptibility to neurological disorders (5).\u003c/p\u003e \u003cp\u003eThe rs1544410 (BsmI) polymorphism of the VDR gene has been associated with genetic predisposition in various diseases. It is hypothesized that this polymorphism may influence VDR gene expression and intracellular signal transduction mediated by vitamin D. Accordingly, VDR rs1544410 variants may also play a role in the development of primary headaches in children (2,3).\u003c/p\u003e \u003cp\u003eIn this study, we aimed to investigate the relationship between headaches and VDR rs1544410 polymorphism in children using both a case-control design and a family-based genetic approach. Evaluating the genotypes of family members was important for revealing possible hereditary characteristics of headache and examining genetic susceptibility from a broader perspective.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e This prospective study was conducted at Turgut \u0026Ouml;zal University Faculty of Medicine with the approval of the Ethics Committee, between November 2013 and October 2014. The study included 8 pediatric patients diagnosed with primary headache (migraine or tension-type headache), 20 family members with identified vitamin D deficiency, and 36 healthy control individuals of similar age.\u003c/p\u003e \u003cp\u003eIndividuals with chronic diseases (such as diabetes mellitus, chronic renal failure, metabolic disorders), genetic syndromes, malabsorption, or immobilization were excluded from the study.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eInformed consent\u003c/strong\u003e \u003cp\u003eforms were obtained from all participants. Peripheral venous blood samples were collected in tubes containing ethylenediaminetetraacetic acid (EDTA), and DNA was isolated using standard protocols.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eThe VDR rs1544410 (BsmI) polymorphism was analyzed using the polymerase chain reaction\u0026ndash;restriction fragment length polymorphism (PCR-RFLP) method. The amplified PCR products were digested with the BsmI enzyme and separated by agarose gel electrophoresis. For the VDR R\u0026thinsp;=\u0026thinsp;G/A (C/T) (rs1544410) polymorphism, the HpyCH4V enzyme (recognition site: TG\u0026rsquo;CA) was used to cut a 300 bp genomic fragment. Genotyping was determined as follows:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eGG\u003c/b\u003e: 189 bp\u0026thinsp;+\u0026thinsp;111 bp\u0026thinsp;=\u0026thinsp;homozygous wild-type\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eAA\u003c/b\u003e: 150 bp\u0026thinsp;+\u0026thinsp;111 bp\u0026thinsp;+\u0026thinsp;39 bp\u0026thinsp;=\u0026thinsp;homozygous polymorphic\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eAG\u003c/b\u003e: 189 bp\u0026thinsp;+\u0026thinsp;150 bp\u0026thinsp;+\u0026thinsp;111 bp\u0026thinsp;+\u0026thinsp;39 bp\u0026thinsp;=\u0026thinsp;heterozygous polymorphic\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using SPSS Statistics 20.0 software (IBM Corp., Armonk, NY, USA). Genotype and allele frequencies were calculated as counts and percentages, and differences between groups were evaluated using the Pearson chi-square test. For cells with expected counts less than 5, Fisher\u0026rsquo;s exact test was applied. Allele frequencies were also analyzed for pairwise group comparisons. Hardy-Weinberg equilibrium (HWE) was tested separately for each group to assess population equilibrium.\u003c/p\u003e \u003cp\u003eThe relationship between vitamin D levels and genotypes was analyzed categorically, and a p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003cp\u003eAdditionally, a secondary analysis was performed to evaluate the distribution of genotypes according to the presence or absence of headache history in family members. The chi-square test and odds ratio comparisons were also used for this analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eBetween November 2013 and October 2014, a total of 64 participants were included in the study: 8 pediatric patients who presented with headaches to the Pediatric Neurology Department of Turgut \u0026Ouml;zal University Faculty of Medicine, 20 family members diagnosed with vitamin D deficiency, and 36 healthy children (control group) who attended routine visits to the general pediatrics outpatient clinics and were found to have vitamin D deficiency.\u003c/p\u003e \u003cp\u003eThe mean age of the patient group was 12.75\u0026thinsp;\u0026plusmn;\u0026thinsp;3.49 years; the mean age of the control group was 8.14\u0026thinsp;\u0026plusmn;\u0026thinsp;3.44 years; and the mean age of the family members was 32.30\u0026thinsp;\u0026plusmn;\u0026thinsp;15.4 years. A statistically significant difference was observed between the patient and control groups in terms of age (p\u0026thinsp;=\u0026thinsp;0.002, \u0026lt; 0.05).\u003c/p\u003e \u003cp\u003eIn the patient group, 3 (37.5%) were male and 5 (62.5%) were female, whereas the control group included 20 (55.6%) males and 16 (44.4%) females. There was no statistically significant difference in sex distribution between the patient and control groups (chi-square\u0026thinsp;=\u0026thinsp;0.855, p\u0026thinsp;=\u0026thinsp;0.448\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eComparison of biochemical data between the patient group and family members showed higher levels of calcium, ALP, and phosphorus in the patients; however, only ALP levels were statistically significantly elevated (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was no statistically significant difference in PTH levels between the two groups.\u003c/p\u003e \u003cp\u003eThe mean serum 25-OH vitamin D levels were 11.46\u0026thinsp;\u0026plusmn;\u0026thinsp;6.91 ng/mL in family members, 9.90\u0026thinsp;\u0026plusmn;\u0026thinsp;3.58 ng/mL in patients, and 18.98\u0026thinsp;\u0026plusmn;\u0026thinsp;7.76 ng/mL in the control group. Patients had lower vitamin D levels compared to their family members, but this difference was not statistically significant. However, the control group had significantly higher vitamin D levels compared to the patient group (p\u0026thinsp;=\u0026thinsp;0.001\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eVitamin D levels were classified as: insufficiency (20\u0026ndash;30 ng/mL), deficiency (10\u0026ndash;19 ng/mL), and severe deficiency (0\u0026ndash;9 ng/mL). No patients had vitamin D insufficiency, while 3 (37.5%) had deficiency and 5 (62.5%) had severe deficiency. In the control group, 17 (47.2%) had insufficiency, 14 (38.9%) had deficiency, and 5 (13.9%) had severe deficiency. Insufficiency was significantly more common in the control group, whereas severe deficiency was significantly more common in the patient group (chi-square\u0026thinsp;=\u0026thinsp;9.833, p\u0026thinsp;=\u0026thinsp;0.003\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eAmong family members, 3 (15%) had insufficiency, 8 (40%) had deficiency, and 9 (45%) had severe deficiency.\u003c/p\u003e \u003cp\u003eThere were no statistically significant differences between the patient and control groups for VDR rs1544410 genotypes (GG wild-type homozygous, AA homozygous polymorphic, AG heterozygous polymorphic) (chi-square\u0026thinsp;=\u0026thinsp;2.073, p\u0026thinsp;=\u0026thinsp;0.453\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Similarly, when the polymorphic genotypes (AG\u0026thinsp;+\u0026thinsp;AA) were grouped together, there was no significant difference (chi-square\u0026thinsp;=\u0026thinsp;0.786, p\u0026thinsp;=\u0026thinsp;0.434\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eWhen comparing the patient group and their family members, there was no statistically significant difference in VDR rs1544410 genotypes (chi-square\u0026thinsp;=\u0026thinsp;0.952, p\u0026thinsp;=\u0026thinsp;0.768\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Combining the polymorphic genotypes (AG\u0026thinsp;+\u0026thinsp;AA) also showed no difference (chi-square\u0026thinsp;=\u0026thinsp;0.233, p\u0026thinsp;=\u0026thinsp;0.472\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThere was no statistically significant association between vitamin D groups and VDR rs1544410 genotypes, either individually or combined (GG vs. AG\u0026thinsp;+\u0026thinsp;AA) (chi-square\u0026thinsp;=\u0026thinsp;1.808, p\u0026thinsp;=\u0026thinsp;0.736\u0026thinsp;\u0026gt;\u0026thinsp;0.05 and chi-square\u0026thinsp;=\u0026thinsp;0.034, p\u0026thinsp;=\u0026thinsp;0.993\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe Hardy-Weinberg equilibrium test indicated that the distribution of alleles in the study population was consistent with equilibrium.\u003c/p\u003e \u003cp\u003eNo statistically significant differences were found between the patient and control groups, or between the patient and family groups, in terms of allele frequencies (p\u0026thinsp;=\u0026thinsp;0.453 and p\u0026thinsp;=\u0026thinsp;0.768, respectively).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe most common primary headache syndromes in children are migraine, tension-type headache, and cluster headache. An accurate diagnosis of primary headaches is essential once secondary causes have been excluded. Since migraine and tension-type headaches can present with overlapping features, distinguishing between them can be challenging, which may impact treatment. According to the literature, the prevalence of headaches in children is approximately 50%, with genetic predisposition recognized as a major risk factor. Studies investigating the hereditary nature of migraine have shown that individuals with first-degree relatives suffering from migraine have a significantly increased risk of developing the condition themselves.\u003c/p\u003e \u003cp\u003eGenome-wide association studies (GWAS) investigating the genetic basis of migraine have identified associations between various genomic regions and migraine. The vitamin D receptor (VDR) gene has emerged as one of the candidate genes, as vitamin D is thought to contribute to headache pathophysiology through its neuroprotective and anti-inflammatory properties.\u003c/p\u003e \u003cp\u003eSeveral studies have also demonstrated associations between VDR polymorphisms and various neurological disorders, including headache, migraine, depression, and epilepsy [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent studies have focused on the relationship between the vitamin D receptor (VDR) BsmI polymorphism (rs1544410) and headache disorders, particularly migraine and cluster headaches, in adults.\u003c/p\u003e \u003cp\u003eA case-control study conducted in Southeastern Europe investigated the association between three common VDR gene variants (FokI, TaqI, and BsmI) and migraine susceptibility. The study reported that individuals with the heterozygous GA genotype of the BsmI polymorphism had a higher risk of developing migraine, particularly migraine without aura, suggesting a possible role of this variant in migraine susceptibility in this population [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother study examining VDR gene polymorphisms and cluster headache (CH) in a Southeastern European Caucasian population found no significant difference in genotype frequencies between CH patients and controls. However, haplotype analysis suggested that the TAC haplotype, which includes the BsmI polymorphism, may be associated with reduced susceptibility to CH. Additionally, the GG genotype of BsmI was associated with more frequent cluster headache attacks, indicating a potential role in disease severity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn a study including 105 fibromyalgia patients and 105 controls, no significant associations were found between the VDR BsmI polymorphism and either genotype or allele frequencies, nor with the clinical symptoms of fibromyalgia (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The authors concluded that larger sample sizes are needed to explore this relationship further [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSch\u0026uuml;rks et al. (2009) investigated 77 polymorphisms in 52 candidate genes in 25,713 women over the age of 45 with and without migraine. Although some polymorphisms (TNF rs673, TGFB1 rs1800469, CCR2 rs1799864) were found to be associated with migraine, the VDR rs1544410 polymorphism showed no statistically significant association [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThese findings suggest a possible association between the VDR BsmI polymorphism and headache disorders in adults. However, further research is needed to fully understand the underlying mechanisms and clinical relevance of these associations. A strength of our study is the inclusion of family members, which allowed us to evaluate hereditary tendencies. Nevertheless, the limited sample size may have prevented the results from reaching statistical significance. Additionally, examining only one polymorphism may have excluded the potential effects of other variants within the VDR gene.\u003c/p\u003e \u003cp\u003eIn conclusion, our study suggests a potential relationship between primary headaches in childhood and the VDR rs1544410 polymorphism. However, further large-scale, multicenter studies examining multiple genetic polymorphisms are needed to confirm this association.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003ePınar \u0026Ccedil;ay Writing \u0026ndash; review and editingFatma M\u0026uuml;jgan S\u0026ouml;nmez - SupervisionEyy\u0026uuml;p \u0026Uuml;\u0026ccedil;tepe- MethodologyEsra G\u0026uuml;nd\u0026uuml;z - Methodology\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlehan F (2003) Prospective evaluation of childhood headaches. J Pediatr Health Dis 46:38\u0026ndash;42\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChampe PC, Harvey RA, Ferrier DR (2007) Biochemistry. 3rd ed. Ulukaya E, editor. Lippincott's Illustrated Reviews. Istanbul: Nobel Medical Publishing\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBringhurst FR, Demay MB, Krane SM, Kronenberg HM (2005) Bone and mineral metabolism in health and disease. In: Kasper DL, Braunwald E, Fauci AS, Hauser SL, Longo DL, Jameson JL (eds) Harrison\u0026rsquo;s Principles of Internal Medicine, 16th edn. McGraw-Hill Companies, New York, pp 2238\u0026ndash;2286\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLabuda M, Fujiwara TM, Ross MV et al (1992) Two hereditary defects related to vitamin D metabolism map to the same region of human chromosome 12q13-14. J Bone Min Res 7:1447\u0026ndash;1453\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u0026Ccedil;akır \u0026Ouml;, Tunalı N (2010) The role of VDR polymorphisms in calcium oxalate kidney stone formation. J Cell Mol Biol 8(2):1\u0026ndash;12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePapasavva M, Vikelis M, Siokas V, Katsarou MS, Dermitzakis EV, Raptis A et al (2023) Genetic variability in vitamin D receptor and migraine susceptibility: a Southeastern European case-control study. Neurol Int 15(3):1117\u0026ndash;1128. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/neurolint15030069\u003c/span\u003e\u003cspan address=\"10.3390/neurolint15030069\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePapasavva M, Vikelis M, Siokas V, Katsarou MS, Dermitzakis E, Raptis A et al (2022) VDR gene polymorphisms and cluster headache susceptibility: case-control study in a Southeastern European Caucasian population. J Mol Neurosci 72(2):382\u0026ndash;392. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12031-021-01892-w\u003c/span\u003e\u003cspan address=\"10.1007/s12031-021-01892-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParvez S, Fatima G, Mehdi F, Hadi NR, Fedacko J (2022) Relationship between vitamin D receptor gene BsmI polymorphism and fibromyalgia syndrome. Cureus 14(7):e27113. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7759/cureus.27113\u003c/span\u003e\u003cspan address=\"10.7759/cureus.27113\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSch\u0026uuml;rks M, Kurth T, Buring JE, Zee RYL (2009) A candidate gene association study of 77 polymorphisms in migraine. J Pain 10(7):759\u0026ndash;766\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"vitamin D receptor, rs1544410 polymorphism, primary headache, children, genetic association, vitamin D deficiency","lastPublishedDoi":"10.21203/rs.3.rs-6641383/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6641383/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Primary headaches are common in childhood and may be influenced by genetic and environmental factors. The vitamin D receptor (VDR) rs1544410 (BsmI) polymorphism has been implicated in neurological disorders, including headaches.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e: To assess the association between VDR rs1544410 polymorphism and primary headaches in children and their families.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: A prospective case-control study was conducted at Turgut Özal University Faculty of Medicine. The study included 8 pediatric patients with primary headaches, 20 family members with vitamin D deficiency and headaches, and 36 age-matched healthy controls. Genotyping was performed by PCR-RFLP. Serum 25-OH vitamin D levels were measured. Results: Patients had significantly lower vitamin D levels compared to controls (p = 0.001). There were no significant differences in VDR rs1544410 genotype distributions between patients, family members, and controls (p \u0026gt; 0.05). Genotype frequencies were consistent with Hardy-Weinberg equilibrium.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Although a potential association between VDR rs1544410 polymorphism and childhood primary headaches is suggested, no statistically significant results were observed. Further studies with larger cohorts are warranted.\u003c/p\u003e","manuscriptTitle":"Evaluation of Vitamin D Receptor rs1544410 Gene Polymorphism in Children and Their Families Presenting With Headache and Low Serum 25-OH Vitamin D Levels to a Pediatric Neurology Outpatient Clinic","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-14 09:47:53","doi":"10.21203/rs.3.rs-6641383/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"de156756-6c29-462e-ac3f-95ddde41a002","owner":[],"postedDate":"May 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-14T09:47:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-14 09:47:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6641383","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6641383","identity":"rs-6641383","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}