Rare variants in restless legs syndrome. E. C. Schulte1,2, M. Kousi3, B. Schormair2,4, F. Knauf2, P. Lichtner2, C. Trenkwalder5,6, B. Högl7, B. Frauscher7, K. Berger8, I. Fietze9, N. Gross1, M. Hornyak1,10,11, K. Stiasny-Kolster12,13, W. Oertel13, C. G. Bachmann14, W. Paulus15, A. Zimprich16, A. Peters17, C. Gieger18, T. Meitinger2,4, B. Müller-Myhsok19, N. Katsanis3, J. Winkelmann1,2,4 1) Neurologische Klinik und Poliklinik, Technische Universität München, Munich, Germany; 2) Institut für Humangenetik, Helmholtz Zentrum München, Munich, Germany; 3) Center for Human Disease Modelling, Department of Cell Biology, Duke University, Durham, NC, USA; 4) Institut für Humangenetik, Technische Universität München, Munich, Germany; 5) Paracelsus Elena Klinik, Kassel, Germany; 6) Klinik für Neurochirurgie, Georg August Universität, Göttingen, Germany; 7) Department of Neurology, Medizinische Universität Innsbruck, Innsbruck, Austria; 8) Institut für Epidemiologie und Schlafmedizin, Westfälische Wilhelms Universität Münster, Münster, Germany; 9) Zentrum für Schlafmedizin, Charité Universitätsmedizin, Berlin, Germany; 10) Interdisziplinäres Schmerzzentrum, Albert-Ludwigs Universität Freiburg, Freiburg, Germany; 11) Algesiologikum München, Munich, Germany; 12) Schlaflabor Marburg, Marburg, Germany; 13) Klinik für Neurologie, Philipps Universität Marburg, Marburg, Germany; 14) Abteilung für Neurologie, Paracelsus Klinikum Osnabrück, Osnabrück, Germany; 15) Abteilung für Neurophysiologie, Georg August Universität, Göttingen, Germany; 16) Department of Neurology, Medizinische Universität Wien, Vienna, Austria; 17) Institute of Epidemiology II, Helmholtz Zentrum München, Munich, Germany; 18) Institute of Genetic Epidemiology, Helmholtz Zentrum München, Munich, Germany; 19) Max-Planck Institut für Psychiatrie München, Munich, Germany.

   Background: Restless legs syndrome (RLS) is a common and genetically complex neurologic disorder characterized by nightly dysesthesias in the legs at rest leading to sleep disturbances. Although GWAS have identified genetic risk factors, these explain only about 6.8% of the total heritability. A portion of the remaining heritability could potentially lie in low-frequency and rare variants (MAF<5%) of strong effect. To date, no such variant is known for RLS. Methods: To assess the role of variants with MAF<5% at the known GWAS loci, we screened the coding regions of MEIS1, PTPRD, BTBD9, MAP2K5, SKOR1, TOX3 and BC034767 in 188 RLS cases and 188 controls by high-resolution melting curve analysis and Sanger sequencing. All identified variants with MAF<5% were genotyped in a sample of 3265 cases and 2944 controls. In a second approach, the coding and untranslated (UTR) regions of MEIS1 were screened in 3760 RLS cases and 3572 controls by the same method. Identified variants in MEIS1 were analysed for functional effects by mRNA in vivo complementation assay in zebrafish embryos using optic tectum area as phenotypic read-out. Results: Across all seven genes, we identified 50 non-synonymous (non-syn), synonymous (syn) and nearsplice variants (MAF<5%) present in 78 patients and 46 controls (p=0.004, McNemar test). When genotyped in 3265 cases and 2944 controls, non-syn variants with MAF<1% (p=3.7x10E-12) and MAF<0.1% (p=9.2x10E-5) across all genes and at solely the MEIS1 locus (p=0.002, all chi-squared test) were more common in cases than controls. In MEIS1, a total of 89 variants with MAF<5% were identified. Overall, variants in the 5`UTR (p=6.2x10E-4) and non-syn variants (p=0.005, both chi-squared test) occurred more frequently in cases than controls. Three single rare MEIS1 variants were significantly associated with RLS prior to correction for multiple testing. In the preliminary assessment of all 17 non-syn MEIS1 variants in zebrafish, null and hypomorphic loss-of-function alleles appear to be more common in the cases than the controls and implicate a loss-of-function as the underlying mechanism. Conclusions: Coding variants with MAF<5% at RLS-GWAS loci are more common in RLS cases than in controls. At the MEIS1 locus, a spectrum of common and rare, non-coding and coding variants appears to contribute to disease development, supporting the concept of allelic series in complex diseases. Several rare variants emerge as candidates for causal RLS variants.

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