title: Functional evaluation of genetic variants associated with endometriosis near GREB1.
abstract: OBJECTIVE
  Do DNA variants in the growth regulation by estrogen in breast cancer 1 (GREB1) region regulate endometrial GREB1 expression and increase the risk of developing endometriosis in women?
  
  
  CONCLUSIONS
  We identified new single nucleotide polymorphisms (SNPs) with strong association with endometriosis at the GREB1 locus although we did not detect altered GREB1 expression in endometriosis patients with defined genotypes.
  
  
  BACKGROUND
  Genome-wide association studies have identified the GREB1 region on chromosome 2p25.1 for increasing endometriosis risk. The differential expression of GREB1 has also been reported by others in association with endometriosis disease phenotype.
  
  
  METHODS
  Fine mapping studies comprehensively evaluated SNPs within the GREB1 region in a large-scale data set (>2500 cases and >4000 controls). Publicly available bioinformatics tools were employed to functionally annotate SNPs showing the strongest association signal with endometriosis risk. Endometrial GREB1 mRNA and protein expression was studied with respect to phases of the menstrual cycle (n = 2-45 per cycle stage) and expression quantitative trait loci (eQTL) analysis for significant SNPs were undertaken for GREB1 [mRNA (n = 94) and protein (n = 44) in endometrium].
  
  
  METHODS
  Participants in this study are females who provided blood and/or endometrial tissue samples in a hospital setting. The key SNPs were genotyped using Sequenom MassARRAY. The functional roles and regulatory annotations for identified SNPs are predicted by various publicly available bioinformatics tools. Endometrial GREB1 expression work employed qRT-PCR, western blotting and immunohistochemistry studies.
  
  
  RESULTS
  Fine mapping results identified a number of SNPs showing stronger association (0.004 < P < 0.032) with endometriosis risk than the original GWAS SNP (rs13394619) (P = 0.034). Some of these SNPs were predicted to have functional roles, for example, interaction with transcription factor motifs. The haplotype (a combination of alleles) formed by the risk alleles from two common SNPs showed significant association (P = 0.026) with endometriosis and epistasis analysis showed no evidence for interaction between the two SNPs, suggesting an additive effect of SNPs on endometriosis risk. In normal human endometrium, GREB1 protein expression was altered depending on the cycle stage (significantly different in late proliferative versus late secretory, P < 0.05) and cell type (glandular epithelium, not stromal cells). However, GREB1 expression in endometriosis cases versus controls and eQTL analyses did not reveal any significant changes.
  
  
  CONCLUSIONS
  In silico prediction tools are generally based on cell lines different to our tissue and disease of interest. Functional annotations drawn from these analyses should be considered with this limitation in mind. We identified cell-specific and hormone-specific changes in GREB1 protein expression. The lack of a significant difference observed following our GREB1 expression studies may be the result of moderate power on mixed cell populations in the endometrial tissue samples.
  
  
  CONCLUSIONS
  This study further implicates the GREB1 region on chromosome 2p25.1 and the GREB1 gene with involvement in endometriosis risk. More detailed functional studies are required to determine the role of the novel GREB1 transcripts in endometriosis pathophysiology.
  
  
  BACKGROUND
  Funding for this work was provided by NHMRC Project Grants APP1012245, APP1026033, APP1049472 and APP1046880. There are no competing interests.
authors: Molecular Epidemiology, QIMR Berghofer Medical Research Institute, Brisbane QLD 4029, Australia jenny.fung@qimrberghofer.edu.au.
authors: Jenny N
authors: JN
authors: Fung
authors: Department of Obstetrics and Gynaecology, Gynaecology Research Centre, University of Melbourne, Royal Women's Hospital, Parkville VIC 3052, Australia.
authors: Sarah J
authors: SJ
authors: Holdsworth-Carson
authors: Molecular Epidemiology, QIMR Berghofer Medical Research Institute, Brisbane QLD 4029, Australia.
authors: Yadav
authors: Y
authors: Sapkota
authors: Molecular Epidemiology, QIMR Berghofer Medical Research Institute, Brisbane QLD 4029, Australia.
authors: Zhen Zhen
authors: ZZ
authors: Zhao
authors: Molecular Epidemiology, QIMR Berghofer Medical Research Institute, Brisbane QLD 4029, Australia.
authors: Lincoln
authors: L
authors: Jones
authors: Department of Obstetrics and Gynaecology, Gynaecology Research Centre, University of Melbourne, Royal Women's Hospital, Parkville VIC 3052, Australia.
authors: Jane E
authors: JE
authors: Girling
authors: Department of Obstetrics and Gynaecology, Gynaecology Research Centre, University of Melbourne, Royal Women's Hospital, Parkville VIC 3052, Australia.
authors: Premila
authors: P
authors: Paiva
authors: Department of Obstetrics and Gynaecology, Gynaecology Research Centre, University of Melbourne, Royal Women's Hospital, Parkville VIC 3052, Australia.
authors: Martin
authors: M
authors: Healey
authors: Molecular Epidemiology, QIMR Berghofer Medical Research Institute, Brisbane QLD 4029, Australia.
authors: Dale R
authors: DR
authors: Nyholt
authors: Department of Obstetrics and Gynaecology, Gynaecology Research Centre, University of Melbourne, Royal Women's Hospital, Parkville VIC 3052, Australia.
authors: Peter A W
authors: PA
authors: Rogers
authors: Molecular Epidemiology, QIMR Berghofer Medical Research Institute, Brisbane QLD 4029, Australia.
authors: Grant W
authors: GW
authors: Montgomery
mesh_terms: D002874:Chromosome Mapping; D019295:Computational Biology; D004715:Endometriosis / Q000235:genetics*; D004717:Endometrium / Q000378:metabolism / Q000473:pathology; D005260:Female; D005786:Gene Expression Regulation; D014644:Genetic Variation; D055106:Genome-Wide Association Study; D005838:Genotype; D006801:Humans; D007150:Immunohistochemistry; D009363:Neoplasm Proteins / Q000235:genetics*; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide; D040641:Quantitative Trait Loci
chemical_list: C416830:GREB1 protein, human; D009363:Neoplasm Proteins
keywords: GREB1; GWAS; eQTLs; endometriosis