Abstract
In order to examine the genetic substrate of the dopamine hypothesis in restless legs syndrome, we analyzed eight genes coding for receptors and enzymes related to dopaminergic transmission, using a population of 92 patients with restless legs syndrome and 182 controls matched for ethnic background. No significant differences were found in the genotypic or allelic distributions between groups. Furthermore, no effect of the loci examined was observed with stratification using clinical parameters such as age at onset or periodic leg movements during sleep index.
Restless legs syndrome (RLS) is a common disorder characterized by an irresistible desire to move the extremities associated with paresthesia. These symptoms, occurring predominantly at rest and relieved by movement, are worst at night, resulting in nocturnal insomnia and chronic sleep deprivation. Despite recurrent reports suggesting a genetic contribution to the etiology of the idiopathic form of the syndrome, few molecular genetic studies have been carried out trying to identify genes that may predispose to RLS.
Although the pathogenesis of RLS remains unknown, a growing literature suggests the involvement of the dopaminergic (DA) system in the etiology of this syndrome. The clinical improvement observed with agents increasing DA transmission (see review1) as well as evidences provided by brain imaging studies2 support the hypothesis of a central dopaminergic dysfunction in RLS.
In an attempt to test for a possible genetic role of the DA system in RLS, we performed a population-based case-control study focusing on the French Canadian population. We examined eight relevant candidate genes involved in dopaminergic transmission and metabolism: DA-receptors D1 to D5, dopamine transporter (DAT), tyrosine hydroxylase (TH), and dopamine β-hydroxylase (DBH) (table 1).
Investigated candidate genes
Methods.
Subjects.
A total of 92 unrelated patients (mean age: 56.47 ± 11.69; range, 34 to 82 years) were recruited from the Sleep Disorder Center at the Hôpital du Sacré-Coeur, Montréal.
Initial diagnoses were made by two physicians with 10 years of experience in the evaluation of sleep disorders and all cases were personally reviewed by one of the co-authors (J.M.). Diagnostic criteria were based on those developed by the International RLS Study Group and included 1) leg paresthesia associated with an urge to move; 2) motor restlessness; 3) increased severity during the evening and/or at night, and 4) worsening of symptoms at rest with partial relief by movement. Exclusion criteria included presence of conditions known to be associated with RLS such as renal failure, peripheral neuropathy, anemia, and rheumatoid arthritis. None of the subjects reported the use of medication known to affect sleep, sensory, or motor functions. The presence of other sleep disorder such as sleep apnea syndrome, REM sleep behavior disorders, or narcolepsy was ruled out by interview and appropriate polysomnographic (PSG) recordings. All patients reported symptoms of RLS at least 3 nights per week for more than 6 months. PSG recordings were carried out on all patients to document sleep parameters and periodic leg movements during sleep (PLMS).
Control subjects consisted of 182 unrelated individuals. Among these, 12% were spouses documented to be free of RLS symptoms. The remainder comprised healthy controls who have not been assessed for the presence of sleep disorders. Only patients and comparison subjects of French Canadian origin were included in the current study. French Canadian ancestry was defined as having four grandparents of French Canadian origin by history. Written informed consent was obtained from all participants and the study was approved by the local ethics committee.
Nocturnal PSG recordings.
PLMS were scored according to standard criteria (i.e., increases in electromyographic [EMG] signal in anterior tibialis muscles occurring in series of at least four consecutive movements lasting 0.5 to 5 seconds with an interval between 4 and 90 seconds). The PLMS index was defined as number of periodic leg movements per hour of sleep.
Genotyping.
Genomic DNA was extracted following standard procedures. DNA was amplified and genotyped by PCR using primer sequences and conditions previously described (see table 1). All PCR and digestion products were separated on a 2% agarose gel and visualized by ethidium bromide staining under ultraviolet light. Genotype readings were carried out blind to affection status.
Statistical analysis.
Patients were categorized into three or more groups on the basis of their genotypes. Different clinical features were considered as dependent variables and we examined their distribution across alleles and genotypes. These parameters include RLS age at onset and PLMS index. The clinical characteristics of the patients regarding allelic frequencies were compared using either the Student’s t-test or the Mann–Whitney U test, whereas associations with respect to genotype frequencies were estimated using either one-way analysis of variance (ANOVA) or Kruskal–Wallis ANOVA, depending on the nature of the data. The χ2 test was performed to compare patients with controls and to compare subgroups of patients as well as to assess deviation of the genotype frequencies from Hardy–Weinberg equilibrium. Bonferroni’s corrections were carried out for multiple analyses.
Results.
Demographic and clinical data regarding the patient population are summarized in table 2. For the population studied, there was no difference between patients and controls with regard to sex ratio (χ2 = 0.957; df = 1; p = 0.328). The mean age at onset of RLS was 28.35 ± 14.31 and the mean PLMS index was 36.8 ± 31.52. Although the mean PLMS index was considered as pathologic for the whole group (i.e., >5/hour of sleep), 10% of patients presented a PLMS index within the normal range whereas 86% showed an index greater than 10. These observations concur with data published previously.
Demographic, clinical, and polysomnographic characteristics for the population studied
For all the loci investigated, genotype distributions did not deviate significantly from the expected values at the Hardy–Weinberg equilibrium for either control or patient groups. As shown in table 3, for all polymorphisms studied, between-group differences in genotype frequencies were small and not significant. Similarly, no significant differences were found with regard to allele frequencies distributions when we compared proband and control groups in the overall sample (table 3).
Genotype and allele frequencies for all genes studied
When investigating the alleles and genotypes distribution for age at onset of RLS and PLMS index, no significant association with any of the genes examined was found according to these clinical features. When examining exclusively patients with positive family history, individuals carrying the DA receptor D3 serine allele presented a higher PLMS index in comparison to those having the glycine allele (U test; p = 0.035). However, this difference becomes nonsignificant following Bonferroni’s correction for multiple testing.
Discussion.
This work represents the first study to examine the influence of DA candidate genes on RLS. Comparison of allele and genotype frequencies between cases and controls suggest that these loci have no major effect on vulnerability to RLS. Moreover, stratification analyses according to age at onset and PLMS index disclosed no significant differences for any of the polymorphisms examined.
These results indicate that, at least in the sample studied, DA receptors and DAT as well as TH and DBH loci are not directly involved in the etiology of RLS. From this, our findings do not provide evidence toward a genetic involvement of the dopaminergic system in RLS symptomatology. However, alternative explanations should also be considered. Indeed, it is possible that DA genes confer only a minor predisposition to RLS. If this is the case, we would have been unable to detect a positive association even though our sample had a power to detect gene effects of relatively small sizes (i.e., assuming a frequency of exposure among controls of around 20%, our study has 80% power to detect an association accounting for an OR of around 2.3).
The importance of DA in the pathogenesis of RLS cannot be ruled out as other loci implicated in the dopaminergic system have not yet been examined. Moreover, the possibility remains that other functional polymorphisms that are not in linkage disequilibrium with those investigated might affect the occurrence or the outcome of the syndrome. However, our conducting this study in a recently founded population makes this explanation unlikely. Further work is currently in progress in our laboratory to investigate other genetic factors that may confer susceptibility to RLS.
Acknowledgments
A.D. and M.M. are supported by CIHR studentships. G.A.R., G.T., and J.M. are supported by the CIHR. This research is supported in part by NIH (1R01NS37754–01A1) and by CIHR (MOP-13365).
Acknowledgment
The authors thank all the subjects who participated in this study. They also thank Sylvie Toupin, Annie Ouellet, Mireille Charron, and Daniel Filipini, MD, for their valuable work with patients’ recruitment, and Jesse Carlevaris for technical assistance.
- Received March 29, 2001.
- Accepted July 14, 2001.
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