Sex- and age-dependent DNA methylation at the 17q12-q21 locus associated with childhood asthma

Abstract

Chromosomal region 17q12-q21 is one of the best-replicated genome-wide association study (GWAS) hits and associated with childhood-onset asthma. However, the mechanism by which the genetic association is restricted to childhood-onset disease is unclear. During childhood, more boys than girls develop asthma. Therefore, we tested the hypothesis that the 17q12-q21 genetic association was sex-specific. Indeed, a TDT test showed that in the Saguenay-Lac-Saint-Jean familial collection, the 17q12-q21 association was significant among male, but not among female asthmatic subjects. We next hypothesized that the bias in the genetic association resulted from sex-specific and/or age-dependent DNA methylation at regulatory regions and determined the methylation profiles of five 17q12-q21 gene promoters using the bisulfite sequencing methylation assay. We identified a single regulatory region within the zona pellucida binding protein 2 (ZPBP2) gene, which showed statistically significant differences between males and females with respect to DNA methylation. DNA methylation also varied with age and was higher in adult males compared to boys. We have recently identified two functionally important polymorphisms, both within the ZPBP2 gene that influence expression levels of neighboring genes. Combined with the results of the present work, these data converge pointing to the same 5 kb region within the ZPBP2 gene as a critical region for both gene expression regulation and predisposition to asthma. Our data show that sex- and age-dependent DNA methylation may act as a modifier of genetic effects and influence the results of genetic association studies.

Local genotype influences DNA methylation at two asthma-associated regions, 5q31 and 17q21, in a founder effect population

“…Correlations have been found between all nine CGs using Pearson's correlation tests (correlation coefficients between 0.5 and 1.0), allowing us to analyse average methylation levels for all nine CGs. Since earlier we have found that ZPBP2 methylation levels depended on the sex of individuals in HapA homozygotes,17 the effect of sex on methylation had to be confirmed for other genotypes. Methylation levels were significantly higher in females compared with males in models considering haplotypes and those results were significant independent of white blood cell counts in agreement with our earlier findings (p=7.6E-13 (previously reported in ref.…”

“…To test the relationship between genotype, DNA methylation and asthma, we analysed promoter methylation levels of those genes where the effect of genotype on expression was previously demonstrated, i.e, ZPBP2 , GSDMB , ORMDL3 and GSDMA, 19 25 27 28 in DNA samples from peripheral blood of subjects from the SLSJ asthma familial collection (table 1). 17 The ORMDL3 promoter was hypomethylated while the GSDMB promoter was hypermethylated in all samples with no evidence for effect of genotype or disease on methylation (see online supplementary table S4). Methylation of the ZPBP2 and GSDMA promoters varied considerably among individuals.…”

“…Methylation levels were significantly higher in females compared with males in models considering haplotypes and those results were significant independent of white blood cell counts in agreement with our earlier findings (p=7.6E-13 (previously reported in ref. 17); figure 1D and table 3). When the effect of genotype on methylation was analysed in males and females separately, higher methylation levels for seven of the nine interrogated CGs were found in homozygous HapB individuals (p values ranging from 7.3E-09 to 5.1E-09 for males and p values ranging from 7.7E-04 to 5.1E-09 for females, Tukey's post hoc tests; table 3 and figure 1D).…”

“…One to two micrograms of DNA were treated with sodium bisulfite as previously described 17. For most samples, at least two independent PCR reactions were done.…”

Sex differences in disease genetics

“…Secondly, the suppression of ZPBP2 activity by 108 epigenetic methylation is greater in females than it is in males, providing a biochemical explanation 109 for why only one sex is affected by the variant (Naumova et al 2013), pending further investigation of 110 tissue-specific methylation profiles. The reason why ZPBP2 is expression is not suppressed in males is 111 possibly because it has an important role in production of viable sperm, but with no known phenotype 112 in females, as determined from gene knock-out studies in mice (Y.-N. Lin et al 2007).…”