title: Integrative DNA methylation and gene expression analysis identifies discoidin domain receptor 1 association with idiopathic nonobstructive azoospermia.
abstract: OBJECTIVE
  To evaluate the association between promoter DNA methylation and discoidin domain receptor 1 (DDR1) gene expression in men with nonobstructive azoospermia (NOA).
  
  
  METHODS
  Comparing fibroblasts cultured from testicular biopsies using a high resolution Infinium 450K methylation array.
  
  
  METHODS
  Basic research laboratory.
  
  
  METHODS
  Men with NOA (n = 16) and with normal spermatogenesis (n = 5).
  
  
  METHODS
  None.
  
  
  METHODS
  Bisulfite clonal sequencing for validation and quantification of CpG methylation of DDR1; gene expression analysis of DDR1 with quantitative polymerase chain reaction and immunohistochemistry to validate the array results at mRNA and protein levels.
  
  
  RESULTS
  We validated promoter methylation, mRNA and protein levels of the CpG sites identified from array results. Differentially methylated CpG sites (∼20K) were identified using an F-test in the NOA samples. We identified 20 genes with >30% difference in DNA methylation within the promoter region of men with NOA and fertile controls. Of the aberrantly methylated genes, 10 were hypomethylated and 10 were hypermethylated genes. From the top 10 hypermethylated genes, six genes (MRI1, DCAF12L1, TMEM95, CECR2, DDR1, and NPHS2) were selected for validation because they were shown to be expressed in the testis. Of the six genes expressed in the fibroblasts cultured from testis, DDR1 showed an abnormal gene expression pattern. Three patients (19%) out of the 16 men with NOA for whom gene expression data were available had lower DDR1 expression levels (1.8x fold decrease) than the fertile men, whereas four (25%) men had higher expression levels (2x fold increase) of DDR1 compared with the levels in fertile men. Quantitative analysis by bisulfite clonal sequencing showed that one of the CpG sites (cg13329862) of DDR1 promoter was hypermethylated in NOA patients compared with fertile controls (53% vs. 15%). Immunohistochemical analysis suggests presence of DDR1 within cytoplasm of germ cells and peritubular connective tissue (in men with hypospermatogenesis) and decreased expression of the protein in men with Sertoli-cell only syndrome.
  
  
  CONCLUSIONS
  Abnormal gene expression of DDR1 is associated with NOA. The functional relevance of aberrant methylation of DDR1 to expression of DDR1 in men with NOA warrants further investigation.
authors: Scott Department of Urology and the Center for Reproductive Medicine, Baylor College of Medicine, Houston, Texas.
authors: Ranjith
authors: R
authors: Ramasamy
authors: Scott Department of Urology and the Center for Reproductive Medicine, Baylor College of Medicine, Houston, Texas.
authors: Alex
authors: A
authors: Ridgeway
authors: Scott Department of Urology and the Center for Reproductive Medicine, Baylor College of Medicine, Houston, Texas.
authors: Larry I
authors: LI
authors: Lipshultz
authors: Scott Department of Urology and the Center for Reproductive Medicine, Baylor College of Medicine, Houston, Texas. Electronic address: dlamb@bcm.edu.
authors: Dolores J
authors: DJ
authors: Lamb
mesh_terms: D053713:Azoospermia / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D016022:Case-Control Studies; D002478:Cells, Cultured; D018899:CpG Islands; D019175:DNA Methylation; D000070819:Discoidin Domain Receptor 1; D044127:Epigenesis, Genetic; D005298:Fertility / Q000235:genetics; D005786:Gene Expression Regulation; D005819:Genetic Markers; D020022:Genetic Predisposition to Disease; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D010641:Phenotype; D011237:Predictive Value of Tests; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA
chemical_list: D005819:Genetic Markers; C092030:DDR1 protein, human; D000070819:Discoidin Domain Receptor 1; D020794:Receptor Protein-Tyrosine Kinases
keywords: Epigenetics; infertility; promoter