A genome-wide scan for attention-deficit/hyperactivity disorder in 155 German sib-pairs

Abstract

Three groups have previously performed genome scans in attention-deficit/hyperactivity disorder (ADHD); linkage to chromosome 5p13 was detected in all of the respective studies. In the current study, we performed a whole-genome scan with 102 German families with two or more offspring who currently fulfilled the diagnostic criteria for ADHD. Including subsequent fine mapping on chromosome 5p, a total of 523 markers were genotyped. The highest nonparametric multipoint LOD score of 2.59 (empirical genome-wide significance 0.1) was obtained for chromosome 5p at 17 cM (according to the Marshfield map). Subsequent analyses revealed (a) a higher LOD score of 3.37 at 39 cM for a quantitative severity score based on symptoms of inattention than for hyperactivity/impulsivity (LOD score of 1.11 at 59 cM), and (b) an HLOD of 4.75 (empirical genome-wide significance 0.001) based on a parametric model assuming dominant inheritance. The locus of the solute carrier 6A3 (SLC6A3; dopamine transporter 1; DAT1) localizes to 5p15.33; the gene has repeatedly been implicated in the etiology of ADHD. However, in our sample the DAT1 VNTR did not show association with ADHD. We additionally identified nominal evidence for linkage to chromosomes 6q, 7p, 9q, 11 q, 12q and 17p, which had also been identified in previous scans. Despite differences in ethnicity, ascertainment and phenotyping schemes, linkage results in ADHD appear remarkably consistent.

Introduction

Attention-deficit/hyperactivity disorder (ADHD) is one of the most heritable disorders in child and adolescent psychiatry; heritability is estimated at approximately 0.81 and the recurrence risk in siblings is in the magnitude of 5.2 A number of candidate genes have been investigated using both case–control and family-based studies in order to identify genetic variation underlying the disorder.3 The most frequently analyzed genes include the dopamine D4 receptor gene (DRD4) and the gene for the dopamine transporter (DAT1, SLC6A3); meta-analyses have yielded significant (P<0.02) results for DRD44, 5 and negative results (P=0.06 and 0.21) for the DAT1 VNTR.5, 6

The first whole-genome scan was performed in a US sample encompassing 126 affected sib-pairs from 104 families,7 which was subsequently extended to 204 families with a total of 270 sib-pairs.8, 9 An independent genome scan based on 164 sib-pairs from 106 families was performed in a Dutch sample.10 A third group performed a genome-wide scan of 16 extended and multigenerational families with ADHD from a genetic isolate located in Columbia.11 These genome scans and subsequent fine mapping studies of peak regions have yielded significant evidence of linkage12 on 4q13.2, 5q33.3,11 6q12,9 11q22,11 16p13,13 and 17p11.9, 11 Chromosomal regions that yielded at least nominal evidence of linkage in two of the three genome scans include 11q22, 17p11, 20q13,8, 9, 11 3q13 and 9q33.10, 11 Nominal evidence of linkage was detected to 5p13 in all three scans.8, 9, 10, 11 Within this region, the highest multipoint MLS scores were localized at markers D5S418 (MLS=2.559) and D5S2500 (MLS=1.43 for the broad phenotype definition of ADHD10); the distance between the two markers is 18.7 Mb.

In the current study, we report the results of a fourth independent genome-wide scan performed in a German sample of 102 families encompassing a total of 229 affected children.

Materials and methods

The 102 families were ascertained and phenotypically characterized by physicians either in the outpatient units of the Departments of Child and Adolescent Psychiatry of the Universities of Aachen, Lübeck, Marburg and Würzburg and of the district Oberpfalz in Regensburg for ADHD. The physicians were in the process of specialization in child and adolescent psychiatry; they were specifically trained for the purpose of the study. Phenotypical assessments were performed either within the respective outpatient units or at the families' homes. Informed parental consent was obtained for all participants and the study was approved by the ethics committees of all participating hospitals.

Families were included if they had at least two affected children with ADHD, one of whom we required to be older than 8 years and the other(s) to be older than 6 years. The children all currently fulfilled diagnostic criteria for ADHD according to DSM-IV;14 index patients were required to fulfill lifetime criteria for the combined type (see Table 1 for clinical features of our sample). In 80, 19 and three families two, three, or four affected children were ascertained, respectively. Of the 155 sib-pairs, 127 were independent; four of the sib-pairs were based on half-sibs. In 94 families, both parents of the index patient were ascertained, and in the remaining eight only the mother was included. The vast majority of parents were of German origin; only 12 parents came from other European countries. All were of Caucasian ethnicity. Socio-economic status (SES) was determined for the whole family based on reported paternal profession and categorized into three groups. A total of 26, 61 and two families had a high, medium and low SES, respectively. For 13 families, the SES could not be determined.

Table 1 Clinical features of ascertained ADHD children

Exclusion criteria (which applied both to index patients and siblings) were (a) general IQ75 as assessed with one of the following IQ tests: the Wechsler Intelligence Scale for Children (WISC-III;15 German version16), the Kaufman Assessment Battery for Children (K-ABC;17 German version18) or the Culture Fair Test (CFT)19, 20, (b) potentially confounding psychiatric diagnoses such as schizophrenia, any pervasive developmental disorder, Tourette's disorder, and primary mood or anxiety disorder (patients with comorbid mood or anxiety disorders were not excluded; in these cases the disorder clearly developed after onset of ADHD), (c) neurological disorders such as epilepsy, (d) a history of any acquired brain damage or evidence of the fetal alcohol syndrome, (e) very preterm birth and/or (f) maternal reports of severe prenatal, perinatal or postnatal complications.

Psychiatric classification was based on the Schedule for Affective Disorders and Schizophrenia for School-Age Children Present and Lifetime version (K-SADS-PL;20 German translation21). The K-SADS-PL is a highly reliable semi-structured interview used to assess a wide range of psychiatric disorders according to DSM III-R,22 DSM-IV14 or ICD-10.23 Mothers received (1) the unstructured introductory interview, (2) the diagnostic screening interview, (3) the supplement completion checklist and upon fulfillment of screening criteria the appropriate diagnostic supplements. Mothers were asked to report the symptoms applicable to their children in the unmedicated state. The child was interviewed with the screening interview of the K-SADS and in case of positive screening for mood or anxiety disorders with the respective supplements of the K-SADS-PL. The rationale for this approach was that internalizing disorders are more reliably assessed by self-reports than by parental ratings, while it turns out to be the other way round for externalizing childhood behavioral disorders.24 Additionally, we employed the Child Behaviour Checklist (CBCL,25 German translation26) and a German teacher rating scale for ADHD (FBB-HKS);27 the questions of the latter instrument directly address the DSM-IV items.

The K-SADS-PL (as based on the maternal interview) was used to assess ADHD both categorically and dimensionally. The sum of nine possible inattentive, nine hyperactive/impulsive or all 18 possible symptoms yielded a severity score of inattention, hyperactivity/impulsivity and a total score, respectively.

‘Best estimate’ diagnoses, which totally overlapped with the diagnosis of ADHD in the K-SADS-PL, were determined for all children after individual review of diagnoses, symptoms and impairment level by senior clinicians (JH, KK, HK, UH) in regular case conferences of participating clinicians scheduled approximately every 6 months throughout the 2-year ascertainment scheme. We consider our index patients as representative of ADHD patients of German child and adolescent psychiatric units. Thus, 161 of the total of 229 affected offspring were currently or had previously been treated with stimulant medication. In 202 children, ADHD had clinically been diagnosed previously by a child and adolescent psychiatrist (155), a pediatrician (31), a psychologist (13) or other physician (3).

Genotyping

We performed a genome-wide scan based on a total of 425 individuals with initially 475 autosomal, 24 X-chromosomal and two pseudoautosomal STR markers spanning the entire genome with an average (maximum) distance of 7(13)cm (adapted from Saar et al.28). Briefly, markers were amplified in multiplex reactions in 384-well microtiter plates on ABI GeneAmp PCR 9700 machines (Applied Biosystems, Darmstadt, Germany). An aliquot of the PCR reaction was submitted to analysis on ABI 3730 sequencers. Semi-automated genotyping was performed with the help of the GeneMapper software version 3.0 (ABI). All genotypes were scored independently by an experienced lab technician and subsequently by one scientist. In all, 16 additional markers (see Table 2) in the major region of interest on chromosome 5 (mostly around the DAT1 (SLC6A3) locus) were subsequently genotyped, including the DAT1 VNTR.5, 6

Table 2 Linkage results based on fine mapping of chromosome 5p: genotyped markers and nonparametric multipoint LOD scores in the chromosome 5 region between 0 and 65 cM (initial markers are in bold, their LOD scores in the initial analysis are in parenthesis)

Statistical analyses

Familial relationships were verified using the GRR program29 on the genome scan markers, which led to the identification of two pairs of identical twins, which were excluded from further analysis (not included in the number of sib-pairs stated above) and two pairs of half-sibs which had been reported to be full-sibs. We checked all markers for Mendelian inconsistencies using PedCheck30 and in case of inconsistencies set the genotypes of all family members for the respective marker and family on missing. Hardy–Weinberg equilibrium was checked for all genetic markers on parental genotypes by χ2 tests as implemented in Mega2 version 3.0,31 and Merlin version 1.0 alpha.32, 33 We checked for likely genotyping errors (e.g., manifesting as close double recombinants) using Merlin32, 33 and set unlikely genotypes on missing for the respective individuals.

Allele frequencies for linkage analysis (relevant only for the few missing parents) were estimated from all parental alleles. Data handling and preparation of files was done with Mega2 version 3.0.31 Our primary linkage analysis was on the qualitative affection status ADHD using the nonparametric Sall statistic.34 For easier comparison of our results with previously published scans, we give both the NPL score and the nonparametric LOD score, using the Kong–Cox transformation35 as implemented in Merlin.32, 33 This transformation is not monotone; NPL scores tend to be highest at genotyped markers, while the Kong–Cox LOD scores usually drop at markers and are higher between markers,35 as parametric LOD scores do as well. We also performed linkage analysis on the quantitative DSM-IV scores (total score, inattentive and hyperactive/impulsive subscores defined as the number of DSM-IV ADHD symptoms present, from the K-SADS-PL) using Merlin-regress36 assuming a heritability of 0.6 and a population mean (variance) of 3(2.5), 1.6 (1.5) and 1.4 (1.5), respectively, as determined previously in Germany.37 This analysis does not require that the phenotypes are normally distributed. Finally, we also performed parametric HLOD linkage analysis with Merlin32, 33 under a dominant model assuming a penetrance of 0.6 (phenocopy rate 0.05) and an allele frequency of 0.01. The empirical genome-wide significance of all linkage results was assessed by simulating 1000 replicates of our data under the null hypothesis of no linkage with the same characteristics regarding family structures, phenotypes, marker spacing and informativity. These simulated scans were analyzed analogously to the real data and the highest nonparametric LOD score and parametric HLOD score in each scan were recorded. The percentage of times that the simulated maximum score exceeds the score obtained in the real data gives the empirical genome-wide P-value.

Post-hoc analyses as to whether the evidence for nonparametric linkage on chromosome 5p could be explained by the investigated DAT1 VNTR was done using the genotype-IBD sharing test GIST,38 which investigates whether the allelic distribution of a polymorphism might be responsible for an observed linkage by testing whether probands with a certain genotype contributed more than expected to the linkage signal. Additionally, we used the pedigree disequilibrium test (PDT),39 a test for allelic association in pedigrees.

Results

The results of the nonparametric linkage analysis are illustrated in Figure 1 and Tables 2 and 3; the highest multipoint LOD score of 2.74 (which corresponds to an NPL score of 3.25) in the initial analysis based on the original 501 STR markers was obtained on chromosome 5p at 3 cM next to the marker D5S2005 (1.72 cM on the Marshfield map, with a LOD score of 2.66) in a broad peak extending beyond D5S1457 (59.3 cM, LOD score 1.35). Other multipoint LOD scores exceeding 1 were obtained for chromosomes 8, 12, 17 and in the pseudoautosomal region of the X and Y chromosomes. Upon genotyping of an additional 16 markers on chromosome 5p, the maximum multipoint LOD score in this region was 2.59 (which corresponds to an NPL score of 3.52; empirical genome-wide P-value 0.1) at 17 cM, close to the marker D5S1953 with a LOD score of 2.58.

Figure 1
figure1

Nonparametric multipoint LOD scores for ADHD.

Table 3 Nonparametric multipoint LOD scores above 0.5 and adjacent markers

The highest multipoint LOD scores for the quantitative DSM-IV scores (obtained upon inclusion of all 521 STR markers) were observed on chromosome 5p for the total score and inattentive subscore (maximum LOD scores of 2.31 at D5S674 (47.09 cM) and 3.37 at D5S502 (39.46 cM), respectively). For the inattentive subscore, the second highest LOD score of 3.26 was obtained on chromosome 12 (D12S1679 at 153.19 cM). The highest linkage peak for the hyperactive/impulsive subscore was on chromosome 8 (maximum LOD score of 1.26 at D8S1778 (110.2 cM)) (Figures 2, 3 and 4).

Figure 2
figure2

Nonparametric multipoint LOD scores for total number of DSM-IV ADHD symptoms.

Figure 3
figure3

Nonparametric multipoint LOD scores for number of DSM-IV inattentive ADHD symptoms.

Figure 4
figure4

Nonparametric multipoint LOD scores for number of DSM-IV hyperactive/impulsive ADHD symptoms.

In the parametric HLOD analysis under a dominant model, we obtained the highest multipoint LOD score of 4.75 again on chromosome 5p, with all families contributing to this LOD score (empirical genome-wide P-value 0.001). HLODs above 1 were also observed on chromosomes 7, 8, 12 and in the pseudoautosomal region on the X and Y chromosomes (Table 4).

Table 4 Results of parametric linkage analysis of ADHD under a dominant model

The DAT1 VNTR did not show an association with ADHD (multiallelic PDT P-value of 0.97). In particular, the 480 bp allele, which had been associated with ADHD in several previous studies (see meta-analyses 5, 6), did not show any relevant over-transmission in our sample (298 times transmitted vs 292 times not transmitted). The linkage signal also did not stem from the VNTR (GIST P-values >0.7 under dominant, additive or recessive models).

Discussion

Our genome-wide scan in ADHD and all three previous scans7, 8, 9, 10, 11 have detected linkage signals in the distal region of chromosome 5p; no other linkage signal has consistently emerged in all scans. The LOD score obtained by Ogdie et al.9 was slightly lower than our score; in both studies the Lander and Krugylak12 criterion for suggestive evidence for linkage was fulfilled; the Bakker et al.10 LOD score of 1.43 was below this criterion. Our HLOD score of 4.75 obtained in the post-hoc parametric analysis even fulfilled the criterion for evidence of linkage for a complex disorder (genome-wide empirical P-value 0.001).

Our initial nonparametric LOD score value of 2.74 next to the marker D5S2005 (1.72 cM on the Marshfield map) was obtained within a broad peak extending from the distal end of the short arm to beyond 60 cM; our subsequent fine-mapping efforts resulted in a nonparametric LOD score of 2.59 at 17 cM. Ogdie et al.9 localized their 5p peak to 59 cM, Bakker et al.10 to 69 cM. In light of the small sample sizes, which lead to considerable stochastic variation of the location of linkage peaks,40 it seems plausible to assume that genetic variation at the same locus underlies the chromosome 5p linkage peaks. The common identification of this region is even more noteworthy in light of the ethnic differences and differences in ascertainment and phenotyping.

Our post-hoc parametric HLOD analysis based on a single model assuming autosomal dominant inheritance with a penetrance of 0.6, a phenocopy rate of 0.05 and an allele frequency of 0.01 resulted in a considerably higher multipoint LOD score of 4.75 at 17 cM. We chose this particular model because we thought that the specified parameters might come close to the real situation in ADHD. Indeed, the HLOD was considerably higher than the nonparametric multipoint LOD score and was significant with an empirical genome-wide P-value of 0.001.

Our analyses based on the quantitative DSM-IV scores again picked up the chromosome 5p region; interestingly, the highest multipoint LOD scores on chromosome 5p differed considerably between the inattentive (3.37 at D5S502, 39.5 cM) and the hyperactivity/impulsivity (1.11 at D5S1457, 59.3 cM) subscores, possibly suggesting that inattention is the phenotype more strongly influenced by the gene(s) in this linkage region (Figure 5). Another likely explanation for this finding is chance, further possibilities include a potentially better temporal stability of symptoms of inattention, their more valid rating by the mothers of our probands and/or a less pronounced contrast (rater) effect (reviewed by Heiser et al.,3) in comparison to symptoms of hyperactivity/impulsivity. Also note that due to our assessment scheme (where the index case is required to have combined subtype), variance for inattentive symptoms was slightly larger than for hyperactive–impulsive symptoms which might have contributed to higher LOD scores for inattentive symptoms. The LOD score differences also need to be interpreted in light of the fact that we had requested the mothers to rate current symptoms as applicable to their unmedicated children.

Figure 5
figure5

LOD scores for ADHD on chromosome 5.

The DAT1 located at the distal end of 5p is an obvious candidate gene within the linkage peaks observed in the different genome scans.7 Stimulant medications that are efficacious in ADHD block binding of dopamine to the dopamine transporter,41 and imaging studies have found reduced dopamine transporter binding in adults with ADHD.42, 43, 44 We had therefore included the DAT1 VNTR and the STR markers D5S2005, D5S1981 and D5S678 in our fine mapping efforts in an attempt to assess the contribution of genetic variation at the DAT1 locus to the linkage signal. Interestingly, in our study allelic variation at the DAT1 VNTR was not responsible for the linkage signal. Previous association and functional studies pertaining to DAT1 have mainly focused on the 3′-UTR VNTR.3 A meta-analysis based on 11 studies with a total of 824 informative meioses yielded an almost significant (P=0.06) pooled odds ratio of 1.27,5 whereas the meta-analysis of Purper-Ouakil et al.,6 based on 13 published family-based association studies, showed that the 10-repeat allele has no significant impact on the risk for ADHD. A recent functional study did not confirm earlier findings that the VNTR has an effect on DAT1 expression levels.45 Instead, it was argued that the previously reported associations with psychiatric phenotypes and in particular ADHD might be mediated via linkage disequilibrium with functionally relevant polymorphisms. This is consistent with our results that show no allelic association of the DAT1 VNTR but imply that other genetic variation in DAT1 or another gene in the 5p region is involved in susceptibility to ADHD.

Quite evidently, further studies are required in an attempt to uncover the genetic variation, that in functional terms underlies our and previous chromosome 5p linkage results and positive DAT1 association studies.3, 5, 6 Efforts should be undertaken to genotype the same DAT1 markers in those samples that have been analyzed for linkage and/or association, thus enabling meta-analyses based on single markers and haplotypes.

It deserves to be pointed out that in addition to linkage to chromosome 5p we identified further regions that potentially coincide with some of those detected in the three previous genome scans for ADHD (Table 5). In particular, our nonparametric multipoint LOD score of 1.39 on chromosome 17 at 1 cM (0.57 Mb) is in the vicinity of peaks detected by both Ogdie et al.8, 9 and Arcos-Burgos et al.11 Similarly, our multipoint LOD score of 0.92 on chromosome 7 at 88 cM (75 Mb) is in the same region as the highest LOD score of 3.04 detected by Bakker et al.10 at 70 cM (49 Mb). Our nonparametric multipoint LOD score of 2.1 on chromosome 12 (166 cM, 130 Mb) coincides with a peak (MLS of 1.09) detected by Fisher et al.7 (165 cM, 125 Mb). These overlapping linkage results are extremely promising. They suggest that specific ADHD genes are common to different populations and that they can be detected in various ADHD samples despite differences in ascertainment, phenotyping and statistical analyses.

Table 5 Overlap of linkage results of current study, Bakker et al.,10 Fisher et al.,7 Ogdie et al.,8, 9 and Arcos-Burgos et al.11

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Acknowledgements

We thank the families for their participation in this study. Seven physicians ascertained the families and performed the phenotypical assessments; we greatly acknowledge their substantial contribution to this study. We thank Regina Pospiech and Inka Szangolies for excellent technical assistance and Gundula Ringler for exceptional data management. The German Ministry for Education and Research (National Genome Research Net; grants 01GS0118, 01GS0482, 01GS0483, 01GR0460) and the Deutsche Forschungsgemeinschaft (grant SCHA 542/10-2) financially supported this study.

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Correspondence to J Hebebrand.

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Electronic-Database Information

The URLs for data presented herein are as follows: Mega2 version 3.0: http://watson.hgen.pitt.edu

Marshfield map: http://research.marshfieldclinic.org

Golden Path (hg17): http://genome.ucsc.edu

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Keywords

  • ADHD
  • 5p13
  • dopamine transporter

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