Table S1. Protocol followed for amplification of target sites.
Gene
Site
rs2236225
MTHFD
(C/T)
rs1051266
RFC1
(G/A)
rs1801131
(A/C)
Amplicon
Primer sequence
5′-3′
Reaction mixture for
PCR
Temperature cycle
Sense:
TCCCAATTCCC
CTGATGTTA.
Antisense:
AGGGCACTCCA
GTGTTTGTC.
Total volume 20 μl: 75100ng template DNA, 20
pmoles of each primer, 1.0
U Taq polymerase, 200
µM dNTP mix, 1X
Thermopol buffer (Genei)
and 2.5 mM MgSO4.
Initial denaturation 95° - 5
min; 35 cycles of denaturation: 95°C - 35 s,
annealing: 60°C - 35 s and
extension: 72°C - 35 s; final
extension 72°C - 5 min
179
Sense:
AGGCACAGTGT
CACCTTCGT.
Antisense:
TGAAGCCGTAG
AAGCAAAGG.
Total volume 20 μl: 75100ng template DNA, 20
pmoles of each primer, 1.0
U Taq polymerase, 200
µM dNTP mix, 1X buffer
B(Genei) and 1.0 mM
MgCl2.
Initial denaturation 95° - 5
min; 35 cycles of denaturation: 95°C - 30 s,
annealing: 59°C - 30 s and
extension: 72°C - 30 s; final
extension 72°C - 5 min
163
Sense:
TTTGGGGAGCT
GAAGGACTA.
Antisense:
CTTTGTGACCA
TTCCGGTTT.
20 μl mix containing 75100ng template DNA, 20
pmoles of each primer, 1.0
U Taq polymerase, 200
µM dNTP mix, 1X buffer
B (Genei) and 1.5 mM
MgCl2.
Initial denaturation 95° - 5
min; 35 cycles of denaturation: 95°C - 35 s,
annealing: 60°C - 35 s and
extension: 72°C - 35 s; final
extension 72°C - 5 min
160
Sense:
GAAGCAGGGA
GCTTTGAGG.
Antisense:
ACGATGGGGCA
AGTGATG-3′.
Total volume 20 μl: 75100ng template DNA, 20
pmoles of each primer, 1.0
U Taq polymerase, 200
µM dNTP mix, 1X buffer
B (Genei) and 1.0 mM
MgCl2.
Initial denaturation 95° - 5
min; 35 cycles of denaturation: 94°C - 30 s,
annealing: 56°C - 30 s and
extension: 72°C - 30 s; final
extension 72°C - 5 min
152
size (bp)
MTHFR
rs1801133
(C/T)
Table S2. Detailed description for the Restriction Fragment Length Polymorphism analysis.
ID
rs2236225
(C/T)
rs1051266
(G/A)
rs1801131
(A/C)
rs1801133
(C/T)
Analytic details
Final volume 20l. 5 μl PCR
amplicon was digested with
1U of MspI and 1X NEBuffer
4 at 37C for overnight.
Products resolved by 12%
PAGE.
Final volume 20l. 3 μl PCR
amplicon was digested with
1U of SfoI and 1X NEBuffer
4 at 37C for overnight.
Products resolved by 12%
PAGE.
Final volume 20l. 5 μl PCR
amplicon was digested with
1U MboII (NEB, USA) and
1X NEB buffer 4 at 37C for
3hrs. Products were resolved
in 20% PAGE.
Final volume 20l. 5 μl PCR
amplicon was digested with
1U of HinfI (NEB, USA) and
1x New England Biolabs
Buffer 4 at 37C for 3hrs.
Products were resolved in
12% PAGE.
Recognition sites
Genotyping
5'-C▼CGG-3'
Wild-type CC, heterozygous CT
and homozygous mutant TT.
Two fragments of 103 and 76bp
from a 179bp product that
carried a C>T mutation.
3'-GGC▲C-5'
5'-GGC▼GCC-3'
3'-CCG▲CGG-5'
5'-GAAGA(N)8▼-3'
3'-CTTCT(N)7▲-5'
5'-G▼ANTC-3'
3'-CTNA▲G-5'
Wild-type GG, heterozygous GA
and homozygous mutant AA.
Two fragments of 130 and 33bp
from a 163bp product that
carried a G>A mutation.
Wild-type AA, heterozygous AC
and homozygous mutant CC.
AA produced four fragments of
54, 30, 28 and 18bp, CC
genotype yielded three
fragments of 82, 30 and 18bp
from 160bp product due to
abolition of one restriction site.
Wild-type CC, heterozygous CT
and homozygous mutant TT.
Two fragments of 96 and 56bp
from a 152bp product that
carried a C>T mutation.
Table S3. Comparative analysis of genotype frequencies in female probands and control .
Gene
Site ID
MTHFD
rs2236225
(C/T)
RFC1
rs1051266
(G/A)
rs1801131
(A/C)
MTHFR
rs1801133
(C/T)
Genotype
Control
(N=20)
Proband
(N=18)
CC
CT
TT
GG
GA
AA
AA
0.18
0.51
0.31
0.25
0.53
0.22
0.29
0.28
0.33
0.39
0.17
0.39
0.44
0.22
AC
0.53
0.67
CC
0.18
0.11
CC
0.74
0.56
CT
0.23
0.44
TT
0.03
0.00
2
(p-value)
6.95 (0.031)
11.0 (0.004)
4.28 (0.117)
12.1 (0.002)
Table S4. Comparative analysis of haplotype frequencies using case-control data.
Control
Gene
Sites
Case
Groups Haplotype
2
(p-value)
OR
A-C
0.42
0.44
0.1909
(0.6622)
1.0
A-T
0.44
0.44
0.0006698
(0.9794)
0.98
C-C
0.12
0.11
0.1599
(0.6892)
0.92
C-T
0.02
0.01
1.068
(0.3013)
0.46
A-C
0.42
0.45
0.2331
(0.6292)
1.0
A-T
0.44
0.44
0.0009066
(0.976)
0.94
C-C
0.13
0.10
0.4685
(0.4937)
0.79
C-T
0.01
0.01
0.01903
(0.8903)
0.95
A-C
0.42
0.34
0.249
(0.6177)
1.0
A-T
0.43
0.44
0.1069
(0.7437)
1.26
C-C
0.12
0.21
1.219
(0.2695 )
2.334
C-T
0.03
1.064e009
0.2466
(0.6195)
4.282e-008
All
rs1801131
MTHFR
-
Male
rs1801133
Female
Table S5. Analysis of gene-gene interaction by Multi Dimensionality Reduction using case-control
dataset.
Group
All
probands
& controls
Male
subjects
Female
subjects
Model
TBA
Prediction
Error
CVC
P- value
Power
1
0.4929
0.5071
7/10
0.8880
100%
23
0.5222
0.4778
7/10
0.6440
100%
124
0.5369
0.4631
9/10
0.4710
100%
1234
0.4701
0.5299
10/10
0.9810-0.9820
100%
1
0.5727
0.4273
10/10
0.2070-0.2080
100%
14
0.5439
0.4561
6/10
0.4720-0.4730
100%
124
0.6102
0.3898
10/10
0.0360-0.0370
100%
1234
0.5372
0.4628
10/10
0.5330
100%
3
0.6000
0.4000
8/10
0.5570
100%
23
0.6333
0.3667
6/10
0.4640
100%
234
0.5278
0.4722
7/10
0.7690
100%
1234
0.6944
0.3056
10/10
0.2500-0.2510
100%
NB. 1- rs2236225, 2- rs1051266, 3-rs1801131, 4-rs1801133; Testing Balance Accuracy- TBA; Cross
validation consistency-CVC.