Association between an AMH promoter polymorphism and serum AMH levels in PCOS patients.

STUDY QUESTION: Do polymorphisms in the anti-Müllerian hormone (AMH) promoter have an effect on AMH levels in patients with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: We have identified a novel AMH promoter polymorphism rs10406324 that is associated with lower serum AMH levels and is suggested to play a role in the mechanism of regulation of AMH gene expression in women. WHAT IS KNOWN ALREADY: Follicle number is positively correlated with serum AMH levels, reflected by elevated AMH levels in women with PCOS. In addition, it is suggested that AMH production per follicle is higher in women with PCOS than in normo-ovulatory women, implying an altered regulation of AMH in PCOS. STUDY DESIGN, SIZE, DURATION: A discovery cohort of 655 PCOS women of Northern European ancestry and both an internal and external validation PCOS cohort (n = 458 and n = 321, respectively) were included in this study. Summary-level data of an AMH genome-wide association study meta-analysis including 7049 normo-ovulatory women was included as a control cohort. A genetic approach was taken through association analysis and in silico analysis of the associated variants in the AMH promoter. In vitro analysis was performed to investigate the functional mechanisms. PARTICIPANTS/MATERIALS, SETTING, METHODS: All common two-allelic single-nucleotide polymorphisms (SNPs) in the region Chr19:2 245 353-2 250 827 bp (Build 37) were selected for the analysis. Linear regression analyses were performed to determine the association between SNPs in the AMH promoter region and serum AMH levels. For the in silico analysis, the webtools 'HaploReg' v4.1 for ENCODE prediction weight matrices and 'atSNP' were used. In vitro analysis was performed using KK1 cells, a mouse granulosa cell line and COV434 cells, a human granulosa tumor cell line. Cells were transfected with the reference or the variant human AMH promoter reporter construct together with several transcription factors (TFs). Dual-Glo® Luciferase Assay was performed to measure the luciferase activity. MAIN RESULTS AND THE ROLE OF CHANCE: Polymorphism rs10406324 was significantly associated with serum AMH levels in all three PCOS cohorts. Carriers of the minor allele G had significantly lower log-transformed serum AMH levels compared to non-carriers (P = 8.58 × 10-8, P = 1.35 × 10-3 and P = 1.24 × 10-3, respectively). This result was validated in a subsequent meta-analysis (P = 3.24 × 10-12). Interestingly, rs10406324 was not associated with follicle count, nor with other clinical traits. Also, in normo-ovulatory women, the minor allele of this variant was associated with lower serum AMH levels (P = 1.04 × 10-5). These findings suggest that polymorphism rs10406324 plays a role in the regulation of AMH expression, irrespective of clinical background. In silico analysis suggested a decreased binding affinity of the TFs steroidogenenic factor 1, estrogen-related receptor alpha and glucocorticoid receptor to the minor allele G variant, however in vitro analysis did not show a difference in promoter activity between the A and G allele. LIMITATIONS, REASONS FOR CAUTION: Functional analyses were performed in a mouse and a human granulosa cell line using an AMH promoter reporter construct. This may have limited assessment of the impact of the polymorphism on higher order chromatin structures. Human granulosa cells generated from induced pluripotent stem cells, combined with gene editing, may provide a method to elucidate the exact mechanism behind the decrease in serum AMH levels in carriers of the -210 G allele. We acknowledge that the lack of follicle number in the external validation and the control cohort is a limitation of the paper. Although we observed that the association between rs10406324 and AMH levels was independent of follicle number in our discovery and internal validation PCOS cohorts, we cannot fully rule out that the observed effects on serum AMH levels are, in part, caused by differences in follicle number. WIDER IMPLICATIONS OF THE FINDINGS: These results suggest that variations in serum AMH levels are not only caused by differences in follicle number but also by genetic factors. Therefore, the genetic context should be taken into consideration when assessing serum AMH levels in women. This may have clinical consequences when serum AMH levels are used as a marker for the polycystic ovarian morphology phenotype. STUDY FUNDING/COMPETING INTEREST(S): No external funding was used. J.S.E.L. has received consultancy fees from the following companies: Ferring, Roche Diagnostics and Ansh Labs and has received travel reimbursement from Ferring. J.A.V. has received royalties from AMH assays, paid to the institute/lab with no personal financial gain. The other authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Parent-of-origin effects on glucose homeostasis in polycystic ovary syndrome.

CONTEXT: Polycystic ovary syndrome (PCOS) is a highly heritable complex trait. Parents of affected women have reproductive and metabolic phenotypes. OBJECTIVE: We tested the hypothesis that there are parental effects on the heritability of fasting dysglycemia in women with PCOS. DESIGN AND SETTING: This was a cross-sectional study at an academic medical center. PARTICIPANTS: PARTICIPANTS included 367 women with PCOS and their parents (1101 total subjects). MAIN OUTCOME MEASURES: We compared maternal and paternal contributions to heritability of fasting dysglycemia and to transmission of the PCOS susceptibility allele of D19S884 within the fibrillin-3 gene (D19S884-A8) on fasting dysglycemia. RESULTS: Fathers had higher fasting glucose levels, prevalence of fasting dysglycemia and proinsulin to insulin molar ratios (P < .0001), a marker of defective insulin processing, compared with mothers. Heritability of fasting dysglycemia was significant in PCOS families (h(2) = 37%, SE = 10%, P = .001). Maternal heritability (h(2) = 51%, SE = 15%, P = .0009) was higher than paternal heritability (h(2) = 23 %, SE = 23%, P = .186) of fasting dysglycemia after adjustment for age and body mass index. Within dysglycemic probands, there was increased maternal compared with paternal transmission of D19S884-A8 (maternal 84% vs paternal 45%, χ(2) = 6.51, P = .011). CONCLUSIONS: There was a sex difference in the parental metabolic phenotype with fathers having an increased risk of fasting dysglycemia and evidence for pancreatic ß-cell dysfunction compared with mothers. However, only maternal heritability had significant effects on the prevalence of fasting dysglycemia in women with PCOS. Furthermore, there were maternal parent-of-origin effects on transmission of D19S884-A8 probands with fasting dysglycemia. These findings suggest that maternal factors, genetic and perhaps epigenetic, contribute to the metabolic phenotype in affected women.