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M.-P. Lopez-Garrido, E. Campos-Mollo, C. Blanco-Marchite, J. García-Feijoo, J. Peralta, J. Belmonte-Martínez, C. Ayuso, J. Escribano; Functional Analysis of Cyp1b1 Mutations in Spanish Patients With Primary Congenital Glaucoma. Invest. Ophthalmol. Vis. Sci. 2009;50(13):868.
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To analyze the contribution of CYP1B1 gene mutations to primary congenital glaucoma (PCG) in Spanish patients.
The promoter region (-1 to-867) and the 3 exons of CYP1B1 gene were analysed by PCR DNA sequencing. The CYP1B1 mutations were generated in vitro by site-directed mutagenesis using as template a cDNA encoding wild type CYP1B1 cloned into the mammalian expression vector pcDNA 3.1, and taged with the myc-epitope at the C-terminal end. To determine the enzymatic activity and protein stability of identified mutations, HEK-293T cells were transiently transfected with the corresponding cDNAs. The time course of the enzymatic activity was analyzed by determining the ethoxyresorufin O-deethylation (EROD) activityin transfected cells using a fluorimetric assay. Protein stability was studied by western blot of transfected cells exposed to cycloheximide for various time points. Recombinant CYP1B1 proteins were detected using an anti-myc antibody. Wild type CYP1B1 was used as a control in all the assays.
We found a total of 16 different mutations in 13 (34.2%) index cases. The identified mutations included 9 missense and 3 nonsense nucleotide changes, 3 small deletions and a short duplication. Eleven probands were compound heterozygotes and two were heterozygotes. Six of the identified mutations were novel. Mutations p.T404fsX30 and p.R355fsX69 were the most prevalent among index cases, and were detected in 6 (23.0%) and 3 (11.5%) patients, respectively. Functional analysis showed that 4 mutations were null-alleles while the remaining mutants were hypomorphic alleles.
Our data indicate that approximately one third of Spanish patients with PCG carry loss-of-function CYP1B1 and show that null alleles are associated with the most severe phenotypes. Hypomorphic alleles may contribute to some cases of incomplete penetrance.
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