Consensus Guidelines on Rodent Models of Restless Legs Syndrome.

Restless legs syndrome (RLS) is a chronic sensorimotor disorder diagnosed by clinical symptoms. It is challenging to translate the diagnostic self-reported features of RLS to animals. To help researchers design their experiments, a task force was convened to develop consensus guidelines for experimental readouts in RLS animal models. The RLS clinical diagnostic criteria were used as a starting point. After soliciting additional important clinical features of RLS, a consensus set of methods and outcome measures intent on capturing these features-in the absence of a face-to-face interview-was generated and subsequently prioritized by the task force. These were, in turn, translated into corresponding methods and outcome measures for research on laboratory rats and mice and used to generate the final recommendations. The task force recommended activity monitoring and polysomnography as principal tools in assessing RLS-like behavior in rodents. Data derived from these methods were determined to be the preferred surrogate measures for the urge to move, the principal defining feature of RLS. The same tools may be used to objectively demonstrate sleep-state features highly associated with RLS, such as sleep disturbance and number and periodicity of limb movements. Pharmacological challenges and dietary or other manipulations that affect iron availability are desirable to aggravate or improve RLS-like behavior and lend greater confidence that the animal model being proffered replicates key clinical features of RLS. These guidelines provide the first consensus experimental framework for researchers to use when developing new rodent models of RLS. © 2020 International Parkinson and Movement Disorder Society.

A novel sleep stage scoring system: Combining expert-based features with the generalized linear model.

In this study, we aim to automate the sleep stage scoring process of overnight polysomnography (PSG) data while adhering to expert-based rules. We developed a sleep stage scoring algorithm utilizing the generalized linear modelling (GLM) framework and extracted features from electroencephalogram (EEG), electromyography (EMG) and electrooculogram (EOG) signals based on predefined rules of the American Academy of Sleep Medicine (AASM) Manual for Scoring Sleep. Specifically, features were computed in 30-s epochs in the time and frequency domains of the signals and were then used to model the probability of an epoch being in each of five sleep stages: N3, N2, N1, REM or Wake. Finally, each epoch was assigned to a sleep stage based on model predictions. The algorithm was trained and tested on PSG data from 38 healthy individuals with no reported sleep disturbances. The overall scoring accuracy reached on the test set was 81.50 ± 1.14% (Cohen's kappa, κ=0.73±0.02 ). The test set results were highly comparable to the training set, indicating robustness of the algorithm. Furthermore, our algorithm was compared to three well-known commercialized sleep-staging tools and achieved higher accuracies than all of them. Our results suggest that automatic classification is highly consistent with visual scoring. We conclude that our algorithm can reproduce the judgement of a scoring expert and is also highly interpretable. This tool can assist visual scorers to speed up their process (from hours to minutes) and provides a method for a more robust, quantitative, reproducible and cost-effective PSG evaluation, supporting assessment of sleep and sleep disorders.

Targeting hypersensitive corticostriatal terminals in restless legs syndrome.

OBJECTIVE: The first aim was to demonstrate a previously hypothesized increased sensitivity of corticostriatal glutamatergic terminals in the rodent with brain iron deficiency (BID), a pathogenetic model of restless legs syndrome (RLS). The second aim was to determine whether these putative hypersensitive terminals could constitute a significant target for drugs effective in RLS, including dopamine agonists (pramipexole and ropinirole) and α2 δ ligands (gabapentin). METHODS: A recently introduced in vivo optogenetic-microdialysis approach was used, which allows the measurement of the extracellular concentration of glutamate upon local light-induced stimulation of corticostriatal glutamatergic terminals. The method also allows analysis of the effect of local perfusion of compounds within the same area being sampled for glutamate. RESULTS: BID rats showed hypersensitivity of corticostriatal glutamatergic terminals (lower frequency of optogenetic stimulation to induce glutamate release). Both hypersensitive and control glutamatergic terminals were significant targets for locally perfused pramipexole, ropinirole, and gabapentin, which significantly counteracted optogenetically induced glutamate release. The use of selective antagonists demonstrated the involvement of dopamine D4 and D2 receptor subtypes in the effects of pramipexole. INTERPRETATION: Hypersensitivity of corticostriatal glutamatergic terminals can constitute a main pathogenetic mechanism of RLS symptoms. Selective D4 receptor agonists, by specifically targeting these terminals, should provide a new efficient treatment with fewer secondary effects. Ann Neurol 2017;82:951-960.

Treatment of restless legs syndrome: Evidence-based review and implications for clinical practice (Revised 2017)§.

The objective of the current review was to update the previous evidence-based medicine review of treatments for restless legs syndrome published in 2008. All randomized, controlled trials (level I) with a high quality score published between January 2007 and January 2017 were reviewed. Forty new studies qualified for efficacy review. Pregabalin, gabapentin enacarbil, and oxycodone/naloxone, which did not appear in the previous review, have accrued data to be considered efficacious. Likewise, new data enable the modification of the level of efficacy for rotigotine from likely efficacious to efficacious. Intravenous ferric carboxymaltose and pneumatic compression devices are considered likely efficacious in idiopathic restless legs syndrome. Bupropion and clonidine were reviewed, but the lack of data determined a rating of insufficient evidence for efficacy. The following interventions continue to be considered efficacious as in 2008: levodopa, ropinirole, pramipexole, cabergoline, pergolide, and gabapentin. Bromocriptine, oxycodone, carbamazepine, and valproic acid are considered likely efficacious. Oral iron is nonefficacious in iron-sufficient subjects, but its benefit for patients with low peripheral iron status has not been adequately evaluated. Restless legs syndrome augmentation has been identified as a significant long-term treatment complication for pramipexole more than pregabalin and possibly for all dopaminergic agents more than α2δ ligands. Therefore, special monitoring for augmentation is required for all dopaminergic medications as well as tramadol. Other drugs also require special safety monitoring: cabergoline, pergolide, oxycodone, methadone, tramadol, carbamazepine, and valproic acid. Finally, we also highlighted gaps and needs for future clinical research and studies of restless legs syndrome. © 2018 International Parkinson and Movement Disorder Society.

Comparison of pregabalin with pramipexole for restless legs syndrome.

BACKGROUND: Dopaminergic medications relieve symptoms of the restless legs syndrome (RLS) but have the potential to cause iatrogenic worsening (augmentation) of RLS with long-term treatment. Pregabalin may be an effective alternative. METHODS: In this 52-week, randomized, double-blind trial, we assessed efficacy and augmentation in patients with RLS who were treated with pregabalin as compared with placebo and pramipexole. Patients were randomly assigned to receive 52 weeks of treatment with pregabalin at a dose of 300 mg per day or pramipexole at a dose of 0.25 mg or 0.5 mg per day or 12 weeks of placebo followed by 40 weeks of randomly assigned active treatment. The primary analyses involved a comparison of pregabalin and placebo over a period of 12 weeks with use of the International RLS (IRLS) Study Group Rating Scale (on which the score ranges from 0 to 40, with a higher score indicating more severe symptoms), the Clinical Global Impression of Improvement scale (which was used to assess the proportion of patients with symptoms that were "very much improved" or "much improved"), and a comparison of rates of augmentation with pregabalin and pramipexole over a period of 40 or 52 weeks of treatment. RESULTS: A total of 719 participants received daily treatment, 182 with 300 mg of pregabalin, 178 with 0.25 mg of pramipexole, 180 with 0.5 mg of pramipexole, and 179 with placebo. Over a period of 12 weeks, the improvement (reduction) in mean scores on the IRLS scale was greater, by 4.5 points, among participants receiving pregabalin than among those receiving placebo (P<0.001), and the proportion of patients with symptoms that were very much improved or much improved was also greater with pregabalin than with placebo (71.4% vs. 46.8%, P<0.001). The rate of augmentation over a period of 40 or 52 weeks was significantly lower with pregabalin than with pramipexole at a dose of 0.5 mg (2.1% vs. 7.7%, P=0.001) but not at a dose of 0.25 mg (2.1% vs. 5.3%, P=0.08). There were six cases of suicidal ideation in the group receiving pregabalin, three in the group receiving 0.25 mg of pramipexole, and two in the group receiving 0.5 mg of pramipexole. CONCLUSIONS: Pregabalin provided significantly improved treatment outcomes as compared with placebo, and augmentation rates were significantly lower with pregabalin than with 0.5 mg of pramipexole. (Funded by Pfizer; ClinicalTrials.gov number, NCT00806026.).

Defining morphology of periodic leg movements in sleep: an evidence-based definition of a minimum window of sustained activity.

OBJECTIVE: Current standard guidelines for scoring periodic leg movements (PLM) define the start and end of a movement but fail to explicitly specify the movement morphology necessary to classify an EMG event as a PLM, rather than some other muscle event. This is currently left to the expert visual scorer to determine. This study aimed to define this morphology to provide a consistent standard for visual scoring and to improve automatic periodic leg movements in sleep scoring. METHODS: A review of expert PLM scoring produced a hypothesized morphology criterion: a window of high EMG activity within the movement lasting at least 0.5 s. Two diverse expert visual scorers were independently presented with images of EMG tracings from candidate leg movements (CLM) that either passed or failed this requirement (aka "full" or "empty" movements, respectively), and indicated whether each should be scored as CLM. The 0.5-s window was compared with alternatives of 0.25 and 0.75 windows. RESULTS: Expert scorers on average identified 94 % of "full" movements as CLM in contrast to only 8.5 % of "empty" movements. The proposed minimum window of 0.5 s also resulted in the highest agreement between visual scorers and between scorers and an automatic program. CONCLUSION: An added criterion requiring 0.5 s of high EMG activity within a valid CLM improves the accuracy of automatic scoring algorithms in relation to the gold standard of expert visual scorers. Our results suggest that this rule is an accurate representation of the morphology feature used by experts. This new rule has the potential to improve consistency and accuracy of visual and automatic scoring of PLM.

Restless legs syndrome/Willis Ekbom disease: evaluation and treatment.

Restless legs syndrome/Willis Ekbom disease (RLS/WED) has been recognized as a significant medical disorder since the 17th century. It was studied mostly in the last 50 years in relation to increasing interest in sleep medicine and health-related quality of life. This led to recognition that the disease is not well characterized as restless feelings in the legs. These symptoms are reported in many situations, but the subjective experience of RLS/WED patients differs from that experienced by others. Thus a new name has been introduced that avoids problems of symptom definition of a disease by naming it after those who first characterized it, i.e. 'Willis Ekbom disease'. This article emphasizes the importance of RLS/WED for psychiatry. The disease carries significant increased risk for depression and anxiety disorders. Treatment requires consideration of these co-morbid disorders. RLS/WED can exacerbate or even engender psychiatric disease, so treatment of psychiatric disease should also include consideration of RLS/WED. The need for attention to RLS/WED is particularly significant for depression. Most anti-depressants exacerbate or can even engender RLS/WED. Thus this article seeks to introduce RLS/WED in relation to psychiatric practice. It presents the RLS/WED disease, its overlap with psychiatry and the current treatment options.

Genome-wide association study identifies novel restless legs syndrome susceptibility loci on 2p14 and 16q12.1.

Restless legs syndrome (RLS) is a sensorimotor disorder with an age-dependent prevalence of up to 10% in the general population above 65 years of age. Affected individuals suffer from uncomfortable sensations and an urge to move in the lower limbs that occurs mainly in resting situations during the evening or at night. Moving the legs or walking leads to an improvement of symptoms. Concomitantly, patients report sleep disturbances with consequences such as reduced daytime functioning. We conducted a genome-wide association study (GWA) for RLS in 922 cases and 1,526 controls (using 301,406 SNPs) followed by a replication of 76 candidate SNPs in 3,935 cases and 5,754 controls, all of European ancestry. Herein, we identified six RLS susceptibility loci of genome-wide significance, two of them novel: an intergenic region on chromosome 2p14 (rs6747972, P = 9.03 × 10(-11), OR = 1.23) and a locus on 16q12.1 (rs3104767, P = 9.4 × 10(-19), OR = 1.35) in a linkage disequilibrium block of 140 kb containing the 5'-end of TOX3 and the adjacent non-coding RNA BC034767.

The prevalence and impact of restless legs syndrome on patients with iron deficiency anemia.

Restless Legs Syndrome (RLS) a common, under-recognized disorder disrupts sleep and diminishes quality of life. Despite a clear relation between low peripheral iron and increased prevalence and severity of RLS, the prevalence and clinical significance of RLS in iron-deficient anemic (IDA) populations is unknown. In this study all new patients referred for anemia to a community-based hematology practice over a 1-year period (March 2011-2012) were included if they had IDA and no RLS treatment. Patients completed a validated questionnaire identifying RLS, blood tests, and a sleep-vitality questionnaire (SVQ). Patients with RLS were compared to patients with no RLS for differences on SVQ, blood tests, baseline characteristics, and sleep quality. Three hundred forty-three patients were evaluated and 251 (89.2% female, average age of 45.6 years) included in the study. The prevalence of clinically significant RLS (RLS sufferers) was 23.9%, nine times higher than the general population. IDA-RLS sufferers reported poorer quality of sleep, decreased sleep time, increased tiredness, and decreased energy during the day compared to patients with IDA without RLS. Blood tests did not relate to RLS diagnosis but RLS was less likely for African-American than Caucasian patients. Clinically significant RLS occurs commonly with IDA producing much greater disruption of sleep and shorter sleep times than does IDA alone. This indicates the need for identification of RLS with IDA and consideration of appropriate therapeutic interventions for this sizeable subgroup: either aggressive iron treatment to reduce the RLS symptoms or medications for RLS or both.

Systems genetic analysis of the effects of iron deficiency in mouse brain.

Iron regulation in the brain is both necessary and highly complex. Too little or too much iron can compromise neurological function, yet we still do not know all of the regulatory processes. In our research, we seek to identify genes and gene networks underlying individual differences in brain iron regulation. To this end, we fed mice from 20+ inbred strains a diet low in iron from weaning to 4 months of age. At sacrifice, we measured iron content in the ventral midbrain (VMB). The VMB contains the substantia nigra, a region particularly vulnerable to iron imbalance. The results showed high, inter-strain variability in dietary iron reduction, from almost no loss to more than 40 % vs. control. When we performed quantitative trait loci (QTL) analysis, we observed a significant area on chromosome 2. Within this QTL, we selected glial high-affinity glutamate transporter 1 (Glt1) as the leading candidate. Expression of this gene is both correlated with VMB iron and is also cis-modulated by local sequence variants that segregate in the BXD family. VMB expression differences of Glt1 in six strains covary with differential susceptibility to VMB iron loss.

Systems genetic analysis of multivariate response to iron deficiency in mice.

The aim of this study was to identify genes that influence iron regulation under varying dietary iron availability. Male and female mice from 20+ BXD recombinant inbred strains were fed iron-poor or iron-adequate diets from weaning until 4 mo of age. At death, the spleen, liver, and blood were harvested for the measurement of hemoglobin, hematocrit, total iron binding capacity, transferrin saturation, and liver, spleen and plasma iron concentration. For each measure and diet, we found large, strain-related variability. A principal-components analysis (PCA) was performed on the strain means for the seven parameters under each dietary condition for each sex, followed by quantitative trait loci (QTL) analysis on the factors. Compared with the iron-adequate diet, iron deficiency altered the factor structure of the principal components. QTL analysis, combined with PosMed (a candidate gene searching system) published gene expression data and literature citations, identified seven candidate genes, Ptprd, Mdm1, Picalm, lip1, Tcerg1, Skp2, and Frzb based on PCA factor, diet, and sex. Expression of each of these is cis-regulated, significantly correlated with the corresponding PCA factor, and previously reported to regulate iron, directly or indirectly. We propose that polymorphisms in multiple genes underlie individual differences in iron regulation, especially in response to dietary iron challenge. This research shows that iron management is a highly complex trait, influenced by multiple genes. Systems genetics analysis of iron homeostasis holds promise for developing new methods for prevention and treatment of iron deficiency anemia and related diseases.

Profile of altered brain iron acquisition in restless legs syndrome.

Restless legs syndrome is a neurological disorder characterized by an urgency to move the legs during periods of rest. Data from a variety of sources provide a compelling argument that the amount of iron in the brain is lower in individuals with restless legs syndrome compared with neurologically normal individuals. Moreover, a significant percentage of patients with restless legs syndrome are responsive to intravenous iron therapy. The mechanism underlying the decreased iron concentrations in restless legs syndrome brains is unknown. We hypothesize that the source of the brain iron deficit is at the blood-brain interface. Thus we analysed the expression of iron management proteins in the epithelial cells of the choroid plexus and the brain microvasculature in post-mortem tissues. The choroid plexus, obtained at autopsy, from 18 neurologically normal controls and 14 individuals who had primary restless legs syndrome was subjected to histochemical staining for iron and immunostaining for iron management proteins. Iron and heavy chain ferritin staining was reduced in the epithelial cells of choroid plexus in restless legs syndrome. Divalent metal transporter, ferroportin, transferrin and its receptor were upregulated in the choroid plexus in restless legs syndrome. Microvessels were isolated from the motor cortex of 11 restless legs syndrome and 14 control brains obtained at autopsy and quantitative immunoblot analyses was performed. Expression of heavy chain ferritin, transferrin and its receptor in the microvessels from restless legs syndrome was significantly decreased compared with the controls but divalent metal protein 1, ferroportin, prohepcidin, mitochondrial ferritin and light-chain ferritin remained unchanged. The presence of an iron regulatory protein was demonstrated in the brain microvasculature and the activity of this protein is decreased in restless legs syndrome; a finding similar to our earlier report in neuromelanin cells from the substantia nigra of restless legs syndrome brains. This study reveals that there are alterations in the iron management protein profile in restless legs syndrome compared with controls at the site of blood-brain interface suggesting fundamental differences in brain iron acquisition in individuals with restless legs syndrome. Furthermore, the decrease in transferrin receptor expression in the microvasculature in the presence of relative brain iron deficiency reported in restless legs syndrome brains may underlie the problems associated with brain iron acquisition in restless legs syndrome. The consistent finding of loss of iron regulatory protein activity in restless legs syndrome brain tissue further implicates this protein as a factor in the underlying cause of the iron deficiency in the restless legs syndrome brain. The data herein provide evidence for regulation of iron uptake and storage within brain microvessels that challenge the existing paradigm that the blood-brain barrier is merely a transport system.

Consensus guidelines on the construct validity of rodent models of restless legs syndrome.

Our understanding of the causes and natural course of restless legs syndrome (RLS) is incomplete. The lack of objective diagnostic biomarkers remains a challenge for clinical research and for the development of valid animal models. As a task force of preclinical and clinical scientists, we have previously defined face validity parameters for rodent models of RLS. In this article, we establish new guidelines for the construct validity of RLS rodent models. To do so, we first determined and agreed on the risk, and triggering factors and pathophysiological mechanisms that influence RLS expressivity. We then selected 20 items considered to have sufficient support in the literature, which we grouped by sex and genetic factors, iron-related mechanisms, electrophysiological mechanisms, dopaminergic mechanisms, exposure to medications active in the central nervous system, and others. These factors and biological mechanisms were then translated into rodent bioequivalents deemed to be most appropriate for a rodent model of RLS. We also identified parameters by which to assess and quantify these bioequivalents. Investigating these factors, both individually and in combination, will help to identify their specific roles in the expression of rodent RLS-like phenotypes, which should provide significant translational implications for the diagnosis and treatment of RLS.

Genetic analysis of iron-deficiency effects on the mouse spleen.

Iron homeostasis is crucial to many biological functions in nearly all organisms, with roles ranging from oxygen transport to immune function. Disruption of iron homeostasis may result in iron overload or iron deficiency. Iron deficiency may have severe consequences, including anemia or changes in immune or neurotransmitter systems. Here we report on the variability of phenotypic iron tissue loss and splenomegaly and the associated quantitative trait loci (QTLs), polymorphic areas in the mouse genome that may contain one or more genes that play a role in spleen iron concentration or spleen weight under each dietary treatment. Mice from 26 BXD/Ty recombinant inbred strains, including the parent C57BL/6 and DBA/2 strains, were randomly assigned to adequate iron or iron-deficient diets at weaning. After 120 days, splenomegaly was measured by spleen weight, and spleen iron was assessed using a modified spectrophotometry technique. QTL analyses and gene expression comparisons were then conducted using the WebQTL GeneNetwork. We observed wide, genetic-based variability in splenomegaly and spleen iron loss in BXD/Ty recombinant inbred strains fed an iron-deficient diet. Moreover, we identified several suggestive QTLs. Matching our QTLs with gene expression data from the spleen revealed candidate genes. Our work shows that individual differences in splenomegaly response to iron deficiency are influenced at least partly by genetic constitution. We propose mechanistic hypotheses by which splenomegaly may result from iron deficiency.

Prevalence and disease burden of primary restless legs syndrome: results of a general population survey in the United States.

To assess prevalence, disease burden, and costs of primary Restless Legs Syndrome (RLS) in the US. In 2007, 61,792 (20%) of 313,000 subjects from a representative US panel completed an online "global opinions" survey identifying respondents reporting all four diagnostic features of RLS. 4,484 met all criteria. 1,400 were randomly selected to complete a questionnaire to exclude those with diagnoses indicating possible secondary RLS. Those that did not have diagnoses associated with secondary RLS were asked to complete the Cambridge-Hopkins RLS questionnaire to exclude RLS mimics. Prevalence was estimated for the following groups: (1) RLS symptomatic, (2) primary RLS, and (3) primary RLS sufferers (symptoms ≥2/wk with moderate-to-severe distress). The primary RLS completed a larger online survey including the IRLS, EuroQol, Work Productivity and Activity Impairment questionnaire, and questions about healthcare resource use. The validated diagnostic tools and exclusion of medical conditions likely to cause RLS provide a very conservative estimate of US census-weighted prevalence of 2.4% for primary RLS and 1.5% for primary RLS sufferers. About 33% of respondents had a physician diagnosis of RLS. Primary RLS sufferers had a mean productivity loss of 1 day/wk. All RLS-related costs increased with RLS symptom severity, with increasingly significant decrements in health status, sleep disturbance, and work productivity. Even this very conservative approach finds RLS in this cohort to be common, under-diagnosed, and carried a significant personal and social burden.

Developing a biomarker for restless leg syndrome using genome wide DNA methylation data.

This study reports on an epigenetic biomarker for restless leg syndrome (RLS) developed using whole genome DNA methylation data. Lymphocyte-derived DNA methylation was examined in 15 subjects with and without RLS (discovery cohort). T-tests and linear regressions were used followed by a principal component analysis (PCA). The principal component model from the discovery cohort was used to predict RLS status in a peripheral blood (N = 24; including 12 cases and 12 controls) and a post-mortem neural tissue (N = 71; including 36 cases and 35 controls) replication cohort as well as iron deficiency anemia status in a publicly available dataset (N = 71, 59 cases with iron deficiency anemia, 12 controls). Using receiver-operating characteristic analysis the optimum biomarker model - that included 49 probes - predicted RLS status in the blood-based replication cohort with an area under the curve (AUC) of 87.5% (confidence interval = 71.9%-100%). In the neural tissue samples, the model predicted RLS status with an AUC of 73.4% (confidence interval = 61.5%-85.3%). An AUC of 83% was found for predictions of iron deficiency anemia. Thus, the blood-based biomarker model reported here and built with epigenome-wide data showed reasonable replicability in lymphocytes and neural tissue samples. A limitation of this study is that we could not determine the metabolic or neurobiological pathways linking epigenetic changes with RLS. Further research is needed to fine-tune this model for prospective predictions of RLS and to enable translation for clinical use.

Randomized, placebo-controlled trial of ferric carboxymaltose in restless legs syndrome patients with iron deficiency anemia.

OBJECTIVE: Intravenous ferric carboxymaltose (FCM) has been shown to be efficacious in treating restless legs syndrome (RLS) symptoms in non-anemic patients. The aim of this study was to evaluate the effectiveness of FCM in treating RLS symptoms in patients who also had an iron deficiency anemia (IDA). METHODS: This is a randomized, double-blinded, placebo-controlled study. Subjects with RLS and IDA were enrolled. Subjects received an infusion of either 1500 mg FCM or placebo in Phase I. The primary outcomes were a change-from-baseline at week six on the International Restless Legs Syndrome Study Group scale (IRLS). Phase II of the study involved long-term (52 weeks) follow-up, for those who responded to treatment in the prior phase, with the potential for further treatment if symptoms returned. RESULTS: We enrolled 29 RLS patients with IDA (15 FCM and 14 placebo). At week six post-infusion, FCM compared to placebo group showed significant improvement from baseline in IRLS score (-13.47 ± 7.38 vs. 1.36 ± 3.59). Among secondary outcome variables, quality of sleep showed significant improvement from baseline in the FCM group. 61% of subjects remained off RLS medications at the Phase II, week-52 endpoint. There were no serious adverse events observed in the study. CONCLUSION: The study showed significant efficacy and safety of FCM 1500 mg treatment both in the short term (6 weeks) and long term (52 weeks) in RLS patients with IDA.