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Restless legs syndrome (RLS, OMIM 102300) is a neurologic condition characterized by a distressing urge to move the legs, usually accompanied by an uncomfortable sensation described as a crawling, muscle ache, or tension. It is usually brought on by rest, worse in the evening or night, and relieved by movement.1 Recently, MEIS1 and BTBD9 have been identified and confirmed as RLS susceptibility genes; however, all the variants found to be associated with disease are located deep in intronic regions and are not thought to be functional.2–4 To date, sequencing of MEIS1 and BTBD9 in RLS samples has not been reported and therefore functional variants responsible for the increased disease risk have not been identified. Here we report sequencing of all MEIS1 and BTBD9 coding exons and exon-intron boundaries in RLS familial probands followed by assessment of segregation of novel variants with disease within families and evaluation of prevalence in patients with RLS and the general population.
Genomic DNA was extracted from peripheral blood lymphocytes using standard protocols. Primer pairs for MEIS1 (NM—002398) and BTBD9 (NM—052893) were used to sequence all coding exons by PCR in a discovery group consisting of 71 RLS familial probands. PCR products were purified using Agencourt bead technology (Beverly, MA) with Biomek FX automation (Beckman Coulter, Fullerton, CA). Prevalence of novel mutations was examined by TaqMan probe in a RLS case-control series consisting of 378 RLS cases (mean age 64.5 ± 13.4 years; onset age 45.3 ± 19.2 years; range 2-86 years) and 853 unrelated controls without evidence of RLS from North America and Europe. In addition, disease segregation was evaluated in affected and unaffected family members. All patients were examined and observed longitudinally and diagnosed with RLS as previously described.4 The ethical review board involved approved the study and all participants provided informed consent.
Sequencing analysis of BTBD9 in RLS probands did not identify any novel variant in coding regions or within 10 bases of exon-intron boundaries. One known synonymous variant (rs41303370; p.V239V) and one variant in the 3′ untranslated region were observed (rs6906771). Sequencing of MEIS1 identified one sample with a novel arginine to histidine (p.R272H; ss107795884) mutation in the first amino acid of the highly conserved homeobox three-amino acid loop extension (TALE) domain (figure, A). Genotyping of this mutation in our RLS case-control series from North America (378 cases and 528 controls) failed to identify p.R272H in any additional individuals. Further genotyping in 325 controls from European populations identified one male mutation carrier from Ireland not diagnosed with a neurologic disease at collection (age 64 years).
Six family members, 3 diagnosed with RLS, were available for segregation analysis of MEIS1 p.R272H; all samples diagnosed with RLS for which DNA was available (II-4, II-7, III-2) and one obligate carrier (II-1; unaffected parent of affected offspring) presented the p.R272H mutation (figure, B).
Sequencing analysis of RLS probands identified one novel MEIS1 nonsynonymous mutation in the highly conserved TALE domain, essential for dimerization and transcription activation.5 Evaluation of the frequency of this mutation in the case-control series identified only one control from Ireland carrying the mutation; therefore, if pathogenic, p.R272H does not appear to be fully penetrant, nor a common cause of disease (1/378 cases).
Segregation analysis of MEIS1 p.R272H is inconclusive due to the small size of the pedigree and having DNA available only for 3 affected family members. However, the presence of the mutation in all affected individuals supports a pathogenic role for this variant in RLS. The existence of an unaffected mutation carrier (II-1; died at 92 years of age) further supports the reduced penetrance of this mutation. Clinically, all affected family members present considerable differences in ages at onset, ranging from 15 (II-4) to 60 (II-7, III-2) years, and disease severity, from 14 (II-7) to 25 (II-4, III-2) points on the IRLSS scale, indicating the high heterogeneity of disease and potentially the presence of modifying factors.
This study reports sequencing of MEIS1 and BTBD9 in patients with RLS. These analyses revealed that nonsynonymous mutations in these genes are not a common cause of RLS in the US population. However, a novel nonsynonymous mutation, p.R272H, in the highly conserved TALE domain of MEIS1 was identified. The importance of homeobox consensus sequences and the detrimental effect of arginine to histidine mutations in this domain have been described in several studies.6,7 However, cosegregation of this mutation and disease within a small pedigree is inconclusive and replication in independent datasets is warranted to evaluate p.R272H as a pathogenic cause of familial RLS.
The authors thank the patients and families who participated in the study.
Supported by Morris K. Udall PD Research Center of Excellence (National Institute of Neurological Disorders and Stroke P50 #NS40256) and a Mayo Clinic Jacksonville Research CR20 grant, “Restless Legs Syndrome: Clinical and Molecular Genetic Studies.”
Disclosure: R.J.U. is funded by the NIH. Z.K.W. is funded by the Morris K. Udall Center, National Institute of Neurological Disorders and Stroke, the NIH, and the Pacific Alzheimer’s Disease Research Foundation. M.J.F. is funded by the NIH, the Morris K. Udall Center, and the National Institute of Neurological Disorders and Stroke. C.V.-G., H.C., B.H.K., J.E.Y., A.R., T.L., J.O.A., and S.-C.L. report no conflicts of interest.
Received December 5, 2008. Accepted in final form March 4, 2009.
Address correspondence and reprint requests to Dr. Carles Vilariño-Güell, Molecular Genetics Laboratory and Core, Morris K. Udall PD Research Center of Excellence, Mayo Clinic, Department of Neuroscience, 4500 San Pablo Road, Jacksonville, FL 32224; ude.oyam@selraC.lleuGoniraliV