| Summary |
Serotonin transporter (SLC6A4) polymorphisms are variously implicated in mediating susceptibility to attention-deficit-hyperactivity disorder (ADHD), a highly heritable and heterogeneous disorder with onset in childhood. Since there has been no survey in this regard from India, a sample of 56 ADHD cases and 174 healthy individuals from Kolkata were genotyped for the SLC6A4 promoter (5-HTTLPR) and intron-2 (STin2) polymorphisms. They report that the observed distribution of allele frequencies is consonant with that expected as per Hardy-Weinberg equilibrium proportions. Pair-wise combination of alleles comprising the 5-HTTLPR and STin2 polymorphic systems exhibit significant linkage disequilibrium of low magnitude. The estimates of haplotype- based haplotype relative risk (HHRR) and transmission disequilibrium test (TDT) statistics using a family-based study design, indicate significant preferential transmission of the STin2.12 (A12) allele to ADHD cases. Maternal inheritance of the A12 allele is significant in terms of the HHRR and TDT suggesting a novel role for epigenetic mechanisms in the etiology of ADHD. Similar analyses yielded no evidence of association between the 5-HTTLPR polymorphism and ADHD. Studies including additional polymorphic markers, ADHD subjects and other ethnic groups are warranted to further substantiate the present findings. |
| Total Sample |
ADHD cases (51 males and 5 females; mean age 7.3,SD=2.4 years; 46 trios, 10 duos) were recruited from the Out-Patient Department of MRIH. They also recruited 174 healthy volunteers (80 males and 94 females; mean age=33.18,SD=8.29 years) for the study. |
| Sample Collection |
The proportions of which in healthy volunteer control samples are as indicated: Brahmins (23.56%), Kayastha (48.28%), Mahisya (2.30%), Baidya (2.29%), Baisya (6.32%), Muslim (3.45%), and Scheduled Caste (13.79%). The estimated proportions in ADHD cases are as indicated: Brahmins (16.07%), Kayastha (50%), Mahisya (3.57%), Baidya (1.79%), Baisya (8.93%), Muslim (7.14%), and Scheduled Caste (12.50%). |
| Diagnosis Description |
Ascertainment of trios (proband and both parents) or duos (proband and one parent) was based on patients meeting the diagnostic criteria as laid out in the Diagnostic and Statistical Manual of Mental Disorders-IV-Text revised version and assessment based on the Conner's rating scale [Conners, 1998; USAn Psychiatric Association, 2000]. Detailed clinical history, information regarding developmental milestones and socioeconomic status were recorded by means of a structured questionnaire. |
| Technique |
Genomic DNA was isolated from whole blood lymphocytes by the salting out procedure of Miller et al. [1988]. Amplification of the 5-HTTLPR polymorphism was performed in the DNA Engine Thermal Cycler (MJ Research, PTC-0200) using forward (5'-ATGCCAGCACCTAACCCCTAATGT-3') and reverse (5'-GGACCGCAAGGTGGGCGGGA-3') primers as described previously [Gelernter et al., 1999]. More details about genotyping and experimental procedures could be found in the original publication. |
| Analysis Method |
Analyses of allele and genotype frequency, heterozygosity and tests for Hardy-Weinberg equilibrium (HWE) were performed with the TFPGA v.1.3 software. The EH+ program v.1.2 was utilized to estimate the haplotype frequencies for different allelic combinations of 5-HTTLPR and STin2 polymorphisms. The 2LD program v.1.00 was used to compute linkage disequilibria values. The family-based study design was used to ascertain association between ADHD and transmission of SLC6A4 polymorphisms and to avoid potentially spurious effects arising from population stratification. The HHRR and TDT statistics were computed for this purpose. The non-matched comparisons of transmitted versus non-transmitted parental alleles are based on X2 statistics. Significance is computed by the standard X2 test. |
| Result Description |
Pair-wise combination of alleles comprising the 5-HTTLPR and STin2 polymorphic systems exhibit significant (X2=14.74, df=1; P-value=0.0001) linkage disequilibrium of low magnitude (D'=0.269). The estimates of haplotype- based haplotype relative risk (HHRR) (X2=4.92, P-value=0.027; R2=1.47; 95% CI=1.01-2.13) and transmission disequilibrium test (TDT) statistics (X2=7.00, P-value=0.008; OR=3.00; 95% CI=1.53-5.90) using a family-based study design, indicate significant preferential transmission of the STin2.12 (A12) allele to ADHD cases. Maternal inheritance of the A12 allele is significant in terms of the HHRR (X2=6.53, P-value=0.011; R2=2.00; 95% CI=1.08-3.72) and TDT (X2=8.07, P-value=0.005; OR=6.50; 95% CI=1.76-23.98) suggesting a novel role for epigenetic mechanisms in the etiology of ADHD. |
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