Methylation of cysteinyl leukotriene receptor 1 genes associates with lung function in asthmatics exposed to traffic-related air pollution

Figures & data

Table 1. Significant correlations with data corrected for cell-type (versus uncorrected data)

Exposure attributable gene correlation	Uncorrected correlation and p-value	Correlation and p-value after correction for cell-type
Log uLTE4 and CysLTR1 cg26848126 methylation	r = 0.54, p = 0.03	r = 0.59, p = 0.02
CysLTR1 cg26848126 methylation and CysLTR1 expression	r = −0.63, p = 0.009	r = −0.49, p = 0.06
Log uLTE4 and CysLTR1 expression	r = −0.30, p = 0.26	r = −0.45, p = 0.08
CysLTR1 cg0081399 methylation and GPR17 cg05361273 methylation	r = −0.36, p = 0.20	r = −0.73, p = 0.001
CysLTR1 expression and GPR17 cg05361273 methylation	r = −0.22, p = 0.42	r = 0.47, p = 0.07
CysLTR1 expression and CysLTR1 cg0081399 methylation	r = 0.10, p = 0.72	r = −0.49, p = 0.06
FEV1 and CysLTR1 expression	r = 0.70, p = 0.003	r = 0.76, p = 0.0007

Figure 1. Exposure-attributable FEV₁ decline pathway correlations. (a) Correlation between exposure-attributable changes (6H–0H) in FEV₁ and log uLTE4 levels. r = −0.51, p = 0.04. (b) Correlation between exposure-attributable changes (6H–0H) in CysLTR1 cg26848126 and log uLTE4 levels. r = 0.59, p = 0.02. (c) Correlation between exposure-attributable changes (6H–0H) in CysLTR1 expression and log uLTE4 levels. r = −0.45, p = 0.08. (d) Correlation between exposure-attributable changes (6H–0H) in CysLTR1 expression and CysLTR1 cg26848126 methylation. r = −0.49, p = 0.06. (e) Correlation between exposure-attributable changes (6H-0H) in FEV₁ and CysLTR1 expression r = 0.76, p = 0.0007

Figure 2. Exposure-attributable FEV₁ increase pathway correlations. (a) Correlation between exposure-attributable changes (6H-0H) in CysLTR1 cg0081399 methylation and GPR17 cg05361273 methylation r = −0.73, p = 0.001. (b) Correlation between exposure-attributable changes (6H-0H) in CysLTR1 expression and GPR17 cg05361273 methylation r = 0.47, p = 0.07. 9 c) Correlation between exposure-attributable changes (6H-0H) in CysLTR1 expression and CysLTR1 cg0081399 methylation r = −0.49, p = 0.06

Figure 3. Biological pathways linking DE exposure to acute FEV₁ changes through methylation and expression changes on CysLTR1-related genes

In the lung function decrease pathway, DE exposure-attributable cysteinyl leukotrienes bind to the CysLTR1 receptor resulting in increased CysLTR1 cg26848126 methylation and decreased CysLTR1 expression. Decreased CysLTR1 expression results in an attenuated antioxidant response and is correlated with FEV₁ decline. In the lung function increase pathway, exposure attributable increases in GPR17 gene methylation are correlated with decreased CysLTR1 cg0081399 methylation which, in turn is correlated with increased CysLTR1 expression. Increased CysLTR1 expression leads to a greater antioxidant response thereby increasing FEV₁.

Table 2. CpG percent methylation by pyrosequencing at baseline and changes after DE, FA

Gene CpG	Percent methylation difference (DE-FA) at 0H, 6H and 6H-0H	Percent methylation at 0H, 6H and 6H-0H after DE exposure	Percent methylation at 0H, 6H and 6H-0H after FA exposure
ALOX5 cg24302529	0H: −1.06 6H: 0.28% 6H-0H: 1.3% (p = 0.13)	0H: 73.9% 6H: 75.8% 6H-0H: 1.9% (p = 0.02)	0H: 75.0% 6H: 75.6% 6H-0H: 0.6% (p = 0.28)
GPR99 cg24663970	0H: −0.56% 6H: −0.23% 6H-0H: 0.32% (p = 0.66)	0H: 73.9% 6H: 75.8% 6H-0H: 1.9% (p = 0.02)	0H: 75.0% 6H: 75.6% 6H-0H: 0.6% (p = 0.28)
CysLTR1 cg26848126	0H: −0.77% 6H: 1.2% 6H-0H: 1.8% (p = 0.17)	0H: 35.2% 6H: 39.6% 6H-0H:4.4% (p = 0.0007)	0H: 35.7% 6H: 37.8% 6H-0H: 2.1% (p = 0.04)
CysLTR1 cg0081399	0H: −0.06% 6H: 0.40% 6H-0H: 0.2% (p = 0.91)	0H: 29.1% 6H: 31.4% 6H-0H: 2.3% (p = 0.13)	0H: 28.6% 6H: 30.2% 6H-0H: 1.6% (p = 0.30)
LTC4 S cg14674859	0H: 0.02% 6H: −0.84% 6H-0H: −0.9% (p = 0.80)	0H: 70.9% 6H: 65.7% 6H-0H: 5.2% (p = 0.006)	0H: 71.7% 6H: 65.7% 6H-0H: 6.1% (p = 0.07)
LTC4 S cg26683398	0H: −2.43% 6H: 1.6% 6H-0H: 4.1% (p = 0.01)	0H: 73.3% 6H: 77.4% 6H-0H: 4.1% (p = 0.0007)	0H: 75.8% 6H: 75.8% 6H-0H:0.0003% (p = 0.99)
LTC4 S cg16361890	0H: 8.2% 6H: −1.6% 6H-0H: −9.8% (p < 0.001)	0H: 65.7% 6H: 58.8% 6H-0H: −6.9% (p < 0.0001)	0H: 57.5% 6H: 60.5% 6H-0H: 3.0% (p = 0.002)
GPR17 cg05361273	0H: −2.0% 6H: 0.62 6H-0H: 2.6% (p = 0.02) *	0H: 52.6% 6H: 57.5% 6H-0H: 4.9% (p < 0.0001)	0H: 54.6% 6H: 56.9% 6H-0H: 2.3% (p = 0.001)

Bold denotes statistically significant ‘exposure-attributable’ changes by pyrosequencing. *Denotes statistically significant ‘exposure-attributable’ changes by Illumina array.

Table 3. Participant characteristics

Variable	Participants clinically reported to have asthma (n = 16)
Male gender, subjects with	7 [44%]
Age (Years)	28.7 ± 6.7 [range: 19.2–46.5]
Ethnicity	Asian 2 [13%] Caucasian 12 [75%] Middle Eastern 1 [6%] South Asian 1 [6%]
Height (cm)	170.0 ± 12.2 [151.0–185.5]
Weight (kg)	71.9 ± 15.5 [53.0–104.6]
BMI	24.8 ± 3.9 [19.8–34.7]
FEV1% predicted	90.3 ± 14.1 [66.0–120.0]
Atopy, subjects with	9 [56%]

Table 4. Primers used for gene expression analysis

Primer	Sequence
CysLTR1 Forward	TTTGGTGACTTCTTGTGCCG
CysLTR1 Reverse	CCTACACACACAAACCTGGC
LTA4 H Forward	ACCCTGCTACCTGATTGCTT
LTA4 H Reverse	CCCATACATACGGTCCTCCC
ALOX5 Forward	ACTGGAAACACGGCAAAAAC
ALOX5 Reverse	TTTCTCAAAGTCGGCGAAGT
ACTB1 Forward	GCCCTGAGGCACTCTTCCA
ACTB1 Reverse	CCAGGGCAGTGATCTCCTT

Table 5. Primers used for methylation analysis

Primer	Sequence
CysLTR1 Forward	GAAAGAGGAAGAAGAATTTGTATATTTG
CysLTR1 Reverse – biotin	AACTTATCCCCAAACTCCTAAAAATT
CysLTR1 cg0081399 Sequencing	TTGGAGTTTGTTTTTGAG
CysLTR1 cg26848126 Sequencing	ATAGGGTATTAAGATATTGTGTA
ALOX5 cg1559007 Forward	GTGGGTATTTTGGTGGGTAAG
ALOX5 cg1559007 Reverse – biotin	TCTACCTCTCCCTCCCTATAC
ALOX5 cg1559007 Sequencing	AAGGTTTTTTTTTTATTTTAAGATT
ALOX5 cg2430202529 Forward	GATGTTTAGAGTTTATTTTTGGTGTTATAT
ALOX5 cg2430202529 Reverse – biotin	CTCCTTCTAAAACCTTTTCTATTTCC
ALOX5 cg2430202529 Sequencing	AGGTAGGGTAGATATTGATTTT
GPR99 cg24663970 Forward – biotin	GGTTGAGGTTTAGAGAAGAGAA
GPR99 cg24663970 Reverse	CCAAAAATTTCACCACTCTCC
GPR99 cg24663970 Sequencing	ATATCCAATTACAACCCT
LTC4 S Forward	ATTGTTTAGATTATAGGGTTTGAGTG
LTC4 S Reverse – biotin	TCCCCATCCAAACTATTCCTAACA
LTC4 S cg14674859 Sequencing	AGTGTGGGGAGGGTA
LTC4 S cg1631890 Sequencing	TGTGGGGTTTAGTGT
LTC4 S cg26683398 Sequencing	GGTATTGGGAAGGGT
GPR17 cg0561273 Forward	GGTTTTTTTGGAGGAAGTGG
GPR17 cg0561273 Reverse – biotin	CAACCCCCAAAACTCTTTAACA
GPR17 cg0561273 Sequencing	TGGTTTGAGTTGAGTTT

Availability of data and materials

The data set analyzed during the current study are available from the corresponding authors on reasonable request.