A patient-enriched MEIS1 coding variant causes a restless legs syndrome-like phenotype in mice.

Restless legs syndrome (RLS) is a neurological disorder characterized by uncomfortable or unpleasant sensations in the legs during rest periods. To relieve these sensations, patients move their legs, causing sleep disruption. While the pathogenesis of RLS has yet to be resolved, there is a strong genetic association with the MEIS1 gene. A missense variant in MEIS1 is enriched sevenfold in people with RLS compared to non-affected individuals. We generated a mouse line carrying this mutation (p.Arg272His/c.815G>A), referred to herein as Meis1R272H/R272H (Meis1 point mutation), to determine whether it would phenotypically resemble RLS. As women are more prone to RLS, driven partly by an increased risk of developing RLS during pregnancy, we focused on female homozygous mice. We evaluated RLS-related outcomes, particularly sensorimotor behavior and sleep, in young and aged mice. Compared to noncarrier littermates, homozygous mice displayed very few differences. Significant hyperactivity occurred before the lights-on (rest) period in aged female mice, reflecting the age-dependent incidence of RLS. Sensory experiments involving tactile feedback (rotarod, wheel running, and hotplate) were only marginally different. Overall, RLS-like phenomena were not recapitulated except for the increased wake activity prior to rest. This is likely due to the focus on young mice. Nevertheless, the Meis1R272H mouse line is a potentially useful RLS model, carrying a clinically relevant variant and showing an age-dependent phenotype.

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.

Reassessment of candidate gene studies for idiopathic restless legs syndrome in a large genome-wide association study dataset of European ancestry.

STUDY OBJECTIVES: Several candidate gene studies have been published for idiopathic restless legs syndrome (RLS) in populations of European ancestry, but the reported associations have not been confirmed in independent samples. Our aim was to reassess these findings in a large case-control dataset in order to evaluate their validity. METHODS: We screened PubMed for RLS candidate gene studies. We used the genome-wide association study (GWAS) dataset of the International EU-RLS-GENE Consortium as our replication sample, which provided genome-wide single-variant association data based on at most 17 220 individuals of European ancestry. We performed additional gene-based tests using the software MAGMA and assessed the power of our study using the genpwr R package. RESULTS: We identified 14 studies conducted in European samples which assessed 45 variants in 27 genes of which 5 variants had been reported as significantly associated. None of these individual variants were replicated in our GWAS-based reassessment (nominal p > 0.05) and gene-based tests for the respective five genes ADH1B, GABRR3, HMOX1, MAOA, and VDR, were also nonsignificant (nominal p > 0.05). Our replication dataset was well powered to detect the reported effects, even when adjusting for effect size overestimation due to winner's curse. Power estimates were close to 100% for all variants. CONCLUSION: In summary, none of the significant single-variant associations from candidate gene studies were confirmed in our GWAS dataset. Therefore, these associations were likely false positive. Our observations emphasize the need for large sample sizes and stringent significance thresholds in future association studies for RLS.

Collagen VI Regulates Motor Circuit Plasticity and Motor Performance by Cannabinoid Modulation.

Collagen VI is a key component of muscle basement membranes, and genetic variants can cause monogenic muscular dystrophies. Conversely, human genetic studies recently implicated collagen VI in central nervous system function, with variants causing the movement disorder dystonia. To elucidate the neurophysiological role of collagen VI, we generated mice with a truncation of the dystonia-related collagen α3 VI (COL6A3) C-terminal domain (CTD). These Col6a3CTT mice showed a recessive dystonia-like phenotype in both sexes. We found that COL6A3 interacts with the cannabinoid receptor 1 (CB1R) complex in a CTD-dependent manner. Col6a3CTT mice of both sexes have impaired homeostasis of excitatory input to the basal pontine nuclei (BPN), a motor control hub with dense COL6A3 expression, consistent with deficient endocannabinoid (eCB) signaling. Aberrant synaptic input in the BPN was normalized by a CB1R agonist, and motor performance in Col6a3CTT mice of both sexes was improved by CB1R agonist treatment. Our findings identify a readily therapeutically addressable synaptic mechanism for motor control.SIGNIFICANCE STATEMENT Dystonia is a movement disorder characterized by involuntary movements. We previously identified genetic variants affecting a specific domain of the COL6A3 protein as a cause of dystonia. Here, we created mice lacking the affected domain and observed an analogous movement disorder. Using a protein interaction screen, we found that the affected COL6A3 domain mediates an interaction with the cannabinoid receptor 1 (CB1R). Concordantly, our COL6A3-deficient mice showed a deficit in synaptic plasticity linked to a deficit in cannabinoid signaling. Pharmacological cannabinoid augmentation rescued the motor impairment of the mice. Thus, cannabinoid augmentation could be a promising avenue for treating dystonia, and we have identified a possible molecular mechanism mediating this.

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.

Therapeutic effectiveness of thalidomide in a patient with treatment-resistant restless legs syndrome.

None: Recent developments in the genetics of restless legs syndrome (RLS) revealed associations of disease risk with genetic loci containing the genes coding cereblon, the protein bound by thalidomide, and its endogenous substrate MEIS2, whose degradation is inhibited by the thalidomide-cereblon interaction. Therefore it was hypothesized that thalidomide may be a potential treatment option for RLS. Here we report on the therapeutic effect of thalidomide in a patient with otherwise treatment-resistant RLS who received 100 mg thalidomide off-label for 3 weeks. The female patient, severely affected by RLS before treatment, experienced significant amelioration of the symptoms, increased self-reported sleep quality, and better daytime functioning during thalidomide treatment. This therapeutic success warrants larger studies investigating the efficacy of drugs of the thalidomide class in RLS.

Identification of Restless Legs Syndrome Genes by Mutational Load Analysis.

OBJECTIVE: Restless legs syndrome is a frequent neurological disorder with substantial burden on individual well-being and public health. Genetic risk loci have been identified, but the causatives genes at these loci are largely unknown, so that functional investigation and clinical translation of molecular research data are still inhibited. To identify putatively causative genes, we searched for highly significant mutational burden in candidate genes. METHODS: We analyzed 84 candidate genes in 4,649 patients and 4,982 controls by next generation sequencing using molecular inversion probes that targeted mainly coding regions. The burden of low-frequency and rare variants was assessed, and in addition, an algorithm (binomial performance deviation analysis) was established to estimate independently the sequence variation in the probe binding regions from the variation in sequencing depth. RESULTS: Highly significant results (considering the number of genes in the genome) of the conventional burden test and the binomial performance deviation analysis overlapped significantly. Fourteen genes were highly significant by one method and confirmed with Bonferroni-corrected significance by the other to show a differential burden of low-frequency and rare variants in restless legs syndrome. Nine of them (AAGAB, ATP2C1, CNTN4, COL6A6, CRBN, GLO1, NTNG1, STEAP4, VAV3) resided in the vicinity of known restless legs syndrome loci, whereas 5 (BBS7, CADM1, CREB5, NRG3, SUN1) have not previously been associated with restless legs syndrome. Burden test and binomial performance deviation analysis also converged significantly in fine-mapping potentially causative domains within these genes. INTERPRETATION: Differential burden with intragenic low-frequency variants reveals putatively causative genes in restless legs syndrome. ANN NEUROL 2020;87:184-193.

Role of MEIS1 in restless legs syndrome: From GWAS to functional studies in mice.

MEIS1 is a transcription factor playing an important role in the development of several organs, including central and peripheral nervous systems. A genetic locus spanning the MEIS1 coding region has been associated with the risk of RLS in genome-wide association studies, with increasing evidence that MEIS1 is the causal RLS gene. The RLS-linked genetic signal has been mapped to an intronic regulatory element within MEIS1. This element plays a role in the ganglionic eminences of the developing forebrain, with the RLS risk allele related to a reduced activation of the enhancer. This suggests that the ganglionic eminences play an important role in the development of genetic susceptibility to RLS. In addition, rare variants within MEIS1 have been shown to contribute to the disease risk. These variants were identified first in RLS families and later found in further RLS cases by targeted sequencing. Some of these variants alone are sufficient to suppress MEIS1 function in neural development, providing further evidence of the importance of neurodevelopmental processes in the pathological mechanism of MEIS1 in RLS. Heterozygous Meis1 inactivation in mice causes hyperactivity at the onset of the inactive period, consistent with human RLS. In addition, these mice revealed an effect of MEIS1 on the dopaminergic system at both the spinal and supraspinal level. More studies are needed in human genetics to determine the exact role of MEIS1 variants in the risk of RLS, as well as in functional genetics and animal studies to further elucidate the pathological mechanism of MEIS1 in RLS.

Restless Legs Syndrome and Other Movement Disorders of Sleep-Treatment Update.

PURPOSE OF REVIEW: The purpose of this review article is to summarize and discuss the recent advances in the treatment of restless legs syndrome (RLS), as well as REM sleep behavior disorder (RBD), and periodic leg movement disorder (PLMD). RECENT FINDINGS: Traditionally, dopaminergic therapy has been considered the sole option for first-line treatment of RLS due to their impressive acute efficacy. Dopamine agonists such as oral pramipexole and ropinirole, as well as transdermal rotigotine are all effective treatment options. However, augmentation of the RLS symptoms is a major limitation of oral dopaminergic therapy. Recently, gabapentinoid agents such as gabapentin enacarbil and pregabalin have shown comparable short-term efficacy to dopaminergics with lower risk of augmentation of the RLS symptoms. Recent evidence on the efficacy of oxycodone-naloxone in treatment-resistant RLS provides an additional therapeutic avenue. The increasing understanding of the role of iron in RLS pathophysiology has led to new options in iron supplementation therapy in RLS, including treatment with ferric carboxymaltose. With emerging evidence of augmentation being a side effect specific to dopaminergic treatment, gabapentinoids are considered a safer option as initial treatment. In severe refractory RLS, oxycodone-naloxone can be used. If iron stores are low, IV iron formulations should be the initial treatment choice. New treatment options are needed to address issues with current therapies.

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.

Sleep disturbance by pramipexole is modified by Meis1 in mice.

Meis homeobox 1 (Meis1) is a transcription factor functioning in the development of the nervous system and the cardiovascular system. Both common and rare variants within the gene have been associated with restless legs syndrome (RLS), while its association with symptoms of insomnia has also been discovered recently. RLS is associated with sleep disturbances, and while Meis1 haploinsufficiency is one of the most promising strategies for an RLS animal model, sleep phenotyping of Meis1 knockout mice has never been conducted. We report a detailed sleep analysis of heterozygous Meis1 knockout mice and challenge it with pramipexole, a dopamine agonist used in the treatment of RLS. At baseline, the Meis1-haploinsufficient mice had a trend towards lower delta power in the electroencephalogram (EEG) during sleep compared to the wild-type littermates, possibly indicating reduced sleep quality, but not sleep fragmentation. Pramipexole had a sleep disrupting effect in both genotype groups. In addition, it exerted differential effects on the EEG power spectra of the two mouse lines, remarkably elevating the theta power of the mutant mice during recovery more than that of the wild-types. In conclusion, Meis1 haploinsufficiency seems to have only a modest effect on sleep, but the gene may interact with the sleep-disrupting effect of dopamine agonists.

Identification of novel risk loci for restless legs syndrome in genome-wide association studies in individuals of European ancestry: a meta-analysis.

BACKGROUND: Restless legs syndrome is a prevalent chronic neurological disorder with potentially severe mental and physical health consequences. Clearer understanding of the underlying pathophysiology is needed to improve treatment options. We did a meta-analysis of genome-wide association studies (GWASs) to identify potential molecular targets. METHODS: In the discovery stage, we combined three GWAS datasets (EU-RLS GENE, INTERVAL, and 23andMe) with diagnosis data collected from 2003 to 2017, in face-to-face interviews or via questionnaires, and involving 15 126 cases and 95 725 controls of European ancestry. We identified common variants by fixed-effect inverse-variance meta-analysis. Significant genome-wide signals (p≤5 × 10-8) were tested for replication in an independent GWAS of 30 770 cases and 286 913 controls, followed by a joint analysis of the discovery and replication stages. We did gene annotation, pathway, and gene-set-enrichment analyses and studied the genetic correlations between restless legs syndrome and traits of interest. FINDINGS: We identified and replicated 13 new risk loci for restless legs syndrome and confirmed the previously identified six risk loci. MEIS1 was confirmed as the strongest genetic risk factor for restless legs syndrome (odds ratio 1·92, 95% CI 1·85-1·99). Gene prioritisation, enrichment, and genetic correlation analyses showed that identified pathways were related to neurodevelopment and highlighted genes linked to axon guidance (associated with SEMA6D), synapse formation (NTNG1), and neuronal specification (HOXB cluster family and MYT1). INTERPRETATION: Identification of new candidate genes and associated pathways will inform future functional research. Advances in understanding of the molecular mechanisms that underlie restless legs syndrome could lead to new treatment options. We focused on common variants; thus, additional studies are needed to dissect the roles of rare and structural variations. FUNDING: Deutsche Forschungsgemeinschaft, Helmholtz Zentrum München-Deutsches Forschungszentrum für Gesundheit und Umwelt, National Research Institutions, NHS Blood and Transplant, National Institute for Health Research, British Heart Foundation, European Commission, European Research Council, National Institutes of Health, National Institute of Neurological Disorders and Stroke, NIH Research Cambridge Biomedical Research Centre, and UK Medical Research Council.

Meis1: effects on motor phenotypes and the sensorimotor system in mice.

MEIS1 encodes a developmental transcription factor and has been linked to restless legs syndrome (RLS) in genome-wide association studies. RLS is a movement disorder leading to severe sleep reduction and has a substantial impact on the quality of life of patients. In genome-wide association studies, MEIS1 has consistently been the gene with the highest effect size and functional studies suggest a disease-relevant downregulation. Therefore, haploinsufficiency of Meis1 could be the system with the most potential for modeling RLS in animals. We used heterozygous Meis1-knockout mice to study the effects of Meis1 haploinsufficiency on mouse behavioral and neurological phenotypes, and to relate the findings to human RLS. We exposed the Meis1-deficient mice to assays of motor, sensorimotor and cognitive ability, and assessed the effect of a dopaminergic receptor 2/3 agonist commonly used in the treatment of RLS. The mutant mice showed a pattern of circadian hyperactivity, which is compatible with human RLS. Moreover, we discovered a replicable prepulse inhibition (PPI) deficit in the Meis1-deficient animals. In addition, these mice were hyposensitive to the PPI-reducing effect of the dopaminergic receptor agonist, highlighting a role of Meis1 in the dopaminergic system. Other reported phenotypes include enhanced social recognition at an older age that was not related to alterations in adult olfactory bulb neurogenesis previously shown to be implicated in this behavior. In conclusion, the Meis1-deficient mice fulfill some of the hallmarks of an RLS animal model, and revealed the role of Meis1 in sensorimotor gating and in the dopaminergic systems modulating it.

Transcutaneous carbon dioxide during sleep-disordered breathing.

Respiratory drive is tightly controlled by the carbon dioxide levels. We tested the hypothesis that sequences of sleep apnoea (obstructive, central or mixed), hypopnoea and flow limitation are characterized by different levels of transcutaneous CO2 (PtcCO2). Polygraphic recordings (n=555) from patients with suspected sleep-disordered breathing (SDB) were retrospectively screened to find sequences (5 min or 10 events) of both SDB and steady breathing. Eighty-eight SDB sequences from 44 patients were included and PtcCO2 and SpO2 values were collected. PtcCO2 values during sequences were normalized by setting wakefulness level as 100%. In terms of PtcCO2, apnoea sequences with central component (central (n=7) and mixed (n=3) apnoea) did not differ from wakefulness (102.0% vs 100%, p=0.122) whereas obstructive apnoea (105.8%, p<0.001) and hypopnoea did (105.4%, p<0.001). PtcCO2 during flow limitation was higher than that during any other sequence, including steady breathing (112.2% vs 108.4%, p=0.022). Continuous PtcCO2 monitoring during sleep adds to the understanding of different SDB phenotypes.

Recessive mutations in the α3 (VI) collagen gene COL6A3 cause early-onset isolated dystonia.

Isolated dystonia is a disorder characterized by involuntary twisting postures arising from sustained muscle contractions. Although autosomal-dominant mutations in TOR1A, THAP1, and GNAL have been found in some cases, the molecular mechanisms underlying isolated dystonia are largely unknown. In addition, although emphasis has been placed on dominant isolated dystonia, the disorder is also transmitted as a recessive trait, for which no mutations have been defined. Using whole-exome sequencing in a recessive isolated dystonia-affected kindred, we identified disease-segregating compound heterozygous mutations in COL6A3, a collagen VI gene associated previously with muscular dystrophy. Genetic screening of a further 367 isolated dystonia subjects revealed two additional recessive pedigrees harboring compound heterozygous mutations in COL6A3. Strikingly, all affected individuals had at least one pathogenic allele in exon 41, including an exon-skipping mutation that induced an in-frame deletion. We tested the hypothesis that disruption of this exon is pathognomonic for isolated dystonia by inducing a series of in-frame deletions in zebrafish embryos. Consistent with our human genetics data, suppression of the exon 41 ortholog caused deficits in axonal outgrowth, whereas suppression of other exons phenocopied collagen deposition mutants. All recessive mutation carriers demonstrated early-onset segmental isolated dystonia without muscular disease. Finally, we show that Col6a3 is expressed in neurons, with relevant mRNA levels detectable throughout the adult mouse brain. Taken together, our data indicate that loss-of-function mutations affecting a specific region of COL6A3 cause recessive isolated dystonia with underlying neurodevelopmental deficits and highlight the brain extracellular matrix as a contributor to dystonia pathogenesis.

Novel metabolic features in Acinetobacter baylyi ADP1 revealed by a multiomics approach.

Expansive knowledge of bacterial metabolism has been gained from genome sequencing output, but the high proportion of genes lacking a proper functional annotation in a given genome still impedes the accurate prediction of the metabolism of a cell. To access to a more global view of the functioning of the soil bacterium Acinetobacter baylyi ADP1, we adopted a multi 'omics' approach. Application of RNA-seq transcriptomics and LC/MS-based metabolomics, along with the systematic phenotyping of the complete collection of single-gene deletion mutants of A. baylyi ADP1 made possible to interrogate on the metabolic perturbations encountered by the bacterium upon a biotic change. Shifting the sole carbon source from succinate to quinate elicited in the cell not only a specific transcriptional response, necessary to catabolize the new carbon source, but also a major reorganization of the transcription pattern. Here, the expression of more than 12 % of the total number of genes was affected, most of them being of unknown function. These perturbations were ultimately reflected in the metabolome, in which the concentration of about 50 % of the LC/MS-detected metabolites was impacted. And the differential regulation of many genes of unknown function is probably related to the synthesis of the numerous unidentified compounds that were present exclusively in quinate-grown cells. Together, these data suggest that A. baylyi ADP1 metabolism involves unsuspected enzymatic reactions that await discovery.

Pramipexole alters thermoregulation in restless legs syndrome.

STUDY OBJECTIVES: Previous studies have associated restless legs syndrome (RLS) with peripheral hypoxia and impaired thermoregulation in the lower extremities. We performed long-term monitoring of skin temperatures in order to investigate whether these findings could be explained by reduced blood flow to the peripheral tissues. METHODS: 96-hour continuous measurements of skin temperature were performed both in the distal and proximal parts of the body of 15 patients with RLS and 14 healthy controls. During the recording, the patients participated in suggested immobilization tests both with and without pramipexole medication. RESULTS: We found no baseline differences in distal or proximal skin temperature between patients and controls in daytime or during immobilization. However, pramipexole significantly increased distal skin temperature in the patient group during immobilization (31.1°C vs. 32.9°C, p < 0.05). Daytime temperatures were not affected by therapy or disease status. CONCLUSIONS: The data suggest that patients with RLS and healthy controls have similar blood flow to the peripheral skin tissue. Pramipexole, however, alters thermoregulation and the previous studies might have been biased by medication. Dopaminergic medication is a major confounding factor when assessing peripheral phenomena in RLS and should be controlled for in the future studies.

Peripheral hypoxia in restless legs syndrome (Willis-Ekbom disease).

OBJECTIVE: A case-control study to measure oxygen and carbon dioxide partial pressures in the legs in order to assess the involvement of peripheral hypoxia or hypercapnia in the pathogenesis of restless legs syndrome (RLS). METHODS: RLS severity was assessed with a standard questionnaire. Suggested immobilization tests were performed twice in 15 patients with RLS and 14 healthy controls. Patients with RLS participated in the tests with and without pramipexole medication. During the tests, peripheral oxygen and carbon dioxide partial pressures were measured noninvasively on the skin of the legs and the chest. RESULTS: During immobilization, the patients with RLS had lower partial pressure of oxygen in their legs (5.54 vs 7.19 kPa, p < 0.01) but not on the chest (8.75 vs 8.20 kPa, p = 0.355). More severe RLS correlated with high chest-to-foot oxygen gradient (ρ = 0.692, p < 0.01). Carbon dioxide levels did not differ between the groups. Pramipexole corrected the peripheral hypoxia toward the levels observed in the controls (from 5.54 to 6.65 kPa, p < 0.05). CONCLUSIONS: Peripheral hypoxia is associated with the appearance of RLS symptoms. Strong correlation with RLS severity suggests a close pathophysiologic link between peripheral hypoxia and the symptoms of RLS. This is further supported by the simultaneous reversal of hypoxia and discomfort by dopaminergic treatment.