Mutational Screening Of Cyp1B1 In Turkish Pcg Families And Functional Analyses Of Newly Detected Mutations

Purpose: 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-luciferine 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.Wo