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

<div><p>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 <i>LHCGR</i> region, <i>STON1-GTF2A1L</i> and <i>LHCGR</i>, were overexpressed in PCOS. In analysis stratified by obesity, <i>LHCGR</i> was overexpressed only in non-obese PCOS women. Although not differentially expressed in the entire PCOS group, <i>INSR</i> was underexpressed in obese PCOS subjects only. Alterations in gene expression in the <i>LHCGR</i>, <i>RAB5B</i> and <i>INSR</i> 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 <i>LHCGR</i> locus and increased methylation in the <i>INSR</i> 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.</p></div

Expression levels of differentially expressed mRNA transcripts.

<p><b>(A)</b> Expression levels of differentially expressed mRNA transcripts between PCOS and controls in the <i>LHCGR</i> and <i>RAB5B/SUOX</i> loci. On the X axis are gene names. On the Y axis are the mean expression levels. Error bars represent standard deviation. <b>(B)</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.</p

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

<p><b>i.</b> 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. <b>ii.</b> 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. <b>iii.</b> UCSC Genome Browser ENCODE tracks show <b>1</b> SNP position from dbSNP143, <b>2</b> poised enhancer activity, <b>3</b> active enhancer activity, <b>4</b> active promoter activity and <b>5</b> transcriptional activity, in 7 Encode reference cell types. <b>iv.</b> meQTL results are shown with box and whisker plots demonstrating mean methylation (Beta level) in each genotype group.</p

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

<p>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.</p

Association of known IBD SNPs.

<p>“Previously reported SNPs” are those IBD snps added to the design of the ImmunoChip; one per locus is shown here. Since 2 phenotypes were tested for this table (CD and UC) and 100 of the SNPs were not in linkage disequilibrium in Puerto Rico controls, the Bonferroni correction for multiple comparisons is 0.05/(2*100) = 0.00025.</p><p>Association of known IBD SNPs.</p

Top Associations with Additional SNPs.

<p>Associations with pvalues <5×10⁻⁵ are listed here. Of the ∼115,000 SNPs tested for this table, ∼20,000 SNPs are not in linkage disequilibrium. Since 3 phenotypes were tested, the Bonferroni correction for multiple comparisons is 0.05/(20,000×3) = 8×10⁻⁷.</p><p>Top Associations with Additional SNPs.</p

Description of study subjects.

<p>Description of study subjects.</p

“Global” continental ancestry in Puerto Rican subjects.

<p>A. Principal components analysis of the combined Puerto Rican and HGDP subjects. B. “Global” continental ancestry was estimated using Admixture 1.2 in an analysis supervised by data from HGDP populations as described in Methods (AFR, sub-Saharan African continent, dark blue; EUR, European continent, red; and AMR, Mexico, Central and South America continents, green). HGDP subjects not originating from these three continents are black. Puerto Rican subjects were sorted left-to-right based on ancestry from the African continent.</p

A favorable cardiometabolic profile is associated with the G allele of the genetic variant rs5068 in African Americans: The Multi-Ethnic Study of Atherosclerosis (MESA)

<div><p>In whites, the minor G allele of the atrial natriuretic peptide (ANP) genetic variant rs5068 is associated with higher circulating levels of ANP and B-type natriuretic peptide (BNP), lower risk of hypertension, higher high-density lipoprotein (HDL) cholesterol plasma levels, and lower prevalence of obesity and metabolic syndrome. The observed phenotype is consistent with the blood pressure lowering and metabolic properties of ANP and BNP. The cardiovascular and metabolic phenotype associated with rs5068 genotypes in African Americans is undefined. We genotyped 1631 African Americans in the Multi-Ethnic Study of Atherosclerosis (MESA) for rs5068 and investigated their phenotype. Genotype frequencies of rs5068 were 93.2% AA (n = 1520), 6.7% AG (n = 110) and 0.1% GG (n = 1). All subsequent analyses are AG + GG versus AA genotype. Using a Bonferroni corrected level of significance of 0.005, the prevalence of metabolic syndrome (23% vs 38%, age-sex-adjusted p = 0.002) and triglycerides plasma values (76 vs 90 mg/dl, age-sex-BMI adjusted p = 0.004) were both significantly lower in the AG+GG genotypes. In the AG+GG genotypes, the prevalence of diabetes (8% vs 18%, age-sex-BMI-adjusted p = 0.02) and insulin plasma levels tended to be lower (4.8 vs 5.7 μU/ml, age-sex-BMI adjusted p = 0.04) whereas HDL-cholesterol levels tended to be higher (55 vs 50 mg/dl, age-sex-BMI-adjusted p = 0.04). No association was found with hypertension. The association between the rs5068 G allele and a favorable metabolic phenotype is now shown in African Americans. The rs5068 AG+GG genotypes are associated with lower prevalence of metabolic syndrome and lower triglycerides values.</p></div

NOD2 haplotypes.

<p>Haplotypes were determined using Phase v2.3; SNPs are, in order, rs2066842 (SNP5), rs2066843 (SNP6), rs2066844 (SNP8), rs2066845 (SNP12), and rs5743293 (SNP13). The association of each haplotype was tested by permutation.</p><p>Footnotes:</p><p>1) IBD susceptibility haplotype in Europeans.</p><p>NOD2 haplotypes.</p