Exploring the genetic link between RLS and ADHD

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Abstract

Attention deficit/hyperactivity disorder (ADHD) is a highly heritable neurodevelopmental disorder of childhood onset. Clinical and biological evidence points to shared common central nervous system (CNS) pathology of ADHD and restless legs syndrome (RLS). It was hypothesized that variants previously found to be associated with RLS in two large genome-wide association studies (GWA), will also be associated with ADHD.

SNPs located in MEIS1 (rs2300478), BTBD9 (rs9296249, rs3923809, rs6923737), and MAP2K5 (rs12593813, rs4489954) as well as three SNPs tagging the identified haplotype in MEIS1 (rs6710341, rs12469063, rs4544423) were genotyped in a well characterized German sample of 224 families comprising one or more affected sibs (386 children) and both parents.

We found no evidence for preferential transmission of the hypothesized variants to ADHD. Subsequent analyses elicited nominal significant association with haplotypes consisting of the three SNPs in BTBD9 (χ2 = 14.8, df = 7, nominal p = 0.039). According to exploratory post hoc analyses, the major contribution to this finding came from the A–A–A-haplotype with a haplotype-wise nominal p-value of 0.009. However, this result did not withstand correction for multiple testing.

In view of our results, RLS risk alleles may have a lower effect on ADHD than on RLS or may not be involved in ADHD. The negative findings may additionally result from genetic heterogeneity of ADHD, i.e. risk alleles for RLS may only be relevant for certain subtypes of ADHD. Genes relevant to RLS remain interesting candidates for ADHD; particularly BTBD9 needs further study, as it has been related to iron storage, a potential pathophysiological link between RLS and certain subtypes of ADHD.

Introduction

Attention deficit/hyperactivity disorder (ADHD) is a common psychiatric disorder in childhood characterized by pervasive and developmentally inappropriate inattention, excessive motor activity, impulsivity, and distractibility. ADHD affects between 2–5% of school-aged children (Swanson et al., 1998). While the heritability of ADHD is estimated at 60–80% (Faraone and Biederman, 1998), the genetic causes of ADHD are still unknown. Multiple theories of ADHD have been proposed, but the most robust evidence points to the dopamine deficit theory (for review, see Swanson et al., 2007). Recent meta-analyses of dopaminergic candidate genes found support for the involvement of DRD4, DRD5, and DAT1 gene polymorphisms (Albayrak et al., 2008, Li et al., 2006, Yang et al., 2007).

Restless legs syndrome (RLS) is a neurological and sleep-related disorder typically characterized by an urge to move and an uncomfortable feeling in the legs. The prevalence of RLS with clinically significant impairment is estimated at 2.7–10% in the general population (Allen et al., 2005). Epidemiologic studies indicate that the prevalence of RLS increases with age. RLS was rarely identified in children until recently. However, retrospective surveys reported that 43% of adults with RLS had symptom onset between the ages of 10–20 years. It is assumed that RLS symptoms in children may go unrecognized by parents and clinicians and mistakenly be attributed to, e.g., insomnia, growing pains, or ADHD (Hoban and Chervin, 2005, Picchietti and Stevens, 2007). Heritability of RLS is estimated at about 50% (Desai et al., 2004). Family history and age at onset appear to differentiate two phenotypes of RLS (Allen and Earley, 2001). Early-onset RLS, in which symptoms occur before age 35, has an autosomal dominant mode of inheritance with a higher prevalence of family history of RLS. Late-onset RLS is characterized by a potentially more rapid symptom progression and a higher prevalence of secondary RLS due to iron deficiency, pregnancy, or end-stage renal disease (Milligan and Chesson, 2002).

Growing evidence suggests an association between RLS and ADHD or ADHD symptoms. The prevalence of RLS or RLS symptoms in subjects with ADHD ranges between 12% and 44%, and the prevalence of ADHD or ADHD symptoms in subjects with RLS is between 11% and 26% (Cortese et al., 2005, Oner et al., 2007). While previous studies do not allow for a precise estimate of the magnitude of this association due to methodological limitations such as small sample size and lack of epidemiological data, most studies suggest that an association between ADHD and RLS does indeed exist. One possible explanation for this association is that RLS might lead to symptoms of ADHD through impairment of the quantity or quality of sleep. A second explanation involves the misclassification of RLS symptoms as hyperactivity in children, i.e. children in need for moving due to leg discomfort during the day might present with inattention and hyperactivity in school. The latter explanation has been criticized (Wagner et al., 2004), since RLS is not commonly associated with inattention. The third possible explanation is that RLS and ADHD might share common central nervous system (CNS) pathology such as dopaminergic function deficits. This hypothesis is supported by evidence suggesting dopaminergic hypoactivity in both ADHD and RLS (Wagner et al., 2004). Notably, iron deficiency has been reported in both, subjects with ADHD and RLS (Konofal et al., 2007, Oner et al., 2007). Iron is a cofactor of tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis. Further, iron deficiency was reported to alter D1 and D2 receptor density and activity in animals (Erickson et al., 2001). These data provided further evidence for the assumption of a common CNS pathology in ADHD and RLS.

Two recently published large genome-wide association studies (GWA) on RLS (Stefansson et al., 2007, Winkelmann et al., 2007) detected an association of variants in four genes, MEIS1, BTBD9, MAP2K5, and LBXCOR1. Two of these genes may be linked to dopaminergic function. MEIS1 appears to be involved in neural crest development and is known to be expressed in adult mouse brain in, amongst other regions, the forebrain and dopaminergic neurons of the substantia nigra (Maeda et al., 2001, Allen Institute for Brain Science, 2004). MAP2K5 is suggested to be involved in pathways important in neuroprotection of dopaminergic neurons (Cavanaugh et al., 2006). LBXCOR1 is a gene located downstream of MAP2K5 and acts as a transcriptional corepressor of LBX1, which appears to be involved in the development of sensory pathways (Gross et al., 2002). Although the function of BTBD9 remains uncertain, its biological plausibility is evidenced by its dose-dependent relationship to periodic limb movements of sleep and decrements in iron stores (Trotti et al., 2008). The latter finding is interesting as there is preliminary evidence that severity of hyperactive and behavioral problems in ADHD may be related to lower ferritin levels (Oner et al., 2008a, Oner et al., 2008b).

These results together with the preliminary clinical and biological body of evidence on shared common CNS pathology of ADHD and RLS stimulated us to explore whether the polymorphisms found in these GWA are relevant to the ADHD phenotype. Accordingly, we hypothesized that variants associated with RLS observed by Winkelmann et al. (2007) and Stefansson et al. (2007) will be over-transmitted to children with ADHD in a large sample of 224 families comprising 386 children with ADHD.

Section snippets

Sample

224 Caucasian families were recruited and phenotypically characterized in 6 child psychiatric outpatient units (Aachen, Marburg, Homburg, Trier, Regensburg, and Würzburg). Families were included if at least one child was diagnosed with ADHD (combined type) according to DSM-IV (APA, 1994). In 96 families one affected child was ascertained, in 98 families two, in 26 families three, and in 4 families four affected children were recruited, respectively. The ascertainment strategy and inclusion

Results

Genotype distributions in the parental generation did not deviate from Hardy–Weinberg equilibrium (all nominal chi square p-values > 0.183). The PDT analyses revealed no significant disequilibrium in allele transmission of the 6 SNPs representing MEIS1, BTBD9, and MAP2K5/LBXCOR1 to children with ADHD (all nominal PDTsum p-values > 0.063, see Table 2). In this sample, only in three SNPs rs9296249, rs3923809, and rs6923737, an over-transmission of the risk alleles detected in the studies by

Discussion

There is clinical and biological evidence on potentially shared common CNS pathology of ADHD and RLS. Accordingly, we hypothesized that the RLS risk alleles (Stefansson et al., 2007, Winkelmann et al., 2007) and the haplotype (Winkelmann et al., 2007), found in two large GWA, are relevant for the ADHD phenotype. However, we found no proof for transmission disequilibrium of the hypothesized polymorphisms or haplotype described by Winkelmann et al. (2007) or Stefansson et al. (2007) in a

Contributors

Authors BGS, SF, AH, and JH developed the research question. SF, AH, CIGV, and SS did the genotyping. BGS, KK, CW, JS, TJR, MR, HP participated in the phenotyping of children with ADHD. TTN performed the data analysis. BGS, SF, AH, TTN, and JH interpreted the data. BGS wrote the first draft of the manuscript. All authors have contributed to and approved the final manuscript.

Role of funding source

The German Ministry for Education and Research (National Genome Research Net 2 and plus, 01GS0820 and 01GS0830 and the German Research Foundation (DFG; KFO 125/1-1, SCHA 542/10-2, ME 1923/5-1, ME 1923/5-3) supported this study. These institutions had no further role in study design, in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the paper for publication.

Conflict of interest

The authors declare there is no conflict of interest relevant to this work.

Acknowledgements

The authors express their gratitude to the patients and their families for participation. We thank Juliane Winkelmann for her helpful comments on an earlier version of this manuscript, Gisela Mueller for excellent technical assistance, and Karin Zamzow and Heiko Zerlaut for exceptional data management.

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