| Summary |
They applied model-based and model-free linkage analyses, as well as the pedigree disequilibrium test, to the results of a genome-wide scan of extended and multigenerational families with ADHD from a genetic isolate. In these families, ADHD is highly comorbid with conduct and oppositional defiant disorders, as well as with alcohol and tobacco dependence. They found evidence of linkage to markers at chromosomes 4q13.2, 5q33.3, 8q11.23, 11q22, and 17p11 in individual families. Fine mapping applied to these regions resulted in significant linkage in the combined families at chromosomes 4q13.2 (LOD=4.44 at D4S3248), 5q33.3 (LOD=8.22 at D5S490 ), 11q22 (LOD=5.77 at D11S1998; multipoint nonparametric linkage [NPL] -log[P-value]=5.49 at ~128 cM), and 17p11 (multipoint NPL -log[P-value]>12 at ~12 cM; multipoint maximum location score 2.48 [alpha=0.10] at ~12 cM; LOD=3.73 at D17S1159). Additionally, suggestive linkage was found at chromosome 8q11.23 (combined two-point NPL -log[P-value] >3.0 at D8S2332). Several of these regions are novel (4q13.2, 5q33.3, and 8q11.23), whereas others replicate already-published loci (11q22 and 17p11). The concordance between results from different analytical methods of linkage and the replication of data between two independent studies suggest that these loci truly harbor ADHD susceptibility genes. |
| Total Sample |
There are more than 500 members in the 16 multigenerational Paisa families presented in this study (79 nuclear families embedded in the extended pedigrees; sibship average size 2.4; range 1-12 sibs). Blood samples for DNA extraction (n=375) and, in some cases, for lymphoblastoid cell lines (n=36) were obtained from all participating family members. Of them, 109 subjects were diagnozed as ADHD. |
| Sample Collection |
The sample was selected from Paisa families from the Medellin metropolitan area in the state of Antioquia, Colombia. Families had to be of Paisa descent, to comprise more than two generations, and to have more than two members affected with ADHD. Individuals were considered to be of Paisa descent if all four grandparents originated from the Paisa region of Colombia (i.e., from the former state of Viejo Caldas). Initial coded pedigrees obtained through a fixed sampling scheme from a parent or grandparent of an index proband were individually reviewed by a genetic statistician to minimize the confounding effects of bilineal transmission of ADHD. Bilineality was defined by the presumptive diagnosis of ADHD in both parents on the basis of an informant's reports of childhood symptoms and/or of academic, occupational, or legal impairment, including alcoholism and related consequences. Full pedigrees that were identified as bilineal were excluded from further study. During the selection phase, pedigrees that contained branches that were bilineal were pruned to preserve the presumptively unilineal branches. Individuals in the selected families were then invited to participate in the present study. |
| Diagnosis Description |
Ascertainment methods, power simulations for both linkage and association, population genetics, demographics, inclusion and exclusion criteria, clinical methods of assessment, and clinical features of the sample have been described elsewhere (Lopera et al. 2001; Arcos-Burgos et al. 2002, 2004). |
| Technique |
A genome scan using automated fluorescent microsatellite analysis was performed at the Center for Inherited Dis-ease Research (CIDR). The current CIDR marker set consists of ~400 primer pairs with an average spacing of 9cM throughout the genome and an average marker heterozygosity of 0.76. Additional details about genotyping, quality control, marker information, and laboratory protocols can be accessed at the Center for Inherited Disease Research Web site. After a genome-wide search was completed at CIDR and the data were analyzed, they genotyped microsatellite loci spanning five regions that exhibited suggestive linkage, with an average resolution of ~1.5 cM. Genotyping was performed by deCODE. Additional details about genotyping, quality control, marker information, and laboratory protocols can be accessed at the deCODE Web site. |
| Analysis Method |
Parametric analysis of linkage was based on the best-fitting genetic model from a previous complex segre-gation analysis of Paisa families with ADHD (Lopera et al. 1999). Two-point LOD score analyses were estimated using FASTLINK. SIMWALK2 was used to perform the parametric multipoint analysis and the heterogeneity analysis and to obtain two-point nonparametric (model-free) linkage (NPL) A-E statistics. Sparse inheritance trees were used for pedigree analysis, as implemented in MERLIN, by trimming affected individuals - mostly single affected cases in a nuclear family - belonging to the central branches of the more extended pedigrees. Additionally, two-point identity-by-descent (IBD)-sharing estimates for the marker loci were obtained with the GENIBD program from the SAGE (Statistical Analysis for Genetic Epidemiology) software package (version 4.5). If the pedigree did not meet the size restriction, IBD-sharing estimates were calculated with the Markov chain¨CMonte Carlo simulation algorithm. With these IBD-sharing estimates, they performed linkage analysis using the conditional logistic model. Two-point analyses were performed for all relative pairs by use of the one-parameter and two-parameter models for the conditional logistic model. They also used the pedigree disequilibrium test (PDT) to search for evidence of linkage disequilibrium (LD) between ADHD and the marker loci. Power analysis and design considerations for applying family-based association tests (FBATs) were used to determine the specific power exhibited by this set of families, as implemented in the Power-Based Association Test software. To test for the presence of linkage heterogeneity with respect to single-marker loci, they used the overall LOD score based on a mixture likelihood, referred to as the heterogeneity LOD score. For this purpose, they used the HOMOG suite of programs and performed two types of heterogeneity testing. To perform family-based association studies involving haplotypes, the HAPLOTYPE module of SIM-WALK2 was used to reconstruct the more probable haplotype arrangements for these two loci. |
| Result Description |
They found evidence of linkage to markers at chromosomes 4q13.2, 5q33.3, 8q11.23, 11q22, and 17p11 in individual families. Fine mapping applied to these regions resulted in significant linkage in the combined families at chromosomes 4q13.2 (LOD=4.44 at D4S3248), 5q33.3 (LOD=8.22 at D5S490 ), 11q22 (LOD=5.77 at D11S1998; multipoint nonparametric linkage [NPL] -log[P-value]=5.49 at ~128 cM), and 17p11 (multipoint NPL -log[P-value]>12 at ~12 cM; multipoint maximum location score 2.48 [alpha=0.10] at ~12 cM; LOD=3.73 at D17S1159). Additionally, suggestive linkage was found at chromosome 8q11.23 (combined two-point NPL -log[P-value] >3.0 at D8S2332). |
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