title: Mutational screening of CYP1B1 in Turkish PCG families and functional analyses of newly detected mutations.
abstract: OBJECTIVE
  To investigate the genetic basis of primary congenital glaucoma (PCG) in a collection of Turkish patients and to assess the pathogenicity of two novel alleles.
  
  
  METHODS
  Intragenic single nucleotide polymorphisms (SNPs) genotyping and mutational screening of CYP1B1, the major PCG causing gene, were performed by PCR amplification and sequencing. PCG cases with either none or a single heterozygous mutation in CYP1B1 were further screened for mutations in myocilin (MYOC), claimed to be a minor contributor to the PCG disease through a digenic mode of inheritance. The subcellular localization and enzymatic activity of the two novel mutant proteins were assessed by immunofluorescent confocal techniques, and by an easy, user-friendly method that we have adapted from toxicity tests that use modified-luciferin substrates.
  
  
  RESULTS
  CYP1B1 mutations were found in 15 out of 35 PCG patients either in the homozygous or heterozygous state. Two novel (p.R117W and p.G329V), as well as six previously reported mutations were identified. No mutation in the MYOC gene was found in any of the PCG cases analyzed. The two new mutant proteins showed considerably reduced enzyme activity as well as a diminished localization in the mitochondria, probably due to a slower traffic rate through the ER compared to the wild-type form.
  
  
  CONCLUSIONS
  The present work provides a mutation and intragenic SNP haplotype profile of the CYP1B1 gene in Turkish PCG families and suggests a modest contribution at best of the MYOC gene to PCG in Turkey. In addition, it describes two new missense mutations and and reports a simple enzymatic assay to assess the pathogenicity of novel variants.
authors: Departament de Genética, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain. sefaet@hacettepe.edu.tr
authors: Sefayet
authors: S
authors: Bagiyeva
authors: Gemma
authors: G
authors: Marfany
authors: Olga
authors: O
authors: Gonzalez-Angulo
authors: Roser
authors: R
authors: Gonzalez-Duarte
mesh_terms: D000483:Alleles; D000818:Animals; D001120:Arginine; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D065633:Cytochrome P-450 CYP1B1; D003598:Cytoskeletal Proteins / Q000235:genetics; D004252:DNA Mutational Analysis; D005136:Eye Proteins / Q000235:genetics; D005838:Genotype; D005901:Glaucoma / Q000151:congenital / Q000201:enzymology* / Q000235:genetics*; D005998:Glycine; D006023:Glycoproteins / Q000235:genetics; D006239:Haplotypes; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D009154:Mutation; D020125:Mutation, Missense; D010375:Pedigree; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution; D014364:Tryptophan; D014421:Turkey; D014633:Valine; D000099179:Myocilin
chemical_list: D001120:Arginine; D001189:Aryl Hydrocarbon Hydroxylases; D065633:Cytochrome P-450 CYP1B1; D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D005998:Glycine; D006023:Glycoproteins; D014364:Tryptophan; D014633:Valine; D000099179:Myocilin; C585496:CYP1B1 protein, human