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-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.

Myocilin mt1 promoter polymorphism in Turkish patients with primary open angle glaucoma.

PURPOSE: To evaluate the association of the myocilin gene promoter variant -1000C>G (MYOC.mt1) with primary open angle glaucoma (POAG) and its possible role on the phenotype and the severity of glaucoma in Turkish patients. METHODS: Eighty eight POAG patients and 123 healthy subjects were included in the study. All subjects were genotyped by PCR-RFLP. Allele and genotype frequencies between healthy subjects and glaucoma patients were compared by the chi2 test. The age at diagnosis, the age at inclusion, the maximum IOP at diagnosis and the number of antiglaucomatous medications were compared between MYOC.mt1 carriers and non-carriers using the Student's t-test; C/D ratio, mean deviation (MD), and pattern standard deviation values were compared with the Mann-Whitney U-test. Statistical significance was defined as p<0.05. RESULTS: MYOC.mt1 genotype and allele frequencies did not differ in POAG and healthy subjects (p=0.204 and p=0.083, respectively). In the control group, 17.1% of the subjects were MYOC.mt1 carriers, while 27.3% of the POAG patients were MYOC.mt1 carriers (p=0.107). The odds ratio for CG was 1.859 (95% CI: 0.9-3.7; p=0.084) and for GG 1.594 (95% CI: 0.31-8.13; p=0.575). The phenotype variables were quite similar in MYOC.mt1 carriers and non-carriers. Gender by itself or with the MYOC.mt1 did not have any effect on IOP, C/D ratio, or MD values (univariate analysis of variance, p>0.05). No significant difference was found in the distribution of genotypes between different stages of glaucoma groups (p=0.93). CONCLUSIONS: Our results suggest that in our Turkish glaucoma patients, MYOC.mt1 is not a risk factor for the development of POAG and is not associated with the phenotype and severity of glaucoma.