Restless leg syndrome is a chronic sensorimotor condition clinically defined and diagnosed by the presence of the following clinical criteria: [1] an urge to move legs, usually but not always associated with uncomfortable sensations, [2] rest worsening symptoms, [3] improvement of symptoms with gyration, movement or activity, [4] evening worsening of symptoms and [5] exclusion/denial of another etiology for symptoms (1). It is a common disorder with studies showing a prevalence in Caucasian populations of between 5-10% depending on the geographical distribution of populations studied, with an incidence of as low as 1% in Asian populations. Women were twice more likely to be affected than men (2-5). 2 phenotypes of the disorder are seen. The first is a familial, early-onset or primary restless leg syndrome with a peak incidence of 20-40 years. The second is a late-onset restless leg syndrome with a peak incidence after 40 years of age and is associated with rapid progression as well as other comorbidities such as Parkinson’s disease (6).
The pathophysiology of restless leg syndrome is not well understood. Studies to elucidate the pathophysiological basis of restless leg syndrome have found abnormalities in several systems. It is likely that there are multiple pathways to this disease. Predominant of these studied abnormalities have been in iron and dopamine. Recently, well-defined genetic factors have been unveiled by genome wide association studies. The increased prevalence of restless leg syndrome in iron deficiency populations (7) and the therapeutic efficacy of dopamine agonists in the treatment of restless leg syndrome (8) have driven extensive studies into the role of these 2 systems in relation to restless leg syndrome pathophysiology. Other systems that have been studied include opioid systems, glutamate, and hormones.
In this essay, we will look at the evidence behind these pathophysiological theories of restless leg syndrome and assess their merit. We will concentrate on the most credible and well-studied of these pathophysiological mechanisms, namely those of iron deficiency, dopamine and genes.
Iron deficiency
Since Norlander (6) first described an association of iron deficiency and restless leg syndrome in 1953, the most consistent observation in pathophysiological studies of restless leg syndrome has been the presence of reduced iron stores in the central nervous system. All conditions that compromise iron levels have been associated with increased risk of restless leg syndrome (eg. Pregnancy, end-stage renal failure). Iron is an essential element that is involved in many functions in the central nervous system including oxygen transport, the synthesis of neurotransmitters, organisation of neural networks and production of myelin (9). Allen et al studied 251 subjects with iron-deficiency anaemia and reported a prevalence clinically significant restless leg syndrome to be 23.9%, 9 times that of the general population (10). Although many patients with restless leg syndrome have normal levels of serum iron, it is the central iron stores that seem to be related to restless leg syndrome. Lower serum ferritin levels correlate with greater restless leg syndrome symptom severity but are also often normal in restless leg syndrome patients. 2 studies were consistent in reporting an inverse correlation between restless leg syndrome severity and serum ferritin levels (11,12). Ferritin is an iron storage and binding protein. In Allen et al’s study, it was suggested that patients with restless leg syndrome are less symptomatic if serum ferritin levels were greater than 50 mcg/l (11). This is a current level that many clinicians still use as a yardstick for treatment.
Reduced iron stores in the central nervous system has been demonstrated in neuroimaging studies, neuropathologic studies and studies analysing cerebrospinal fluid (CSF). In 2001, Allen et al used MRI measurements to assess iron concentrations in the brains of 10 subjects (5 with restless leg syndrome and 5 controls). In subjects with restless leg syndrome, there was reduced iron concentration seen in the substantia nigra and in the putamen. The reduction of iron stores in these regions were proportional to symptom severity (13). A subsequent study done by the same group in 41 subjects with restless leg syndrome bore consistent results, especially in early onset restless leg syndrome subjects (14). A similar MRI study performed by Astrakas et al in 2008 on 25 restless leg syndrome reported consistent results (15). Brain ultrasonography utilising iron echogenicity first reported by Gouda et al, has also been able to identify changes consistent with reduced iron stores in the substantia nigra (16, 17) Mutiple studies from different groups using different imaging modalities have identified consistent results in reduction of iron concentration in the brain. The most consistent regions showing reduced iron are the substantia nigra, and to a lesser extent, the putamen and caudate. Recently, Rizzo et al demonstrated similar findings of reduced iron in the thalamus (51). These findings suggest that iron deficiency is more regional than global and the affected areas include iron rich regions such as the substantia nigra and iron poor areas such as the thalamus. The pathophysiology would then seem to be a regional iron deficiency in the brain in an otherwise normal iron status.
Postmortem neuropathological studies would also be consistent with these findings. In 2003, Connor et al performed a neuropathologic evaluation of 7 brains from subjects with restless leg syndrome and 5 age-matched controls. They found that iron and H-ferritin (iron storage protein) staining was greatly reduced in the substantia nigra of restless leg syndrome patients (18). Interestingly, the levels of L-ferritin was not increased. H-ferritin is responsible for iron transport and storage, more so than L-ferritin. A subsequent study on neuromelanin cells from the substantia nigra of 4 brains from patients with restless leg syndrome and 4 control brains again showed reduced iron and H-ferritin staining, but also increased transferrin stains and reduced transferrin receptors in restless leg syndrome neuromelanin cells compared with controls (19). The decreases were mainly in iron intake and storage proteins. There was no change in ferropotin, a protein responsible for iron export. Additionally, the authors analysed the activity and concentration of iron regulatory proteins (IRP) in patient and control samples. They found a decrease in total IRP activity, IRP1 activity, and IRP1 protein levels in patient samples. IRB1 is responsible for the regulation of ferritin molecule production and transferrin receptor mRNA stability. The reduction of IRB1 protein levels would therefore affect the normal homeostatic mechanism of intracellular iron regulation leading to reduced iron store seen in the CNS. There is a pattern of impaired iron transport into the brain cells and specifically the neuromelanin cells of the substantia nigra (52).
CSF levels of ferritin have also been shown to be decreased in restless leg syndrome patients (20). Clardy et al used immunoblot analysis of the CSF of restless leg syndrome subjects and controls. They found significant reduction of ferritin levels in early onset restless leg syndrome subjects. This led the authors to propose that CSF levels of ferritin may be used as a biomarker to assist the diagnosis of restless leg syndrome. CSF transferrin has also been observed to be raised in restless leg syndrome subjects (21).
One theory behind these findings is that in restless leg syndrome, regional brain iron deficiency is caused by an inability to transport iron across the blood-brain barrier, and also the inability to import iron into important regulatory cells such as the neuromelanin cells in the substantia nigra. While the reduction of iron stores in the CNS of restless leg syndrome subjects has been established, what has not been established is a clear pathophysiological pathway of how this reduction of iron would lead to the symptoms of RLS. It may very well be that the reduction of iron is a secondary effect rather than a causal one. 2 theories have been put forth: the hypoxia model and the myelin loss model. Iron plays a critical role in oxygen transport and the hypothesis is that iron deficiency should lead to a reduction of oxygen transport and hypoxia. One neuropathological study showed hypoxia inducible factor-1α levels were raised in the neurons of the substantia nigra in 5 of 6 restless leg syndrome patients as compared with controls (53). This was evidence that hypoxic pathways are activated in the substantia nigra of restless leg syndrome subjects. However, as stated earlier, whether the activation of the hypoxic pathway led to iron deficiency or result from iron deficiency remains unclear. Iron is involved in myelin synthesis and a postmortem and imaging study showed reduction in white matter volume of restless leg syndrome subjects in the precentral gyrus, anterior cingulum and corpus collosum. Using Western blot analysis the authors also showed reduction of transferrin and H-ferritin in the myelin shealths of restless leg syndrome subjects as well as a 25% reduction in myelin proteins (54). There may be a role of myelin deficit in the symptomatology of restless leg syndrome.
Dopamine
Iron is an essential cofactor of tyrosine hydroxylase (TH), the rate limiting enzyme of L-dopa synthesis. Therefore iron and dopamine metabolism in the brain are closely related to each other. The dramatic clinical improvement in restless leg syndrome symptoms to dopamine-based medications have been well studied and has led to a belief that restless leg syndrome is due to a deficiency in brain dopamine. Scholz et al published the results of a meta-analyses of 35 placebo controlled and 3 active controlled studies to assess the efficacy of dopamine agonists in the treatment of RLS. They found that mean reduction on the International RLS Severity Rating Scale (IRLS) was significantly lower (5.7 points) in dopamine agonist treatment groups compared to placebo. There was also statistically significant improvement in self-reported quality of sleep and disease specific quality of life (22). By this observation, we could infer that RLS is characterised by a hypodopaminergic state. However, there is no evidence to suggest that there is a dopaminergic deficiency in RLS patients. In fact there may be evidence in human studies that suggest that RLS is a hyperdopaminergic state. If iron is a co-factor of tyrosine hydroxylase, one would expect that iron deficiency in the brain would result in a decrease in tyrosine hydroxylase. In a human autopsy study done by Connor et al on 8 RLS patient brains, they found that there was an unexpected increase in tyrosine hydroxylase in the substantia nigra of restless leg syndrome patients. With regards to the quantitative profile of the other components of the dopaminergic system, there were no significant differences in dopamine D1 receptor, dopamine transporters or vesicular monoamine transporter. However, dopamine D2 receptor levels were significantly lower in the putamen of RLS patients. The increase in tyrosine hydroxylase and decrease in dopamine D2 receptors suggest a hyperdopaminergic state with increased production of L-dopa and downregulation of dopamine receptors (23). Other neuropathological studies had similar findings and did not show any reduction of dopamine cells or dopamine in the striatum of substantia nigra (18, 24).
Nulcear imaging studies of dopamine activity in the brain reveal inconsistent results. In 1995, Staedt et al performed a single photon emission tomography (SPET) study using a highly specific dopamine D2 receptor ligand in 20 patients with periodic limb movement in sleep and 10 controls. There was lower binding of this ligand in the striatal structures compared to controls, indicating a downregulation of dopamine receptors in these patients (25). Trenkwalder et al performed a FDG and FDOPA positron emission tomography study on 6 patients with restless leg syndrome and matched them with controls. They found no significant differences for regional blood flow values or for any binding constants (26). In another PET scan study done by Turjanski et al on 13 RLS patients, there was a reduction in the mean dopamine D2 receptor binding in the caudate and putamen of restless leg syndrome patients as compared to controls (27). However, these findings were not consistent with a 2004 study by Tribl at al who found no difference in dopamine 2 receptor function and levels in restless leg syndrome and control groups (28). These neuroreceptor studies have not provided any consistent findings as to the status of dopamine precursors, dopamine transporters or dopamine receptors in the brains of patients with restless leg syndrome.
CSF analysis studies have shown increased levels of 3 ortho-methyldopa (3-OMD) in restless leg syndrome patients. Allen et al studied the CSF of 49 restless leg syndrome patients and 36 matched controls and found significantly raised levels of 3-OMD in the CSF of restless leg syndrom patients. The increase in 3-OMD levels were proportionate with severity of symptoms (29). Since 3-OMD is a product of the metabolism of L-dopa, these findings indicate an increase in tyrosine hydroxylase activity and dopamine metabolism, which would support the theory of a hyperdopaminergic state. The same authors found in a separate study that CSF 3-OMD levels in restless leg syndrome patients also demonstrated a circadian pattern. While in normal controls, 3-OMD levels were found to be slightly increased at night, restless leg syndrome patients exhibited a reduction of 3-OMD levels of about one-third. This reduction of 3-OMD would indicate a relative dopamine deficiency state on a background of a hyperdopaminergic state in restless leg syndrome patients (30).
However, if RLS is a hyperdopaminergic state, why does treatment with dopamine agonists reduce RLS symptoms? A dopamine dysregulation theory exists to explain this phenomenon. This theory is heavily based on the findings of a strong circadian pattern of both dopaminergic activity and restless leg syndrome symptoms. In a hyperdopaminergic state, the increased dopaminergic activity would lead to a downregulation of dopamine receptors and internal cellular function. This was observed in some of the neuropathological and nuclear imaging studies mentioned above, where decreased dopamine d2 receptors were observed. Although a baseline hyperdopaminergic state exists, there is a circadian pattern to dopaminergic activity in restless leg syndrome, with a relative reduction of activity in the evening and night, reflecting the increase in severity of symptoms. The increase in dopaminergic activity in the day is adequate to resolve any symptoms. However, the dopamine receptor downregulation and the nocturnal low dopamine activity combine to create a temporary state of dopamine deficiency at night. Treatment with dopamine agonists helps relieve this deficiency and hence improve symptoms. However the treatment with dopamine agonists may not provide lasting symptom improvement. With exogenous dopamine agonists supplementing dopamine activity, dopamine receptors may be downregulated even further, leading greater dopamine deficiency at night and the worsening and prolongation of symptoms. This will lead to increased exogenous dopamine requirements. This theory may be the pathophysiological basis for the observation of augmentation. Augmentation in restless leg syndrome is the clinical phenomenon where continuing treatment with dopamine agonists leads to a worsening of symptoms and symptoms occurring earlier in the day. The dopamine dysregulation theory and augmentation were described in papers by Earley and Allen (31, 32).
Genetic pathophysiology
Due to a well-defined phenotype and diagnostic criteria, we know that RLS is often familial, with a family history being present in 40-60% of cases (33, 34). Most pedigrees show an autosomal dominant pattern of inheritance (35-37), and there is a high concordance rate in twin studies (38,39). Recent genome wide association studies (GWAS) have shown positive genetic findings in RLS. They have discovered significant associations for single nucleotide polymorphisms (SNPs) in the genomic regions for MEIS1, BTBD9, PTPRD, MAP2k5/SKOR1, and TOX3/BC034767 (40-42). Periodic leg movements in sleep (PLMS) was also found to be associated with SNPs in the above-mentioned genes related to RLS (40). PLMS commonly occurs in patients with RLS and has been described by Winkelman as an endophenotype of RLS (43). We will examine 2 well studied genes and the evidence that they are linked to RLS.
The BTBD9 gene was initially discovered to be associated with RLS through a GWAS conducted on study populations in both Iceland and the United States (44). There was a observed genome wide significant association with a common variant in the BTBD9 gene. In this study, the variant was associated with a population attributable risk of RLS with PLMS of approximately 50%. Later GWAS by other groups on different populations in Canada and Europe would confirm this association of BTBD9 to RLS (41,45). This variant was located in an intron of BTBD9 on chromosome 6p21.2. The precise role of BTBD9 is not understood, but it has been implicated in a wide range of cellular functions including cytoskeleton dynamics, transcriptional regulation, ion channel assembly and ubinquination of proteins (46). Animal models have been used to study the implications of BTBD9 gene abnormalities. Of particular interest were the effects on dopamine and iron systems. DeAndre et al generated BTBD9 mutations in mice and observed motor restlessness, sensory alterations in rest phase with reduced sleep and increased wakefulness in these mice. They also found altered serum iron levels and monoamine neurotransmitter systems. There was also relief of the sensorineural symptoms with the administration of a dopaminergic agonist (47). Further studies were performed on Drosophilia flies utilising mRNA interference knockdown of the BTBD9 gene. The observed effects were sleep fragmentation and restlessness with a 50% reduction of brain dopamine seen in these knockdown flies compared to wild strains. However, isolated knockdown of dopaminergic neurons did not yield the same effect, implying an indirect process that is not completely understood. Again, as was seen in mice, when given a dopamine agonist, there was reversal of symptom levels to that of control subjects (48).
Another gene that has been associated with a 50% attributable risk of RLS is the ME1S1 gene (41). ME1S1 has been found to be involved in leukomogenesis and haematopoiesis
and is located on the short arm of chromosome 2 (2p14) (49). In an animal study performed on worms, mRNA interference knockdown of the ME1S1 gene increases ferritin expression in RLS brain tissues. The same group also found that human cells cultured under iron-deficient conditions show reduced MEIS1 expression.
Discussion
We have detailed the 3 most well-studied pathophysiologic factors involved in restless leg syndrome. These are the key points:
Brain iron deficiency occurs in restless leg syndrome
Hyperdopaminergic states exist in restless leg syndrome
There are well-defined genetic factors in restless leg syndrome
While the evidence for central iron deficiency is credible, the pathophysiology of the occurrence of this observation is not. We still do not understand how these states of iron deficiency and increased dopamine activity develop. Recently, with GWAS, genetic factors have been identified. It is rare in sleep disorders that such well-defined genetic factors have been identified and this presents an excellent opportunity to study. The presence of 5 associated specific genomic regions would mean that restless leg syndrome is likely to be more complicated than what has been presented. It is likely to be an interplay of different pathways, multiple biologic systems and more than one or two molecules.
Future directions of research should include
The presence or absence of circadian rhythm of brain iron
Iron treatment for restless leg syndrome
The interaction of other biological systems with dopaminergic systems (eg. glutamatergic, adenosinergic)
Anatomical locus for symptoms
Further genetic knockdown models involving genes other than BTBD9 and MES1S.
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