2. Materials and Methods
2.1. Study participants and baseline study
In the Hokkaido Birth Cohort Study on Environment and Children’s Health,
the participants of 20,296 pregnant women were enrolled from 2003 to
2012 in Hokkaido, Japan. During the first trimester, participants
completed the baseline questionnaire on maternal and paternal
characteristics. Maternal peripheral blood samples were taken during
pregnancy without fasting. Medical birth records from the delivery
hospital were collected for birth weight, height, sex, and other medical
conditions (Kishi et al., 2013; 2017).
The institutional ethics board for epidemiological studies at Hokkaido
University Graduate School of Medicine and Hokkaido University Center
for Environmental and Health Sciences approved the study protocol
(approval number 69). In this study, informed consent was obtained from
all study participants before enrollment.
2.2. Follow-up study until age 7 and saliva samples collection.
We have collected data regarding the wheezing symptoms of children at 1,
2, 4, and 7 years of age using a modified section of the Japanese
version of the International Study of Asthma and Allergies in Childhood
(ISAAC) Phase Three questionnaire (Asher et al., 1995). We defined
childhood wheeze as a maternal positive answer to the question ”Has your
child had wheezing or whistling in the chest in the past 12 months?,”
based on each ISAAC questionnaire administered when the child was 1, 2,
4, and 7 years of age (Figure 1). Moreover, we collected information
child’s history of infectious diseases (Goudarzi et al., 2018).
In a case-control study, we selected all 314 children with wheezing and
randomly selected 374 control children without any allergic symptoms
(extraction rate 24%) from the ISAAC questionnaire at 7 years age.
Salivary samples (2 ml) were collected from children of case and control
using The Oragene® OG-300 DNA Self-Collection kit (DNA Genotek Inc.,
Ottawa, Ontario, Canada). Eventually, we used 275 salivary samples to
analyze DNA methylation (Figure 1).
2.3. Quantification of DNA Methylation
Genomic DNA was extracted from saliva using a Maxwell 16 DNA
Purification Kit (Promega, Madison, WI, USA). The DNA (500 ng) was then
subjected to bisulfite conversion using Epitect Plus Bisulfite Kit
(Qiagen, Venlo, The Netherlands). Bisulfite pyro sequence was performed
as described previously (Murphy et al., 2012; Bollati et al., 2007). We
analyzed DNA methylation in genes related to asthma for five of the CpG
sites of IKZF3 (cg16293631, cg13432737), ORMDL3(cg02305874, cg14647739), and GSDMB (cg12360886). Pyrosequencing
was performed using Pyromark Q24 system (Qiagen) and data were analyzed
using the Pyro Q-CpG Software (Qiagen). Eventually, we completed the
quantification of DNA methylation only three CpG sites of ORMDL3(cg02305874), and IKZF3 (cg16293631, cg13432737). Other PCR
primers of two CpG sites including GSDMB (cg12360886) andORMDL3 (cg14647739) did not work. Conditions of primers were
described in Supplementary Table 1. Average methylation levels of each
CpG site that were analyzed in duplicate were used in statistical
analyses.
2.4. Maternal folate and cotinine measurements
Folate is quantified by direct chemiluminescent acridinium ester
technology. This technique has an acceptable imprecision of less than
10.0%, with an advanced Quality Control package. It has an analytical
sensitivity of 0.91 nmol/l. Specimen preparations, shipping, and assays
were performed in batches, depending on new recruitments. We have
previously reported the significance of the inverse association between
maternal folate and cotinine levels (Kishi et al., 2013; Yila et al.,
2012). The details of serum folate and plasma cotinine measurements are
described in our previous report (Sasaki et al., 2011).
2.5. Statistical analysis
We analyzed the association between participant’s characteristics and
maternal folate level using Mann–Whitney’s U-test and rank correlation
test. In logistic regression analysis, we evaluated odds ratios (ORs)
and 95% confidence interval (CI) of childhood wheeze, according to
maternal folate level, respectively. Because a biologically relevant
threshold of folate on wheezing is not elucidated, we used two types of
maternal folate levels as follows;
first, ORs and 95% CI for the risk of wheezing were calculated for
three categorical maternal folate levels in the folate suboptimal
(6.80–13.59 nmol/l) or optimal (≥ 13.60 nmol/l) and compared to those
in folate deficiency (< 6.80 nmol/l) with reference to the WHO
guideline (Sauberlich, 1999). Second, ORs and 95% CI for the risk of
wheezing were calculated for 4 categorical maternal folate levels in the
second, third, and the fourth quartiles and compared to those in the
lowest quartiles as reference by the distribution of their quartiles.
For the calculation of P for trend, we handled categorical values
of maternal folates as ordinal variables.
In a case-control study, we used two types of maternal folate levels,
including categorical of quartile, and two categorical with reference to
the WHO guideline, which were optimal (≥ 13.60 nmol/l) or under
suboptimal category (< 13.60 nmol/l) (Supplementary Table 2).
In liner regression analysis, we evaluated β and 95% CI for DNA
methylation, including, three CpG sites of IKZF3 (cg16293631,
cg13432737) and ORMDL3 (cg02305874), according to maternal folate
levels. In logistic regression analysis, we evaluated odds ratios (ORs)
and 95% CI of childhood wheezing at 7 years age according to DNA
methylation.
In both logistic and liner regression analysis, we used adjusted factors
as potential confounders, including maternal age, parity, delivery year,
alcohol drinking during pregnancy, log10-transformed cotinine levels,
maternal allergic history, paternal allergic history, annual household
income, and sex of child (model 1). Furthermore, to confounding
variables of the model 1, we used adjusted factors which were collected
after birth, including current maternal and paternal smoking status,
breast feeding, and day care attendance (yes, no) (model 2). Pvalue of less than 0.05 was considered statistically significant. All
statistical analyses were performed with SPSS software for Windows
(version 21.0J; IBM, Armonk, NY, USA).