Linkage And Association Studies Of Restless Legs Syndrome

Restless legs syndrome (RLS) is a common neurological disorder characterized by an urge to move the limbs, and is usually accompanied by uncomfortable sensations that worsen with rest, improve with movement, and are worse in the evening or night. It can impair sleep and adversely affects health and quality of life. Epidemiological studies of RLS revealed a prevalence rate of 5%-24% in Caucasian populations, which is much higher than the rate in Asian and African populations (0.1%-3.9%), suggesting a possible role for ethnic factors in the occurrence of RLS. Furthermore, RLS is twice more common in females than in males. RLS is associated with a positive family history in 45% to 65% of patients and twin studies showed a high concordant rate in monozygotic twins4. All these results suggest that genetic factors play an important role in the pathogenesis of RLS. Seven genetics loci for RLS have been reported. These loci were identified by linkage analysis in families, and include RLS1 on chromosome 12ql2-q21, RLS2 on 14q13-21, RLS3 on 9p24-p22, RLS4 on 2q33, RLS5 on 20p13, RLS6 on 19p13 and RLS7 on 16p12.1. To date, no disease-causing mutations from these loci have been reported. Recent genome-wide association studies (GWAS) using 500,000 to a million single nucleotide polymorphisms (SNPs) identified four additional genetic loci that are associated with RLS. The identified GWAS loci are represented by SNPs on chromosome 2p14 (MEIS1), 6p21.2 (BTBD9),15q23(MAP2K5/SKOR1 or MAP2K5/LBXCOR1), and 9p24.1-p23 (PTPRD) which overlaps with RLS3. And the association studies in linkage region RLS1 identified nNOS as a gene being associated with RLS.In this project, I have carried out molecular genetics studies for RLS using linkage analysis and association studies. There are three major areas for this project.Part 1 Linkage analysis was performed in 15 U.S. RLS familiesI performed a genome-wide genetic linkage scan in 15 multiplex families using 400 marker loci with a mean spacing of 10 cM. I used Genehunter Plus to generate linkage statistics, expressed as heterogeneity (HLOD) scores, under dominant and recessive genetic models. I then used fine mapping to characterize the positive linkage regions with additional markers within these regions. Genome-wide linkage analysis showed a linkage signal on chromosome 4 with a recessive inheritance model (HLOD>3) and a suggestive linkage on chromosome 3 and 5 with a dominant inheritance model (HLOD>2). After fine mapping, the 4q34.5-35.1 keeps linkage was defined within a region from 194.3cM to 200cM (HLOD>3). The peak multipoint HLOD was 3.55 and single point HLODmax was 2.54 at D4S171; the maxima multipoint HLOD was 2.2 in the suggestive linkage region on 3q25.1(166.5-169cM) and single point HLODmax was 2.36 at D3S1299; the maxima multipoint HLOD for 5p15.33 locus(1.7-7.7cM) was 2.79 and single point HLODmax was 2.54 at D5S2005. This study identifies one novel RLS susceptibility locus on 4q34.5-35.1 and two suggestive RLS loci on 3q25.1and 5p15.33.Part 2 Association study between PTPRD and RLS in 15 U.S. RLS families and 189cases/560controlsThe PTPRD gene encoding protein tyrosine phosphatase receptor type D became a strong candidate gene for RLS at the RLS3 locus. To test whether PTPRD is associated with RLS, I performed population-based association study using two intronic SNPs rs1975197 and rs4626664 in PTPRD in a case control cohort of 189 patients and 560 controls; I also performed a family-based association study in 15 multiplex families to assess the association between PTPRD and RLS in an American Caucasian population. Moreover direct DNA sequence analysis was carried out to screen coding exons and exon-intron boundaries of PTPRD for rare mutations in 15 probands. The association between RLS and rs 1975197 was significant in the population-based case control association study (allelic P=0.0004, odds ratio=1.68; genotypic P=0.0013 and 0.0003 for an additive and dominant model, respectively). SNP rs4626664 is associated with RLS only in the allelic association study (P=0.023). A family-based sibling transmission disequilibrium test showed association of RLS with SNP rs1975197 (P=0.015), but not with rs4626664 (P=0.622). One rare p.E1639D variant was identified in exon 39 in kindred RLS40005. The rare D1639 allele did not co-segregate with RLS in the family, suggesting that p.E1639D variant is not a causative mutation. This represents the first independent study to validate the association between PTPRD variants and RLS. Both family-based and population-based association studies suggest that PTPRD variant rs1975197 confers risk of RLS.Part 3 Association study between MEIS1, BTBD9, MAP2K5/SKOR1 and RLS in 38 U.S. RLS families and 189 cases/560 controlsRecent GWAS identified three additional loci for RLS. But, GWAS results need to be replicated in as many populations as possible. We assessed these GWAS loci using the same population as used for the PTPRD study. We investigated three variants, rs2300478 in MEIS1, rs9357271 in BTBD9, and rs1026732 in MAP2K5/SKOR1 in 38 RLS families and 189 RLS patients/560 controls from the U.S. for their association with RLS. Both family-based and population-based case-control association studies were carried out. Case-control association studies showed significant association between all three variants and RLS (P=0.0001/OR=1.65, P=0.0021/OR=1.11, P=0.0011/OR=1.14 for rs2300478, rs9357271, and rs1026732, respectively). The family-based study showed that SNP rs1026732 in MAP2K5/SKOR1 was significantly associated with RLS (P=0.01). In conclusion, variants in MEIS1, BTBD9, and MAP2K5/SKOR1 confer a significant risk of RLS in a U.S. population. The study provided evidences to support the association of MEIS1, BTBD9, and MAP2K5/SKOR1 and RLS in an American Caucasian population. This is the firstly family-based association study to identify the association between MAP2K5/SKOR1 and RLS.