More about the proteins identified in the new research study by Patton, et.al. There is a lengthy discussion of the methods at the following link:http://www.fluidsbarrierscns.com/content/10/1/20
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Restless Legs Syndrome/Willis-Ekbom Disease (RLS/WED) is a sensorimotor disorder that causes patients to experience overwhelming and distressing sensations in the legs compelling the patient to move their legs to provide relief. The purpose of this study was to determine if biomarkers in the cerebrospinal fluid can distinguish RLS/WED patients from neurological controls.
We obtained CSF samples by lumbar puncture from 5 early-onset RLS/WED patients and 5 controls. We performed 2-dimensional difference in-gel electrophoresis (2D-DIGE). Proteins that were significantly altered were identified by Student’s t-test. Protein spots that were differentially expressed (p ≤ 0.05, Av. Ratio ≥ 2.0) between RLS/WED and control CSF samples were identified using MALDI-TOF-MS. Statistical analyses of the validation immunoblot assays were performed using Student’s t-test.
In this discovery study we identified 6 candidate CSF protein markers for early-onset RLS/WED. Four proteins (Cystatin C, Lipocalin-type Prostaglandin D2 Synthase, Vitamin D binding Protein, and β-Hemoglobin) were increased and 2 proteins (Apolipoprotein A1 and α-1-acid Glycoprotein) were decreased in RLS/WED patients.
Our results reveal a protein profile in the RLS/WED CSF that is consistent with clinical findings of disruptive sleep, cardiovascular dysfunction and painful symptoms. Moreover, protein profiles are consistent with neuropathological findings of activation of hypoxia inducible factor (HIF) pathways and alterations in dopaminergic systems. These data indicate the CSF of RLS/WED patients may provide information relevant to biological basis for RLS/WED, treatment strategies and potential new treatment targets.
Sleep disorders; Restless legs syndrome/Willis-Ekbom disease; Nitric oxide; Hypoxia inducible factor; Pain
Restless Legs Syndrome/Willis-Ekbom Disease (RLS/WED) is a sensorimotor disorder that affects between 5-10% of the population . Patients who suffer with RLS/WED experience an overwhelming and distressing sensation that forces them to move their legs . Those with moderate to severe symptoms report significant disability, chronically reduced sleep times, 20% reduction in their work productivity, diminished quality of life, and increased depression and anxiety . RLS/WED has also been identified as a risk factor for cardiovascular disease [4-7]. RLS/WED prevalence increases with age and it affects women twice as frequently as men.
Iron dysregulation in the pathogenesis of RLS/WED is supported by a substantial volume of research including several studies reporting decreased ferritin in cerebrospinal fluid (CSF) and decreased brain iron particularly in the substantia nigra using MRI, ultrasound imaging and brain autopsy analysis . A number of studies to date support the concept of diminished brain iron in RLS/WED, thus providing the basis for the hypothesis that RLS/WED occurs as a result of low brain iron content [8-10]. We still do not understand, however, the full consequences that low brain iron may have on neural systems in patients with RLS/WED. We performed proteomic analyses in order identify neural systems and pathways that are altered in those suffering with RLS/WED and possibly identify novel avenues for potential therapeutic intervention.
CSF was utilized for this biomarker study because it is most likely to reflect changes in CNS metabolic status due to its proximity to the brain. An advantage of CSF as the biological fluid for proteomic analyses over blood is that the CSF is sequestered behind both the blood–brain and brain-CSF barriers. This isolation permits the identification of biomarkers that are specific to CNS disease processes. Hence, our goal for this study was to identify CSF biomarkers for RLS/WED using two-dimensional difference-in-gel electrophoresis (2D-DIGE) together with tandem Mass Spectrometric (MS) analysis. To further validate the identified biomarkers, we used immunoblot analyses to quantify differences in protein levels between early-onset RLS/WED and age- and gender-matched control subjects. In this study, we identify six proteins whose levels are altered in early-onset RLS/WED subjects.
Our results reveal a protein profile in the RLS/WED CSF that is consistent with iron deficiency, dopamine dysregulation and inflammation. The APO-A1 finding may be of relevance outside of CNS given the recently reported increased risk of cardiovascular disease in patients with RLS and cardiovascular dysfunction and reports of painful symptoms. The profiles in the CSF are also consistent with neuropathological findings of activation of HIF pathways and alterations in dopaminergic systems. The data indicate that the CSF protein profile, if confirmed in larger sample sizes, may provide support for existing hypotheses about a biological basis for RLS/WED which could prove clinically meaningful in evaluating therapeutic strategies and identifying new targets.
Dr. Allen has in the last 2 years served as a consultant for Boehringer Ingelheim, GlaxoSmithKline, Luitpold Pharmaceuticals, Pfizer, EMD Serono, Pharmacosmos, Neurogen, Jazz Pharmaceuticals and UCB Pharma. He also received research support from GlaxoSmithKline, Pharmacosmos and the USA National Institutes of Health. Dr. Earley was a member of the Data Safety Committee for Phase III clinical trial by Merck. Dr. Connor discloses that he is a paid consultant for GlaxoSmithKline and the International Copper Association and has consulted for Neurogen. Drs. Patton and Cho, and Mr. Clardy report no disclosures.
SMP carried out the 2D-DIGE analyses, identification of biomarker proteins, and drafted the manuscript, YWC recruited, diagnosed and obtained cerebrospinal fluid samples from RLS/WED and control subjects, TC performed the DeCyder-DIA proteein analyses of 2D-DIGE images, RPA participated in study design and coordination, and assisted in the draft of the manuscript, CE participated in study design and assisted in the draft of the manuscript, and JRC participated in the study design and assisted in the draft of the manuscript. All authors have read and approved the final version of the manuscript.
This work was supported by grants from GlaxoSmithKline (#109851) (JRC).