SUPPORTING INFORMATION
Methylation of IL-2 promoter at birth alters the risk of asthma
exacerbations during childhood
John A Curtin PhD1#, Angela Simpson MD1#, Danielle Belgrave MSc1, Aida Semic-Jusufagic
MD1, Adnan Custovic MD1*, Fernando D Martinez MD2*
1
The University of Manchester, Manchester Academic Health Science Centre, University
Hospital of South Manchester NHS Foundation Trust, Manchester, UK
2
Arizona Respiratory Center, Tucson, AZ, USA
#
Equal contributors
*Joint last authorship
Corresponding Author: Professor Adnan Custovic, School of Translational Medicine, 2nd
Floor ERC, UHSM, Manchester, M23 9LT, UK; adnan.custovic@manchester.ac.uk, (T) +44
(0)161 275 5802, (F) +44 (0)161 291 5730
1
METHODS
Study population
The Manchester Asthma and Allergy Study is an unselected, population-based prospective
study which follows the development of asthma and other atopic disorders in a cohort of
children. The setting is the maternity catchment area of Wythenshawe and Stepping Hill
Hospitals, comprising of 50 square miles of South Manchester and Cheshire, UK, a stable
mixed urban-rural population. Study was approved by the Local Research Ethics
Committee. Informed consent was obtained from all parents. The study is registered as
ISRCTN72673620 (www.controlled-trials.com/isrctn/pf/72673620).
Screening & Recruitment
All pregnant women were screened for eligibility at 'Booking' antenatal visits (8th-10th week of
pregnancy). The study was explained to the parents, and informed consent for initial
questionnaires and skin prick testing was obtained. Both parents completed a questionnaire
about their and their partner’s history of asthma and allergic diseases and smoking habits.
If the pregnant woman’s partner was not present at the antenatal clinic visit, an invitation
was sent for him to attend an open-access evening clinic for skin prick testing and
questionnaire. Once both parents had completed questionnaires and skin prick testing, a full
explanation of the proposed future follow-up for the child was given.
Of the 1499 couples who met the inclusion criteria (<10 weeks of pregnancy, maternal age
>18 years, questionnaire and skin test data available for both parents), 288 declined to take
part in the study. Of 1211 couples who initially agreed to take part, 1085 had a successful
full-term pregnancy (>36 weeks gestation) and gave consent to a further follow-up.
Follow-up
Clinical follow-up
The children have been followed prospectively, and attended review clinics at ages 1, 3, 5
and 8 years (±4 weeks).
Wheeze: A validated ISAAC questionnaire was interviewer-administered to collect
information on parentally reported symptoms and physician-diagnosed illnesses.
2
Atopic sensitization: Ascertained by skin prick testing at age 1, 3, 5 and 8 years (D
pteronyssinus, cat, dog, grasses, moulds, milk, egg [Bayer, Elkahrt, IN, USA]). We defined
sensitization as a mean weal diameter 3mm greater than negative control to at least one of
the allergens tested. We also measured specific serum IgE to mite, cat, dog, grasses, milk,
egg and peanut by ImmunoCAPTM (Phadia, Uppsala, Sweden) collected at 3, 5 and 8. The
detection limit of the assay was 0.2 kUA/L, and sensitization defined specific IgE value>0.35
kUA/L to at least one of the allergens tested.
Data from primary care medical records
The United Kingdom health care system is unique as it is free at point of delivery for all
residents and there are no financial barriers to access primary care. Therefore almost the
entire UK population is registered with a General Practitioners (GP). The medical record
follows the patient as they change GP and provides a full record of all health care contacts.
GPs are legally required to maintain accurate records of all medical encounters of their
patients, including retention of hospital admission discharge letters, outpatient appointments
and all prescriptions. We extracted data from GP-held medical records including
prescriptions, acute wheeze episodes, oral steroid prescriptions and hospital admissions for
asthma or wheeze during the first 8 years of life.
Data Extraction
Eligible GP practices were informed about the study and we sought permission to access
medical records of consenting children. Data extraction commenced in January 2006 and
ended in February 2008. Data access and manual extraction were performed during
arranged visits to each GP practice. GP practices which refused access to premises or had
only one study child registered contributed by sending copies of subjects’ medical records.
Timing, type of visit, symptoms, indication and prescriptions for each encounter were noted.
Home visits and assessment of environmental allergen exposure
After recruitment (between 12th and 16th week of pregnancy) a home visit was made, when
dust sampling was performed and an environmental questionnaire completed. Dust samples
3
from the parental bed, neonate's bed, bedroom floor, nursery floor, living room floor and
upholstered furniture were collected using a Medivac dust sampler (Medivac Plc, Wilmslow,
UK) with air-flow rate 45 L/ s through a 355-m diameter mesh screen on to a 5-m vinyl
filter, enabling the collection of fine dust samples. The filters were supported in a plastic dust
trap located behind the cleaner nozzle. A 1-m2 area of carpet and mattress (upper part) were
each sampled for 2 minutes. Samples were transferred into a preweighed Petri dish,
weighed, coded and stored at 8oC until extraction. One hundred mg of fine dust was
extracted with 2 mL borate-buffered saline with 0.1% Tween 20, pH 8.0 (BBS-T). For
samples less than 50 mg 1 mL BBS-T was added. Samples between 50 mg and 100 mg
were extracted in the proportional amount of BBST (1:20). The dust was resuspended using
a vortex mixer. Samples were then mixed end over end on an orbital rotator for 2 hours at
25oC before being centrifuged for 20 min at 1100 g at 4oC. Supernatants were removed and
stored at -35oC for future analysis of allergen content.
ELISA measurement of indoor allergens
Der p 1 was assayed using a two-site immunometric ELISA. The standard used to establish
the control curve for Der p 1 assay (UVA 93/02) was considered to contain 2500 ng Der p
1/mL (relative to WHO/IUIS D. pteronyssinus standard NIBSC 82/518).
Cotinine Measurement
Mothers who were taking part in the study had their antenatal notes flagged, and at the time
of delivery, a sample of cord blood was obtained (see below). Cotinine was measured using
a capillary column gas-liquid chromatographic method, with the lower detection limit of the
method of 0.1 ng/ml.
Assessment of stimulated proliferative responses in cord blood mononuclear cells
Mothers of children at high-risk (both parents atopic) and low-risk (both parents non-atopic)
provided cord blood samples. Umbilical cord blood was drawn within 10 min of birth and cell
separation commenced within the following 45 min. Cord blood, with 1% heparin as anticoagulant, was diluted (1:1) with AIM V serum-free medium (Gibco BRL, Life Technologies
Ltd, Paisley, UK) and twice separated by density gradient centrifugation (Ficoll Paque,
4
Amersham Pharmacia, Uppsala, Sweden). The resulting mononuclear cells were stimulated
(triplicate wells) at a density of 105 cells/well in primary culture (round-bottomed microtitre
plates, 37oC, 5% CO2). Proliferative responses were assessed by 3H-Thymidine (1 mCi/well)
incorporation after 3 days stimulation by the mitogen phytohaemaglutinin (PHA, Murex, 1
mg/mL); or 6 days stimulation by Der p 1 (affinity purified using a 4C1 monoclonal antibody,
30 mg/mL; analysed to exclude the possibility of endotoxin contamination).
Pyrosequencing
PCR primers were designed to amplify ~80bp fragments of bisulfite-treated DNA. The
primers were designed to avoid potential CpG methylation sites in order to minimize
amplification biases for methylated or unmethylated using the BioTage PSQ HS 96 Assay
Design Software Version 1.0.6 (Biotage Inc, Uppsala, Sweden). The PCR products were
analysed on a BioTage PSQ HS 96 Pyrosequencer according to the manufacture’s
instructions. Methylation quantification was performed using the provided software.
IL-2
A 25l PCR was carried out using 50 pmol of the forward primer (5’GGAGGAAAAATTGTTTTATATAGAAGG -3), 50 pmol of biotinylated reverse primer (5’CCTCTTTATTACATTAACCCACACT -3’) and ~50 ng of bisulfite treated genomic DNA.
0.8 U of HowStar Taq DNA polymerase (Qiagen), 1XPCR buffer, 200 μM each dNTP
(Bioline).The reverse primers was biotin-labeled so the PCR product can be purifed using
Sepharose beads. PCR cycling conditions consisted of an initial denaturation step of 95°C
for 10 minutes, followed by 50 cycles of 95°C for 20 s, 50°C for 20 s and 72°C for 20 s. The
PCR product was analysed on a BioTage PSQ HS 96 Pyrosequencer according to the
manufacture’s instructions. Briefly, the biotinylated PCR product was purifed and made
single-stranded to act as a template in a pyrosequencing reaction as recommended by the
manufacturer using the Pyrosequencing Vacune Prep Tool (Biotage Inc, Uppsala, Sweden).
The PCR product was bound to Streptavidin Sepharose HP (Amersham Biosciences,
Uppsala, Sweden) and the Sepharose beads containing the immobilized PCR product were
purified, washed, denatured using a 0.2 M NaOH solution, and washed again. Then, 0.3 mM
5
pyrosequencing primer (5’- AAATTGTTTTATATAGAAGG -3’) was annealed to the purified
single-stranded PCR product and pyrosequencing was performed using the PSQ HS 96
Pyrosequencing System (Biotage Inc, Uppsala, Sweden). Methylation quantification was
performed using the provided software.
LINE-1
We measured LINE-1 using the PyroMark LINE-1 kit (Biotage, Uppsala, Sweden) which is a
commercially available kit. This measures methylation at 4 CpG sites, we averaged the DNA
methylation levels across the four LINE-1 CpG cites within individuals. The nucleotide
dispensation order was: ACT CAG TGT GTC AGT CAG TTA GTC TG. Complete conversion
of cytosine at a non-CpG site ensured successful bisulfite conversion. The amount of C
relative to the sum of the amounts of C and T at each CpG site was calculated as
percentage. The average of the relative amounts of C in the 4 CpG sites was used as overall
LINE-1 methylation level in a given sample.
6
RESULTS
Table E1. Characteristics of children with or without available cord blood
Missing
Measurement
Frequency
%
Maternal asthma
No
Yes
Maternal atopy
No
Yes
Cat owner
No
Yes
Dog owner
No
Yes
Pet owner
No
Yes
Socio-economic status;
Paternal income, £/annum
< 10000
10K - 20K
20K - 30K
30K - 40K
Current wheeze age 1
No
Yes
Current wheeze age 3
No
Yes
Current wheeze age 5
No
Yes
Current wheeze age 8
No
Yes
Sensitised at age 1 years
No
Yes
Sensitised at age 3 years
No
Yes
Sensitised at age 5 years
No
Yes
Measurement
Frequency
%
260
68
53.0
48.6
231
72
47.0
51.4
96
232
55.2
50.8
78
225
44.8
49.2
279
49
52.2
50.5
255
48
47.8
49.5
282
46
51.7
53.5
263
40
48.3
46.5
245
83
52.7
50.0
220
83
631
p-value
0.36
631
0.32
631
0.75
631
0.76
631
0.55
563
0.32
516
0.43
495
0.06
469
0.57
449
0.06
504
0.45
462
0.26
425
0.52
47.3
50.0
55
109
75
39
52.9
47.0
54.3
43.8
49
123
63
50
47.1
53.0
45.7
56.2
144
99
45.7
49.3
171
102
54.3
50.7
185
51
45.7
56.7
220
39
54.3
43.3
174
44
47.2
44.0
195
56
52.8
56.0
171
40
45.1
57.1
208
30
54.9
42.9
208
29
46.4
51.8
240
27
53.6
48.2
163
57
46.2
52.3
190
52
53.8
47.7
135
71
47.4
50.7
150
69
52.6
49.3
7
Total
Sensitised at age 8 years
No
117
Yes
77
Asthma/wheeze hospital admission
after 1st year of life
No
175
Yes
10
Asthma/wheeze hospital admission
after 3rd year of life
No
180
Yes
5
Wheeze phenotypes
Never wheezed
100
Transient wheezers
47
Intermittent wheezers
21
Late-onset wheezers
18
Persistent wheezers
25
43.8
52.4
150
70
56.2
47.6
46.1
47.6
205
11
53.9
52.4
46.4
38.5
208
8
53.6
61.5
45.0
47.0
38.2
69.2
54.3
122
53
34
8
21
55.0
53.0
61.8
30.8
45.7
414
0.10
401
0.89
401
0.57
449
0.10
Table E2. Number of asthma/wheeze exacerbations and hospital admissions in the first 8
years of life for each year
Age
1
2
3
4
5
6
7
8
TOTAL
Asthma/Wheeze Exercabation
No
Yes
208
8
210
6
210
6
206
10
209
7
213
3
211
5
212
4
49
8
Asthma/Wheeze Hospital Admission
No
Yes
210
6
213
3
213
3
211
5
215
1
215
1
213
3
216
0
22
Table E3. Association between methylation levels and severe exacerbation and hospital
admission for asthma (multiple logistic regression analysis adjusted for gender, maternal
asthma and maternal smoking)
IL-2-CpGsite1
OR [95% CI]
p-value
IL-2-CpGsite7
OR [95% CI]
p-value
LINE-1
OR [95% CI]
p-value
Severe asthma exacerbations
after age 1
1.07 [1.01 - 1.14]
0.03
0.92 (0.70 - 1.21)
0.56
1.02 (0.75 - 1.38)
0.92
Severe asthma exacerbations
GEE 2-8
1.09 [1.01 - 1.17]
0.02
0.92 [0.71 - 1.19]
0.52
1.29 [0.96 - 1.74]
0.09
Hospital admission for asthma
after age 1
1.12 [1.04 - 1.20]
0.002
1.01 [7.74 - 1.37]
0.96
1.24 [0.88 - 1.75]
0.22
Hospital admission for asthma
GEE 2-8
1.10 [1.10 - 1.21]
0.02
1.04 [0.67 - 1.61]
0.88
1.46 [0.91 - 2.35]
0.12
Table E4. Mean methylation in cord blood in relation to hospital admissions for
asthma/wheeze and severe asthma exacerbations after the first year of life
Hospital admission
Yes
No
mean (95% CI)
59.0 (55.3-62.7)
53.0 (51.9-54.2)
Exacerbation
Yes
No
55.2 (52.1-58.3)
53.1 (51.9-54.3)
9
Table E5. Conditional odds ratios for the change in the risk of hospital admission with a unit
increase in methylation level for each age (univariate analysis)
Age (years)
Odds ratio (95% CI)
No (n)
Yes (n)
1
0.96 (0.90-1.02)
210
6
2
0.98 (0.92-1.06)
213
3
3
1.01 (0.95-1.08)
213
3
4
1.03 (0.97-1.10)
211
5
5
1.06 (0.99-1.13)
215
1
6
1.09 (1.02-1.16)
215
1
7
1.12 (1.04-1.19)
213
3
8
1.14 (1.07-1.22)
216
0
Table E6. Conditional odds ratios for the change in the risk of severe asthma exacerbations
with a unit increase in methylation level for each age (univariate analysis)
Age (years)
Odds ratio (95% CI)
No (n)
Yes (n)
1
0.94 (0.87-1.02)
208
8
2
0.96 (0.90-1.04)
210
6
3
0.98 (0.92-1.07)
210
6
4
1.00 (0.94-1.10)
206
10
5
1.02 (0.97-1.12)
209
7
6
1.04 (0.99-1.15)
213
3
7
1.06 (1.02-1.18)
211
5
8
1.08 (1.04-1.21)
212
4
10
Table E7. Association between methylation levels and wheeze phenotypes
Adjusted for gender, maternal asthma and maternal smoking at age
* reference group non-wheezers
Adjusted Estimates
OR (95% CI)
p-value
IL-2 Site 1
GEE Wheeze 1-8
1.01 (0.98 - 1.04)
0.42
Transient Wheeze*
1.01 (0.96 - 1.05)
0.79
Intermittent Wheeze*
0.99 (0.94 - 1.03)
0.57
Late-onset Wheeze*
1.04 (0.94 - 1.15)
0.46
Persistent Wheeze*
1.02 (0.96 - 1.09)
0.53
GEE Wheeze 1-8
0.98 (0.88 - 1.09)
0.7
Transient Wheeze*
1.05 (0.87 - 1.25)
0.62
Intermittent Wheeze*
0.85 (0.69 - 1.04)
0.11
Late-onset Wheeze*
1.37 (0.82 - 2.26)
0.23
Persistent Wheeze*
0.77 (0.58 - 1.01)
0.06
GEE Wheeze 1-8
1.10 (0.97 - 1.24)
0.14
Transient Wheeze*
1.01 (0.84 - 1.21)
0.93
Intermittent Wheeze*
1.12 (0.90 - 1.39)
0.32
Late-onset Wheeze*
1.15 (0.75 - 1.76)
0.52
Persistent Wheeze*
1.15 (0.88 - 1.51)
0.32
IL-2 Site 7
LINE-1
11
Table E8. Perinatal factors and methylation of CpG sites (continuous variables)
B represents the effect that alters with a unit change in methylation
Gestational age
IL-2 Site1
IL-2 Site7
LINE-1
B [95% CI]
-0.308 [-0.853 - 0.237]
-0.059 [-0.220 - 0.101]
0.056 [-0.078 - 0.190]
p-value
0.270
0.467
0.415
Maternal age
IL-2 Site1
IL-2 Site7
LINE-1
B [95% CI]
0.67 [-0.125 - 0.258]
0.041 [-0.018 - 0.100]
-0.018 [-0.064 - 0.029]
0.494
0.171
0.455
Birth weight
IL-2 Site1
IL-2 Site7
LINE-1
B [95% CI]
-0.144 [-2.106 - 1.818]
-0.237 [-0.358 - 0.832]
0.174 [-0.290 - 0.638]
0.885
0.433
0.461
Birth length
IL-2 Site1
IL-2 Site7
LINE-1
B [95% CI]
0.015 [-0.277 - 0.0307]
-0.003 [-0.094 - 0.088]
0.024 [-0.047 - 0.094]
0.921
0.941
0.508
Der p 1 exposure
IL-2 Site1
IL-2 Site7
LINE-1
B [95% CI]
0.089 [-0.500 - 0.678]
-0.050 [-0.237 - 0.138]
0.092 [-0.048 - 0.231]
0.766
0.603
0.197
Cord cotinine levels
IL-2 Site1
IL-2 Site7
LINE-1
B [95% CI]
-0.022 [-0.065 - 0.021]
-0.017 [-0.031 - -0.003]
0.000 [-0.011 - 0.010]
0.319
0.016
0.938
12
Figure E1. CpG sites in IL-2 promoter in which methylation status was assessed.
IL-2 CpG Site1 (green) is the first CpG site upstream of IL-2 and is located 251 bps upstream
of the IL-2 Transcription Start Site (TSS); GenBank Accession: M13879).
IL-2 CpG Site7 (red) lies just outside the promoter–enhancer region of IL-2 and is the 7th CpG
site upstream of the IL-2 gene and is located 938 bps IL-2 TSS (GenBank Accession:
M13879).
Figure E2. Conditional odds ratios (95% CI) for the change in the risk of hospital admission
for reasons other than asthma with a unit increase in methylation level of IL-2 Site1
13
0
.1
.2
.3
.4
a
.5
Figure E3: The proportion of children sensitised in quartiles of methylation
1
3
5
8
Age (Years)
4th Quartile IL-2 Site1
.5
2nd Quartile IL-2 Site1
3rd Quartile IL-2 Site1
0
.1
.2
.3
.4
b
1st Quartile IL-2 Site1
1
3
5
8
Age (Years)
2nd Quartile IL-2 Site7
3rd Quartile IL-2 Site7
4th Quartile IL-2 Site7
.3
.2
.1
0
c
.4
.5
1st Quartile IL-2 Site7
1
3
5
8
Age (Years)
1st Quartile LINE-1
2nd Quartile LINE-1
3rd Quartile LINE-1
4th Quartile LINE-1
14
Figure E4. DNA methylation and immunoproliferation of CBMCs with PHA
(a) IL-2 Site1 (b) IL-2 Site7 and (c) LINE-1.
a
b
c
15
Figure E5. IL-2 Site1 methylation and mononuclear cell lymphoproliferative response at birth
16
Figure E6. DNA methylation and immunoproliferation of CBMCs with Der p 1
(a) IL-2 Site1 (b) IL-2 Site7 and (c) LINE-1.
a
b
c
17