The transcription factor MEIS1 belongs to the family of highly conserved TALE homeobox genes and interacts with PBX and HOX proteins to increase the affinity and specificity of HOX proteins [74] as well as CREB1 [75] in DNA binding. In Xenopus laevis, meis1 is known to be involved in neural crest development [76]. Murine Meis1 is essential for proximo-distal limb patterning [77] and plays a role in the Hox transcriptional regulatory network that specifies spinal motor neuron pool identity and connectivity [78]. In the CNS of the adult mouse, it is known to be expressed in cerebellar granule cells, the forebrain, and the substantia nigra. While MEIS1 was initially identified in the context of acute myeloid leukemia [79, 80], in recent years, a role in murine heart development has also been recognized [81], and SNPs in intron 8 (but in weak LD with the known RLS SNPs) play a role in determining atrioventricular conduction velocity as reflected by the length of the PR interval of the electrocardiogram in both Europeans and African-Americans [82, 83]. Meis1 ^−/− mice develop ocular and vascular defects, fail to produce megakaryocytes, and display extensive hemorrhaging. They also die by embryonic day 14.5 [84]. Meis1 ^−/+ mice, however, survive into adulthood and exhibit hyperactivity reminiscent of the human RLS phenotype [85].

A second independent association signal is located in an intergenic region approximately 1.3 Mb downstream of MEIS1 and potentially possesses long-range regulatory function with MEIS1 and ETAA1 as potential target genes [72].

Several rare non-synonymous variants in MEIS1 have been identified in RLS patients. However, coding variants in MEIS1 are very rare in general (13 out of approximately 4,250 individuals with a non-synonymous variant in the NHLBI-ESP exomes [86]), possibly owing to the fact that MEIS1 represents one of the most highly conserved genes in the human genome, and, therefore, remain ambiguous with regard to possible causality of the RLS phenotype [87–90]. On the whole, however, non-synonymous variants with MAF <0.1 % were found significantly more frequently in individuals with RLS compared to the general population [90]. Functional annotation using an in vivo complementation assay in zebra fish further revealed that variants harboring a loss of MEIS1 function were significantly enriched in individuals with RLS [90]. The same study also identified a low-frequency variant in the 3′ untranslated region (UTR) of MEIS1 (rs11693221; MAF_cases = 13.55 % vs. MAF_controls = 3.58 %; p = 1.27 × 10⁻⁸⁹, OR = 4.42) associated with RLS [90]. Yet, at present, it cannot be determined whether this or any other variant tagged by rs11693221 represents the causal factor underlying this association signal. What does become clear, though, is the fact that, at the MEIS1 locus, an allelic series of genetic variants of different frequencies and different effect sizes contributes to the genetic framework of RLS.

Since the publication of the first GWAS, which identified common variants in MEIS1 as susceptibility factors for RLS, three studies have been reported which examine the functional differences brought about by the RLS-associated intronic variants. In the first, a significant decrease in MEIS1 mRNA and protein expression was found in lymphoblastoid cell lines and brain tissue (pons and thalamus) from homozygous carriers of the risk haplotype when compared to homozygous carriers of the non-risk haplotype [89]. In a second study, knockdown of the MEIS1 orthologue unc-62 by RNA interference in Caenorhabditis elegans was related to increased ferritin expression and an extended life span. In thalamus but not in pons samples of RLS patients homozygous for the MEIS1 risk haplotype (n = 9), ferritin light and heavy chains as well as divalent metal transporter 1 (DMT1) mRNA and protein expression were significantly increased when compared to RLS patients carrying the protective haplotype (n = 7). Several other key players in the iron metabolism such as transferrin, the transferrin receptors 1 and 2, aconitase 1, iron-responsive element binding protein 2, ceruloplasmin, hepcidin, and ferroportin were unchanged [91]. The authors argue that these data are in support of a disruption of physiological iron transport into the brain and—in conjunction with the also observed decrease of MEIS1 expression in in vitro cell models of iron deprivation—provide a functional link between the RLS gene MEIS1 and the iron metabolism, which is believed to play a role in RLS pathogenesis [91]. In the third study, it was shown that the risk allele of the best-associated SNP (rs12469063) in MEIS1 from the GWAS reduces enhancer activity in the Meis1 expression domain of the ganglionic eminences, which constitute the primordial basal ganglia, in mouse embryos at E12.5 [85]. In vitro studies suggest that CREB1 binds more strongly to this enhancer when the risk allele is present [85].

Another locus encompasses both mitogen-activated protein kinase MAP2K5 and transcriptional corepressor, SKOR1. MAPK pathways are highly conserved among different species and are activated in response to signals that mediate the transduction of extracellular signals to the cytoplasmic nuclear effectors [92]. More specifically, MAP2K5 phosphorylates and activates ERK5 in response to oxidative stress, hyperosmolarity, and growth factors. It is expressed in the heart and skeletal muscle and critical in muscle cell differentiation [92]. Interestingly, the MAP2K5/ERK5 pathway has also been implicated in neuroprotection of dopaminergic neurons [93]. Not much is known about the physiologic function of SKOR1. It acts as a transcriptional corepressor of homeobox gene LBX1, which has been recognized as a factor in the development of pain and touch relay via sensory pathways in the dorsal horn of the spinal cord [94]. The genomic locus comprising MAP2K5 and SKOR1 was shown to harbor nine blood-based cis-eSNPs, that is, common variants that are located within ± 500 kb of the lead SNP, which alter blood-based gene expression [95]. None of these affected the expression of SKOR1, and only two affected the expression of MAP2K5, while seven altered expression of CALML4, thus highlighting the possibility that other genes in the vicinity of the current candidate genes at a given locus could also play a role in RLS pathophysiology [95].