| Summary |
Using the transmission disequilibrium test (TDT), the current study assesses for linkage disequilibrium between polymorphisms in the serotonin HTR2A receptor gene and ADHD. One hundred and fifteen families with a total of 143 children diagnosed with ADHD (DSM-IV) were genotyped for the His452Tyr and the T102C polymorphisms in the serotonin HTR2A receptor gene. TDT analysis revealed a preferential transmission of the 452Tyr allele to the affected offspring (P=0.03), suggesting linkage disequilibrium of this polymorphism with ADHD. This may open a new door in ADHD molecular genetics research, expanding the existing view of a catecholaminergic hypothesis to include a serotonergic hypothesis and should help elucidate the complex interplay among the neurotransmitter systems in the etiology of ADHD. |
| Total Sample |
The sample consisted of 115 families containing probands between the ages of 7 and 16. Twenty-four of these families consisted of one affected sibling in addition to the proband, and two families consisted of two affected siblings in addition to the proband, for a total sample of 143 children who met DSM-IV criteria for ADHD. One hundred and twelve of these children were males and 31 were females. The majority of families (94%) included in this study were of mixed European Caucasian descent describing themselves as English, Scottish, Irish, German, Italian, Polish, and Dutch. The remaining 6% of families were of mixed or non-Caucasian decent including African Canadian and Native Canadian. Of the 115 families included in this study, 93 families had genotypes available from both parents and the remaining 22 families had genotypes available from one parent. Of the total of 143 affected cases meeting DSM-IV criteria for ADHD, 57% of the children were combined type (CT), 19% were hyperactive/impulsive type (HIT), and 24% were primarily inattentive type (PIT). In addition, 39% of the children were diagnosed with comorbid ODD, and 11% with comorbid CD. |
| Sample Collection |
Probands from 115 families were referred to the Hospital for Sick Children in Toronto, Ontario for the clinical assessment of attention, behavior and learning problems. All participants gave informed written consent and the protocol was approved by the Hospital Research Ethics Board. |
| Diagnosis Description |
Diagnosis was based on measures with established reliability and validity. Semi-structured interviews for parents (Parent Interview for Child Symptoms (PICS-IV)) and teachers (Teacher Telephone Interview-IV (TTI)) were conducted to establish the persistence of ADHD symptoms in two different settings. Interviews were supplemented with information about behavior, development, and medical history derived from standardized parent and teacher questionnaires including the Conners Parent and Teacher Rating Scales-Revised and the Ontario Child Health Survey Scales-Revised. Children were assessed for anxiety (Children's Manifest Anxiety Scale), depression (Children's Depression Inventory), intelligence (Wechsler Intelligence Scale for Children, 3rd edn), academic achievement in reading, arithmetic and spelling (Wide Range Achievement Test-III), and receptive and oral language skills (Clinical Evaluation of Language Fundamentals, 3rd edn). Children were included if they met DSM-IV criteria for one of the three ADHD subtypes (inattentive, hyperactive/impulsive, combined) and were excluded if they scored below 80 on both the Performance and Verbal Scales of the WISC-III, had evidence of neurological or complex medical illness, Tourette syndrome, chronic multiple tics, bipolar affective disorder, psychotic symptoms, or had comorbid anxiety, depressive or developmental disorder that could better account for the behaviors (as specified by DSM-IV). Children were free of medication for 24 h when assessed. |
| Technique |
All family members including available parents, probands, and affected siblings were genotyped for the HTR2A T102C and His452Tyr polymorphisms. The high salt extraction method of Miller and colleagues was used to isolate DNA from whole blood lymphocytes. The HTR2A T102C polymorphism was amplified using a modified protocol of Warren et al. The HTR2A His452Tyr polymorphism was amplified using the PCR protocol of Ozaki et al. |
| Analysis Method |
Following genotype assignment, statistical analysis was performed using the extended transmission disequilibrium program (ETDT version 1.8). The TDT was used to assess for non-random transmission of alleles from heterozygous parents to an affected offspring by comparing the frequency of transmitted alleles to non-transmitted alleles. TDT analysis of HTR2A T102C and His452Tyr marker haplotypes was analyzed using the program Transmit Version 2.5. Linkage disequilibrium among marker haplotypes in the HTR2A gene was assessed using the Equilibrium Haplotype (EH) linkage utility program. |
| Result Description |
Under the null hypothesis of random transmission of alleles, TDT results for the HTR2A His452Tyr polymorphism provided evidence for preferential transmission of the 452Tyr allele to the affected child. Of the 42 informative transmissions, in which at least one parent was heterozygous, results indicated that the 452Tyr allele was passed 28 times and the 452His allele was passed 14 times, a statistically significant result (P=0.030). There was no evidence for biased transmission of alleles of the HTR2A T102C polymorphism to the ADHD proband. Assessment of linkage disequilibrium (LD) between HTR2A T102C and His452Tyr revealed very low levels of LD among marker haplotypes in unrelated parents (P=0.04). Using a haplotype TDT analysis, no evidence was found for preferential transmission of any HTR2A haplotype to the affected child. |
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