Medicine® OBSERVATIONAL STUDY Association Between Vitamin D Receptor rs731236 (Taq1) Polymorphism and Risk for Restless Legs Syndrome in the Spanish Caucasian Population Fe´lix Javier Jime´nez-Jime´nez, MD, PhD, Elena Garcı´a-Martı´n, MD, PhD, Hortensia Alonso-Navarro, MD, PhD, Carmen Martı´nez, MD, PhD, Martı´n Zurdo, MD, Laura Turpı´n-Fenoll, MD, PhD, Jorge Milla´n-Pascual, MD, Teresa Adeva-Bartolome´, MD, PhD, Esther Cubo, MD, PhD, Francisco Navacerrada, MD, Ana Rojo-Sebastia´n, MD, Lluisa Rubio, MD, Sara Ortega-Cubero, MD, Pau Pastor, MD, PhD, Marisol Calleja, CN, Jose´ Francisco Plaza-Nieto, Bele´n Pilo-De-La-Fuente, MD, PhD, Margarita Arroyo-Solera, MD, Esteban Garcı´a-Albea, MD, PhD, and Jose´ A.G. Agu´ndez, MD, PhD Abstract: Several recent works suggest a possible role of vitamin D carrying the allelic variant rs731236G had an earlier age at onset, and deficiency in the etiology or restless legs syndrome (RLS). We analyzed those carrying the rs731236GG genotype had higher severity of RLS, the possible relationship of 2 common single nucleotide polymorphisms although these data disappeared after multivariate analyses. None of (SNPs) in the vitamin D3 receptor (VDR) gene with the risk for RLS. the SNPs studied was related with the positivity of family history of We studied the genotype and allelic variant frequencies of VDR RLS. rs2228570 and VDR rs731236 SNPs in 205 RLS patients and 445 These results suggest a modest, but significant association between healthy controls using a TaqMan essay. VDR rs731236 SNP and the risk for RLS. The frequencies of the rs731236AA genotype and the allelic variant (Medicine 94(47):e2125) rs731236A were significantly lower in RLS patients than in controls (P < 0.005 and < 0.01, respectively). Restless legs syndrome patients Abbreviations: CSF = cerebrospinal fluid, DNA = deoxyribonucleic acid, DOPAC = dihydroxyphenylacetic acid, ET Editor: Maohua Xie. = essential tremor, GABA = gamma-hydroxybutyric acid, iRLS = Received: September 16, 2015; revised: October 20, 2015; accepted: idiopathic restless legs syndrome, IRLSSGRS = International October 28, 2015. Restless Legs Syndrome Study Group Rating Scale, MAO-A = From the Section of Neurology, Hospital Universitario del Sureste, Arganda del Rey (FJJ-J, HA-N, FN, MC, JFP-N, BP-D-LF, MA-S); monoamine oxidase A, PD = Parkinson’s disease, RLS = restless Department of Pharmacology, University of Extremadura, Ca´ceres; legs syndrome, RR = relative risk, SNP = single nucleotide Department of Medicine-Neurology, Hospital ‘‘Prı´ncipe de Asturias’’, polymorphism, VDR = vitamin D3 receptor, WED = Willis-Ekbom Universidad de Alcala´, Alcala´ de Henares, Madrid (AR-S, LR, EG-A, FJJ- Disease. J, HA-N); Department of Pharmacology, Universidad de Extremadura, Ca´ceres (EG-M, JAGA); Department of Pharmacology, University of Extremadura, Badajoz (CM); Section of Neurology, Hospital Virgen del Puerto, Plasencia, Ca´ceres (MZ); Section of Neurology, Hospital La INTRODUCTION Mancha-Centro, Alca´zar de San Juan, Ciudad Real (LT-F, JM-P); Unit of enetic factors are likely to be very important in the etiology Neurology, Clı´nica Recoletas, Zamora (TA-B); Section of Neurology, Hospital Universitario de Burgos, Burgos (EC); CIBERNED,Centro de G of restless legs syndrome (RLS or Willis-Ekbom disease— Investigacio´n Biome´dica en Red de Enfermedades Neurodegenerativas, WED), but all the responsible gene(s) remain(s) to be identified Instituto de Salud Carlos III (SO-C, PP); Neurogenetics Laboratory, (revised in reference).1 Genome Wide Association Studies Division of Neurosciences, Center for Applied Medical Research, (showed association between the risk for RLS and variants Universidad de Navarra, Pamplona (SO-C, PP); Department of Neurology, Clı´nica Universidad de Navarra, University of Navarra School of Medicine, of several genes, including Protein Tyrosine Phosphatase Pamplona (SO-C, PP); and Department of Neurology, Hospital Universitari Receptor Type Delta (PTPRD, chromosome 9p24.1–p23) Mutua de Terrassa, Terrassa, Barcelona, Spain (PP). BTB/POZ Domain Containing Protein 9 (BTBD9, chromosome Correspondence: Fe´lix Javier Jime´nez-Jime´nez, Hospital Universitario del 6p21), MEIS1 (chromosome 2p14p13), mitogen-activated Sureste, Arganda del Rey (Madrid)Arganda del Rey, Madrid, Spain (e-mail: [email protected]; [email protected]). protein kinase 5/SKI family transcriptional corepressor Funding: this work was supported in part by Grants PI12/00241, PI12/00324, 1 (MAP2K5/SKOR1, chromosome 15q23), and the variants and RETICS RD12/0013/0002 from Fondo de Investigacio´n Sanitaria, rs6747972 at chromosome 2p14, and rs3104767 at chromosome Instituto de Salud Carlos III, Madrid, Spain, and GR15026 from Junta de 16q12.1.1 Whole exome sequencing studies found association Extremadura, Me´rida, Spain, and PRIS10016 (Fundesalud, Me´rida,S- or RLS risk with some variants in the protocadherin alpha pain), and by grants from the Spanish Ministry of Science and Innovation 1 SAF2006-10126 (2006–2009) and SAF2010-22329-C02-01 (2011- 3 (PCDHA3, chromosome 5q31) in a German family. 2013) to PP and by the ‘‘Unio´nTe´cnica de Empresas’’ (UTE) project The pathophysiology of idiopathic RLS (iRLS) is not well FIMA to PP and project from the Centro de Investigaciones Me´dicas understood as well. Whereas iron deficiency and dopaminergic Aplicadas (CIMA), Spain. Partially funded with FEDER funds. The authors have no conflicts of interest to disclose. dysfunction are the main pathogenic hypothesis, recent works Copyright # 2015 Wolters Kluwer Health, Inc. All rights reserved. suggest the possible implication of neurotransmitters and/or This is an open access article distributed under the Creative Commons neuromodulators such as aspartate, glutamate, opiates, or Attribution License 4.0, which permits unrestricted use, distribution, and gamma-hydroxybutyric acid (GABA) (revised in reference).2 reproduction in any medium, provided the original work is properly cited. ISSN: 0025-7974 Moreover, in the last years, several interesting reports pointed DOI: 10.1097/MD.0000000000002125 out a possible role of vitamin D deficiency (which eventually Medicine Volume 00, Number 00, Month 2015 www.md-journal.com | 1 Jime´nez-Jime´nez et al Medicine Volume 94, Number 47, November 2015 could cause alterations in the development of the dopaminergic system),3,4 in the etiology of iRLS: TABLE 1. Demographic Data of the Series Studied (1) A case-control study found decreased serum 25-hydro- RLS Patients Controls xyvitamin D in female with iRLS, which was inversely Group (n ¼ 205) (n ¼ 445) correlated with disease severity.5 (2) Increased prevalence of RLS described in patients with Age, years, mean 53.8 (15.0) 63.6 (14.6) musculoskeletal symptoms with relatively lower when (SD) compared with those patients of the same cohort with Age range, years 20–92 17–104 higher serum 25-dihydroxyvitamin levels.6 Age at onset years, 43.9 (16.7) NA (3) Increased levels of vitamin D-binding protein in the CSF of mean (SD) RLS patients found in a preliminary case-control study Female % 170 (82.9%) 369 (82.9%) using proteomic analysis of the CSF.7 NA ¼ not available, RLS ¼ restless legs syndrome, SD ¼ standard (4) Improvement in the severity of RLS symptoms with deviation. administration of vitamin D supplements to patients with vitamin D deficiency found in an open-label study 8 involving 12 patients. Restless legs syndrome severity was assessed by using the International RLS Study Group Rating Scale (IRLSSGRS).13 Vitamin D3 (1,25-dihydroxyvitamin D3) receptor gene The 445 controls were healthy Caucasian Spanish indi- (VDR, NR1I1 or PPP1R163; chromosome 12q13.11; Gene viduals (none of them having RLS, tremor or other movement ID 7421; MIM 601769) encodes the nuclear hormone receptor disorders, or systemic diseases) matched by gender (275 of them for vitamin D3. This receptor has a similar sequence to the were recruited from the Infanta Cristina University Hospital, thyroid and steroid receptors, belongs to a family of trans-acting Badajoz, Spain; and the remaining 270 were recruited at the transcriptional regulatory factors and acts as a receptor for the Clı´nica Universitaria de Navarra; Pamplona, Spain). lithocolic acid. It is involved in mineral metabolism and other metabolic pathways involved in cancer and in the immune Ethical Aspects response (link http://www.ncbi.nlm.nih.gov/gene/7421). All the participants were included in the study after giving VDR gene shows 2 common single nucleotide polymorph- their written informed consent when the procedure of the study isms (SNP) in Caucasians: the SNP rs2228570 (Fok1), which was full explained. The Ethics Committees of Clinical Inves- causes the amino acid substitution (Met 1 Thr), and the synon- tigation of the University Hospital ‘‘Prı´ncipe de Asturias’’ ymous SNP rs731236 (Taq1), which does not cause amino acid (University of Alcala´, Alcala´ de Henares, Madrid, Spain), the substitution (Ile 352 Ile), but it has been studied with regard to Clı´nica Universitaria de Navarra (Pamplona, Spain), the Infanta several clinical conditions. These SNPs have been associated Cristina University Hospital (Badajoz, Spain), and the Province with the risk for multiple sclerosis by several studies, although a of Ca´ceres (Ca´ceres, Spain), approved the study, that was meta-analysis of these studies did not confirm such association.9 conducted according with the principles enumerated in the The rs731236 and other 2 SNPs in the VDR gene showed lack of Helsinki Declaration of