Systems Genetics Reveals the Functional Context of PCOS Loci and Identifies Genetic and Molecular Mechanisms of Disease Heterogeneity

Abstract

Genome wide association studies (GWAS) have revealed 11 independent risk loci for polycystic ovary syndrome (PCOS), a common disorder in young women characterized by androgen excess and oligomenorrhea. To put these risk loci and the single nucleotide polymorphisms (SNPs) therein into functional context, we measured DNA methylation and gene expression in subcutaneous adipose tissue biopsies to identify PCOS-specific alterations. Two genes from the LHCGR region, STON1-GTF2A1L and LHCGR, were overexpressed in PCOS. In analysis stratified by obesity, LHCGR was overexpressed only in non-obese PCOS women. Although not differentially expressed in the entire PCOS group, INSR was underexpressed in obese PCOS subjects only. Alterations in gene expression in the LHCGR, RAB5B and INSR regions suggest that SNPs in these loci may be functional and could affect gene expression directly or indirectly via epigenetic alterations. We identified reduced methylation in the LHCGR locus and increased methylation in the INSR locus, changes that are concordant with the altered gene expression profiles. Complex patterns of meQTL and eQTL were identified in these loci, suggesting that local genetic variation plays an important role in gene regulation. We propose that non-obese PCOS women possess significant alterations in LH receptor expression, which drives excess androgen secretion from the ovary. Alternatively, obese women with PCOS possess alterations in insulin receptor expression, with underexpression in metabolic tissues and overexpression in the ovary, resulting in peripheral insulin resistance and excess ovarian androgen production. These studies provide a genetic and molecular basis for the reported clinical heterogeneity of PCOS.

Fig 1. Expression levels of differentially expressed mRNA transcripts.

(A) Expression levels of differentially expressed mRNA transcripts between PCOS and controls in the LHCGR and RAB5B/SUOX loci. On the X axis are gene names. On the Y axis are the mean expression levels. Error bars represent standard deviation. (B) Expression levels of mRNA transcripts differentially expressed between PCOS and controls, stratified by obesity. On the X axis is the obesity status of the subjects, non-obese and obese subjects analyzed separately. On the Y axis are the mean expression levels. * Denotes results that remained significant after correction for multiple testing. Error bars represent standard deviation.

Fig 2. Methylation (Beta) levels at CpG sites with significantly different methylation between PCOS and controls.

On the X axis are the significant CpG sites in the windows around the PCOS GWAS SNPs. On the Y axis is the methylation status, measured as the mean beta level. Error bars represent standard deviation.

Fig 3. PCOS risk loci contain alterations in gene regulation and expression in PCOS adipose tissue.

i. Chromosomal co-ordinates, gene structure and gene expression profile (grey = not expressed in adipose, black = expressed in adipose). The index PCOS GWAS risk SNP is marked by a filled black triangle and is labeled with rs number. ii. Methylation sites are shown as open (unmethylated), grey filled (semi-methylated) or black (fully methylated) circles, and meQTL relationships between these sites and local SNPs are shown with a green arrow. eQTL results are shown by an orange star marking the gene and orange arrows marking SNP position of independent signals. iii. UCSC Genome Browser ENCODE tracks show 1 SNP position from dbSNP143, 2 poised enhancer activity, 3 active enhancer activity, 4 active promoter activity and 5 transcriptional activity, in 7 Encode reference cell types. iv. meQTL results are shown with box and whisker plots demonstrating mean methylation (Beta level) in each genotype group.