Computational protocol: Disruption of an AP-2α binding site in an IRF6 enhancer is strongly associated with cleft lip

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Protocol publication

[…] Sequences of the genomic region encompassing the IRF6 gene for human and organisms listed in were compiled from publicly available whole-genome datasets. Orthologous sequences from rabbit, pig, bat, armadillo, and elephant were generated specifically for this study using targeted bacterial artificial chromosome (BAC)-based mapping and sequencing strategy (). MultiPipMaker was used to align these sequences with the single coverage option that eliminates some matches caused by duplications and the search both strand option. MCSs were identified using the WebMCS program, which calculates a conservation score for each base in the reference sequence by analyzing windows of 25nt across MultiPipMaker-generated multispecies sequence alignment based in the algorithm developed by Margulies et al. [...] Primers used to amplify MCSs were designed with Primer 3 (v.0.3.0) program and are available in online. Cycle sequencing was performed on 1µl of 1/10 diluted PCR product in 10µl reaction by using 0.25µl of ABI Big Dye Terminator sequencing reagent (v. 1.1), 0.5µl of 5μM sequencing primer, 0.5µl of DMSO, 1µl of 5X sequencing buffer and 6.75µl of ddH2O. Following a denaturation step at 96°C for 30 sec, reactions were cycle sequenced at 96°C for 10 sec, at primer Tm for 5 sec, and at 60°C for 4 min for 40 cycles. Unincorporated dyes and reaction buffers were removed by Sephadex™ G-50 columns (GE Healthcare) or magnetic beads (Agencourt) and the sequencing reaction was then injected on an ABI 3730 capillary sequencer (Applied Biosystems). Chromatograms were transferred to a Unix workstation, base called with PHRED (v.0.961028), assembled with PHRAP (v. 0.960731) scanned by POLYPHRED (v. 0.970312) and the results viewed with the CONSED program (v. 4.0) [...] Genotyping was carried out by using TaqMan® SNP Genotyping Assays on the ABI Prism 7900HT machine and analyzed with SDS 2.2 software (Applied Biosystems). The genotyping success rate was extremely high (>98%) and the Mendelian error rate was less than 2% (some of which may be due to potential deletions in this region). For the family data, SNP and haplotype TDT analyses were performed with the FBAT program (v.1.7.3), using the –e option of the program to account for multiple sibs in a family or multiple nuclear families in a pedigree. With a Bonferroni correction, the alpha level for significance of the association analyses was calculated as 0.05/32=0.0016. In order to assess possible dosage effects of the associated allele, log-linear models with genotype and imprinting effects were fit to the proband-parent triads using the SAS version of the LRT program. Likelihood ratio tests of the model parameter effects were then used to estimate the RR (with 95% CI) for over-transmission of 1 versus 2 associated alleles (A allele) at SNP rs642961. In these same proband-parent triads plus proband siblings if available (ie proband nuclear families), we compared the risk associated with haplotype V-A to that of haplotype V-G, as well as the risks associated with haplotypes I-G vs. V-G. We also estimated the OR of the effects due to a particular haplotype with respect to a specific reference haplotype. The 95% CI associated with the OR were also estimated. The haplotype RR and OR were calculated using LRT methods, as implemented in the software Unphased. In the case-control data, OR with Fisher’s exact P-values were calculated using SAS v.9.1.3 (Cary, NC) software. The population attributable risk percentage (PAR%) was calculated based on the following formula, PAR%= [Pe (RR-1)/RR] ×100, where Pe is the prevalence of the allele in cases, and RR is the relative risk. The OR was used as a proxy for the RR. The Danish case-control data was from the singleton cases and controls that were ascertained separately from the cleft family data. The Norwegian case-control data consisted of the probands from the cleft family data and the probands from a control family data. […]

Pipeline specifications

Software tools MultiPipMaker, PolyPhred, FBAT, UNPHASED
Applications Miscellaneous, Sanger sequencing, GWAS
Organisms Mus musculus
Diseases Cleft Lip, Cleft Palate, Craniofacial Abnormalities