title: Identification of novel functional sequence variants in the gene for peptidase inhibitor 3.
abstract: BACKGROUND
  Peptidase inhibitor 3 (PI3) inhibits neutrophil elastase and proteinase-3, and has a potential role in skin and lung diseases as well as in cancer. Genome-wide expression profiling of chorioamniotic membranes revealed decreased expression of PI3 in women with preterm premature rupture of membranes. To elucidate the molecular mechanisms contributing to the decreased expression in amniotic membranes, the PI3 gene was searched for sequence variations and the functional significance of the identified promoter variants was studied.
  
  
  METHODS
  Single nucleotide polymorphisms (SNPs) were identified by direct sequencing of PCR products spanning a region from 1,173 bp upstream to 1,266 bp downstream of the translation start site. Fourteen SNPs were genotyped from 112 and nine SNPs from 24 unrelated individuals. Putative transcription factor binding sites as detected by in silico search were verified by electrophoretic mobility shift assay (EMSA) using nuclear extract from Hela and amnion cell nuclear extract. Deviation from Hardy-Weinberg equilibrium (HWE) was tested by chi2 goodness-of-fit test. Haplotypes were estimated using expectation maximization (EM) algorithm.
  
  
  RESULTS
  Twenty-three sequence variations were identified by direct sequencing of polymerase chain reaction (PCR) products covering 2,439 nt of the PI3 gene (-1,173 nt of promoter sequences and all three exons). Analysis of 112 unrelated individuals showed that 20 variants had minor allele frequencies (MAF) ranging from 0.02 to 0.46 representing "true polymorphisms", while three had MAF < or = 0.01. Eleven variants were in the promoter region; several putative transcription factor binding sites were found at these sites by database searches. Differential binding of transcription factors was demonstrated at two polymorphic sites by electrophoretic mobility shift assays, both in amniotic and HeLa cell nuclear extracts. Differential binding of the transcription factor GATA1 at -689C>G site was confirmed by a supershift.
  
  
  CONCLUSIONS
  The promoter sequences of PI3 have a high degree of variability. Functional promoter variants provide a possible mechanism for explaining the differences in PI3 mRNA expression levels in the chorioamniotic membranes, and are also likely to be useful in elucidating the role of PI3 in other diseases.
authors.affiliation: Center of Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI, USA. mchowdh@med.wayne.edu
authors.forename: Mahboob A
authors.initials: MA
authors.lastname: Chowdhury
authors.forename: Helena
authors.initials: H
authors.lastname: Kuivaniemi
authors.forename: Roberto
authors.initials: R
authors.lastname: Romero
authors.forename: Samuel
authors.initials: S
authors.lastname: Edwin
authors.forename: Tinnakorn
authors.initials: T
authors.lastname: Chaiworapongsa
authors.forename: Gerard
authors.initials: G
authors.lastname: Tromp
mesh_terms: D001665:Binding Sites; D053492:Elafin; D005838:Genotype; D006239:Haplotypes; D006367:HeLa Cells; D006801:Humans; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D053492:Elafin; C506806:PI3 protein, human; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D014157:Transcription Factors