| Summary |
Prior work has shown reduced serotonin transmission to be associated with impulsivity and behavioral problems. The current study assessed the association between ADHD and two variants of the serotonin transporter gene: the 44-bp deletion/insertion polymorphism (5-HTTLPR) and the 17 bp-repeat polymorphism in intron 2 (STin2.VNTR). They hypothesized that ADHD phenotypes associated with impulsivity would show an association with these variants. Two-hundred and ninety-three ADHD trios were genotyped and analyzed using transmission disequilibrium test (TDT) analysis and haplotype analysis. They found no association between the STin2.VNTR and ADHD, but did find preferential transmission of the S allele of the 5-HTTLPR polymorphism (P-value=0.016) to probands with ADHD. Haplotype analysis found the L/10 haplotype was over-transmitted (P-value=0.013), while L/12 was under-transmitted to probands with ADHD (P-value=0.027). |
| Total Sample |
293 unrelated complete trios were recruited. Of the 293 ADHD trios, 120 (41.0%) probands met criteria for ADHD-C, 19 (6.5%) met criteria for ADHD-HI and 154 (52.5%) met criteria for ADHD-I. Within the total sample, 223 (76.1%) probands met diagnostic criteria for other disorders: 38.6% had comorbid oppositional defiant disorder (ODD) and/or conduct disorder (CD), 9.6% had an emotional disorder, 17.4% had a tic disorder, 9.6% had an affective disorder, 8.5% had obsessive-compulsive disorder (OCD), and 36.9% had a learning disability (LD). The full scale IQ ranged from 72 to 142 (mean=100.2, SD=13.7). |
| Sample Collection |
293 unrelated complete trios, composed of ADHD probands and both biological parents were recruited from the child psychiatric clinics at Peking University Institute of Mental Health from September 1999 to December 2002. |
| Diagnosis Description |
Affected children were diagnosed by experienced clinicians according to the diagnostic and statistical manual of mental disorders (DSM-IV) [USAn Psychiatric Association, 1994] criteria for ADHD. Children were excluded if they showed evidence of bipolar affective disorder, childhood schizophrenia, autism, mental retardation, or other neurological or complex medical illnesses. All non-Han families were excluded. All subjects agreed to participate and parents signed informed consent. Both the informed consent and research protocol were reviewed and approved by the Ethics Committee of the Institute of Mental Health, Peking University (6th affiliated Hospital of Peking University). Diagnoses of ADHD and subtypes of ADHD were based on the USAn Clinical Diagnostic Interview Scales (CDIS) [Barkley, 1998]. Two psychiatrists, one of whom was of senior status, interviewed all parents separately. Additionally, teachers completed Rutter's Scale to evaluate the children's behaviors at school. Based upon data collected from the CDIS, parent interviews and teacher reports, consensus diagnoses were assigned to all probands. The intelligence quotient (IQ) was estimated using the Chinese-Wechsler Intelligence Scale for Children (C-WISC, standardized by Gong Yaoxian). Other supplementary assessment instruments included the Achenbach Child Behavior Checklist (CBCL), the Rutter Child Behavior Questionnaires, Conner's Parent and TeacherRating Scales, and a child mental health scale. |
| Technique |
5-HTTLPR polymorphism typing: The venous blood samples were refrigerated at 4oC in EDTA-test tubes. Genomic DNA was extracted using standard method within 2 weeks. Forward primer and reverse primer were used to generate 484 bp and 528 bp fragments of 5-HTTLPR polymorphism. A 25-ul reaction was performed according to the protocol previously described [Collier et al., 1996] on a PE-9700 or a PE-2400 thermal cycle (Perkin Elmer, USA). Please refer to the original publication for details on PCR procedure. PCR products were analyzed with Gel Doc 2000 imaging system to detect and record the genotype of each sample. The L variant represented the fragment of 528 bp and the S allele represented the fragment of 488 bp. Genotypes were read by at least two researchers, ambiguous or unidentifiable results were ream-plified and re-scored. Samples that continued to amplify poorly were eliminated from the study. STin2.VNTR typing: Forward primer and reverse primer were used to generate 300 bp and 267 bp fragments of STin2.VNTR polymorphism. A 25-ul reaction was performed according to the protocol previously described [Lesch et al., 1994]. Please refer to the original publication for details on PCR procedure. The PCR products were run out on a 2% agarose gel stained with ethidium bromide for 3 hr. The 12-repeat variant represented the fragment of 300 bp and the 10-repeat variant represented the fragment of 267 bp. |
| Analysis Method |
The transmission disequilibriumtest (TDT) [Spielman et al., 1993] was performed to explore the possible linkage and association of the two polymorphisms with ADHD. In addition, haplotype analysis was also performed by using the TRANSMIT program [version 2.5; Clayton, 1999]. |
| Result Description |
The allele and genotype frequencies described in the current study were consistent with other Chinese research, but were significantly different with research about European-USAn Caucasians (P-value<0.05). Preferential transmission of the 5-HTTLPR S allele to ADHD offspring was found (P-value=0.016) but analyses of subtypes did not reach statistical significance. Due to the small sample size, they did not do the TDT analysis in ADHD-HI trios. No significant biased transmission of any STin2.VNTR allele was observed for ADHD or subtypes of ADHD. For the diagnosis of ADHD, the global chi-square test for haplotype transmission was not significant (P-value=0.10). The haplotype analysis was also not significant for ADHD-C offspring (P-value=0.12) and ADHD-I offspring (P-value=0.31). Moreover, the haplotype of L/10 was over-transmitted (P-value=0.013), while the haplotype of L/12 was under-transmitted (P-value=0.027) to probands of ADHD. As for three subtypes of ADHD, under-transmission of haplotype L/12 (P-value=0.027) was found in families with probands of ADHD-C. |
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