Summary |
In this study, they examined the genotypes of two polymorphisms, 5-HTTLPR and the intron 2 VNTR, in 126 Korean ADHD families and investigated linkage disequilibrium (LD) between SLC6A4 and ADHD, using the transmission disequilibrium test (TDT) and haplotype analysis. Additionally, association with quantitative measures of inattention, hyperactivity-impulsivity, and overall severity was tested using logistic regression and QTDT analysis. TDT of both polymorphisms and haplotype analysis failed to detect LD. However, after excluding ADHD NOS subtype, TDT revealed nominally significant LD between 5- HTTLPR and ADHD. QTDT revealed positive association between 12 repeats of the intron 2 VNTR and attention, but case-control and TDT logistic regression analyses were negative. These markers have low heterozygosity in the Korean population, which would be expected to reduce the power of association. |
Total Sample |
One hundred twenty-six children with ADHD were ascertained by K-SADS-PL-K. Among 126 families, 19 trios did not have complete genotyping, because one or two of family members did not complete blood collection (1 family with missing proband, 15 families with 1 missing parent, 3 families with 2 missing parents). Three trios were incompatible for the 5- HTTLPR and one trio for the intron 2 VNTR. Dropping these trios from further analysis resulted in 104 completed trios for the 5-HTTLPR and 106 trios for the intron 2 VNTR. Normal control N=211 (Kim et al. [2004a] (Korean)), N=208 (Pae et al. [2003] (Korean)), N=252 (Kim et al. [2000] (Korean)) |
Sample Collection |
Consecutive subjects consenting to participate in a family-based association study for ADHD, and their biological parents, were studied between September 2000 and August 2002 at four, university-based, child psychiatry outpatient clinics (Seoul, Anyang, and Jinju) in South Korea. |
Diagnosis Description |
The included children met DSM-IV diagnostic criteria on the Diagnostic Interview Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime-Korean Version (K-SADS-PL-K). The ADHD subtypes included inattentive type 27.8% (n=35), hyperactive-impulsive type 7.9% (n=10), combined type 28.6% (n=36), and NOS 35.7% (n=45). |
Technique |
DNA was extracted from whole blood using the PureGene DNA isolation procedure. PCR for 5-HTTLPR was carried out in a 10 ul volume containing 50 ng of genomic template, 0.5 uM of each primer, 200 uM of each dNTP, 1X PCR buffer, 1.5 mM MgCl2, and 0.3 U of DyNAzymeTM EXT DNA polymerase, with 0.5MGC-melt. For the intron 2 VNTR, 0.25 U of HotStar Taq DNA polymerase was used instead of DyNAzymeTM EXT DNA polymerase and 0.5 M GC-melt. Primer sequences were as follows: For 5-HTTLPR, 5'-FAM-CTGAATGCCAGCACCTAACCCCTAATGT- 3' and 5'-GGGGAATACTGGTAGGGTGCAAGGAGAA- 3'. For the intron 2 VNTR, primer sequences were 5'-HEX-TGGATTTCCTTCTCTCAGTGATTGG-3' and 5'- TCATGTTCCTAGTCTTACGCCAGTG-3'. Post PCR products were injected and detected by laser-induced fluorescence on an ABI PRISM 3700 Genetic Analyzer at the University of Chicago DNA Sequencing and Genotyping Core. Electropherograms were processed and alleles called with Genotyper software. |
Analysis Method |
Sib_tdt test was used for TDT, and TDTPHASE was used for the haplotype analysis. The odds ratio (OR) was calculated using the method of Lohmueller et al. [2003]. They assumed hypothetical control group that is very large population (10,000) with equal numbers of each allele (to reflect the expected 50:50 transmission ratio). For the quantitative analysis, symptom severity was computed from each ADHD item in K-SADS-PL-K. The quantitative traits were the independent variables and the dependent variable was either the genotype an individual carried (case-control) or the allele that was transmitted from a heterozygous parent (TDT). Additionally, quantitative transmission disequilibrium test (QTDT) was performed to examine the association between the genotypes and the ADHD subscale severity scores. |
Result Description |
TDT of both polymorphisms and haplotype analysis failed to detect LD. However, after excluding ADHD NOS subtype, TDT revealed nominally significant LD between 5-HTTLPR and ADHD (X2=4.9, P=0.036). QTDT revealed positive association between 12 repeats of the intron 2 VNTR and attention (P=0.031), but case-control and TDT logistic regression analyses were negative. |