| Summary |
They identified using single stranded conformational polymorphism analysis (SSCP) four DNA sequence variants in the 39 untranslated region of the human SNAP-25 gene. They tested for linkage of this gene and ADHD using two of the identified polymorphisms that change a restriction enzyme recognition site. They examined the transmission of the alleles of each of these polymorphisms and the haplotypes of both polymorphisms using the transmission disequilibrium test in a sample of 97 small nuclear families consisting of a proband with ADHD, their parents, and affected siblings. Biased transmission of the haplotypes of the alleles of these two polymorphisms was observed. These findings are suggestive of a role of this gene in ADHD. |
| Total Sample |
A sample of 97 nuclear families was used to test for linkage. The majority (96%) of families in this sample described themselves as mixed European Caucasian descent. The most common ethnic groups were English, Scottish, Irish, German, French, Italian, Polish, and Dutch. Five families described themselves as being of mixed or non-Caucasian descent. The Non-Caucasian ethnic groups consisted of African Canadians and Native Canadians. Specifically, 83 families consisted of a proband and both parental DNAs genotyped and 14 families with a proband genotyped for a single parent. Haplotypes could be determined unambiguously in 66 of these families. |
| Sample Collection |
This protocol was approved by The Hospital for Sick Children (Toronto, Ontario) Research Ethics Board and informed written consent was obtained for all participants. Subjects were recruited from referrals for clinical assessment of attention, behavior and learning problems to two clinics at an urban pediatrics hospital. |
| Diagnosis Description |
The assessment and characteristics of the subjects for this study have been described previously (Barr CL et al., 1999). Briefly, subjects were included if they met the following DSM-IV criteria for ADHD: six symptoms of inattention and/or hyperactivity-impulsiveness either in the home or school setting as determined by clinical interview, evidence of pervasiveness defined as a minimum of four symptoms in the non-criterion setting, onset before 7 years of age. Exclusion criteria were subjects with scores below 80 on both the Performance and Verbal Scales of the WISC-III, had evidence of neurological or chronic medical illness, bipolar affective disorder, psychotic symptoms, Tourette syndrome, or chronic multiple tics. Information for the diagnosis of ADHD and comorbid conditions was based on a semi-structured interview for parents (Parent Interview for Child Symptoms, PICS-IV; Schachar & Ickowicz, unpublished) and teachers (Teacher Telephone Interview-IV, TTI; Tannock & Schachar, unpublished), supplemented with the following questionnaires and child assessments: Conners Parent and Teacher Rating Scales-Revised, the Ontario Child Health Survey Scales-Revised, Wide Range Achievement Test III, Clinical Evaluation of Language Fundamentals, 3rd Edition, Children's Depression Inventory, and Children's Manifest Anxiety Scale. Children were free of medication for a minimum of 24 h before their assessment. |
| Technique |
DNA was extracted from blood lymphocytes using a high salt extraction method. The 261-bp fragment of the 39 untranslated region was amplified. DNA sequence variants were identified using single strand conformational polymorphism (SSCP) analysis. For genotyping the two polymorphisms, five ul of PCR product were digested with five units of either MnlI or DdeI. The restriction enzyme fragments were electrophoresed on 3% agarose gels followed by ethidium bromide staining to detect the alleles. |
| Analysis Method |
The transmission disequilibrium test was used for analysis to avoid the uncertainty in the mode of inheritance. Statistical analysis was performed using the ETDT program. Linkage disequilibrium between the two markers was estimated with the EH program. |
| Result Description |
By SSCP analysis they identified several different shifts in mobility of the 261bp PCR fragment of the 39 untranslated region. Sequence analysis revealed four different base pair changes from the published sequence, G1026A, G1038A, T1065G, and T1069C. Two of these sequence changes, T1065G and T1069C, result in a change in restriction enzyme recognition site and were further pursued. The T to G change at 1065 results in a gain of the MnlI restriction site and the T to C change at 1069 results in a gain of a DdeI restriction site. Significant disequilibrium for the distribution of the genotypes (P=0.000001) of these two alleles was observed. The TDT test was not significant for either allele of the MnlI polymorphism. For the DdeI polymorphism, allele 2 (C) was transmitted 48 times compared to allele 1 (T) that was passed 32 times. The TDT chi-square showed a trend but not significant (P=0.074). For the haplotypes of these two polymorphisms, the haplotype of the absence of the site for the MnlI polymorphism (T, allele 1) and the presence of the DdeI site (C, allele 2) was significant (P=0.030). |
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