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DAT1 GENE AND ADHD IN CHINESE CHILDREN

which was standardized by Gong Yaoxian. Subjects were excluded if their IQ scored below 70, or showed evidence of neurological or chronic medical illness, bipolar affective disorder, psychotic symptoms, Tourette syndrome, or chronic physical disability. Children were free of medication for a minimum of 24hr before their assessment. DAT1 genotyping

Peripheral blood was drawn from the ante cubical vein, and genomic DNA was extracted from whole blood using a standard DNA isolation procedure from 54 ADHD cases and 108 their biological parents, in addition to 66 controls. DNA was resuspended in 20 μl of water and stored at 4°C (concentration range 25-100 ng L-1).

The primer sequence used to amplify the 40-bp sequence of the VNTR polymorphic loci was as follows [15,18,22]: PCR amplification of the DAT1 40-bp VNTR in the 3’-UTR was carried out using the following pair of primers, upstream: 5’-TGT GGT GTA GGG AAC GGC CTG AG-3’ and downstream: 5’-CTT CCT GGA GGT CAC GGC TCA AGG-3’. Forty cycles were conducted consisting of denaturation at 95°C for 30 sec, annealing at 68°C for 30 sec, and extension at 72°C for 90 sec. An initial denaturing step at 95°C for 5 min and a last extension step at 72°C for 7 min were also added. The reactions were 25 μl that consisted of 100 ng genomic DNA, 10 pmol of each primer, 20 mM dNTPs, 2 U of Taq polymerase, 10×Taq buffer, and distilled water. 2 μl of PCR products were run on 2% agarose gel. A 50-bp DNA ladder was used to identify the various repeat alleles by size: 7-repeat (360bp), 8-repeat (400bp), 9-repeat (440bp), 10-repeat (480bp), and 11-repeat (520bp). Statistical analysis

The allele frequencies were estimated by counting, and the Hardy-Weinberg equilibrium was calculated based on these allele frequencies, using the Chi-square test. The association of DAT1 with ADHD was tested by two methods; in the case-control association analysis, allele and genotype frequencies in different groups of subjects were compared using the Chi-square test. For the family-based analysis, we used the transmission disequilibrium test (TDT)[23,24], and the haplotype-based haplotype relative risk (HHRR)[15] method to avoid any potential population stratification. All statistical tests were carried out using SPSS for Windows (release 11.0).

RESULTS

For this study, 54 ADHD cases and all 108 of their parents as well as 66 controls were successfully genotyped. Among all subjects (ADHD cases, parents and controls), five DAT1 alleles were identified: 7-repeat, 8-repeat (only in parents group), 9-repeat, 10-repeat and 11-repeat alleles. The genotype distribution of DAT1 gene was in Hardy-Weinberg equilibrium for each of the groups. Tables 1 and 2 show allele and genotypes frequencies, respectively, of the DAT1 40-bp VNTR for both ADHD and control subjects. There were no significant differences between the ADHD cases and the controls (Table1: χ2=0.588, df =3, p=0.899; Table 2: χ2=0.635, df =3, p=0.888).

7-Repeat

9-Repeat

10-Repeat

11-Repeat

Total

ADHD

2 (1.9)

5 (4.6)

100 (92.6)

1 (0.9)

108 (100)

Table 1. Allele frequencies for DAT1 in ADHD and control group (100%)

NS: no significance

Controls

4 (3.0)

P value

NS

7 (5.3) NS

132 (100)

119 (90.2)

2 (1.5)

NS

NS

329

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