SNPs were chosen with a minor allele frequency (MAF) > 0.15 and pair-wise linkage disequilibrium (LD) of r2 < 0.8 using the HapMap CEU population. Seven SNPs within a 132 kb region of CHRM2 were investigated. Two SNPs are located in intron 3 (rs1424569, rs1424387), two in intron 4 (rs1824024, rs2061174), one in intron 5 (rs324650) and two in Exon 8 (rs8191992, rs8191993) (Entrez gene). The CHRM2 genotyping was completed using a Biotage PSQ 96MA Pyrosequencer (Biotage AB, Uppsala, Sweden). An amplimer containing the polymorphism was generated by PCR in 96-well plates in a 50 uL total reaction volume, containing 10 ng of human genomic DNA; 1X GeneAmp® PCR Gold Buffer; 2.5 mM magnesium chloride; 200 uM dNTPs; 1 unit of AmpliTaq Gold™ taq polymerase; 1pmol each of the unmodified forward primer and the biotinylated reverse primer.
