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Primer Name / Primer Location / Primer Sequence (5-3) / Annealing Temperature
Resequencing Primers
I1F(-179) M13 / Intron 1 / TGAGGCCTTCTGCTGTCTCT / 60C
I2R311 M13 / Intron 2 / AGATCCCACTCTCTGCTGGA
I2F233 M13 / Intron 2 / TCCCCTCTGTGGCCCTAG / 55C
I2R735 M13 / Intron 2 / TGTGGCCTTTTCATTGAGTG
I2F662 M13 / Intron 2 / AGTGGGTCTGTCCTGGTGTG / 58C
I2R1211 M13 / Intron 2 / AGTGCTCTGTGCTCCTCCTG
I2F1112 M13 / Intron 2 / CTGGCATTTCTGAACCTTGC / 60C
I3R72 M13 / Intron 3 / TCCTGTAAGGGCTTTGATGC
I3F698 M13 / Intron 3 / GTTCCCCTCTCTCCACCTGT / 60C
I4R211 M13 / Intron 4 / CTTGGCAGTTTACCCAGAGC
I4F83 M13 / Intron 4 / CAGGTGTTCACACCACGTTC / 62C
I5R168 M13 / Intron 5 / CTCCTCAGTCCCCCACACT
I5F(-46) M13 / Intron 5 / GTTCTCTGGGCACCTCTGAC / 62C
DR56 M13 / 3-FR / GGAATGGCAGTTGAGAAGGA
Site-Directed Mutagenesis
F110 / Exon 3 / CTGGAACGAGTTCATCCTGC / 58C
R948 / Exon 6 / ACGTCTGGCACCTTCAGTAC
F292V / Exon 4 / AAGATCGTGGACACCGTGATT / 58C
R312V / Exon 4 / AATCACGGTGTCCACGATCTT / 58C
F464W / Exon 4 / TCGCTGGCGTGAAG / 58C
R479W / Exon 4 / TCCTTCACGCCAGCGAAAT / 58C

Table 1: PCR primers for COMT resequencing and site directed mutagenesis. M13 tags were added to the 5-ends of each forward and reverse primer used during the gene resequencing studies. In the primer designations, “F” represents forward; “R”, reverse; “D”, downstream; “I”, intron; “FR”, flanking region; “W”, wild type; “V”, variant; and “M13” indicates the addition of an M13 tag. The underlined nucleotide in the site-directed mutagenesis primers indicates the “mutated” base. Brackets indicate primer pairs used to perform specific amplifications. Temperatures are the annealing temperatures used during the PCR amplifications. The numbering scheme for primers located within exons and the 5 and 3-untranslated regions are based on the MB-COMT cDNA sequence, with the “A” in the translation initiation codon designated (+1). Nucleotide positions located 5 to that position were assigned negative numbers, while those located 3 were assigned positive numbers. Intron primers were numbered on the basis of nucleotide positions relative to the splice junctions, with the first 5 nucleotide in an intron designated (+1). For introns of ambiguous length, negative numbers were assigned beginning with the initial 3 nucleotide (-1). This numbering scheme was also used to designate SNP positions (Table 2).

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Polymorphism Pair
/ African-Americans / Caucasian-Americans
D / p / D / p
I2(51) / 186 / 0.91 / <110-6 / --- / ---
I2(201) / I2(832) / --- / --- / -0.96 / <110-6
I2(201) / I2(1140) / --- / --- / -0.96 / <110-6
I2(201) / 186 / --- / --- / 1 / <110-6
I2(201) / 408 / --- / --- / -0.96 / <110-6
I2(201) / 472 / --- / --- / 1 / <110-6
I2(201) / I5(82) / --- / --- / 1 / 5.810-6
I2(832) / I2(935) / 1 / 110-3 / --- / ---
I2(832) / I2(1140) / 0.96 / <110-6 / 1 / <110-6
I2(832) / 186 / -1 / 110-6 / -1 / <110-6
I2(832) / 219 / 1 / 210-6 / --- / ---
I2(832) / 408 / --- / --- / 1 / <110-6
I2(832) / 472 / --- / --- / -1 / <110-6
I2(832) / I5(82) / --- / --- / -1 / 7.410-4
I2(935) / I2(1140) / 1 / 8.110-4 / --- / ---
I2(935) / 219 / 1 / <110-6 / --- / ---
I2(1140) / 186 / -1 / <110-6 / -1 / <110-6
I2(1140) / 219 / 1 / 110-6 / --- / ---
I2(1140) / 408 / --- / --- / 1 / <110-6
I2(1140) / 472 / --- / --- / -1 / 4.210-4
I2(1140) / I5(82) / --- / --- / -1 / <110-6
186 / 408 / --- / --- / -1 / <110-6
186 / 472 / 0.91 / <110-6 / -0.96 / <110-6
186 / I5(82) / --- / --- / 1 / 1.910-5
219 / 304 / 1 / 5.610-3 / --- / ---
219 / 408 / -1 / 2.610-3 / --- / ---
408 / 472 / -1 / 8.010-4 / -1 / <110-6
408 / I5(82) / -1 / 2.710-3 / -1 / 3.710-4
472 / I5(82) / --- / --- / 1 / 2.810-5
975 / D23 / 1 / 9.010-6 / --- / ---

Table 2: COMTlinkage analysis. The table lists results for all COMT polymorphisms observed during the gene resequencing studies. Values are shown for all polymorphism pairs with an absolute D value greater than 0.9 (p < 0.05). Dash lines indicate either that the polymorphisms in that pair were not tightly linked (i.e., D value < 0.9), or that one of the SNPs was either present at a low frequency or was not detected in that population. SNPs tightly linked with the nucleotide responsible for the Val108/158Met polymorphism are shown in bold type.

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Construct / COMT activity, (%WT) / COMT immunoreactive protein, (%WT)
COS-1, (N=12) / HEK293, (N=8) / COS-1, (N=6) / HEK293, (N=6)
WT / 100 / 100 / 100 / 100
Met108 / 62.1  8.8* / 64.6  8.1* / 12.7  3.8** / 36.3  15.6*
Thr52 / 107.0  11.9 / 127.0  16.3 / 160.0  32.7* / 142.0  25.8
Empty vector / 1.0  0.5** / 0.2  0.1** / 0 / 0

Table 3: Recombinant COMT activity and immunoreactive protein levels. Recombinant WT and the 2 variant allozymes were expressed in COS-1 and HEK293 cells, and levels of COMT enzyme activity and immunoreactive protein were measured. All results are listed as percentages of average values for the WT allozyme  SEM. (*) p < 0.05 and (**) p < 0.005 as compared with results for the WT allozyme.