| Summary |
The DAT1 gene has additional common polymorphisms in intron 9 and exon 9. They investigated the possibility of linkage of DAT1 and ADHD using the VNTR polymorphism and two additional common polymorphisms in 102 nuclear families with an ADHD proband. Using the transmission disequilibrium test, they examined the transmission of the alleles of each of these polymorphisms, as well as the haplotypes of the polymorphisms. They did not observe significant evidence for the biased transmission of the alleles of either the VNTR or the additional two polymorphisms when examined individually, although there was a trend for the biased transmission of the 480-bp allele of the VNTR. When they examined the haplotypes of the three polymorphisms they found significant evidence for biased transmission of one of the haplotypes containing the 480-bp VNTR allele. They also genotyped six additional DNA sequence variants of the DAT1 gene. However, these variants were not sufficiently polymorphic in their sample to be informative. Two of the DNA variants that result in an amino acid change, Ala559Val and Glu602Gly, were not observed in their sample. |
| Total Sample |
They genotyped 333 individuals from 102 nuclear families (ADHD probands with both parents) identified from the Toronto area. Twenty-seven affected siblings were also genotyped and used in the analysis. Twenty-one of the families had two ADHD children, and three families had three ADHD children. |
| Sample Collection |
The assessment and characteristics of the subjects for this study have been previously described (Barr et al 1999, 2000a, 2000b, 2000c). They genotyped 333 individuals from 102 nuclear families (ADHD probands with both parents) identified from the Toronto area. |
| Diagnosis Description |
All subjects in this study met the following DSM-IV criteria for ADHD; Information for the diagnosis of ADHD and comorbid conditions was based on a semi-structured interview for parents and teachers. The interviews were supplemented with the following questionnaires and child assessments: Conners Parent and Teacher Rating Scales (revised; Conners 1997), the Ontario Child Health Survey Scales (revised; Boyle et al 1993), Wide Range Achievement Test (third edition; Wilkinson 1993), Clinical Evaluation of Language Fundamentals (third edition; Semel et al 1995), Children's Depression Inventory (Kovacs 1995), and Children's Manifest Anxiety Scale (Reynolds and Richmond 1985). Of the subjects who were observed in this study, 57% of the children were of the combined subtype, 19% were of the hyperactive/impulsive subtype, and 24% were of the primarily inattentive subtype. |
| Technique |
DNA was extracted from blood lymphocytes using a high-salt extraction method (Miller et al 1988). The VNTR polymorphism in DAT1 was genotyped as previously described (Vandenberghet al 1992). For the other polymerase chain reactions (PCRs), the PCR used 40 ng of each of the primers, 0.05 mmol/L deoxynucleotides, 1.5 mmol/L magnesium chloride, and 0.5 units Taq polymerase in a total reaction volume of 20 uL. For restriction enzyme analyses 5-10 uL of PCR product was digested with 5 units per reaction of the appropriate restriction enzyme for each polymorphism for 1.5 hours. For single-strand conformational polymorphism (SSCP) analysis, 1 uL of PCR product was denatured in 12 uL of SSCP dye (98% formamide, 0.1% sodium dodecyl sulfate, 10 mmol/L EDTA, 0.1% bromphenol blue, and 0.1% xylene cyanole) at 94oC for 4 min, chilled on ice, and electrophoresed on 4-20% TBE minigels (Novex, San Diego) at 20oC, 10oC, or 4oC to increase the sensitivity of detection of mobility shifts. |
| Analysis Method |
They used the TDT to search for linkage of the alleles and haplotypes to ADHD. The TDT statistic was calculated with the extended TDT (ETDT) program (Sham and Curtis 1995). For the analysis of the haplotypes, they used two approaches to analyze the data. For the first method, they determined the haplotypes by inspection of the pedigrees. They also analyzed the haplotypes using the TRANSMIT program (version 2.5; Clayton 1999). |
| Result Description |
They did not observe significant evidence for the biased transmission of the alleles of either the VNTR or the additional two polymorphisms when examined individually, although there was a trend for the biased transmission of the 480-bp allele of the VNTR. When they examined the haplotypes of the three polymorphisms they found significant evidence for biased transmission of one of the haplotypes containing the 480-bp VNTR allele. They also genotyped six additional DNA sequence variants of the DAT1 gene. However, these variants were not sufficiently polymorphic in their sample to be informative. Two of the DNA variants that result in an amino acid change, Ala559Val and Glu602Gly, were not observed in their sample. |
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