*These authors contributed equally to this work.
Restless legs syndrome (RLS) is a highly heritable and common neurological sensorimotor disease disturbing sleep. The objective of study was to investigate significant gene for RLS by performing GWA and replication study in a Korean population.
We performed a GWA study for RLS symptom group (n=325) and non-RLS group (n=2,603) from the Korea Genome Epidemiology Study. We subsequently performed a replication study in RLS and normal controls (227 RLS and 229 controls) to confirm the present GWA study findings as well as previous GWA study results.
In the initial GWA study of RLS, we observed an association of rs11645604 (OR=1.531, p=1.18×10−6) in
From our sequential GWA and replication study, we could hypothesize rs9390170 polymorphism in
Restless legs syndrome (RLS), also known as Willis-Ekbom disease (WED), is a common neurological sensorimotor disease that disables sensory and motor elements. RLS is diagnosed by: 1) an urge to move the legs usually but not always accompanied by or felt to be caused by uncomfortable and unpleasant sensations in the legs; 2) the urge to move the legs and any accompanying unpleasant sensations begin or worsen during periods of rest or inactivity such as lying down or sitting; 3) the urge to move the legs and any accompanying unpleasant sensations are partially or totally relieved by movement, such as walking or stretching, at least as long as the activity continues; 4) the urge to move the legs and any accompanying unpleasant sensations during rest or inactivity only occur or are worse in the evening or night than during the day; and 5) the occurrence of the above features is not solely accounted for as symptoms primary to another medical or behavioral condition (e.g., myalgia, venous stasis, leg edema, arthritis, leg cramps, positional discomfort, habitual foot tapping).
A familial inclination has been reported, particularly in early-onset RLS.
Although there have been various genetic association studies of RLS from various ethnic groups, all of the GWA studies of RLS were performed only in Caucasian populations. Because there are possible ethnic differences in genetic influence, it would be advantageous to perform GWA studies of RLS in ethnically diverse populations.
We performed a GWA study of RLS to find susceptibility genes for RLS in Korean cohort samples, and tried to replicate the results from our study as well as the previous GWA studies in Caucasian populations in the independent clinical samples of RLS and non-RLS controls in a Korean population.
Subjects for the GWA study were collected from the Ansung and Ansan cohorts of the Korea Association Resource (KARE) project: 7,515 participants ranging in age from 40 to 69 years. These cohorts were investigated as part of the Korea Genome Epidemiology Study (KoGES). Epidemiological information of the cohorts contains more than 260 traits, including demographic information, medical and family disease histories, health condition, and lifestyle such as sleep pattern.
Genomic DNAs for the GWA study were extracted from peripheral blood obtained from Ansung and Ansan cohort participants for genotyping with the Affymetrix Genome-Wide Human SNP array 5.0. A total of 7,515 samples were genotyped, leading to an initial identification of 500,568 SNPs. An exclusion process was subsequently performed based on the quality control results such as high missing genotype call rate (>4%), high heterozygosity (>30%), gender inconsistencies, individuals who had developed any type of cancer, and higher computed average pairwise identity-by-state values than those estimated from first-degree relatives of Korean sibpair samples (>0.80). In addition, markers with high missing gene call rate (>5%), significant deviation from Hardy-Weinberg equilibrium (p<1×10−6), or low minor allele frequency (<0.01) were excluded for quality control. After the quality control process, a total of 352,228 markers in 6,675 subjects remained.
A total of 467 subjects were genotyped for the replication study of GWA study findings. Blood was collected in 5-mL EDTA vacutainers (REF 367525, BD, Plymouth, UK) from study participants and stored at -80℃ until genotyping. Genomic DNA was extracted using the Blood DNA Purification Kit (CMB0115, Cosmo Genetech Co. Ltd., Seoul, Korea). After quality assessment with the NanoDrop spectrophotometer (ND-1000, NanoDrop Technologies, Wilmington, DE, USA) and polymerase chain reaction (PCR), 250 ng of each DNA were genotyped by high resolution melting (HRM) analysis using a real-time PCR system with SsoFast EvaGreen Supermix (172-5205AP, Bio-Rad Laboratories, Hercules, CA, USA) and appropriate primer pairs for each SNP. The Hardy-Weinberg equilibrium test was performed to assess the goodness of fit of the data.
We identified the RLS symptom group and non-RLS group for GWA. Among the 6,675 individuals remaining after quality control, the case and control groups were identified based on answers to a questionnaire regarding RLS symptoms. Individuals were asked four RLS-related questions based on the following situations: 1) an urge to move the legs, usually accompanied or caused by uncomfortable and unpleasant sensations in the legs, 2) the urge to move or unpleasant sensations beginning or worsening during periods of rest or inactivity such as lying or sitting, 3) the urge to move or unpleasant sensations are partially or totally relieved by movement, such as walking or stretching, at least as long as the activity continues, and 4) the urge to move or unpleasant sensations are worse in the evening or nighttime than during the day or only occur in the evening or night.
Our replication study samples were consistent with RLS clinical case and non-RLS matched controls. All the RLS cases and controls were assessed using the International Restless Legs Syndrome Study Group diagnostic criteria. RLS subjects with medical history that may cause RLS-like symptoms similar to those above were excluded from sample collection in the replication study. Finally, there were 227 subjects in the RLS cases and 229 control subjects.
To infer the genotypes for missing markers, the data were imputed with HapMap3 (release #2, Feb 2009) reference panel by using IMPUTE2 software.
Linkage disequilibrium (LD) blocks were identified from 1,000 Genomes Phase3 JPT+CHB [PMID:26432245] using Haploview software [PMID:15297300]. The proxy SNPs with regional recombination rates were assessed using SNAP [PMID: 18974171] (ver. 2.2; the Genomics Platform at the Broad Institute, Cambridge, MA, USA).
For the genes harboring SNPs with significant association (p<0.005), we performed functional enrichment analysis using Gene Ontology categories in DAVID software.
We performed a GWA of RLS symptom group vs. non-RLS group to identify SNPs that are associated with RLS in a Korean population. A total of 96 associated SNPs with p<10−4 were found in the present GWA study. Of these, rs11645604 [OR=1.531, p=1.18×10−6 (permuted p=1.0×10−6)], rs1918752 [OR=0.6582, p=1.93×10−6 (permuted p=2.0×10−6)], and rs9390170 [OR=0.6778, p=7.67×10−6 (permuted p=5.0×10−6)] showed the most significant associations (
Next, we identified two genomic regions of interest (ROIs) on chromosomes 6 and 16, defined as regions containing at least two SNPs with p<10−4 and adequate support for association (i.e. p<10−3) from surrounding SNPs within 100 kb. From the identified ROI of the present GWA study, ROI1 and ROI2 contain the most associated genes,
Next, we performed functional enrichment analysis to determine whether the associated SNPs show enrichment in certain functional pathways using 887 genes from SNPs associated with p<0.005. As shown in
To confirm the results of the present GWA study, we performed a case-control association analysis for RLS within independent samples that were collected from our research centers. We selected rs11645604 (OR=1.531; p=1.18×10−6) in
The KARE project has been conducted to identify genetic factors associated with various diseases and traits in Korea. We performed a GWA study for RLS within the KARE project, and confirmed the results of the present GWA study by performing a replication study based on the results of variant genes identified in the initial GWA study. There were several findings of the GWA study for RLS that are worthy of close attention. However, virtually all previous GWA findings were performed and replicated within Western subject populations, and no GWA studies had been conducted for RLS in Asian populations. It is crucial to note that this is the first GWA study for RLS with a replication study conducted within an Asian population. The reliability of the replication study increased because it confirmed the results of the present GWA study and simultaneously verified the previous GWA study findings.
We found a novel susceptibility locus for RLS, rs9390170 in
Three primary methods to determine the pathophysiology of RLS have typically been performed: 1) localizing the areas of abnormal central nervous system (CNS) function, 2) finding the neurotransmitter systems associated with medications (dopaminergic), and 3) finding the relationship between iron deficiency and RLS.
Winkelmann et al.
There are several limitations. First, experts did not make a diagnosis of RLS by face-to-face treatment. The questionnaire was used to determine whether or not RLS symptoms were present. Based on our clinical experience, it is important for RLS diagnosis to be identified directly by the clinician. When using a simple subjective questionnaire for RLS, there might be problems of false-positive or -negative because it is difficult for patients to report their RLS symptoms clearly. This study was conducted for a large-scale cohort, and we classified the RLS symptoms group according to the core symptoms of RLS criteria, after exclusion of medical conditions which may show symptoms similar to RLS. Therefore, it should be noted that the results of this study are limited in interpretation. However, since the study has been replicated in different sample, we believe that the results are valuable. Second, the replication sample was relatively small in size. Although it has modest power, subsequent studies with a larger number of samples are needed to increase the statistical power of genetic studies. Third, the significant results from the replication study lose their statistical power after using Bonferroni correction for multiple testing. Although genetic studies performed with sex and age matched healthy controls, the interpretation is limited because it is an uncorrected result for multiple testing.
To our knowledge, this is the first GWA and replication study performed in Asia for RLS. Although there have been no investigations into the direct relationship between utrophin and RLS, we can hypothesize that utrophin is involved in the pathogenesis of RLS, particularly as it relates to the neuromuscular system. Moreover, we anticipate that utrophin can be the link between the distinct known pathogenesis of RLS such as dopamine neurotransmission and iron deficiency. Further investigation into the function and role of utrophin as a causative factor in the pathogenesis of RLS should confirm our findings and hypothesis. Based on the results of the first GWA study and the replication study of RLS in a Korean population taken together with the evidence for a potential relationship between utrophin and RLS, we suggest the existence of a susceptibility locus, rs9390170 on 6q24, for RLS.
The GWAS samples were provided with biospecimens and data from Korean Genome Analysis Project (4845-301), the Korean Genome and Epidemiology Study (4851-302), and Korea Biobank Project (4851-307, KBP-2013-000) that were supported by the Center for Disease Control and Prevention, Republic of Korea.
This study was supported by the Korea Health 21 R&D Project funded by the National Research Foundation of Korea (NRF-2016M3C7A1904345).
The online-only Data Supplement is available with this article at
Regions of interest (ROIs) with enriched association in the RLS symptom group vs. non-RLS group analysis
GO: gene ontology
*p<0.05. SNP: single-nucleotide polymorphism, RLS: restless legs syndrome
*p<0.05. RLS: restless legs syndrome