| Summary |
They have identified a novel microsatellite repeat in SNAP-25 located between the 5'UTR and the first coding exon, and tested for association with ADHD. Case-control analyses suggest there may be a role of this polymorphism in ADHD, with one allele over-represented in controls and another over-represented in probands. Within-family tests of linkage and association confirmed these findings. Further work is needed to ascertain the role of SNAP-25 in ADHD and assess the functional significance of this polymorphism. |
| Total Sample |
In total 159 clinical subjects and 177 control individuals were included in this study. For family-based analysis, DNA from 107 parent-child trios were used. Out of 132 cases collected at the IOP, 120 had the combined subtype, nine had the hyperactive/impulsive subtype and three the inattentive subtype. Out of 30 cases at UB, 24 had the combined subtype, four the hyperactive/ impulsive subtype and two the inattentive subtype. Axis 1 comorbidity consisted of seven cases with an affective disorder and two cases with Tourettes syndrome. Two independent epidemiologically obtained and ethnically matched control samples were used in this study. Initial control data were generated from 100 children, aged 3-5 years who were part of the Twins Early Development Study (TEDS). A second control sample comprised children aged 5-15 years with low hyperactivity scores on a parent rating scale. The five-item hyperactivity scale from the SDQ was used with selected controls scoring 0 or 1 on a 10-point scale. In addition, DNA from both parents were collected whenever possible, for within-family tests of association and linkage. Complete trio data were available from all 30 of the UB cases and 55 of the IOP cases. At the IOP, DNA was also collected from 89 siblings of ADHD probands. |
| Sample Collection |
Cases' were clinically-referred individuals diagnosed using semi-structured research interviews and rating scales, from two centres (the Institute of Psychiatry and University of Birmingham). 'Controls' were drawn from two independent epidemiological samples, matched for ethnicity. Two clinical samples were used in this study collected at the Institute of Psychiatry (IOP) and the University of Birmingham (UB). Subjects were identified from child behavioural clinics in London, Horsham, Southampton and Birmingham. |
| Diagnosis Description |
All cases fulfilled criteria for a diagnosis ADHD-combined subtype (DSM IV) with no significant Axis Icomorbidity apart from oppositional defiant disorder (ODD) and conduct disorder (CD). Cases were referred for assessment if they were thought by experienced clinicians to have a diagnosis of the combined subtype of ADHD under DSM-IV criteria, with no significant Axis I comorbidity apart from oppositional defiant disorder (ODD) and conduct disorder (CD). Parents of referred cases were interviewed with an abbreviated version of the Child and Adolescent Psychiatric Assessment (CAPA). Information on ADHD symptoms at school were obtained using the Conners questionnaire. Following the IOP assessments, HYPESCHEME data sheets were completed using data gathered from the research interview, questionnaire and where necessary review of case notes. HYPESCHEME diagnoses were checked against researcher applied DSM-IV criteria and discrepancies reviewed by two researchers (PA and SR). Where consensus could not be reached, cases were brought to case conference and final consensus agreement made with a senior clinical researcher (ET). In the UB, DSM-IV criteria were applied directly by the researcher (LK) and consensus diagnosis agreed at case conference. All the subjects used in this study were free of neurological disease and damage, and did not have any congenital disorders known to cause hyperactivity. They were all Caucasian, aged between 5 and 15 at the time of first assessment and had an IQ above 60 (mean=98.8, SD=18.6, range 60-139). Cases were included in this study if they had a diagnosis of ADHD under DSM-IV criteria. |
| Technique |
The genomic sequence spans clones bA416N4 (GenBank accession number AL354824) and HS1068F16 (GenBank accession number AL023913). The sequence was screened using a web-based program called Tandem Repeats Finder (Benson, 1999), and detected an (ATTT)n repeat between the first transcribed exon and the 5'UTR of the SNAP-25 gene. The SNAP-25 microsatellite was amplified by PCR. The fluorescently-tagged products were separated on an ABI3100 Genetic Analyzer (PE Applied Biosystems, Foster City, CA) and analyzed using GENOTYPER (PE Applied Biosystems) software. |
| Analysis Method |
Case-control association was assessed using CLUMP [Sham and Curtis, 1995], a program that tests for the overall allele frequency differences between two groups as well as testing one allele against the rest clumped together. The transmission disequilibrium test (TDT) was applied to the sub-set of probands with DNA available from both parents for genotyping. |
| Result Description |
Seven alleles of the SNAP-25 microsatellite were detected in current samples. Case-control association analysis: Comparing the allele frequencies between the probands and controls gave a near significant difference (P=0.08). Using CLUMP they found a significant difference between probands and controls when one allele was taken compared to the rest clumped together (P=0.01). When a similar analysis was performed for each allele individually, a significant difference was found for allele 2 (P=0.01) with this allele being at a lower frequency in probands as compared to controls and a near-significant difference was found for allele 5 (P=0.06) that was more prevalent in probands as compared to controls. The odds ratio for allele 2 is 0.54, highlighting its protective effect, and that for allele 5 is 1.34 suggesting it is a weak risk allele. Within-family TDT analysis: Results from TDT analysis on the two alleles nominated from the case-control analysis showed that both are marginally significant (allele 2: P=0.05; allele 5: P=0.04), and transmissions are in the direction predicted from case-control analysis. |
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