Supplementary Information
Materials and Methods
Participants
Exclusion criteria for analyses were: 1) major psychiatric illnesses including anxiety, affective and psychotic disorders, ADHD (self report or CAARS-S:L1, H sub-scale >65), autism spectrum disorders, drug-induced psychosis or a first-degree relative with same; 2) neurologic illnesses including CVA, CNS tumor, head trauma, multiple sclerosis, epilepsy, movement disorders or migraine in treatment; 3) drug dependency on alcohol or illicit substances; 4) coagulopathies, superficial or deep vein thrombosis or peripheral vascular diseases that would contraindicate an MRI and, 5) current use of respiratory, cardiovascular or anticonvulsant drugs.
Stop-Signal task - “staircase” method (cohort 1)
100 participants in the behavioural testing session (and all of the participants in the fMRI component) completed a “staircase” version of the stop signal task. Each participant completed one 128 trial practice block and four 128 trial test blocks. In all blocks, twenty five percent of all trials were “stop” trials (divided equally between the two targets and presented in random order). SSD was initially set at 250 ms and progressively adjusted by a dynamic tracking algorithm which manipulated the delay between the onset of the “go” and “stop” signals in 50ms increments (SSD) based on the participants success in the previous trial. Successful inhibitions were followed by a longer SSD (making the subsequent stop trial more difficult) while failed inhibitions were followed by a shorter SSD (making the subsequent stop trial easier). Emphasis was placed on both speed and accuracy. Stop-Signal Reaction Time (SSRT; ms) was calculated as the difference between Mean Go RT and the SSD at which participants could inhibit 50% of the “stop” trials 2.
Stop-Signal task - “sigmoid” method (cohort 2)
305 participants in the behavioural testing session completed a “method-of-constants” version of the task. Each participant completed three training blocks and two experimental blocks. Blocks one and two of training (36 and 72 trials respectively) contained only “go” trials and served to establish a pre-potent “go” response. The final training block (72 trials) introduced “stop” trials (25%, divided equally between the two targets and presented in random order) in order to allow participants to practice inhibition. Both experimental blocks (432 trials each) maintained the same “stop”/“go” trial ratio and randomisation as the final training block. In blocks with “stop” trials, three SSDs for each set of 24 trials were calculated as mean go reaction time from the previous set -150, -200 and -250 ms. Within these 24 trial sets, each of the three SSD’s occurred with equal frequency and in random order. SSRT was interpolated using a previously reported three-parameter sigmoidal function, which maps the probability of inhibition 3.
Genotyping
For the DRD4 VNTR polymorphism, the 48 base pair repeat sequence located in the exon 3 region of the DRD4 cDNA was amplified using 1U of Taq DNA polymerase (New England biolabs). After initial denaturation at 95°C for 9.5 mins, the following Polymerase Chain Reaction (PCR) cycling protocol was performed in the presence of the primers; forward: 5’-GCGACTACGTGGTCTACTCG-3’, reverse 5’-AGGACCCTCATGGCCTTG-3’ (75ng each): 40 cycles of denaturation at 95°C for 15 secs, annealing at 58°C for 15 secs and extension at 72°C for 15 secs. This process was followed by a final 10 min extension at 72°C. For theDAT1 3’UTR polymorphism, the 40 base pair repeat sequence located in the 3’-untranslated region of the DAT cDNA was amplified using 1U of Taq DNA polymerase. After Initial denaturation at 94°C for 2 mins, the following PCR cycling protocol was performed in the presence of the primers; forward: 5’-TGTGGTGTAGGGAACGGCCTGAG-3’; reverse: 5’-CTTCCTGGAGGTCACGGCTCAAGG-3’ (50ng each): 40 cycles of denaturation at 94°C for 15 secs, annealing at 62°C for 15 secs and extension at 72°C for 45 secs. This process was followed by a final 5 min extension at 72°C. For theDAT1i8 polymorphism, the 30 base pair repeat sequence located in the intron 8 region of the DAT cDNA was amplified using 1U of Taq DNA polymerase. After Initial denaturation at 98°C for 5 mins, the following PCR cycling protocol was performed in the presence of the primers; forward: 5’-GCAGAAACAAGGAGGAGCAG-3’; reverse: 5’-TCCGTGTACTCCTTCGACTC-3’ (50ng each): 40 cycles of denaturation at 98°C for 10 secs, annealing at 64°C for 30 secs and extension at 72°C for 9 secs. This process was followed by a final 10 min extension at 72°C. For each VNTR, amplification products were visualised on 1.5% agarose gels using 10μl of SYBRsafe per 100ml of gel.
fMRI acquisition and processing
The BOLD effects of each test condition were recorded using echoplanar imaging (EPI) performed in a 1.5T Siemens Sonata scanner fitted with a circular polarised headcoil (TR: 2000ms; TE: 30ms; FA: 90 deg; slice thickness: 3.6mm; no. of slices: 29; gap: 0.4mm; FOV: 230 x 230; matrix: 64 x 64 voxels at 3.6 x 3.6 mm resolution; bandwidth: 2004 Hx/pix). For individual data registration, a high-resolution 3D T1-weighted coronal image was acquired (MP_RAGE: TR: 1930ms; TE: 3.93ms; TI: 1000ms; FA: 15 deg; FOV: 240 x 40; matrix: 256 x 256; bandwidth:130 Hz/pix; voxel size: 0.9 x 0.9 x 0.9mm).
Functional data were analysed with statistical parametric mapping (SPM5, Wellcome Trust Centre for Neuroimaging, London, UK) implemented in Matlab 7.6 (Mathworks, Sherborne, MA, USA). The first three dummy scans were discarded to allow for T1 equilibration effects. Pre-processing involved realignment using the first volume as the reference scan. A mean realigned image was created for each subject and then co-registered to the corresponding structural (T1) image. Each individual’s T1 was then normalized to the SPM5 MNI template using the unified segmentation procedure 4. The resulting spatial normalization parameters were applied to the EPI time series data, and re-sliced to 3 x 3 x 3mm voxels. Images were then spatially smoothed using a full width half maximum isotropic Gaussian kernel of 9mm.
Results
Genotyping
Seven individuals were excluded from the study as they had successful genotyping completion rates below 80%. SNPs that were excluded from analyses included: rs3787441 and rs60593443 (ADRA1D and ADRA1A genes respectively) which had successful completion rates below 90%, rs8118409 (ADRA1D gene) which showed deviation from Hardy Weinberg Equilibrium (HWE) under a critical value of p=.01, rs3730287, rs11568324, rs62388321 and rs77943502 (ADRA1A, SLC6A2, DRD1, DRD2 genes respectively) which were not suitable for additive analyses since no participants were homozygous for the minor allele and rs12718529, rs1800544 and rs7194256 (DDC, ADRA2A, SLC6A2 genes) which were in perfect LD in our sample with another SNP in their respective genes. 141 SNPs remained after this quality control process and for each of these polymorphisms the minor allele frequency exceeded 5% (see Table 1).
Genotypic analysis for the DRD4 marker indicated that the 7-repeat allele was present with a frequency of 0.12 and other alleles were present with frequencies of 0.11 [2R], 0.05 [3R] and 0.70 [4R]. Rare variants [5R, 6R, 8R] were each present with frequencies ≤.01. For the SLC6A3 3’UTR VNTR the 10-repeat allele was present with a frequency of 0.74, the 9-repeat allele with a frequency of 0.25 and rare variants [7R and 11R] with frequencies <0.01. For the SLC6A3 intron 8 VNTR the 6-repeat allele was present with a frequency of 0.80, the 5-repeat allele with a frequency of 0.20 and a rare variant [4R] with a frequency <0.01.
Table 1 The influence of common Catecholamine gene variations on Stop Signal Reaction Time (SSRT), Go reaction time (Go RT) and Go RT variability (SD Go RT).
Chromosome/Gene / Marker ID / MAF / p valueSSRT / Go RT / SD Go RT
2/ADRA2B / rs2312955 / .3 / .92 / 0.52 / 0.14
3/DRD3 / rs3732790 / .41 / .37 / 0.51 / 0.44
rs2134655 / .25 / .32 / 0.23 / 0.18
rs963468 / .41 / .45 / 0.65 / 0.61
rs324036 / .12 / .31 / 0.61 / 0.41
rs167771 / .17 / .44 / 0.3 / 0.39
rs324029 / .29 / .14 / 0.055 / .042*
rs11706283 / .1 / .12 / 0.003* / 0.071
rs7633291 / .2 / .055 / 0.028* / .03*
rs6280 / .33 / .47 / 0.13 / 0.11
rs9825563 / .3 / .95 / 0.024* / 0.022*
rs17605608 / .2 / .085 / 0.0001** / .0009*
rs6762200 / .34 / .34 / 0.038* / 0.078
4/DRD5 / rs10033951 / .32 / .86 / 0.12 / 0.14
rs1967550 / .37 / .95 / 0.32 / 0.34
5/SLC6A3 / rs40358 / .14 / .21 / 0.043* / 0.56
rs37020 / .45 / .0002** / 0.31 / 0.11
rs10053602 / .23 / .49 / 0.57 / 0.51
rs393795 / .22 / .0012* / 0.065 / .037*
rs11737901 / .36 / .007* / 0.57 / 0.72
rs460000 / .23 / .0004** / 0.086 / 0.02
5/ADRB2 / rs1042713 / .34 / .039* / 0.0065* / 0.72
rs1042714 / .46 / .64 / 0.1 / 0.67
5/ADRA1B / rs2030373 / .23 / .28 / 0.34 / 0.38
rs6884105 / .34 / .87 / 0.68 / 0.75
rs756275 / .1 / .39 / 0.5 / 0.12
rs6892282 / .43 / .68 / 0.91 / 0.42
rs6888306 / .26 / .45 / 0.93 / 0.5
rs13162302 / .17 / .5 / 0.43 / 0.57
rs7737796 / .4 / .41 / 0.45 / 0.99
rs17057305 / .15 / .95 / 0.18 / 0.3
rs12653825 / .32 / .065 / 0.31 / 0.87
rs952037 / .32 / .25 / 0.38 / 0.73
rs11953285 / .12 / .62 / 0.74 / 0.31
5/DRD1 / rs4867798 / .33 / .34 / 0.32 / 0.12
rs686 / .39 / .92 / 0.29 / 0.062
rs5326 / .14 / .3 / 0.082 / 0.72
rs265981 / .39 / .92 / 0.22 / 0.076
7/DDC / rs4947510 / .31 / .99 / 0.52 / 1
rs11575553 / .11 / .76 / 0.67 / 0.099
rs11238131 / .34 / .36 / 0.95 / 0.53
rs17634958 / .13 / .8 / 0.47 / 0.95
rs6592952 / .46 / .77 / 0.65 / 0.74
rs3807566 / .43 / .36 / 0.84 / 0.75
rs880028 / .24 / .86 / 0.19 / 0.58
rs1817074 / .37 / .35 / 0.75 / 0.82
rs12535064 / .25 / .3 / 0.98 / 0.45
rs3735273 / .22 / .89 / 0.35 / 0.83
rs11575334 / .48 / .24 / 0.44 / 0.41
rs2329340 / .34 / .95 / 0.9 / 0.69
rs10249420 / .24 / .72 / 0.5 / 0.53
rs7807335 / .24 / .93 / 0.34 / 0.4
rs12666409 / .23 / .89 / 0.48 / 0.37
8/ADRA1A / rs1157690 / .16 / .1 / 0.056 / 0.22
rs17055923 / .22 / .21 / 0.32 / 0.11
rs4732641 / .37 / .3 / .023* / 0.47
rs1048101 / .47 / .57 / 0.23 / 0.66
rs472151 / .45 / .41 / 0.83 / 0.99
rs2322333 / .4 / .3 / 0.2 / 0.43
rs10503800 / .31 / .53 / 0.38 / 0.3
rs574647 / .28 / .57 / 0.95 / 0.71
rs577366 / .24 / .25 / 0.59 / 0.21
rs472865 / .11 / .49 / 0.099 / 0.34
rs523816 / .35 / .92 / 0.19 / .024*
rs486354 / .3 / .52 / 0.87 / 0.064
rs2046186 / .19 / .88 / 0.46 / 0.54
rs580739 / .28 / .8 / 0.089 / .0063*
rs13274396 / .28 / .77 / 0.57 / 0.63
rs563097 / .15 / .87 / 0.99 / .026*
rs528257 / .42 / .34 / 0.57 / 0.53
rs3808585 / .25 / .45 / 0.77 / 0.22
8/ADRB3 / rs9694197 / .08 / .85 / 0.87 / 0.74
rs4998 / .08 / .83 / 0.85 / 0.76
rs4994 / .06 / .95 / 0.69 / 0.61
9/DBH / rs1611115 / .19 / .21 / 0.28 / 0.7
rs2797849 / .34 / .25 / 0.12 / 0.61
rs1548364 / .44 / .78 / .024* / .03*
rs2519152 / .49 / .45 / 0.091 / 0.086
rs2797853 / .32 / .2 / 0.35 / 0.78
rs6479643 / .37 / .46 / 0.8 / 0.69
rs77905 / .49 / .92 / 0.83 / 1
rs10761412 / .4 / .87 / 0.44 / 0.89
rs6271 / .09 / .33 / 0.55 / 0.24
rs129882 / .17 / .46 / 0.38 / 0.89
10/ADRA2A / rs521674 / .3 / .84 / 0.3 / 0.092
rs11195419 / .14 / .25 / 0.61 / 0.71
rs602618 / .31 / .89 / 0.28 / 0.078
10/ADRB1 / rs1801253 / .25 / .47 / 0.14 / 0.46
rs3813720 / .38 / .042* / .033* / 0.39
11/DRD4 / rs3758653 / .18 / .33 / 0.29 / 0.82
rs752306 / .06 / .36 / 0.058 / 0.43
rs11246226 / .48 / .75 / 0.4 / 0.59
rs7395429 / .49 / .62 / 0.48 / 0.54
11/TH / rs3842727 / .32 / .27 / 0.97 / 0.36
rs2070762 / .46 / .048* / 0.0005* / .0008*
rs7483056 / .43 / .31 / 0.003* / .0035*
rs6356 / .37 / .95 / 0.92 / 0.23
rs10840489 / .15 / .61 / 0.65 / 0.55
11/ANKK1 / rs10891545 / .12 / .092 / 0.42 / 0.74
rs17115439 / .34 / .95 / 0.21 / 0.28
rs7118900 / .19 / .24 / 0.71 / 0.41
rs2734849 / .49 / .11 / 0.84 / 0.42
rs1800497 / .2 / .068 / 0.67 / 0.69
11/DRD2 / rs6279 / .29 / .88 / 0.44 / 0.45
rs1076560 / .16 / .39 / 0.81 / 0.91
rs2283265 / .16 / .39 / 0.8 / 0.88
rs2440390 / .12 / .36 / 0.9 / 0.9
rs2471851 / .17 / .19 / 0.4 / 0.94
rs12364051 / .38 / .48 / 0.74 / 0.8
rs17115583 / .1 / .21 / 0.57 / 0.91
rs4245146 / .48 / .83 / 0.51 / 0.8
rs4460839 / .11 / .72 / 0.15 / 0.48
rs7131056 / .44 / .53 / 0.73 / 0.53
rs4350392 / .14 / .022* / 0.48 / 0.22
16/SLC6A2 / rs2397771 / .39 / .0032* / 0.96 / 0.54
rs3785143 / .1 / .04* / 0.93 / 0.96
rs36024 / .39 / .041* / 0.84 / 0.98
rs36021 / .44 / .33 / 0.49 / 0.89
rs36017 / .46 / .35 / 0.87 / 0.41
rs1345429 / .47 / .5 / 0.89 / 0.65
rs3785157 / .34 / .57 / 0.46 / 0.3
rs5568 / .34 / .041* / 0.069 / 0.58
rs8047672 / .18 / .097 / 0.76 / 0.46
rs36009 / .11 / .67 / 0.21 / 0.53
rs1800887 / .21 / .4 / 0.84 / 0.64
rs8049681 / .15 / .28 / 0.75 / 0.57
rs2242447 / .33 / .47 / 0.58 / 0.77
rs9930182 / .21 / .48 / 0.55 / 0.54
20/ADRA1D / rs709024 / .42 / .36 / 0.35 / 0.36
rs8183794 / .19 / .46 / 0.14 / 0.44
rs6116268 / .5 / .094 / 0.35 / 0.99
rs1556832 / .49 / .17 / 0.6 / 0.81
rs4815670 / .45 / 1.00 / 0.057 / 0.46
rs6133098 / .27 / .95 / 0.35 / 0.87
rs6084670 / .27 / .67 / 0.49 / 0.44
rs3787442 / .47 / .92 / 0.45 / 0.24
rs6052456 / .22 / .43 / 0.6 / 0.98
22/COMT / rs737866 / .29 / .94 / 0.055 / 0.0091*
rs740603 / .48 / .46 / 0.19 / 0.15
rs4680 / .5 / .88 / 0.45 / 0.89
rs9332377 / .16 / .72 / 0.83 / 0.91
5/SLC6A3 / DAT1intron8VNTRc / - / .17 / 0.43 / 0.9
DAT13’UTRVNTRb / - / .64 / 0.51 / 0.88
11/DRD4 / DRD4VNTRexon3a / - / .6 / 0.98 / 0.56
abcFor the purposes of analyses on the VNTR variables; a, b and c, each variable number repeat was coded as 6 repeat or other, 10 repeat or other and 7 repeat or other respectively. The reference in each of these analyses was the most common genotype, namely; 6/6, 10/10 and other/other respectively. MAF: minor allele frequency. *Significant at uncorrected level, critical α=.05; ** significant at corrected level, critical α=5.04x10-4