Disease-causing mutations in proteins: structural analysis of the CYP1B1 mutations causing primary congenital glaucoma in humans.

In this communication, we report an in-depth structure-based analysis of the human CYP1b1 protein carrying disease-causing mutations that are discovered in patients suffering from primary congenital glaucoma (PCG). The "wild-type" and the PCG mutant structures of the human CYP1b1 protein obtained from comparative modeling were subjected to long molecular dynamics simulations with an intention of studying the possible impact of these mutations on the protein structure and hence its function. Analysis of time evolution as well as time averaged values of various structural properties--especially of those of the functionally important regions: the heme binding region, substrate binding region, and substrate access channel--gave some insights into the possible structural characteristics of the disease mutant and the wild-type forms of the protein. In a nutshell, compared to the wild-type the core regions in the mutant structures are associated with subtle but significant changes, and the functionally important regions seem to adopt such structures that are not conducive for the wild-type-like functionality.

Correlations of genotype with phenotype in Indian patients with primary congenital glaucoma.

PURPOSE: To establish the genotype-phenotype correlations of various CYP1B1 (human cytochrome P450) mutations in patients in India with primary congenital glaucoma (PCG). METHODS: The study cohort comprised 146 patients with PCG from 138 pedigrees. Patients were analyzed for six distinct CYP1B1 mutations by sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. A severity index for grading various PCG phenotypes was constructed based on clinical parameters. RESULTS: Six mutations were identified in 45 patients analyzed and genotype-phenotype correlations were established for 43 of them. The percentages of severe phenotypes associated with various mutations in at least one eye were: frameshift, 100%; G61E, 66.7%; P193L, 62.5%; E229K, 80%; R368H, 72%; R390C, 83.3%. The frameshift mutation resulted in blindness. Based on the severity index, the disease severity was graded from normal to severe and the prognosis from good to very poor (blind). De novo mutation was identified in one family. CONCLUSIONS: This is the first study to attempt to devise a severity index for grading various PCG phenotypes and to use genotype as an indicator to predict the prognoses of the disorder. This index may help guide therapy and counseling of the afflicted family regarding the progression of the disorder. All patients with severe phenotypes showed poor prognoses (r = 0.976; P < 0.0001). The data derived from this study could be used as an added clinical tool in disease management. Integrated management of PCG that makes use of a genetic approach could yield better results than medical, surgical, and rehabilitation interventions alone.

Identification of R368H as a predominant CYP1B1 allele causing primary congenital glaucoma in Indian patients.

PURPOSE: To investigate the predominant mutation in the CYP1B1 gene in patients in India with primary congenital glaucoma (PCG), using PCR-restriction fragment length polymorphism (RFLP) methods and to characterize the molecular defect in two generations of an affected family. METHODS: DNA samples from 146 patients with PCG from 138 pedigrees were analyzed for several distinct mutations in CYP1B1 by PCR-RFLP. RESULTS: PCR-RFLP screening revealed that 30.8% of patients were positive for any one of the six mutations (376insA, 528G-->A, 923C-->T, 959G-->A, 1449G-->A, and 1514C-->A), and 17.8% of the patients were found to have the rarely reported mutation R368H (1449G-->A). All mutations were confirmed by DNA sequencing. CONCLUSIONS: The results suggest extensive allelic heterogeneity in the Indian patients with PCG, with the predominant allele being R368H among the 146 Indian patients tested. It appears possible to use this approach for carrier detection in pedigrees with a positive family history and in population screening. The approach also offers a method for rapid screening of potential carriers and affected individuals.

Congenital glaucoma associated with 22p+ variant in a dysmorphic child.

A case of congenital glaucoma with developmental delay and several dysmorphic features showing 22p+ chromosomal variant is reported.

Novel mutation in FOXC1 wing region causing Axenfeld-Rieger anomaly.

PURPOSE: To determine the possible molecular genetic defect underlying Axenfeld-Rieger anomaly (ARA) and to identify the pathogenic mutation causing this anterior segment dysgenesis in an Indian pedigree. METHODS: The FOXC1 gene was amplified from genomic DNA of members of an ARA-affected family and control subjects using four novel sets of primers. The amplicons were directly sequenced, and the sequences were analyzed to identify the disease-causing mutation. RESULTS: A heterozygous novel missense mutation was identified in the coding region of the FOXC1 gene in all three patients in this family. Consistent with the autosomal dominant inheritance pattern, the mutation segregated with the disease phenotype and was fully penetrant. The mutation was found in the wing region of the highly conserved forkhead domain of the FOXC1 gene and resulted in a very severe phenotype leading to blindness. CONCLUSIONS: This is the first study to demonstrate that a mutation in the FOXC1 wing region can cause an anterior segment dysgenesis of the eye. This mutation resulted in blindness in the ARA-affected family, and the findings suggest that the FOXC1 wing region has a functional role in the normal development of the eye. Moreover, this is the first study from India to report the genetic etiology of Axenfeld-Rieger anomaly. Genotype-phenotype correlations of FOXC1 may help in establishing the disease prognosis and also in understanding the clinical and genetic heterogeneity associated with various anterior segment dysgenesis caused by this gene.

Identification of novel mutations causing familial primary congenital glaucoma in Indian pedigrees.

PURPOSE: To determine the possible molecular genetic defect underlying primary congenital glaucoma (PCG) in India and to identify the pathogenic mutations causing this childhood blindness. METHODS: Twenty-two members of five clinically well-characterized consanguineous families were studied. The primary candidate gene CYP1B1 was amplified from genomic DNA, sequenced, and analyzed in control subjects and patients to identify the disease-causing mutations. RESULTS: Five distinct mutations were identified in the coding region of CYP1B1 in eight patients of five PCG-affected families, of which three mutations are novel. These include a novel homozygous frameshift, compound heterozygous missense, and other known mutations. One family showed pseudodominance, whereas others were autosomal recessive with full penetrance. In contrast to all known CYP1B1 mutations, the newly identified frameshift is of special significance, because all functional motifs are missing. This, therefore, represents a rare example of a natural functional CYP1B1 knockout, resulting in a null allele (both patients are blind). CONCLUSIONS: The molecular mechanism leading to the development of PCG is unknown. Because CYP1B1 knockout mice did not show a glaucoma phenotype, the functional knockout identified in this study has important implications in elucidating the pathogenesis of PCG. Further understanding of how this molecular defect leads to PCG could influence the development of specific therapies. This is the first study to describe the molecular basis of PCG from the Indian subcontinent and has profound and multiple clinical implications in diagnosis, genetic counseling, genotype-phenotype correlations and prognosis. Hence, it is a step forward in preventing this devastating childhood blindness.