Heterozygous loss-of-function variants in CYP1B1 predispose to primary open-angle glaucoma.

PURPOSE: Although primary congenital glaucoma (PCG)-associated CYP1B1 mutations in the heterozygous state have been evaluated for association with primary open-angle glaucoma (POAG) in several small studies, their contribution to the occurrence of POAG is still controversial. The present study was conducted to determine whether heterozygous functionally characterized CYP1B1 mutations are associated with the disease in a large cohort of German patients with POAG. METHODS: The frequency of CYP1B1 variants on direct sequencing of the entire coding region was compared in 399 unrelated German patients with POAG (270, POAG; 47, JOAG; and 82, NTG) and 376 control subjects without any signs of glaucoma on ophthalmic examination. In vitro functional assays were performed and relative enzymatic activity of the CYP1B1 variants embedded in their respective background haplotypes and not previously unambiguously classified were determined, to assess their possible causative role. RESULTS: Apart from known polymorphic variants, 11 amino acid substitutions in CYP1B1 reported before, both in PCG and POAG cases, were identified. After in vitro functional assay, variants P52L and R368H showed marked reduction of activity, confirming their role as loss-of-function mutations similar to previously determined variants G61E, N203S, and G329V. In contrast, variants G168D, A443G, and A465V showed no relevant effects and were thus classified as polymorphisms. Overall, seven functionally impaired variants were present in 13 (3.6%) patients and in 1 (0.2%) control subject (P = 0.002, OR = 5.4). Reanalysis of previous studies reporting CYP1B1 mutations in patients with POAG based on updated functional validation showed a significant excess of carriers among patients compared to controls (OR = 3.85; P = 2.3 x 10(-7)). CONCLUSIONS: Heterozygous CYP1B1 mutations with absent or reduced relative enzymatic activity can be considered a risk factor for POAG.

Heterozygous NTF4 mutations impairing neurotrophin-4 signaling in patients with primary open-angle glaucoma.

Glaucoma, a main cause of blindness in the developed world, is characterized by progressive degeneration of retinal ganglion cells (RGCs), resulting in irreversible loss of vision. Although members of the neurotrophin gene family in various species are known to support the survival of numerous neuronal populations, including RGCs, it is less clear whether they are also required for survival and maintenance of adult neurons in humans. Here, we report seven different heterozygous mutations in the Neurotrophin-4 (NTF4) gene accounting for about 1.7% of primary open-angle glaucoma patients of European origin. Molecular modeling predicted a decreased affinity of neurotrophin 4 protein (NT-4) mutants with its specific tyrosine kinase receptor B (TrkB). Expression of recombinant NT-4 carrying the most frequent mutation was demonstrated to lead to decreased activation of TrkB. These findings suggest a pathway in the pathophysiology of glaucoma through loss of neurotrophic function and may eventually open the possibility of using ligands activating TrkB to prevent the progression of the disease.

Profiling of WDR36 missense variants in German patients with glaucoma.

PURPOSE: Mutations in WDR36 were recently reported in patients with adult-onset primary open-angle glaucoma (POAG). In this study, the prevalence of WDR36 variants was investigated in patients with glaucoma who were of German descent with diverse age of onset and intraocular pressure levels. METHODS: Recruited were 399 unrelated patients with glaucoma and 376 healthy subjects of comparable age and origin, who had had repeated normal findings in ophthalmic examinations. The frequency of observed variants was obtained by direct sequencing of the entire WDR36 coding region. RESULTS: A total of 44 WDR36 allelic variants were detected, including 14 nonsynonymous amino acid alterations, of which 7 are novel (P31T, Y97C, D126N, T403A, H411Y, H411L, and P487R) and 7 have been reported (L25P, D33E, A163V, H212P, A449T, D658G and I264V). Of these 14 variants, 6 were classified as polymorphisms as they were detected in patients and control individuals at similar frequencies. Eight variants present in 15 patients (3.7%) but only 1 control individual (0.2%) were defined as putative disease-causing variants (P = 0.0005). Within this patient group, 12 (80%) presented with high and 3 (20%) with low intraocular pressure. Disease severity and age of onset showed a broad range. CONCLUSIONS: The occurrence of several rare putative disease-causing variants in patients with glaucoma suggests that WDR36 may be a minor disease-causing gene in glaucoma, at least in the German population. The large variability in WDR36, though, requires functional validation of these variants, once its function is characterized.

Novel CYP1B1 and known PAX6 mutations in anterior segment dysgenesis (ASD).

PURPOSE: The study intended to define the underlying genetic defects for 21 index patients affected with different forms of anterior segment dysgenesis. Sequence analysis for the PAX6, PITX2, FOXC1, and CYP1B1 genes has been implemented for this purpose. METHODS: Ten patients affected with Peters anomaly, 8 with Rieger anomaly, and 3 with aniridia were included in this study. All patients underwent a complete eye examination, including anterior segment evaluation, with slit-lamp microsocopy, fundoscopy, tonography, and gonioscopy. Twenty-one intronic primer pairs were used to amplify the coding exons of the FOXC1, CYP1B1, PITX2, and PAX6 genes for sequence analysis on an automated sequencer (ABI 3730). RESULTS: We were able to detect mutations in 5 of 21 patients with anterior segment malformations. We found mutations in individuals suffering from Rieger anomaly and aniridia, in CYP1B1 and PAX6, respectively. None of the 10 Peters anomaly patients had causative mutations in any of the 4 genes we screened. CONCLUSIONS: Our results suggest primary congenital glaucoma and the anterior segment dysgenesis disorders may share a common molecular pathophysiology in the CYP1B1 pathway.

Primary congenital glaucoma and Rieger's anomaly: extended haplotypes reveal founder effects for eight distinct CYP1B1 mutations.

PURPOSE: Mutations in the cytochrome P450 1B1 (CYP1B1) gene are a frequent cause of primary congenital glaucoma (PCG) in different ethnic groups. Cytochrome P450 proteins are monooxygenases, which catalyze many reactions involved in the metabolism of drugs as well as steroids and other lipids. The repeated occurence of several mutations in various ethnic groups raises the question if founder effects or mutation-prone sites in CYP1B1 are the cause for this observation. METHODS: A total of 30 individuals (26 PCG patients, three Rieger's anomaly patients, and one variant carrier), presenting 17 variants in CYP1B1 (15 mutations and two variations) were included in our study. We sequenced the entire genomic region of CYP1B1 and analyzed microsatellites flanking the gene in all individuals and constructed haplotypes for all variations using a combination of single nucleotide polymorphisms and microsatellites. RESULTS: For the CYP1B1 genomic region, we identified five extended haplotypes associated with 17 variations. These haplotypes were complemented with microsatellite information from the region surrounding this gene. A total of eight CYP1B1 mutations were found more than once, each of them presenting one identical haplotype in different individuals. Six mutations were represented in different ethnic groups. CONCLUSIONS: Our results confirm founder effects for most of CYP1B1 mutations. Most of these mutations must have occurred as unique events in the past.

Primary congenital glaucoma: a novel single-nucleotide deletion and varying phenotypic expression for the 1,546-1,555dup mutation in the GLC3A (CYP1B1) gene in 2 families of different ethnic origin.

PURPOSE: To present new molecular genetic data on primary congenital glaucoma from 2 families, 1 isolated case and 3 familial cases due to mutations in the cytochrome P-450 1B1 (CYP1B1) gene. METHODS: All diagnoses were made by slit-lamp biomicroscopy, gonioscopy, cornea and optic disk measurements, ultrasound-biometry, and automated static threshold perimetry where possible. Mutation screening was performed by direct sequence analysis of DNA extracted from peripheral blood of the patients and their relatives. RESULTS: For the isolated case, a child of 4 years, a homozygous nucleotide deletion within a tetrad of cytosines (nt622-625, 622delC) was found leading to a predicted nonsense codon 93 truncating the protein by 450 amino acids. For the familial cases, the 3 affected members showed a homozygous mutation 1,546-1,555dupTCATGCCACC for which 9 healthy relatives proved to be heterozygous. The phenotypic expression of these 3 patients varied widely. CONCLUSION: Our results confirm the crucial role of CYP1B1 mutations for congenital glaucoma.

Novel mutations in the MYOC/GLC1A gene in a large group of glaucoma patients.

Mutations at the myocilin (MYOC) gene within the GLC1A locus have been revealed in 2-4% of patients suffering primary open angle glaucoma (POAG) worldwide. In our ongoing glaucoma study six hundred eighty two persons have been screened for MYOC mutations. The first group consisted of 453 patients from a long-term clinical study diagnosed either with juvenile OAG (JOAG), POAG, ocular hypertension (OHT) or normal tension glaucoma (NTG) plus 22 cases of secondary glaucoma. This group, and additional 83 healthy controls, is part of a long term study with repeated clinical examinations at the University of Erlangen-Nurnberg. An additional sample of 124 glaucoma patients or at risk persons referred from other sources were included in the mutation screening. Five novel mutations, namely Gly434Ser, Asn450Asp, Val251Ala, Ile345Met and Ser393Asn, could be identified as cause of preperimetric POAG, JOAG, normal tension POAG and POAG. Myocilin mutations were identified similar with previous reports with other ethnic populations at the rate of 11/341 (3.2%) probands.