Exploring the genetic link between RLS and ADHD

Journal of Psychiatric Research 43 (2009) 941–945

Contents lists available at ScienceDirect

Journal of Psychiatric Research journal homepage: www.elsevier.com/locate/jpsychires

Exploring the genetic link between RLS and ADHD B.G. Schimmelmann a,*,1, S. Friedel a,1, T.T. Nguyen b, S. Sauer c, C.I. Ganz Vogel a, K. Konrad d, C. Wilhelm d, J. Sinzig e, T.J. Renner f, M. Romanos f, H. Palmason h, A. Dempfle b, S. Walitza f,g, C. Freitag i, J. Meyer h, M. Linder j, H. Schäfer b, A. Warnke f, K.P. Lesch k, B. Herpertz-Dahlman d, A. Hinney a, J. Hebebrand a a

Department of Child and Adolescent Psychiatry and –Psychotherapy, University of Duisburg-Essen, LVR Klinikun Essen, Virchowstr. 174, 45147 Essen, Germany Institute of Medical Biometry and Epidemiology, Philipps-University Marburg, Marburg, Germany c Max Planck Institute for Molecular Genetics, Otto Warburg Laboratory, Berlin, Germany d Department of Child and Adolescent Psychiatry, Technical University of Aachen, Aachen, Germany e Department of Child and Adolescent Psychiatry, University of Cologne, Cologne, Germany f Department of Child and Adolescent Psychiatry, Julius-Maximilians-University Wuerzburg, Wuerzburg, Germany g Department of Child and Adolescent Psychiatry, University of Zürich, Switzerland h Department of Neurobehavioral Genetics, University of Trier, Trier, Germany i Department of Child and Adolescent Psychiatry, Saarland University Hospital, Homburg, Germany j Department of Child and Adolescent Psychiatry, Bezirksklinik Regensburg, Regensburg, Germany k Department of Psychiatry and Psychotherapy, Julius-Maximilians-University Würzburg, Würzburg, Germany b

a r t i c l e

i n f o

Article history: Received 19 August 2008 Received in revised form 13 January 2009 Accepted 13 January 2009

Keywords: Attention deficit/hyperactivity disorder RLS Genome-wide association study

a b s t r a c t 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 (v2 = 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. Ó 2009 Elsevier Ltd. All rights reserved.

1. 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), * Corresponding author. Tel.: +49 201 7227 251; fax: +49 201 7227 311. E-mail address: [email protected] (B.G. Schimmelmann). 1 These authors contributed equally. 0022-3956/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jpsychires.2009.01.003

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

942

B.G. Schimmelmann et al. / Journal of Psychiatric Research 43 (2009) 941–945

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,b). 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. 2. Materials and methods 2.1. 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 criteria have been described previously (Friedel et al., 2007; Schimmelmann et al., 2007). All families were of Caucasian ethnicity. The sample characteristics are displayed in Table 1. 2.2. SNP selection and genotyping The aim of this study was to cover all confirmed association results by Winkelmann et al. (2007) and Stefansson et al. (2007), as well as the haplotype findings resulting from fine mapping on chromosome 2 (chr. 2, Winkelmann et al., 2007). Confirmed association results by Winkelmann et al. (2007) refer to those SNPs, which were confirmed in three sample sets and withstood genome-wide correction for multiple testing in the joint analysis of these three samples. On chr. 2 (MEIS1), we selected rs2300478. Additionally, we genotyped SNPs rs6710341, rs12469063, and rs4544423 representing the relevant haplotype in MEIS1. On chr. 6 (BTBD9 genomic region), we genotyped rs9296249 (representing the other confirmed SNP in BTBD9, i.e. rs9357271, with r2 = 1). We further genotyped SNPs rs3923809 and rs6923737 in BTBD9 reported by Stefansson et al. (2007). On chr. 15 (MAP2K5/LBXCOR1 genomic region), we selected rs12593813 and rs4489954 (representing another three confirmed SNPs reported by Winkelmann et al. (2007) with r2 > 0.78). In summary, six SNPs and one haplotype (in MEIS1) were hypothesized to be associated with ADHD. Furthermore, we performed two exploratory haplotype analyses

Table 1 Clinical characteristics of offspring with ADHD (N = 386). Probands n (%) or mean (SD) Sex Male Female Age Subtypes Predominantly inattentive Predominantly hyperactive-impulsive Combined Comorbiditiesa,b Oppositional defiant disorder Conduct disorder Mood disordersc Anxiety disordersd Tic disorder a

284 (73.58%) 102 (26.42%) 10.9 (SD 3.2) 78 (20.2%) 13 (3.4%) 295 (76.4%) 137 (35.6%) 30 (7.8%) 22 (5.7%) 48 (12.5%) 30 (7.8%)

Current DSM-IV diagnosis according to K-SADS or Kinder-DIPS. Multiple scoring possible. Diagnoses: major depressive disorder or dysthymic disorders. d Diagnoses: generalized anxiety disorder, separation anxiety disorder, social phobia, specific phobias. b c

943

B.G. Schimmelmann et al. / Journal of Psychiatric Research 43 (2009) 941–945

of the genotyped SNPs in BTBD9 and MAP2K5/LBXCOR1, respectively. Genotyping of SNPs rs2300478, rs4544423, rs12469063, rs9296249, rs6923737 rs12593813 and rs4489954 was performed by multiplexed primer extension based assays and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) detection using the iPlex technology from Sequenom (San Diego, USA). Data analysis was done as described recently (Sauer et al., 2006) and applying the Typer FS software (Sequenom). SNPs rs6710341 and rs3923809 were genotyped using TaqManÒ assays (Applied Biosystems, Foster City, CA, USA). All TaqManÒ probes and primers were purchased from applied biosystems (assays on demand; ).

respectively. The power calculation assumes a sample of a specified number of independent case-parent trios, while our sample comprises a high percentage of families with two and more affected sibs. Assuming only 224 independent trios with just one affected child from each family would therefore under-estimate true power, while treating the 386 affected children like independent trios would overestimate power. Therefore, the true power lies in between the following two power estimates. Assuming a sample size of 224 independent case-parent trios, the power was 64% for all seven tests, four of them (RLS associated haplotype, rs2300478, rs3923809 and rs6923737) actually had power of about 80%. Assuming a sample size of 386 independent case-parent trios, the power was 89% for all tests.

2.3. Statistical analysis

3. Results

Mendelian inconsistent genotype data were detected and removed using PedCheck (O’Connell and Weeks, 1998) resulting in the 224 families with 386 children. Genotype distributions in the parents were tested for departure from Hardy–Weinberg equilibrium by chi square test. For the six SNP-wise association analyses the pedigree disequilibrium test was used (PDT, Martin et al., 2000). Haplotype analyses were carried out by means of UNPHASED (), which provides chi square tests for the comparison of transmission- and non-transmission rate of individual haplotypes taking into account the fact that a nuclear family can have more than one affected offspring (Dudbridge, 2008). For exploratory haplotype analyses of SNPs in BTBD9 and MAP2K5/LBXCOR1 the overall likelihood ratio test for association of at least one haplotype was taken as a criterion. All reported p- values are nominal and two-sided. The comparison-wise significance level after correction for multiple testing was set at 0.007 (= 0.05/7, with 7 is the total number of hypotheses tested). We used QUANTO () for power calculation. The power was calculated for seven planned tests at the one-sided significance level of 0.05/7 under log additive inheritance mode with an ADHD prevalence of 5%. In an attempt to estimate the power of this sample we equivalated the hypothesized effect sizes for ADHD to the effect sizes reported for RLS by Winkelmann et al. (2007) and Stefansson et al. (2007). We assumed risk allele frequencies according to the control sample by Winkelmann et al. (2007) and haplotype frequency according to HapMapEU (); these risk haplotype/allele frequencies and corresponding reported effects were: 0.10–2.36, 0.23–1.74, 0.2–1.67, 0.74–1.8, 0.68–1.7, 0.31–1.50, 0.28–1.51,

Genotype distributions in the parental generation did not deviate from Hardy–Weinberg equilibrium (all nominal chi square pvalues > 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 Winkelmann et al. (2007) and by Stefansson et al. (2007; see Table 2) were observed. Additionally, there was no evidence for an over-transmission of the at-risk haplotype in MEIS1 (G–A–G, 51 transmissions versus 59 non-transmissions, nominal p-value = 0.381). We additionally performed exploratory haplotype analyses for all SNPs in BTBD9 and MAP2K5/LBXCOR1 to explore potential associations with ADHD. The overall test of haplotype main effects detected nominal significance for the haplotypes in BTBD9 (v2 = 14.8, df = 7, nominal p = 0.039). The major contribution to this finding came from the A–A–A- and A–A–G haplotypes with a haplotypewise nominal p-value of 0.009, resp. 0.010 (see Table 3). However, this finding resulted from exploratory post hoc analyses unadjusted for multiple testing. Under the assumption that transmission disequilibrium may be more pronounced among 295 children with combined subtype, we performed the same analyses again for this extreme subgroup. We observed similar non-significant results for the two SNPs in MAP2K5, the BTBD9 SNP rs3923809 as well as for the at-risk haplotype in MEIS1, yet a nominally significant over-transmission in two risk alleles of the SNPs rs9296249 and rs6923737 in BTBD9 detected in the studies by Winkelmann et al. (2007) and by Stefansson et al. (2007) (nominal p-values = 0.045 and 0.037,

Table 2 PDT results for the 6 RLS associated SNPs in 386 ADHD children of 224 families. SNP /gene rs2300478 /MEIS1 rs9296249 /BTBD9 rs3923809 /BTBD9 rs6923737 /BTBD9 rs12593813 /MAP2K5/ LBXCOR1 rs4489954 /MAP2K5/ LBXCOR1

Triadsc 373 355 379 350 356 348

Allele T G T C A G A G A G T G

RLS risk allele a

G T

a

A

b

Ab Ga a

G

Transmitted

Non-transmitted

v2(df = 1)

p-valuesd

595 151 553 157 520 238 481 219 252 460 229 467

578 168 530 180 488 270 445 255 242 470 229 467

1.23

0.267

1.91

0.167

2.47

0.116

3.45

0.063

0.33

0.564

0.00

1.000

Bold alleles represent over-transmitted alleles. a Winkelmann et al. (2007). b Stefansson et al. (2007). c Number of triads deviate from total 386 triads due to missing genotype data (mean missing rate across all SNPs 1.88%) or mendelian inconsistencies (mean rate 0.94%). d Nominal two-sided p-values.

944

B.G. Schimmelmann et al. / Journal of Psychiatric Research 43 (2009) 941–945

Table 3 Exploratory analysis of haplotype transmission in BTBD9–386 children with ADHD. rs9296249

rs3923809

rs6923737

No. transmitted/ non-transmitted

% in Transmitted/non-transmitted

v2

p-values

A A A A G G G G

A A G G A A G G

A G A G A G A G

370/322 21/38 10/17 35/34 3/6 1/1 12/17 84/101

69.0/60.1 3.9/7.1 1.9/3.2 6.5/6.3 0.6/1.1 0.2/0.2 2.2/3.2 15.7/18.8

6.73 6.59 2.32 0.01 NA NA 0.73 1.34

0.009 0.010 0.127 0.907 NA NA 0.393 0.246

NA = not applicable due to small frequency. In bold haplotypes nominal p-values < 0.05 were detected.

respectively). The overall test for BTBD9 haplotypes remained similar (v2 = 15.53, df = 7, nominal p = 0.030) with the major contribution of now three haplotypes A–A–A (p = 0.0035), A–A–G (p = 0.020) and A–G–A (p = 0.039). 4. 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 Caucasian sample comprising 386 children with ADHD and their parents (224 independent families). We further checked results of the genome-wide association scan ‘GAIN International Multi-Center ADHD Genetics Project’ pre-computed by NCBI and recently published online (). In three of the 9 RLS SNPs the TDT results were available and non-significant for ADHD (rs12469063 on chr. 2 as well as rs9296249 and rs6923737 on chr. 6. The respective number of trios tested and p-values were 523–0.501, 542–0.875, and 623– 0.916). Since 6 RLS SNPs were not tested in GAIN, we checked all available GAIN SNPs in the genomic regions capturing ±200 kb of the RLS SNPs. If GAIN SNPs in these regions were associated with ADHD at nominal p < 0.05, we checked for linkage disequilibrium (LD) with the RLS SNPs in HapMapEU (). This procedure revealed no LD between significant GAIN SNPs and RLS SNPs. Furthermore, two SNPs in a fourth genomic region were recently identified to be associated with RLS, i.e. PTPRD on chr. 9, this gene presumably being involved in axon guidance and termination of mammalian motorneurons during embryonic development. (Schormair et al., 2008). Again, GAIN data were checked and revealed no relevant association between or LD with these two SNPs and ADHD. Generally, large sample size and replication is mandatory in genetic studies to minimize the risk of false positive or negative findings. The GAIN study was not powered to particularly address the relevance of RLS SNPs for ADHD, and the sample is more heterogeneous compared to our study. The power of this study was sufficient to detect association of the respective polymorphisms with ADHD assuming they have the same effect size as reported for RLS. In light of the observed trends for over-transmission of the RLS risk alleles and the trend for haplotype effects in BTBD9 in our study, we should point out that the effect size of these risk alleles and haplotypes may be lower for ADHD than for RLS rendering this study under-powered. Furthermore, it cannot be excluded

that other variants in MEIS1, BTBD9, and MAP2K5/LBXCOR1 are relevant for ADHD. Additionally, the negative findings may result from genetic heterogeneity of ADHD, i.e. risk alleles for RLS may only be relevant to certain subtypes of ADHD. The finding of stronger trends towards over-transmission of BTBD9 risk alleles to the more extreme subgroup with ADHD combined subtype points in this direction. Although derived from secondary analyses, the latter finding is interesting as BTBD9 risk alleles have been linked to decrements in iron stores (Trotti et al., 2008), and there is preliminary evidence that severity of hyperactive and behavioral problems in ADHD may be related to lower ferritin levels (Oner et al., 2008a and b). Notably, in a small randomized placebo-controlled pilot trial by Konofal et al. (2008), iron supplementation (80 mg/ day) improved ADHD symptoms in 23 children with low serum ferritin levels (without anaemia) adding to the body of evidence that iron deficiency may be involved in the aetiology of a subgroup of patients with ADHD (Cortese et al., 2008). Accordingly, genes relevant to RLS, particularly BTBD9, remain interesting candidates for ADHD due to potential clinical and neurobiological overlap of the two phenotypes rendering future investigations mandatory. 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

B.G. Schimmelmann et al. / Journal of Psychiatric Research 43 (2009) 941–945

Mueller for excellent technical assistance, and Karin Zamzow and Heiko Zerlaut for exceptional data management. References Allen RP, Earley CJ. Restless legs syndrome: a review of clinical and pathophysiologic features. Clinical Neurophysiology 2001;18(2):128–47. Allen Institute for Brain Science. Allen Brain Atlas. 2004. Allen RP, Walters AS, Montplaisir J, Hening W, Myers A, Bell TJ, et al. Restless legs syndrome prevalence and impact: REST general population study. Archives of Internal Medicine 2005;165(11):1286–92. Albayrak O, Friedel S, Schimmelmann BG, Hinney A, Hebebrand J. Genetic aspects in attention-deficit/hyperactivity disorder. Journal of Neural Transmission 2008;115(2):305–15. American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 4th ed. Washington, DC: American Psychiatric Association; 1994. Cavanaugh JE, Jaumotte JD, Lakoski JM, Zigmond MJ. Neuroprotective role of ERK1/2 and ERK5 in a dopaminergic cell line under basal conditions and in response to oxidative stress. Journal of Neuroscience Research 2006;84(6):1367–75. Cortese S, Konofal E, Lecendreux M, Arnulf I, Mouren MC, Darra F, et al. Restless legs syndrome and attention-deficit/hyperactivity disorder: a review of the literature. Sleep 2005;28(8):1007–13. Cortese S, Lecendreux M, Bernardina BD, Mouren MC, Sbarbati A, Konofal E. Attention-deficit/hyperactivity disorder, tourette’s syndrome, and restless legs syndrome: the iron hypothesis. Medical Hypotheses 2008;70(6):1128–32. Desai AV, Cherkas LF, Spector TD, Williams AJ. Genetic influences in self-reported symptoms of obstructive sleep apnoea and restless legs: a twin study. Twin Research 2004;7(6):589–95. Dudbridge F. Likelihood-based association analysis for nuclear families and unrelated subjects with missing genotype data. Human Heredity 2008;66:87–98. Erickson KM, Jones BC, Hess EJ. Iron deficiency decreases dopamine D1 and D2 receptors in rat brain. Pharmacology Biochemistry Behaviour 2001;69:409–18. Faraone SV, Biederman J. Neurobiology of attention-deficit hyperactivity disorder. Biological Psychiatry 1998;44(10):951–8. Friedel S, Saar K, Sauer S, Dempfle A, Walitza S, Renner T, et al. Association and linkage of allelic variants of the dopamine transporter gene in ADHD. Molecular Psychiatry 2007;12(10):923–33. Gross MK, Dottori M, Goulding M. Lbx1 specifies somatosensory association interneurons in the dorsal spinal cord. Neuron 2002;34:535–49. Hoban TF, Chervin RD. Pediatric sleep-related breathing disorders and restless legs syndrome: how children are different. Neurologist 2005;11(6):325–37. Konofal E, Cortese S, Marchand M, Mouren MC, Arnulf I, Lecendreux M. Impact of restless legs syndrome and iron deficiency on attention-deficit/hyperactivity disorder in children. Sleep Medicine 2007;8(7–8):693–4. Konofal E, Lecendreux M, Deron J, Marchand M, Cortese S, Zaim M, et al. Effects of iron supplementation on attention deficit hyperactivity disorder in children. Pediatric Neurology 2008;38(1):20–6. Li D, Sham PC, Owen MJ, He L. Meta-analysis shows significant association between dopamine system genes and attention deficit hyperactivity disorder (ADHD). Human Molecular Genetics 2006;15(14):2276–84. Maeda R, Mood K, Jones TL, Aruga J, Buchberg AM, Daar IO. Xme is 1, a protooncogene involved in specifying neural crest cell fate in Xenopus embryos. Oncogene 2001;20(11):1329–42.

945

Martin ER, Monks SA, Warren LL, Kaplan NL. A test for linkage and association in general pedigrees: the pedigree disequilibrium test. American Journal of Human Genetics 2000;67:146–54. Milligan SA, Chesson AL. Restless legs syndrome in the older adult: diagnosis and management. Drugs Aging 2002;19(10):741–51. O’Connell JR, Weeks DE. Pedcheck: a program for identification of genotype incompatibilities in linkage analysis. American Journal of Human Genetics 1998;63:259–66. Oner P, Dirik EB, Taner Y, Caykoylu A, Anlar O. Association between low serum ferritin and restless legs syndrome in patients with attention deficit hyperactivity disorder. Tohoku Journal of Expert Medicine 2007;213(3):269–76. Oner P, Oner O. Relationship of ferritin to symptom ratings children with attention deficit hyperactivity disorder: effect of comorbidity. Child Psychiatry and Human Development 2008a;39(3):323–30. Oner O, Alkar OY, Oner P. Relation of ferritin levels with symptom ratings and cognitive performance in children with attention deficit-hyperactivity disorder. Pediatrics International 2008b;50(1):40–4. Picchietti DL, Stevens HE. Early manifestations of restless legs syndrome in childhood and adolescence. Sleep Medicine 2007;[ Epub ahead of print Nov. 15]. Sauer S, Reinhardt R, Lehrach H, Gut IG. Single-nucleotide polymorphisms: analysis by mass spectrometry. Nature Protocols 2006;1(4):1761–71. Schimmelmann BG, Friedel S, Dempfle A, Warnke A, Lesch KP, Walitza S, et al. No evidence for preferential transmission of common valine allele of the val66met polymorphism of the brain-derived neurotrophic factor gene (BDNF) in ADHD. Journal of Neural Transmission 2007;114(4):523–6. Schormair B, Kemlink D, Roeske D, Eckstein G, Xiong L, Lichtner P, et al. PTPRD (protein tyrosine phosphatase receptor type delta) is associated with restless legs syndrome. Nature Genetics 2008;40:946–8. Stefansson H, Rye DB, Hicks A, Petursson H, Ingason A, Thorgeirsson TE, et al. A genetic risk factor for periodic limb movements in sleep. New England Journal of Medicine 2007;357(7):639–47. Swanson JM, Sergeant JA, Taylor E, Sonuga-Barke EJ, Jensen PS, Cantwell DP. Attention-deficit hyperactivity disorder and hyperkinetic disorder. Lancet 1998;351(9100):429–33. Swanson JM, Kinsbourne M, Nigg J, Lanphear B, Stefanatos GA, Volkow N, et al. Etiologic subtypes of attention-deficit/hyperactivity disorder: brain imaging, molecular genetic and environmental factors and the dopamine hypothesis. Neuropsychology Reviews 2007;17(1):39–59. Trotti LM, Bhadriraju S, Rye DB. An update of the pathophysiology and genetics of restless legs syndrome. Current Neurology and Neuroscience Reports 2008;8(4):281–7. Wagner ML, Walters AS, Fisher BC. Symptoms of attention-deficit/ hyperactivity disorder in adults with restless legs syndrome. Sleep 2004;27(8):1499–504. Winkelmann J, Schormair B, Lichtner P, Ripke S, Xiong L, Jalilzadeh S, Fulda S, Pütz B, Eckstein G, Hauk S, Trenkwalder C, Zimprich A, Stiasny-Kolster K, Oertel W, Bachmann CG, Paulus W, Peglau I, Eisensehr I, Montplaisir J, Turecki G, Rouleau G, Gieger C, Illig T, Wichmann HE, Holsboer F, MüllerMyhsok B, Meitinger T. Genome-wide association study of restless legs syndrome identifies common variants in three genomic regions. Nature Genetics 2007;39(8):1000–6. Yang B, Chan RC, Jing J, Li T, Sham P, Chen RY. A meta-analysis of association studies between the 10-repeat allele of a VNTR polymorphism in the 3´UTR of dopamine transporter gene and attention deficit hyperactivity disorder. American Journal of Medical Genetics Part B 2007;144B:541–50.