Table S1. Details of genotyping protocol used.
Site
rs1799732
rs6278
DRD4
Exon3
VNTR
rs916455
rs28363170
rs3836790
Oligonucleotide sequences
Sense
5’TCCTCTCCTTGCCTGACTTG- 3’
Antisense
5’CCACCAAAGGAGCTGTACCT- 3’
PCR amplification
20 µl reaction mix containing 75–100
ng genomic DNA, 1X Thermopol
buffer (Genei, India), 1.5 mM MgSO4,
200mM dNTP mix, 10 pmoles of each
primer and 1 U Taq polymerase (Genei,
Bangalore, India).
Sense
5- 20 µl reaction mix containing 75–100
CCGATGTGGACCTCTACTTTC - 3’
ng genomic DNA, 1X Taq B buffer
Antisense
5’- (Genei, India), 1.0 mM MgCl2, 200mM
TGTGAGAAGGGATACATTGCAG - 3’ dNTP mix, 10 pmoles of each primer
and 1 U Taq polymerase (Genei,
Bangalore, India).
Sense
5’- 20 µl reaction mix containing 75–100
GCGACTACGTGGTCTACTCG - 3’
ng genomic DNA, 1X Taq B buffer
Antisense
5’- (Genei, India), 1.0 mM MgCl2, 200mM
AGGACCCTCATGGCCTTG - 3’
dNTP
mix,
0.01%
Gelatin,
100%DMSO, 10 pmoles of each primer
and 1 U Taq polymerase (Genei,
Bangalore, India)
Sense
5’- 20 µl reaction mix containing 75–100
GGAGGTTTTGCCAGATACCA - 3’
ng genomic DNA, 1X Taq B buffer
Antisense
5’- (Genei, India), 1.0 mM MgCl2, 200mM
CGAAGTAAGGGCAGAGAAAC- 3’
dNTP mix, 8 pmoles of each primer
and 1 U Taq polymerase (Genei,
Bangalore, India)
Sense
5’- 20 µl reaction mix containing 75–100
TGTGGTGTAGGGAACGGCCTGA- 3’ ng genomic DNA, 1X Taq B buffer
Antisense
5’- (Genei, India), 1.0 mM MgCl2, 200mM
CTTCCTGGAGGTCACGGCTCAAGdNTP mix, 0.01% Gelatin, 2% Sucrose
3’
10 pmoles of each primer and 1 U Taq
polymerase (Genei, Bangalore, India)
Sense
5’- 20 µl reaction mix containing 75–100
GCATGTGGATGTGTTCTTGC- 3’
ng genomic DNA, 1X Taq A buffer
Antisense
5’- (Genei, India), 200mM dNTP mix,
GATCAGAGGGCTTTGGTTCA- 3’
0.01% Gelatin, 100%DMSO, 10
pmoles of each primer and 1 U Taq
polymerase (Genei, Bangalore, India)
Temperature cycle
Initial denaturation 94°C5mins;
35
cycles
of
denaturation at 94oc- 30sec,
annealing at 60°C-30 sec,
extension at 72°C-30 sec; final
extension at 72°C for 5 mins.
Initial denaturation 95°C5mins;
35
cycles
of
denaturation at 94oc- 30sec,
annealing at 58°C-30 sec,
extension at 72°C-30 sec; final
extension at 72°C for 5 mins.
Initial denaturation 95°C7mins;
35
cycles
of
denaturation at 94oc- 30sec,
annealing at 59°C-30 sec,
extension at 72°C-1min30 sec;
final extension at 72°C for 7
mins.
Initial denaturation 95°C5mins;
35
cycles
of
denaturation at 95oc- 40sec,
annealing at 58°C-40 sec,
extension at 72°C-40 sec; final
extension at 72°C for 5 mins
Initial denaturation 95°C5mins;
35
cycles
of
denaturation at 94oc- 20sec,
annealing at 73°C-30 sec,
extension at 72°C-20 sec; final
extension at 72°C for 5 mins
Initial denaturation 95°C5mins;
35
cycles
of
denaturation at 94oc- 30sec,
annealing at 63°C-35 sec,
extension at 72°C-30 sec; final
extension at 72°C for 5 mins
Genotyping
5 µl PCR product digested
at 60°C for 16 hrs in 10l
reaction mixture containing
1U BstNI, 1X NEB Buffer
2, 1X BSA (NEB) and
resolved in 10% PAGE.
5 µl PCR product digested
at 37°C for 16 hrs in 10l
reaction mixture containing
1U BbvI, 1X NEB Buffer 2,
1X BSA (NEB) and
resolved in 12% PAGE.
5 µl PCR product resolved
in 12% PAGE and repeats
analyzed based on sizes.
5 µl PCR product digested
at 37°C for 16 hrs in 10l
reaction mixture containing
1U RsaI, 1X NEB Buffer 4,
1X BSA (NEB) and
resolved in 12% PAGE.
5 µl PCR product resolved
in 12% PAGE was used for
VNTR analysis
5 µl PCR product resolved
in 12% PAGE was used for
VNTR analysis
Table S2. HHRR performed in nuclear families with ADHD probands (N=148).
Gene
SNP
rs1799732
DRD2
rs6278
Exon3
DRD4
rs916455
rs28363170
SLC6A3
rs3836790
Allele
Transmitted
C
Del
G
T
≤4R
≥4R
C
T
9R
10R
5R
6R
0.87
0.13
0.79
0.21
0.93
0.07
0.96
0.04
0.18
0.82
0.26
0.74
Not
transmitted
0.84
0.16
0.72
0.28
0.95
0.05
0.94
0.06
0.13
0.87
0.23
0.76
Chi
square
P value
(df=1)
0.34
0.55
2.09
0.15
1.43
0.3
1.6
0.2
2.83
0.09
0.24
0.63
Table S3. Comparative analysis on haplotypic frequencies in controls (120), probands (160) and their parents (N=297).
Gene
DRD2
DRD4
SLC6A3
Haplotypes
Control
Proband
CG
CT
DelG
DelT
≤4R -C
≤4R -T
>4 R -C
>4 R -T
9R-5R
9R-6R
10R-5R
10R-6R
0.56
0.3
0.08
0.06
0.91
0.05
0.04
0.0
0.02
0.04
0.17
0.77
0.55
0.19
0.16
0.1
0.86
0.07
0.07
0.002
0.08
0.13
0.19
0.6
Chisquare
P
value
6.15
0.1
1.29
0.52
10.6
Statistically significant differences are presented in bold.
0.01
Father
0.65
0.2
0.13
0.02
0.88
0.05
0.07
0.07
0.08
0.19
0.66
Chisquare
P
value
5.86
0.12
0.868
0.648
6
0.11
Mother
0.59
0.24
0.11
0.06
0.89
0.04
0.07
0.006
0.09
0.12
0.17
0.62
Chisquare
P value
1.22
0.75
0.95
0.62
10.1
0.02
Table S4. Linkage Disequilibrium analyzed between the studied sites.
Proband
Father
Mother
D (r2)
D( r2 )
D( r2 )
Gene
Marker 1
Marker 2
Control
DRD2
rs6278
rs1799732
0.075 (0.002)
0.131 (0.011)
DRD4
EX 3 VNTR
rs916455
1.0 (0.002)
0.558 (0.002)
0.098 (0.008)
0.063 (0.002)
SLC6A3
rs28363170
rs3836790
0.245 (0.017)
0.148 (0.016)
0.018 (0.0)
0.197 (0.032)
0.526 (0.014)
0.065 (0.002)
Table S5: Independent and interactive effects of different sites and phenotypic attributes analyzed by MDR.
Best Combination in
each dimension
1
1-4
-1-2-4
P
3-P
Cognitive deficit
1-3-P
1-5-6-P
P
1-P
Hyperactivity
1-6-P
1-5-6-P
Trait
TBA
PE
CVC
P value
Power
0.6450
0.7123
0.6567
0.9403
0.9360
0.9224
0.8793
0.9497
0.9455
0.9224
0.9150
0.355
0.2877
0.3433
0.0597
0.064
0.0776
0.1207
0.0503
0.0545
0.0776
0.085
8
10
8
10
8
7
7
10
9
7
9
0.0010-0.0020
0.0000-0.0010
0.0010-0.0020
0.0000-0.0010
0.0000-0.0010
0.0000-0.0010
0.0000-0.0010
0.0000-0.0010
0.0000-0.0010
0.0000-0.0010
0.0000-0.0010
100%
Nodal No. 1- DRD2 rs1799732, 2- DRD2 rs6278, 3- DRD4 ex 3, 4- DRD4 rs916455, 5- SLC6A3 rs28363170, 6- SLC6A3
rs3836790, P-Phenotypic attributes (cognitive deficit & hyperactivity); No. of attributes= 7; No. of Cross-validation (CV) Intervals
chosen= 10; TBA=Testing balance accuracy; PE= Prediction error; CVC= Cross-validation contingency. The model with the
maximum TBA, a CVC>5 out of 10 and a minimum PE for that comparison is considered as best model.
Table S6. Independent and interactive effects of different sites on various co-morbidities analyzed by MDR.
Co-morbidities
LD
MD
ODD
CD
Best combination
in each dimension
1-5
2-4-5
4
1-6
1-2-6
1-2-4-6
1
1-6
1-4-5
4-5
2-4-5
2-4-5-6
TBA
PE
CVC
P value
Power
0.6503
0.6523
0.6436
0.7712
0.6905
0.6830
0.6205
0.6402
0.6105
0.7101
0.7031
0.6204
0.3497
0.3477
0.3564
0.2288
0.3095
0.317
0.3793
0.3598
0.3895
0.2899
0.2969
0.3796
6
8
10
10
9
9
10
7
6
10
10
7
0.0010-0.0020
0.0010-0.0020
0.0010-0.0020
0.0000-0.0010
0.0000-0.0010
0.0000-0.0010
0.0300-0.0310
0.0010-0.0020
0.0300-0.0310
0.0000-0.0010
0.0000-0.0010
0.0130-0.0140
100%
Nodal No. 1- DRD2 rs1799732, 2- DRD2 rs6278, 4- DRD4 rs916455, 5- SLC6A3 rs28363170, 6- SLC6A3 rs3836790. No. of
attributes= 6; No. of Cross-validation (CV) Intervals chosen= 10; TBA=Testing balance accuracy; PE= Prediction error; CVC= Crossvalidation contingency. The model with the maximum Testing BA, a CVC>5 out of 10 and a minimum PE for that comparison is
considered as the best model.