title: Endometrial vezatin and its association with endometriosis risk.
abstract: OBJECTIVE
  Do endometriosis risk-associated single nucleotide polymorphisms (SNPs) found at the 12q22 locus have effects on vezatin ( ITALIC! VEZT) expression?
  
  
  CONCLUSIONS
  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.
  
  
  BACKGROUND
  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).
  
  
  METHODS
  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.
  
  
  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.
  
  
  RESULTS
  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)).
  
  
  CONCLUSIONS
  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.
  
  
  CONCLUSIONS
  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.
  
  
  BACKGROUND
  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.
  
  
  BACKGROUND
  N/A.
authors.affiliation: Department of Obstetrics and Gynaecology, University of Melbourne, Gynaecology Research Centre, Royal Women's Hospital, Level 7, 20 Flemington Road, Parkville, Victoria 3052, Australia.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Holdsworth-Carson
authors.affiliation: Molecular Epidemiology, QIMR Berghofer Medical Research Institute, Brisbane, Queensland 4029, Australia.
authors.forename: Jenny N
authors.initials: JN
authors.lastname: Fung
authors.affiliation: Molecular Epidemiology, QIMR Berghofer Medical Research Institute, Brisbane, Queensland 4029, Australia.
authors.forename: Hien T T
authors.initials: HT
authors.lastname: Luong
authors.affiliation: Molecular Epidemiology, QIMR Berghofer Medical Research Institute, Brisbane, Queensland 4029, Australia.
authors.forename: Yadav
authors.initials: Y
authors.lastname: Sapkota
authors.affiliation: Molecular Epidemiology, QIMR Berghofer Medical Research Institute, Brisbane, Queensland 4029, Australia.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Bowdler
authors.affiliation: Molecular Epidemiology, QIMR Berghofer Medical Research Institute, Brisbane, Queensland 4029, Australia.
authors.forename: Leanne
authors.initials: L
authors.lastname: Wallace
authors.affiliation: Department of Obstetrics and Gynaecology, University of Melbourne, Gynaecology Research Centre, Royal Women's Hospital, Level 7, 20 Flemington Road, Parkville, Victoria 3052, Australia.
authors.forename: Wan Tinn
authors.initials: WT
authors.lastname: Teh
authors.affiliation: The Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia Centre for Neurogenetics and Statistical Genomics, Queensland Brain Institute, University of Queensland, St Lucia, Queensland 4072, Australia.
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Powell
authors.affiliation: Department of Obstetrics and Gynaecology, University of Melbourne, Gynaecology Research Centre, Royal Women's Hospital, Level 7, 20 Flemington Road, Parkville, Victoria 3052, Australia.
authors.forename: Jane E
authors.initials: JE
authors.lastname: Girling
authors.affiliation: Department of Obstetrics and Gynaecology, University of Melbourne, Gynaecology Research Centre, Royal Women's Hospital, Level 7, 20 Flemington Road, Parkville, Victoria 3052, Australia.
authors.forename: Martin
authors.initials: M
authors.lastname: Healey
authors.affiliation: Molecular Epidemiology, QIMR Berghofer Medical Research Institute, Brisbane, Queensland 4029, Australia.
authors.forename: Grant W
authors.initials: GW
authors.lastname: Montgomery
authors.affiliation: Department of Obstetrics and Gynaecology, University of Melbourne, Gynaecology Research Centre, Royal Women's Hospital, Level 7, 20 Flemington Road, Parkville, Victoria 3052, Australia parogers@unimelb.edu.au.
authors.forename: Peter A W
authors.initials: PA
authors.lastname: Rogers
mesh_terms: D001315:Australia; D002352:Carrier Proteins / Q000235:genetics*; D015331:Cohort Studies; D004715:Endometriosis / Q000235:genetics* / Q000378:metabolism; D004717:Endometrium / Q000378:metabolism*; D005260:Female; D056726:Genetic Association Studies; D020022:Genetic Predisposition to Disease; D005838:Genotype; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D009520:New Zealand; D020641:Polymorphism, Single Nucleotide; D040641:Quantitative Trait Loci
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; C494547:VEZT protein, human
keywords: GWAS; endometriosis; expression quantitative trait loci; genetics; vezatin