Computational protocol: DRD2 and PPP1R1B (DARPP-32) polymorphisms independently confer increased risk for autism spectrum disorders and additively predict affected status in male-only affected sib-pair families

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Protocol publication

[…] Four polymorphisms (rs1799732 Ins/Del, rs1079597 G/A, rs1800498 T/C and rs1800497 C/T) were studied at the DRD2 locus (Figure ). The DRD2 gene contains four haplotype blocks [Haploview 4.1; available at http://hapmap.ncbi.nlm.nih.gov] []. Three of the blocks are small (<4 kb) and one block is 20 kb in size. One of four polymorphisms used in this study, rs1079597, is part of the HapMap dataset. both this SNP and and rs1800498 are located within the larger haplotype block and were examined in previous studies of other neuropsychiatric disorders. Primer sequences, PCR and digestion conditions are shown in Table . All PCR reactions were carried out using 5 ng of template DNA; digestion products were separated on either 2% (rs1079597, rs1800498 and rs1800497) or 2.5% (rs1799732) agarose gels and visualized using ethidium bromide and UV illumination. In order to minimize genotyping errors, DNAs from affected individuals and family members were randomly arranged on 96-well plates, and all results were independently scored and tabulated by two persons. [...] Prior to carrying out analyses, Mendelian errors were checked in the family cohort using the FBAT program, v1.5.5 []. DRD2 marker data on five families and PPP1R1B marker data on two families were excluded from the analyses due to identified Mendelian errors. Single gene analyses were performed as previously described []. To avoid allele and genotype frequency distortion from using related individuals in case–control comparisons, one affected individual was selected at random from each family using SPSS v14.0 (SPSS, Chicago, IL) with the same cohort of randomly chosen individuals used for single marker allele and genotype frequency comparisons for all polymorphisms as well as general discriminant analyses. Family-based association tests (FBAT), including quantitative disequilibrium tests (QTDT), were done using FBAT v1.5.5. Because FBAT v1.5.5 can accommodate multiple affected individuals from each family, all affected individuals including those used for case–control comparisons, were included for FBAT and QTDT analyses. The domains, ‘reciprocal social interaction’, ‘communication’ and ‘repetitive stereotyped behaviours’ used for QTDT analyses were derived from the total scores from the ‘Qualitative Abnormalities in Reciprocal Social Interaction’ (A1 to A4), ‘Qualitative Abnormalities in Communication’ (B1, B2(V), B3(V) and B4) and ‘Restricted, Repetitive, and Stereotyped Patterns of Behaviour’ (C1 to C4) subdomains in the ADI-R diagnostic algorithm [].General Discriminant Analysis (GDA) was used to evaluate the predictive value of our single gene findings in discriminating between individuals with and without autism, as well as to test for evidence of interaction effects between genes. Genotypes were coded as categorical variables and GDA was performed using Statistica 9.1 [Statsoft, Tulsa, OK, USA]. […]

Pipeline specifications

Software tools Haploview, FBAT, Statistica
Applications Miscellaneous, GWAS
Organisms Homo sapiens
Chemicals Dopamine