Endometrial vezatin and its association with endometriosis risk.

STUDY QUESTION: Do endometriosis risk-associated single nucleotide polymorphisms (SNPs) found at the 12q22 locus have effects on vezatin ( ITALIC! VEZT) expression? SUMMARY ANSWER: The original genome-wide association study (GWAS) SNP (rs10859871), and other newly identified association signals, demonstrate strong evidence for ITALIC! cis-expression quantitative trait loci (eQTL) effects on ITALIC! VEZT expression. WHAT IS KNOWN ALREADY: GWAS have identified several disease-risk loci (SNPs) associated with endometriosis. The SNP rs10859871 is located within the ITALIC! VEZT gene. ITALIC! VEZT expression is altered in the endometrium of endometriosis patients and is an excellent candidate for having a causal role in endometriosis. Most of the SNPs identified from GWAS are not located within the coding region of the genome. However, they are likely to have an effect on the regulation of gene expression. Genetic variants that affect levels of gene expression are called expression quantitative trait loci (eQTL). STUDY DESIGN, SIZE, DURATION: Samples for genotyping and ITALIC! VEZT variant screening were drawn from women recruited for genetic studies in Australia/New Zealand and women undergoing surgery in a tertiary care centre. Coding variants for ITALIC! VEZT were screened in blood from 100 unrelated individuals (endometriosis-dense families) from the QIMR Berghofer Medical Research Institute dataset. SNPs at the 12q22 locus were imputed and reanalysed for their association with endometriosis. Reanalysis of endometriosis risk-association was performed on a final combined Australian dataset of 2594 cases and 4496 controls. Gene expression was performed on 136 endometrial samples. eQTL analysis in whole blood was performed on 862 individuals from the Brisbane Systems Genetics Study. Endometrial tissue-specific eQTL analysis was performed on 122 samples (eutopic endometrium) collected following laparoscopic surgery. VEZT protein expression studies employed ITALIC! n = 56 (western blotting) and ITALIC! n = 42 (immunohistochemistry) endometrial samples. PARTICIPANTS/MATERIALS, SETTING, METHODS: The women recruited for this study provided blood and/or endometrial tissue samples in a hospital setting. Genomic DNA was screened for common and coding variants. SNPs of interest in the 12q22 region were genotyped using Agena MassARRAY technology or Taqman SNP genotyping assay. Gene expression profiles from RNA extracted from blood and endometrial tissue samples were generated using Illumina whole-genome expression chips (Human HT-12 v4.0). Whole protein extracted from endometrium was used for VEZT western blots, and paraffin sections of endometrium were employed for VEZT immunohistochemistry semi-quantitative analysis. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 11 coding variants of ITALIC! VEZT (including one novel variant) were identified from an endometriosis-dense cohort. Polymorphic coding and imputed SNPs were combined with previous GWAS data to reanalyse the endometriosis risk association of the 12q22 region. The disease association signal at 12q22 was due to coding variants in ITALIC! VEZT or ITALIC! FGD6 (FYVE, RhoGEF and PH domain-containing 6) and SNPs with the strongest signals were either intronic or intergenic. We found strong evidence for ITALIC! VEZT cis-eQTLs with the sentinel SNP (rs10859871) in blood and endometrium, where the endometriosis risk allele (C) was associated with an increase in ITALIC! VEZT expression. We could not demonstrate this genotype-specific effect on VEZT protein expression in endometrium. However, we did observe a menstrual cycle stage specific increase in VEZT protein expression in endometrial glands, specific to the secretory phase ( ITALIC! P = 2.0 × 10(-4)). LIMITATIONS, REASONS FOR CAUTION: In comparison to the blood sample datasets, the study numbers of endometrial tissues were substantially reduced. Protein studies failed to complement RNA results, also likely a reflection of the low study numbers in these experiments. ITALIC! In silico prediction tools used in this investigation are typically based on cell lines different to our tissues of interest, thus any functional annotations drawn from these approaches should be considered carefully. Therefore, functional studies on VEZT and related pathway components are still warranted to unequivocally implicate a causal role for VEZT in endometriosis pathophysiology. WIDER IMPLICATIONS OF THE FINDINGS: GWAS have proven to be very valuable tools for deciphering complex diseases. Endometriosis is a text-book example of a complex disease, involving genetic, lifestyle and environmental influences. Our focused investigation of the 12q22 region validates an association with increased endometriosis risk. Endometriosis risk SNPs (including rs10859871) located within this locus demonstrated evidence for ITALIC! cis-eQTLs on ITALIC! VEZT expression. By examining women who possess an enhanced genetic risk of developing endometriosis, we have identified an effect on ITALIC! VEZT expression and therefore a potential gene/gene pathway in endometriosis disease establishment and development. STUDY FUNDING/COMPETING INTERESTS: Funding for this work was provided by NHMRC Project Grants GNT1012245, GNT1026033, GNT1049472 and GNT1046880. G.W.M. is supported by the NHMRC Fellowship scheme (GNT1078399). S.J.H.-C. is supported by the J.N. Peters Bequest Fellowship. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

No evidence for genetic association with the let-7 microRNA-binding site or other common KRAS variants in risk of endometriosis.

STUDY QUESTION: Is there a contribution of the minor allele at the KRAS single nucleotide polymorphism (SNP) rs61764370 in the let-7 microRNA-binding site to endometriosis risk? SUMMARY ANSWER: We found no evidence for association between endometriosis risk and rs61764370 or any other SNPs in KRAS. WHAT IS KNOWN ALREADY: The rs61764370 SNP in the 3' untranslated region of the KRAS gene is predicted to disrupt a complementary binding site (LCS6) for the let-7 microRNA, and was recently reported to be at a high frequency (31%) in 132 women of varying ancestry with endometriosis compared with frequencies in a database of population controls (up to 7.6% depending on ancestry), suggesting a strong effect of this KRAS SNP in the aetiology of endometriosis. STUDY DESIGN, SIZE AND DURATION: This was a case-control study with a total of 11 206 subjects. The study was performed between February 2012 and July 2012. PARTICIPANTS/MATERIALS, SETTINGAND METHODS: We first investigated a possible association between common markers in KRAS and endometriosis risk from our genome-wide association (GWA) data in 3194 surgically confirmed endometriosis cases and 7060 controls of European ancestry. Although rs61764370 was not genotyped on the GWA arrays, five SNPs typed in the study were highly correlated with this variant. The rs61764370 and two SNPs highly correlated with rs61764370 were then genotyped in 933 endometriosis cases and 952 controls using the Sequenom MassARRAY platform. MAIN RESULTS AND THE ROLE OF CHANCE: There was no evidence for an association between rs61764370 and endometriosis risk P = 0.411 and odds ratio = 1.10 (95% confidence intervals: 0.88-1.36). We also found no evidence for an association between the highly correlated SNP rs17387019 and endometriosis. Their minor allele frequencies in cases and controls were of 0.087-0.091 similar to the population frequency reported previously for this variant in controls. Analyses of endometriosis cases with revised American Fertility Society stage III/IV disease also showed no evidence for an association between these SNPs and endometriosis risk. LIMITATIONS AND REASONS FOR CAUTION: The GWA and genotyped data sets were not independent since individuals and cases from some families overlap. Controls in our GWA study were not screened for endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: The key SNP, rs61764370, was genotyped in a subset of samples. Our results do not support the suggestion that carrying the minor allele at rs61764370 contributes to a significant number of endometriosis cases and rs61764370 is, therefore, unlikely to be a useful marker of endometriosis risk. STUDY FUNDING/COMPETING INTEREST(S): The research was funded by grants from the Australian National Health and Medical Research Council and Wellcome Trust. None of the authors has competing interests for the study.

Variation in BMPR1B, TGFRB1 and BMPR2 and control of dizygotic twinning.

Genes in the TGF9 signaling pathway play important roles in the regulation of ovarian follicle growth and ovulation rate. Mutations in three genes in this pathway, growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and the bone morphogenetic protein receptor B 1 (BMPRB1), influence dizygotic (DZ) twinning rates in sheep. To date, only variants in GDF9 and BMP15, but not their receptors transforming growth factor ß receptor 1 (TGFBR1), bone morphogenetic protein receptor 2 (BMPR2) and BMPR1B, have been investigated with respect to their roles in human DZ twinning. We screened for rare and novel variants in TGFBR1, BMPR2 and BMPR1B in mothers of dizygotic twins (MODZT) from twin-dense families, and assessed association between genotyped and imputed variants and DZ twinning in another large sample of MODZT. Three novel variants were found: a deep intronic variant in BMPR2, and one intronic and one non-synonymous exonic variant in BMPRB1 which would result in the replacement of glutamine by glutamic acid at amino acid position 294 (p.Gln294Glu). None of these variants were predicted to have major impacts on gene function. However, the p.Gln294Glu variant changes the same amino acid as a sheep BMPR1B functional variant and may have functional consequences. Six BMPR1B variants were marginally associated with DZ twinning in the larger case-control sample, but these were no longer significant once multiple testing was taken into account. Our results suggest that variation in the TGF9 signaling pathway type II receptors has limited effects on DZ twinning rates in humans.