Revision Bottema et al
IL13, CD14, pet and tobacco smoke influence atopy in 3 Dutch birth cohorts; The
Allergenic study
Renske W.B. Bottema, Naomi E. Reijmerink, Marjan Kerkhof, Gerard H. Koppelman, Foekje
F. Stelma, Jorrit Gerritsen, Carel Thijs, Bert Brunekreef, Constant P. van Schayck, Dirkje S.
Postma
Online Data Supplement
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Summarized description of the cohorts presented in the study
PIAMA
The primary aim of the PIAMA study is to investigate the incidence and risk factors, and
prevention of asthma and inhalant atopy[1]. The study includes a natural history part and an
intervention part, i.e. a double-blind placebo-controlled study on the primary preventive effect
of the use of mattress covers. At baseline, 4,146 children were included, 1,327 children of
allergic mothers and 2,819 children of non-allergic mothers. Recruitment took place during
the first trimester of pregnancy with a validated short screening questionnaire[2] and was
conducted by 52 midwife practices in 3 different regions in the Netherlands: north (Groningen
and surroundings), central (Bilthoven and Wageningen and surroundings), and southwest
(Rotterdam and surroundings). Women reporting any of the following self-reported symptoms
were defined as allergic (i.e. a history of asthma, current hay fever, current atopy to house
dust house dust mite, or pets) and their children were defined as ‘high-risk’. Women reporting
none of these symptoms were defined as nonallergic and their children were defined as ‘lowrisk’. 472 high-risk children and 2,819 low-risk children were included in the natural history
part of the study. 855 high-risk children were included into the intervention part of the study.
The participating children were born between May 1996 and December 1997.
PREVASC
The PREVASC study addresses the primary prevention of asthma in infants and small
children. The objective of this study is to investigate whether a multifaceted prenatally started
intervention strategy in high-risk infants prevents asthma development[3, 4]. ‘High-risk’ for
asthma and atopy was defined as at least one first-degree family member affected with asthma
as registered by their general practitioner. At baseline, 476 children were recruited by primary
caregivers (general practitioners and midwives) and advertisements, and randomised to either:
Revision Bottema et al
1) a control group (n=234), receiving usual care; or 2) an intervention group (n=242) in which
families followed the primary prevention program. The latter included house dust mite
impermeable bed coverings, education on breast and hypoallergenic feeding, timing of solid
food introduction and smoking cessation. In addition, a separate group of 317 children at
‘low-risk’ for asthma were followed without intervention for the natural history of asthma and
atopy. ‘Low-risk’ was defined as the absence of asthma in first-degree family members of the
children. Children participating in the intervention study were born between July 1997 and
July 2000 and the ‘low-risk’ children were born between March 1999 and October 2002.
KOALA
The primary aim of the KOALA Birth Cohort Study[5] is to identify factors that influence the
clinical expression of atopic disease, the main focus being lifestyle, dietary habits and
infections during the first year of life and gene-environment interaction. At baseline, 2,343
children were recruited among pregnant women who were invited for a prospective cohort
study on pregnancy-related pelvic girdle pain[6]. In addition, a group of 491 children was
recruited among pregnant women who had an ‘alternative lifestyle’ with regard to child
rearing practices, dietary habits, vaccination schemes and use of antibiotics, through organic
food shops, anthropologic doctors and midwives, Steiner schools, and magazines. All children
were enrolled between 14 and 18 weeks of gestation and were born between February 2001
and August 2003.
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Genotyping and quality assessment
Genomic DNA was extracted from buccal swabs or blood using standard methods[7]. DNA
was amplified by using REPLI-g UltraFast technology (Qiagen™). Genotyping was
performed by Competitive Allele-Specific PCR using KASPar™ genotyping chemistry,
performed under contract by K-Biosciences[8]. Quality of genotype data was guaranteed by
standards of K-Biosciences and verified. We verified the genotyping quality by three steps: 1)
16 samples were genotyped in both genomic and amplified DNA; 2) inheritance of alleles
between parents and children was checked using FBAT (http://biostat.harvard.edu), data of
both parents were available for 331 children; 3) genotype data were analyzed for deviations
from Hardy-Weinberg equilibrium using χ2 statistics. Comparison of genotypes between
genomic and amplified DNA and evaluation of inheritance patterns between parents and
children revealed an excellent quality of the genotypes with a genotyping error of <1%. All
IL13 and CD14 genotypes were in Hardy-Weinberg equilibrium (p>0.01).
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Table E1. Definitions of the Allergenic study, potential confounders and environmental exposures.
Variable
Definition
Dutch Ethnicity
Mother born in the Netherlands and has the Dutch Nationality
(PIAMA)
Mother and >1 parent of the mother born in the Netherlands
(KOALA)
Father and mother Caucasian (PREVASC)
Education level mother
Low: primary, lower vocational and lower general
Intermediate: senior high school and intermediate vocational
High: higher vocational and university
Family history
Atopy mother / father
Asthma ever or current hay fever or current allergy to pets or
house dust (mite)
Asthma mother / father
Self-reported ‘asthma ever’
Breast feeding
Never: never breast feeding
< 3 months: duration breast feeding < 13 weeks
> 3 months: duration breast feeding > 13 weeks
ETS at home first year
Family members smoking > 1 cigarette / day or visitors smoking
> 1 time / week in the home at age 3 months or 1 year (PIAMA)
Mother or her partner smoking in the home or visitors smoking > 1
time / week in the first year (PREVASC)
Somebody > 1 hour /week smoking in the presence of the child at
age 3 or 7 months or 1 year (KOALA).
Day care at age 1
> 4 hour / week day-care attendance at age 1 (PIAMA and
KOALA)
> 4 hour / week day-care attendance in > 7 of the last 13 weeks or
> 26 weeks of the first year of life (PREVASC)
Pet (dog and/or cat) first
year
Dog and / or cat at home at 3 months (PIAMA), 7 months
(KOALA) or after delivery (PREVASC)
Presence older siblings at
birth
Other children in the household at 3 months (PIAMA), 7 months
(KOALA) or at inclusion during pregnancy (PREVASC)
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Table E2. Association of IL-13 SNPs with total serum IgE levels at all ages in (A) Pooled data (Allergenic), and in (B) PIAMA, (C) PREVASC
and (D) KOALA separately.
(A) Pooled data (Allergenic)
1 year
SNP
3’UTR
Arg130Gln
-1512 A/C
-1111 C/T
Genotype
IgE*
2 years
4 years
8 years
N
P
IgE*
N
P
IgE*
N
P
0.0000002
10.00
643
0.000001
27.54
571
0.03
IgE*
N
P
54.95
448
0.0004
CC
5.75
783
CT
8.32
362
14.79
297
37.15
249
87.10
219
TT
12.30
61
27.54
55
38.90
45
114.82
35
GG
5.75
727
10.00
597
26.92
555
54.95
444
GA
8.32
350
14.79
291
37.15
247
87.10
218
AA
11.22
62
26.92
54
38.90
45
104.71
35
AA
6.03
746
10.47
617
28.18
556
58.88
448
CA
7.76
377
14.13
306
34.67
270
81.28
222
CC
8.71
48
26.92
38
38.90
33
77.62
29
CC
6.03
755
10.47
624
28.18
551
60.26
443
CT
7.94
374
14.45
308
33.88
277
74.13
228
TT
9.55
42
28.84
35
48.98
28
81.28
29
0.000001
0.008
0.001
0.000003
0.0002
0.00007
*Geometric mean IgE values (IU/ml). N=number of children per genotype.
0.01
0.13
0.08
0.001
0.04
0.23
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(B) PIAMA
1 year
SNP
3’UTR
Arg130Gln
-1512 A/C
-1111 C/T
Genotype
IgE*
2 years
4 years
8 years
N
P
IgE*
N
P
IgE*
N
P
0.03
-
-
-
31.62
441
0.08
IgE*
N
P
54.95
448
0.0004
CC
6.03
222
CT
9.55
108
-
-
-
43.65
196
87.10
219
TT
9.33
17
-
-
-
42.66
32
114.82
35
GG
5.89
219
-
-
-
30.20
434
54.95
444
GA
9.33
106
-
-
-
43.65
193
87.10
218
AA
8.13
18
-
-
-
42.66
32
104.71
35
AA
6.46
224
-
-
-
33.88
431
58.88
448
CA
8.71
107
-
-
-
38.02
210
81.28
222
CC
6.76
14
-
-
-
42.66
27
77.62
29
CC
6.46
220
-
-
-
33.11
428
60.26
443
CT
8.71
105
-
-
-
38.02
210
74.13
228
TT
7.24
13
-
-
-
48.98
25
81.28
29
0.04
0.23
0.21
0.03
0.53
0.36
* Geometric mean IgE values (IU/ml). N= number of children per genotype. - = No IgE measurements.
0.0009
0.04
0.23
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(C) PREVASC
1 year
SNP
3’UTR
Arg130Gln
-1512 A/C
-1111 C/T
Genotype
IgE*
2 years
4 years
8 years
N
P
IgE*
N
P
IgE*
N
P
IgE*
N
P
0.006
10.47
222
0.040
16.60
130
0.20
-
-
-
CC
6.76
141
CT
11.75
61
13.18
99
20.42
53
-
-
-
TT
15.49
13
22.91
19
31.62
13
-
-
-
GG
6.76
130
10.47
210
16.60
121
-
-
-
GA
12.02
62
13.18
101
20.89
54
-
-
-
AA
15.49
13
22.91
19
31.62
13
-
-
-
AA
6.76
134
10.23
224
14.79
125
-
-
-
CA
10.72
69
13.49
100
24.55
60
-
-
-
CC
17.78
6
30.20
8
25.70
6
-
-
-
CC
6.61
128
10.47
220
15.49
123
-
-
-
CT
12.02
78
14.45
108
23.99
67
-
-
-
TT
41.69
3
25.70
6
44.67
3
-
-
-
0.005
0.02
0.0005
0.04
0.03
0.05
0.21
0.05
0.06
* Geometric mean IgE values (IU/ml). N= number of children per genotype. - = No IgE measurements.
Revision Bottema et al
(D) KOALA
1 year
SNP
3’UTR
Arg130Gln
-1512 A/C
-1111 C/T
Genotype
IgE*
2 years
N
P
0.0001
IgE*
4 years
8 years
N
P
IgE
N
P
IgE
N
P
9.77
421
0.00002
-
-
-
-
-
-
CC
5.25
420
CT
7.08
193
15.85
198
-
-
-
-
-
-
TT
12.88
31
30.90
36
-
-
-
-
-
-
GG
5.37
378
9.77
387
-
-
-
-
-
-
GA
6.92
182
15.85
190
-
-
-
-
-
-
AA
12.02
31
29.51
35
-
-
-
-
-
-
AA
5.75
388
10.47
393
-
-
-
-
-
-
CA
6.46
201
14.13
206
-
-
-
-
-
-
CC
8.51
28
26.30
30
-
-
-
-
-
-
CC
5.62
407
10.47
404
-
-
-
-
-
-
CT
6.31
191
14.45
200
-
-
-
-
-
-
TT
9.33
26
29.51
29
-
-
-
-
-
-
0.0009
0.17
0.12
0.00007
0.006
0.002
* Geometric mean IgE values (IU/ml). N= number of children per genotype. - = No IgE measurements.
Revision Bottema et al
Table E3. Interaction of CD14 genotypes and combined pet exposure (cat and/or dog) with
respect to total serum IgE levels at ages 4 and 8 years.
Pet exposure†
No pet exposure†
Age
CD14 SNP Genotype
Interaction
IgE*
4 years
8 years
N
P
IgE*
N
P
P‡
3’UTR
CC+CA
AA
29.4
41.3
304
24
0.30
30.1
38.6
481
44
0.33
0.79
-159 C/T
CC + CT
TT
31.6
28.1
257
69
0.58
28.5
40.2
431
99
0.05
0.04
-1145 T/C
TT + CT
CC
31.7
25.7
256
74
0.32
28.4
40.2
429
99
0.05
0.01
-1619 T/C
TT + CT
CC
32.0
23.8
255
48
0.23
29.0
39.8
422
69
0.13
0.06
-550 C/T
CC
CT
TT
30.5
28.1
35.2
192
118
13
0.84
33.1
29.8
17.9
279
202
31
0.12
0.15
3’UTR
CC+CA
AA
57.7
83.1
240
22
0.31
73.2
55.4
402
34
0.35
0.25
-159 C/T
CC + CT
TT
59.2
65.6
209
53
0.68
64.8
99.1
341
92
0.03
0.17
-1145 T/C
TT + CT
CC
58.4
62.3
209
56
0.79
65.1
94.8
344
95
0.05
0.18
-1619 T/C
TT + CT
CC
60.6
62.4
212
37
0.91
70.4
86.7
347
62
0.36
0.48
-550 C/T
CC
CT
TT
55.7
54.7
82.3
152
98
13
0.68
71.4
72.3
49.0
248
168
19
0.62
0.63
* Geometric mean IgE values. N= number of children per genotype. †Pet exposure = pet (dog
and/or cat) exposure at home in the first year of life; No pet exposure = neither cat nor dog
exposure at home in the first year of life; ‡ P-values were adjusted for gender, atopy mother,
atopy father, siblings, breastfeeding and ETS exposure.
Revision Bottema et al
Table E4. Interaction of CD14 genotypes with combined pet exposure (cat and/or dog) in the
development of sensitisation to any allergen at age 8 years.
CD14 SNP Genotype
Sensitisation to any allergen§
Interaction
yes / no* (proportion)
Pet exposure †
No pet exposure †
P‡
3’UTR
CC+CA
AA
87 / 153 (0.57)
12 / 9 (1.33)
193 / 207 (0.93)
10 / 24 (0.42)
0.002
-159 C/T
CC + CT
TT
89 / 119 (0.75)
13 / 40 (0.33)
153 / 187 (0.82)
50 / 41 (1.22)
0.002
-1145 T/C
TT + CT
CC
88 / 120 (0.73)
12 / 44 (0.27)
154 / 189 (0.81)
50 / 44 (1.14)
0.001
-1619 T/C
TT + CT
CC
88 / 123 (0.72)
10 / 27 (0.37)
159 / 188 (0.85)
34 / 27 (1.26)
0.03
-550 C/T
CC
CT
TT
58 / 93 (0.62)
37 / 61 (0.61)
6 / 7 (0.86)
119 / 128 (0.93)
78 / 90 (0.87)
6 / 13 (0.46)
0.60
§ Specific IgE >0.35 IU/ml to house dust mite, cat, dog, Dactylis glomerata, Betula
verrucosa, Alternaria alternata, egg, or milk allergens. * Number of children sensitised /
number of non-sensitised children; †Pet exposure = pet (cat and/or dog) exposure at home in
the first year of life; No pet exposure = neither cat nor dog exposure at home in the first year
of life; ‡ P-value for interaction, adjusted for gender, atopy mother, atopy father, siblings,
breastfeeding and ETS exposure.
Revision Bottema et al
Table E5. Interaction of CD14 genotypes with dog exposure with respect to total serum IgE
levels at age 4 and 8 years.
Age
CD14 SNP Genotype
Dog exposure
No dog exposure
Interaction
IgE*
4 years
8 years
N
P
IgE*
N
P
P†
3’UTR
CC+CA
AA
30.2
23.4
127
11
0.59
29.5
43.7
658
57
0.08
0.20
-159 C/T
CC + CT
TT
33.9
18.6
103
33
0.04
28.8
40.7
585
135
0.03
0.005
-1145 T/C
TT + CT
CC
34.7
17.0
102
36
0.01
28.8
39.8
583
137
0.04
0.002
-1619 T/C
TT + CT
CC
33.1
14.1
106
21
0.01
29.5
38.9
571
96
0.13
0.02
-550 C/T
CC
CT
TT
21.9
38.9
117.5
83
44
7
0.004
34.7
27.5
15.8
388
276
37
0.008
0.00001
3’UTR
CC+CA
AA
55.0
74.1
85
5
0.70
69.2
64.6
557
51
0.76
0.52
-159 C/T
CC + CT
TT
60.3
43.7
68
21
0.42
63.1
95.5
482
124
0.01
0.08
-1145 T/C
TT + CT
CC
60.3
38.0
67
25
0.21
63.1
93.3
486
126
0.01
0.04
-1619 T/C
TT + CT
CC
67.6
24.5
74
11
0.03
66.1
89.1
485
88
0.13
0.03
-550 C/T
CC
CT
TT
35.5
81.3
134.9
52
31
7
0.01
70.8
63.1
47.9
348
235
25
0.42
0.006
* Geometric mean IgE values. N= number of children per genotype. † P-values were adjusted
for gender, atopy mother, atopy father, siblings, breastfeeding and ETS.
Revision Bottema et al
Table E6. Interaction of CD14 genotypes with dog exposure in the development of
sensitisation to any allergen at age 8 years.
CD14 SNP Genotype
Sensitisation to any allergen§
Interaction
yes / no* (proportion)
Dog exposure †
No dog exposure †
P‡
3’UTR
CC+CA
AA
31 / 54 (0.57)
3 / 2 (1.5)
249 / 306 (0.81)
19 / 31 (0.61)
0.20
-159 C/T
CC + CT
TT
30 / 38 (0.79)
4 / 17 (0.24)
212 / 268 (0.79)
59 / 64 (0.92)
0.04
-1145 T/C
TT + CT
CC
30 / 37 (0.81)
4 / 21 (0.19)
212 / 272 (0.78)
58 / 67 (0.87)
0.02
-1619 T/C
TT + CT
CC
32 / 42 (0.76)
2 / 9 (0.22)
215 / 269 (0.80)
42 / 45 (0.93)
0.13
-550 C/T
CC
CT
TT
14 / 38 (0.37)
17 / 14 (1.21)
3 / 4 (0.75)
163 / 183 (0.89)
98 / 137 (0.72)
9 / 16 (0.56)
0.04
§ Specific IgE >0.35 IU/ml to house dust mite, cat, dog, Dactylis glomerata, Betula
verrucosa, Alternaria alternata, egg, or milk allergens. * Number of children sensitised /
number of non-sensitised children; †Dog exposure = dog exposure at home in the first year
of life; No dog exposure = no dog exposure at home in the first year of life. ‡ P-value for
interaction, adjusted for gender, atopy mother, atopy father, siblings, breastfeeding and ETS
exposure.
Revision Bottema et al
Table E7. Interaction of CD14 genotypes and cat exposure with respect to total serum IgE
levels at ages 4 and 8 years.
Cat exposure†
No cat exposure†
Age
CD14 SNP Genotype
Interaction
IgE*
4 years
8 years
N
P
IgE*
N
P
P‡
3’UTR
CC+CA
AA
29.4
71.4
200
17
0.02
30.0
32.4
585
51
0.74
0.19
-159 C/T
CC + CT
TT
32.1
37.5
175
41
0.56
28.8
33.8
513
127
0.31
0.72
-1145 T/C
TT + CT
CC
32.0
34.0
175
44
0.82
28.8
33.0
510
129
0.39
0.56
-1619 T/C
TT + CT
CC
32.8
33.3
172
30
0.96
29.2
31.9
505
87
0.64
0.67
-550 C/T
CC
CT
TT
39.0
24.2
11.6
128
81
7
0.02
29.8
31.1
24.7
343
239
37
0.71
0.04
3’UTR
CC+CA
AA
59.1
95.6
176
20
0.20
70.2
52.4
466
36
0.31
0.20
-159 C/T
CC + CT
TT
60.7
75.4
157
39
0.44
63.4
89.1
393
106
0.06
0.52
-1145 T/C
TT + CT
CC
59.4
75.9
158
40
0.38
63.8
83.1
395
111
0.14
0.72
-1619 T/C
TT + CT
CC
58.8
85.0
157
30
0.25
69.7
73.3
402
69
0.82
0.53
-550 C/T
CC
CT
TT
67.2
48.0
60.4
116
74
8
0.35
64.1
73.4
60.5
284
192
24
0.65
0.30
* Geometric mean IgE values. N= number of children per genotype. ‡ P-values were adjusted
for gender, atopy mother, atopy father, siblings, breastfeeding and ETS exposure.
Revision Bottema et al
Table E8. Interaction of CD14 genotypes with cat exposure in the development of
sensitisation to any allergen at age 8 years.
CD14 SNP Genotype
Sensitisation to any allergen§
Interaction
yes / no* (proportion)
Cat exposure †
No cat exposure †
P‡
3’UTR
CC+CA
AA
62 / 114 (0.54)
11 / 8 (1.38)
218 / 246 (0.89)
11 / 25 (0.44)
0.005
-159 C/T
CC + CT
TT
66 / 90 (0.73)
10 / 29 (0.34)
176 / 216 (0.81)
53 / 52 (1.02)
0.02
-1145 T/C
TT + CT
CC
65 / 92 (0.71)
9 / 31 (0.29)
177 / 217 (0.82)
53 / 57 (0.93)
0.02
-1619 T/C
TT + CT
CC
63 / 93 (0.68)
9 / 21 (0.43)
184 / 218 (0.84)
35 / 33 (1.06)
0.13
-550 C/T
CC
CT
TT
47 / 68 (0.69)
25 / 49 (0.51)
3 / 5 (0.60)
130 / 153 (0.85)
90 / 102 (0.88)
9 / 15 (0.60)
0.61
§ Specific IgE >0.35 IU/ml to house dust mite, cat, dog, Dactylis glomerata, Betula
verrucosa, Alternaria alternata, egg, or milk allergens. * Number of children sensitised /
number of non-sensitised children; †Cat exposure = cat exposure at home in the first year of
life; No cat exposure = no cat exposure at home in the first year of life; ‡ P-value for
interaction, adjusted for gender, atopy mother, atopy father, siblings, breastfeeding and ETS
exposure.
Revision Bottema et al
Table E9. Interaction of CD14 genotypes and environmental tobacco smoke exposure (ETS)
with respect to total serum IgE levels at age 4 years.
ETS exposure†
No ETS exposure†
CD14 SNP Genotype
Interaction
IgE*
4 years
3’UTR
N
P
IgE*
N
P
P‡
CC+CA
AA
29.0
14.8
170
16
0.12
29.6
47.6
575
49
0.04
0.04
-159 C/T
CC + CT
TT
26.6
28.8
145
42
0.77
30.1
34.6
507
119
0.38
0.79
-1145 T/C
TT + CT
CC
26.1
27.8
143
44
0.82
30.2
33.1
506
122
0.56
0.84
-1619 T/C
TT + CT
CC
24.7
30.1
141
30
0.55
31.0
31.1
501
82
0.98
0.59
-550 C/T
CC
CT
TT
24.3
33.1
13.9
113
59
13
34.2
27.5
26.0
341
243
26
0.22
0.64
0.17
* Geometric mean IgE values. N= number of children per genotype. †ETS = environmental
tobacco smoke exposure at home in the first year of life; ‡ P-values were adjusted for gender,
atopy mother, atopy father, siblings, breastfeeding, cat and dog exposure.
Revision Bottema et al
Reference List
1. Brunekreef B, Smit J, de JJ, Neijens H, Gerritsen J, Postma D, Aalberse R, Koopman L,
Kerkhof M, Wilga A, van SR. The prevention and incidence of asthma and mite allergy
(PIAMA) birth cohort study: design and first results. Pediatr Allergy Immunol 2002; 13
Suppl 15: 55-60.
2. Lakwijk N, Van Strien RT, Doekes G, Brunekreef B, Gerritsen J. Validation of a
screening questionnaire for atopy with serum IgE tests in a population of pregnant
Dutch women. Clin Exp Allergy 1998; 28: 454-458.
3. Schonberger HJ, Dompeling E, Knottnerus JA, Maas T, Muris JW, van WC, van
Schayck CP. The PREVASC study: the clinical effect of a multifaceted educational
intervention to prevent childhood asthma. Eur Respir J 2005; 25: 660-670.
4. Kuiper S, Maas T, van Schayck CP, Muris JW, Schonberger HJ, Dompeling E, Gijsbers
B, van WC, Knottnerus JA. The primary prevention of asthma in children study: design
of a multifaceted prevention program. Pediatr Allergy Immunol 2005; 16: 321-331.
5. Kummeling I, Thijs C, Penders J, Snijders BE, Stelma F, Reimerink J, Koopmans M,
Dagnelie PC, Huber M, Jansen MC, de BR, van den Brandt PA. Etiology of atopy in
infancy: the KOALA Birth Cohort Study. Pediatr Allergy Immunol 2005; 16: 679-684.
6. Bastiaanssen JM, de Bie RA, Bastiaenen CH, Heuts A, Kroese ME, Essed GG, van den
Brandt PA. Etiology and prognosis of pregnancy-related pelvic girdle pain; design of a
longitudinal study. BMC Public Health 2005; 5: 1.
7. Sambrook, J. and Russell, D. Molecular Cloning. 3rd Edition 2007. Preparation of
plasmid DNA by lysis with SDS.
Revision Bottema et al
8.
K-Biosciences, Cambridge, UK; URL: http://www.kbiosciences.co.uk