| Summary |
They performed the first systematic genome-wide linkage scan for loci influencing ADHD in 126 affected sib pairs, using a ~10-cM grid of microsatellite markers. Allele-sharing linkage methods were used to exclude any loci with a lambdas of >=3 from 96% of the genome and those with a lambdas of >=2.5 from 91%, indicating that there is unlikely to be a major gene involved in ADHD susceptibility in their sample. Under a strict diagnostic scheme all screened regions of the X chromosome could be excluded for a locus-specific lambdas of >=2 in brother-brother pairs, demonstrating that the excess of affected males with ADHD is probably not attributable to a major X-linked effect. Qualitative trait maximum LOD score analyses pointed to a number of chromosomal sites that may contain genetic risk factors of moderate effect. None exceeded genome-wide significance thresholds, but LOD scores were >1.5 for regions on 5p12, 10q26, 12q23, and 16p13. Quantitative-trait analysis of ADHD symptom counts implicated a region on 12p13 (maximum LOD 2.6) that also yielded a LOD >1 when qualitative methods were used. A survey of regions containing 36 genes that have been proposed as candidates for ADHD indicated that 29 of these genes, including DRD4 and DAT1, could be excluded for a ls of 2. Only three of the candidates-DRD5, 5HTT, and CALCYON-coincided with sites of positive linkage identified by this screen. Two of the regions highlighted in the present study, 2q24 and 16p13, coincided with the top linkage peaks reported by a recent genome-scan study of autistic sib pairs. |
| Total Sample |
The genome-scan sample comprised 104 families. Seven families included only one parent; the remaining 97 families in cluded both. Several families included more than two affected siblings, yielding a total of 126 possible ASPs for analysis. The mean full scale IQ was 105 (SD 14). Five subjects scored in the 70-80 range, but none of these were from the same ASP family. The majority (94%) of the ASP members met definite criteria for ADHD, and each family included at least one child with a definite diagnosis. The distribution of subtypes among the affected siblings was similar to that observed in epidemiological studies of ADHD, as were the frequencies of comorbid psychiatric disorders (Brown et al. 2001). The 126 ASPs included 66 brother-brother, 54 brother-sister, and 6 sister-sister pairs. |
| Sample Collection |
Families were identified through clinics, hospitals, schools, and community organizations in the greater Los Angeles area, as part of an ongoing molecular genetic study of ADHD. The majority of families were initially ascertained through advertisements requesting the partic-ipation of families with at least two children >=5 years of age showing symptoms of ADHD. An additional 18 families were selected from a previous family study of ADHD because they were each known to contain an affected sib pair. The sample was largely white (84%), with the greatest representation of families in socioeconomic classes II and III (67%). |
| Diagnosis Description |
Assessment of psychiatric disorders, including ADHD, was performed using a semistructured interview, the Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime version (KSADS-PL). This was administered to the mother and was followed by a direct interview with the child if he or she was >=8 years of age. Additional measures employed included the parent and teacher versions of the SNAP-IV, the Child Behavior Checklist, and the Teacher's Report Form. A best estimate procedure, using all available information, was employed to determine diagnoses, with senior psychiatrists reviewing positive diagnoses in a weekly case-review meeting. The mean weighted kappa for psychiatric diagnoses was .84 (SD=.14), with values of 1.0, .93, and 1.0 for ADHD, ODD, and CD, respectively. ADHD was diagnosed by DSM-IV criteria. Other psychiatric disorders were based on lifetime diagnoses made by use of DSM-IV criteria (or DSM-IIIR, in the case of the 18 families drawn from the earlier family study). Families were excluded from the study if a child affected with ADHD also met criteria for schizophrenia or autism. However, other psychiatric diagnoses (such as ODD or CD) and/or evidence of specific learning disa-bilities were not grounds for exclusion. Full scale IQ was determined by use of the WISC-III, and academic achievement was assessed using the Peabody Individual Achievement Test-Revised. Children with full-scale IQs <70 were excluded from the study. A more detailed description of the sample and mea-sures is given by Smalley et al. (2000) |
| Technique |
In children and parents from each family, 404 highly polymorphic markers, spanning all 22 autosomes and the X chromosome, were genotyped. The majority of autosomal markers were taken from the ABI PRISM LMS2-MD10 panels (Applied Biosystems), whereas the X-chromosome markers came from the CHLC (Cooperative Human Linkage Center)/Weber Human Screening Set Version 6 (Research Genetics). Sex-averaged marker maps were derived primarily from the CHLC, were supplemented with data from Genethon (Dib et al. 1996), and were verified by comparison to maps estimated from the family sample. For autosomal markers, semiautomated fluorescent genotyping was performed by use of standard techniques, as described by Fisher et al. (1999). For Xlinked genotyping, pooled PCR products were electro-phoresed on a LICOR apparatus GeneReadIR 4200, and gel images were analyzed with Saga Genotyping Software version 1.0 (University of Washington). Raw allelesize data were checked for Mendelian inheritance and were converted to LINKAGE format by use of the GAS software package (version 2.0) (A. Young, Oxford University). The Discovery Manager (Genomica) database system was used for storage of genotypic and phenotypic data and for exporting files in the appropriate format for statistical analysis. As a final check on genotyping quality, marker haplotypes were generated from the data by use of Genehunter version 2.0 (Kruglyak et al. 1996), to identify any chromosomes showing an excessive number of recombination events. Allele frequencies were estimated from all founders in the sample. |
| Analysis Method |
Singlepoint and multipoint sib-pair based linkage analyses of genotype data were performed under two qualitative classification schemes: broad (all sibs have a probable or definite DSM-IV diagnosis of ADHD, 126 pairs) and narrow (all sibs met a definite diagnosis, 110 pairs). Autosomal markers were analyzed by use of the Mapmaker/SIBS options available in version 2.0 of the Genehunter software package. Linkage was assessed by the maximum LOD score (MLS) method. Exclusion mapping was performed under a series of locus-specific values of lambdas, under the assumption of no dominance variance. For X-chromosome markers, MLS and exclusion-mapping methods were run using the X-linked estimate and exclude options of the Mapmaker/SIBS software package. All analyses utilized all possible sibling pairs with no weighting schemes. Multipoint methods employed a 1-cM increment for IBD scanning. Quantitative Trait Linkage (QTL) analysis was performed via traditional Haseman-Elston (HE) regression of sib-pair squared trait differences against estimated IBD sharing, using the Genehunter 2.0 package. X-linked HE analyses were performed by use of Mapmaker/SIBS. As for qualitative analyses, the quantitative approach employed all possible sibling pairs with no weighting schemes and a 1-cM increment was used for IBD scanning. The present study reported precise P values without adjustment for multiple comparisons. |
| Result Description |
In single-point MLS analysis, one marker (D5S418 on 5p12) yielded a LOD score of >2, whereas additional markers on 2q, 4p, 7p, 9q, 10q, 12p, 12q, 13q, and Xp gave LODs >1. Multipoint data implicated the same regions of 5p, 9q, 10q, 12q, 13q, and Xp as those highlighted by single-point analyses. Multipoint LOD scores also exceeded 1 on chromosomes 11q and 16p. Three of the regions suggested by multipoint analyses--10q26, 12q23, and 16p13--yielded peak LOD scores of >1.5 under at least one diagnostic scheme. For X-linked MLS analyses the overall LOD score is taken as the sum of those separately calculated for brother-brother, brother-sister, and sister-sister pairings. The Xp22 linkage identified in the ADHD ASP sample arose exclusively from the brother-sister pairings. Investigation of the D4-TOT quantitative measure yielded LOD scores >1 for markers on 3q, 8p, 12p, 13q, 16q, and 21q in single-point analysis. The 8p, 12p, and 13q regions also yielded LODs >1 when multipoint data were used, with a peak LOD score of 2.6 in 12p13. Sib-pair exclusion mapping involves calculation of the expected allele sharing proportions of a particular locus, under the assumption of a given locus-specific lambdas. The likelihood of the genotype data assuming this lambdas is compared to the likelihood under the null hypothesis of no linkage. If the resulting LOD score is <-2, this is traditionally taken as evidence for exclusion of the region being investigated. Using multipoint data from all 126 ASPs, 96% of all regions scanned from containing a locus with a lambdas of >=3, and 91% from containing a locus with a lambdas of >=2.5 could be excluded. The investigated regions of chromosomes 18, 19, 21, and 22 could be entirely excluded from containing a locus with a lambdas of >=2 under each of the diagnostic schemes. The X chromosome could be completely excluded for a lambdas of >=2 under the narrow diagnostic scheme. This includes the Xp22 region implicated by MLS analyses of brother-sister pairs, but the evidence for exclusion of this region came exclusively from the brother-brother pairings. |
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