Supplementary online information
Subjects
In the present study, asthmatic patients (n=189, 37.86  17.08 years) and controls (n=270,
34.74  14.4 years) were recruited from our collaborating centers following clinician’s
diagnosed asthma and inclusion/exclusion criterion of American thoracic society (ATS)
guidelines after obtaining ethical clearance from our institute and the participating
centers. A written consent was obtained from individuals for participating in the study,
performing SPT (skin prick test) and obtaining blood samples. A standard questionnaire
was filled by the candidates giving clinical details, migration status, environmental
history, family history of diseases etc. The clinical test used for ascertaining asthma
phenotypes were pulmonary function test (PFT) FEV1, bronchial reversibility (>15%)
test using 2-agonist inhaler (albuterol/salbutamol) and skin prick test (wheal reaction
>3mm diameter) to a panel of 15 local environmental allergens and total serum IgE. Only
individuals with self reported history of breathlessness and wheezing with positive family
history were included in the study. Normal controls were recruited from general
population taking details of migration status, ethnicity and no self reported history of
asthma or allergic diseases. All individuals with smoking history and parasitic/helminthic
infections in the past were excluded from the study. SPT was done for all the individuals
while PFT was done wherever consent was obtained. For the family based association
studies 137 families were collected as ascertained through probands (15.5  8.04 years)
who were asthmatics along with both the parents affected/unaffected. Families were
further extended wherever consents were obtained and a total of 625 individuals were
1
thus recruited with average family size of 4.5 (range from 3 to 12 members). The disease
status was confirmed in a manner similar to case-control study.
The genetic homogeneity between patients and controls was confirmed by genotyping
loci as yet unknown to be associated with atopy or asthma (P> 0.05, data not shown). The
panels of unlinked markers used were: D20S117, D6S1574, D20S196, D6S470,
D12S368, D16S404, D6S446, D16S3136, D6S441, D8S264, D8S258, D8S1771,
D8S285, D8S260, D8S270, D8S1784, D8S514, D8S284, D8S272, D5S406, D5S416,
D5S419, D5S426, D5S418, D5S407, D5S647, D5S424, D5S641, D5S428, D5S2027,
D5S471, D5S2115, D5S436, D5S422, D5S408, D6S281, D6S308, D6S264 and D6S287.
Identification of polymorphisms
NCBI data search was carried out to identify polymorphism already reported in other
populations. Polymorphisms were selected based on their frequency and efforts were
made to select them uniformly through the gene (Schematic diagram, Figure1 and
supplementary information). There was no polymorphic loci in the promoter region (-350
to +5bp) of this gene in Indian population as reported earlierS1 while a CA repeat (intron
1) was found to be polymorphicS1. Six single nucleotide polymorphisms SNPs ss1 (T/C,
rs2069705) (promoter), ss2 (A/G, rs1861494), ss3 (T/C, rs1861493), ss4(C/T, rs2069718)
(intron 3),ss5 (A/G, rs2069727) and ss6 (G/A, rs2069728) (3’ UTR) were further
selected from the database and found to be polymorphic in our study population after
sequencing 40 individuals.
Genotypic DNA preparation, PCR amplification, sequencing and genotyping
DNA was isolated from peripheral white blood cells using the modified salting out
method and was stored at -20C until further analysis. PCR amplification was done using
2
primers and conditions as listed (see Supplementary information Table B). PCR products
were then sequenced on an ABI 3100 capillary sequencer (Applied Biosystems, Foster
City, CA, USA) by Big Dye terminator kit V 3.1 using both forward and reverse primers
(see supplementary information Tables B). Sequences were aligned and analyzed using
SeqManTm II.
CA repeat was genotyped using PCR with Tet-labeled forward primer and unlabelled
reverse primer followed by separation by electrophoresis on ABI 3100 genetic analyzer
(PE Applied Biosystems, Foster city, Calif) along with internal size standard. Fragment
lengths were determined using the GeneMapper software version 3.7 (Applied
Biosystems, Foster City, USA). SNPs were genotyped using SNaPShot ddNTP Primer
Extension Kit (Applied Biosystems, Foster City, USA). 1U of calf intestinal phosphatase
(CIP) (New England Biolabs, Beverly, MA) was used to clean up the primer extension
reaction. These samples were subsequently electrophoresed using the ABI Prism 3100
Genetic Analyzer as per the manufacturer’s instructions. The results were analyzed using
the program ABI Prism GeneMapper v3.7 (Applied Biosystems, Foster City, USA).
Cell Culture:
The Jurkat T cells (Jurkat E 6.1) were procured from National Centre for Cell Science
(NCCS), Pune, India. Cells were grown and maintained in RPMI 1640 medium
supplemented with 17.83mM NaHCO3, 10% heat-inactivated fetal calf serum and 1X
Antibiotic-Antimycotic solution (Sigma, St. Louis, MO). At cell concentration of 1-1.5
million/ml, the cells were subcultured, by centrifugation at 300g and then reconstituted in
medium as mentioned above. The viability of cells and cell count was determined by
trypan blue staining.
3
Preparation of Nuclear Extracts:
Nuclear extracts were prepared using a modification of previously published methods
(S2). Briefly, Jurkat T cells (1 × 106 cells/ml), without LPS or incubated with 10ng/ml
LPS for 45 minutes were pelleted by centrifugation at 300g. The cells were given two
washes with PBS and then pelleted. The pellets were resuspended in cell lysis buffer [10
mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM phenylymethylsulfonyl
fluoride, 1 mM dithiothreitol (DTT), 0.5% Nonidet P40, 0.1 mM EGTA, and 0.1 mM
EDTA] and allowed to swell on ice for 10 min. This was followed by centrifugation at
3300g for 15 min. The supernatant was stored as cytoplasmic extract and the nuclear
pellet resuspended in nuclear extraction buffer (20 mM HEPES, 25% glycerol, 1.5 mM
MgCl2, 420 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM phenylymethylsulfonyl
fluoride, and 1 mM DTT) and incubated for 30 min at 4°C. The extracted nuclei were
pelleted at 25,000g for 15 min at 4°C and the supernatant was collected as nuclear
extract. The protein concentration was estimated using the bicinchoninic acid method.
The nuclear and cytoplasmic extracts were stored at
70°C.
Electrophoretic Mobility Shift Assay:
IFN
A
(5’
AGGAAGAAGCAGGGAGTACTG
3’
and
5’
CAGTACTCCCTGCTTCTTCCT) 3’ IFN G (5’ AGGAAGAAGCGGGGAGTACTG
3’ and 5’ CAGTACTCCCCGCTTCTTCCT 3’) were synthesized, end labeled with
32
P-
ATP and used for binding assays. In vitro binding reactions between labeled doublestranded oligonucleotides and nuclear extracts (as described in Online Repository) were
performed in a total volume of 20 l, containing 2 l of 10X binding buffer (12 mM
HEPES, 50 mM NaCl, 10 mM TrisCl [pH 7.5], 10% glycerol, 1 mM EDTA, and 1 mM
4
DTT), 1 l of 1.0 g/l poly dI-dC (Sigma, St. Louis, MO), and 20 g of nuclear protein.
This reaction was allowed to proceed at 25–28ºC for 30 min before the addition of 2 l of
nondenaturing loading buffer (0.2% bromophenol blue, 20% glycerol). The samples were
electrophoresed on 1.5-mm-thick 6% polyacrylamide gel using Tris-glycine buffer (pH
8.5), and visualized by autoradiography.
References
S1 Nagarkatti R, B Rao C, Rishi JP, Chetiwal R, Shandilya V, Vijayan V et al.
Association of IFNG gene polymorphism with asthma in the Indian population. J
Allergy Clin Immunol. 2002; 110(3): 410-2.
S2 Dignam JD, Lebovitz RM and Roeder RG. Accurate transcription initiation by RNA
polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids
Res 1983; 11:1475-89.
5
Supplementary Table A: Demographic profile of the patient and the control groups in
case-control and family studies.
Patients
(N = 189)
Controls
(N = 270)
Families
(Proband)
(N= 137)
Native Place-
Indian
Indian
Indian
Mean Age (years):
37.86 ( 17.08)
34.74 ( 14.4)
15.5 ( 8.04)
Sex ratio (M Vs F):
57:43
56:44
55:45
Familial history of
Asthma/ Atopy†:
All
None
All
Smoking History¶:
None
None
None
% Reversibilty from
baseline FEV1
(after 2-agonist usage):
21.68(5.9)
ND
Log10-mean serum
total IgE (IU/ml):
2.80 ( 0.67)
2.31 ( 0.60)
2.9 ( 0.66)
Self reported history
of allergies:
All
None
All
* Patients
†
19.3 (4.17)
and controls were recruited from Delhi, Lucknow (UP), and Mumbai.
Control individuals were subjected to a questionnaire so as to eliminate all individuals having atopic
disorders or family history of atopic disorders. Only those patients who have at least one first-degree
relative affected with atopy and/or asthma were included for the study.
¶
Patients and controls known to have experienced smoking in the past three years, or suffering from
parasitic infections, were excluded from the study.
Parenthesis contains the values for standard deviation (SD). ‘ND’ denotes that the test is not done.
6
Supplementary Table B. Primers and PCR conditions used for PCR amplification and genotyping IFN gene polymorphisms.
Primer Name
IFNG_CA_FP
IFNG_CA_RP
IFNG_1FP
IFNG_1RP
IFNG_2FP
IFNG_2RP
IFNG_3FP
IFNG_3RP
IFNG_4FP
IFNG_4RP
IFNG_ss1
IFNG_ss2
IFNG_ss3
IFNG_ss4
IFNG_ss5
IFNG_ss6
Fragment/Polymorphism
CA Repeat
CA Repeat
ss1
ss1
ss2, ss3 (SNPs)
ss2, ss3(SNPs)
ss4
ss4
ss5, ss6 (SNPs)
ss5, ss6 (SNPs)
SNP
SNP
SNP
SNP
SNP
SNP
Primer Sequence
5' FAM GCT CTC ATA ATA ATA TTC AGA C 3'
5' CCA CCC CAC TAT AAA ATA C 3'
5' GAGCCGAGATCGCGCCACTACACT3'
5'AGGGGCACAAATGAGGGGAAAAAGAAC3'
5' TTG CGT AAA GAC AGG TGA GTT GAC A 3'
5' CTG GGG GCT TAC ATG AGA GGA GT 3'
5' CTCATGTAAGCCCCCAGAA 3'
5' ACTAAGCTCCCCAGGTGATT 3'
5' CAT TTT GTT GGC TTT CGC TTT TC 3'
5' CCC TCC CAC TCC ACC ATA CAT AG 3'
5' TACTATCTAGCTATATGATTGTGAGTTA 3'
5' GAA GAG GAA GTA GGT GAG GAA GAA GC 3'
5' TCA TCT TAA AAG GAA ACT GTT AGG 3'
5' GGCCATTTAGATGATGCTTCATAA 3'
5' TCC ACC TTC CTA TTT CCT CCT TC 3'
5' AAT TTC CCT GTC CTG CTC T 3'
7
Product size
198-214
198-214
1082
1082
828
828
820
820
668
668
29
27
25
25
23
19
Annealing Temperature 0C (Tm)
59
59
62
62
58
58
62
62
58
58
55
55
55
55
55
55
Supplementary Table C. Family based association tests for IFN CA repeat.
Alleles
(Repeat size)
Transmitted
TDT-sTDT
NonTransmitted
FBAT
z'
Value
p
Value
Allele
frequency
10
13
11
0.204
0.84
11
108
109
-0.011 0.99
12
92
100
0.63
0.53
13
16
15
0
1
14
62
55
0.555
0.58
15
0
1
0
1
17
1
1
-0.707
0.48
The p-values are nominal and have not been corrected for multiple testing.
* NA denotes statistics not available due to lesser frequency.
8
No. of
families
z'Value
p
Value
0.03
0.383
0.378
0.032
0.173
11
119
114
19
74
-0.192
0.467
0.07
1.3
0.09
0.85
0.64
0.94
0.19
0.92
0
1
NA
NA
0.003
2
NA
NA
Global p
Value
0.88
Supplementary Table D: A three locus sliding window haplotypic analysis in casecontrols. Statistically significant results are in bold.
Loci
global chi-square
global p
value
ss1
CA repeat
ss2
16.92 (7)
0.02
CA Repeat
ss2
ss4
19.47(6)
0.003
ss2
ss4
ss5
15.99(4)
0.003
ss4
ss5
ss6
4.05 (4)
0.39
The p-values are nominal and have not been corrected for multiple testing.
9