Screening glaucoma genes in adult glaucoma suggests a multiallelic contribution of CYP1B1 to open-angle glaucoma phenotypes.

BACKGROUND: Despite increasing knowledge of the genetic pathophysiology of glaucoma, mutations in known genes account for less than 15% of disease. Gene screening predominantly remains a research tool rather than an essential part of the clinical work-up. We aimed to determine the mutational spectrum and frequency in the genes implicated in glaucoma, in a range of glaucoma and 'glaucoma suspect' (GS) participants, with a positive family history. METHODS: Observational large case series. One hundred fifteen patients recruited from public hospital and private clinics had diagnoses of GS, ocular hypertension, pseudoexfoliative glaucoma (PXG) or primary open-angle glaucoma (POAG), and at least one affected family member. In a university laboratory, DNA samples were screened for mutations in all coding exons of MYOC and CYP1B1, and OPTN (exons 4, 5 and 16). WDR36 (exons 1, 4, 5, 8, 11, 13 and 17) was screened in those with CYP1B1 changes. LOXL1 risk variants were screened in PXG pedigrees. Cascade screening of family members was undertaken. RESULTS: Seven out of one hundred fifteen (6.1%) individuals had at least one pathogenic or hypomorphic CYP1B1 allele associated with GS, POAG (5) and PXG phenotypes, including two novel sequence variations (p.Ser6Gly, p.Val243Leu). No pathogenic MYOC change was detected. Five individuals (4.3%) carried an OPTN sequence variation. Three of the seven with CYP1B1 changes had polygenic changes. CONCLUSIONS: Mutational analysis of known glaucoma genes in a mixed glaucoma population replicates the reported frequency of pathogenic CYP1B1 changes. Heterozygous CYP1B1 changes occurred at a greater frequency than other genes. Glaucoma pathogenesis in the clinic setting is genetically heterogeneous and may be polygenic.

TGFBI mutational analysis in a New Zealand population of inherited corneal dystrophy patients.

AIM: The corneal dystrophies represent a group of clinically and genetically heterogeneous, inherited diseases, often resulting in bilateral opacification of the cornea, and may require penetrating keratoplasty. Mutations in the transforming growth factor beta-induced (TGFBI) gene segregate with a wide range of phenotypically heterogeneous corneal dystrophies. Many of the other dystrophies remain without molecular characterisation. This study aimed to characterise the molecular basis for corneal disease in a New Zealand population. METHODS: Nineteen unrelated individuals affected with a corneal dystrophy (granular, fleck, lattice, posterior polymorphous) and their family members were recruited, a pedigree obtained and their dystrophy extensively phenotyped. After informed consent, samples were taken for DNA extraction. PCR and sequencing of all coding exons of TGFBI was undertaken. RESULTS: All five patients with granular dystrophy had the R555W mutation, and H626P was identified in an intermediate dystrophy of Bowman layer pedigree. No other mutations were detected including in the stromal dystrophy cases. CONCLUSION: Mutational analysis of TGFBI in a small population has identified sequence changes consistent with previously identified genotype-phenotype correlations. A new genotype-phenotype association was also characterised. No mutations were identified in some individuals/pedigrees suggesting greater genetic heterogeneity than is currently known in this group of disorders.

Exclusion of known corneal dystrophy genes in an autosomal dominant pedigree of a unique anterior membrane corneal dystrophy.

PURPOSE: With advances in phenotyping tools and availability of molecular characterization, an increasing number of phenotypically and genotypically diverse inherited corneal dystrophies are described. We aimed to determine the underlying causative genetic mechanism in a three-generation pedigree affected with a unique anterior membrane corneal dystrophy characterized by early onset recurrent corneal erosions, small discrete focal opacities at the level of Bowman layer and anterior stroma, anterior stromal flecks, and prominent corneal nerves. METHODS: Twenty affected and unaffected members of a three-generation family were examined and extensively clinically characterized including corneal topography and in vivo confocal microscopy, and biological specimens were collected for DNA extraction. Mutational analysis of two corneal genes (TGFBI [Transforming Growth factor-beta induced] and ZEB1 [zinc finger E box-binding homeobox 1]) was undertaken, in addition to testing with the Asper Corneal Dystrophy gene chip (Asper Ophthalmics, Tartu, Estonia). Subsequent Genotyping To 11 Known Corneal Gene Loci (COL8A2 [Collagen, Type VIII, Alpha-2], TACSTD2 [Tumor-Associated Calcium Signal Transducer 2], PIP5K3 [Phosphatidylinositol-3-Phosphate 5-Kinase, Type III], GSN [Gelsolin], KERA [Keratocan], VSX1 [Visual System Homeobox Gene 1], COL6A1 [Collagen, Type VI, Alpha-1], MMP9 [Matrix Metalloproteinase 9], KRT3 [Keratin 3]), and two putative loci, 3p14-q13 and 15q22.33-24) was undertaken using polymorphic markers, and haplotypes constructed. Multipoint linkage analysis was performed to generate LOD scores and produce LOD plots across the candidate intervals. RESULTS: No pathogenic sequence variations were detected in TGFBI or ZEB1 of the proband nor on the Asper Corneal Dystrophy gene chip (302 mutations in 12 genes). Multipoint linkage analysis of 11 known corneal genes and loci generated negative LOD plots and was able to exclude all genes tested including PIP5K3. CONCLUSIONS: Exclusion of linkage to candidate corneal loci combined with an absence of pathogenic mutations in known corneal genes in this pedigree suggest a different genetic causative mechanism in this dystrophy than the previously documented corneal genes. This unique phenotype of an anterior membrane dystrophy may therefore provide an opportunity to identify a new gene responsible for corneal disease.

A novel phenotype-genotype relationship with a TGFBI exon 14 mutation in a pedigree with a unique corneal dystrophy of Bowman's layer.

PURPOSE: Corneal dystrophy of Bowman's layer (CDB) belongs to a group of dystrophies associated with mutations in the transforming growth factor-beta-induced (TGFBI) gene. CDB is further divided into a geographic variant (CDB1/Reis Bücklers, RBCD), and a honeycomb variant (CDB2/Thiel Behnke, TBCD). We undertook mutational analysis of TGFBI in a family with an unusual CDB variant and describe a novel phenotype-genotype association. METHODS: Individuals from a pedigree with CDB underwent extensive phenotyping, including laser scanning in vivo confocal microscopy, and histological examination of four corneal buttons obtained at penetrating keratoplasty. Transmission electron microscopy of an excised allograft cornea from one affected individual was also performed. Following informed consent, DNA samples were collected. Polymerase chain reaction (PCR) and sequencing of all coding exons of TGFBI was performed. Family members were recruited with subsequent phenotyping and genotyping, and paternity testing. RESULTS: Clinical examination and other phenotypic information confirmed a diagnosis of CDB, with various features either more suggestive of CDB1 or of CDB2. A mutation in exon 14, H626P, segregated with the disease in this pedigree. This mutation was confirmed with NlaIII restriction enzyme digest, and was not seen in 100 control chromosomes. CONCLUSIONS: Within this pedigree, CDB segregates with an H626P mutation, which is previously described occurring in lattice corneal dystrophy. The majority of mutations in TGFBI previously described segregating with CDB1 and CDB2 are R124L and R555Q, respectively. Although a Bowman's layer dystrophy, the phenotype in this pedigree does not closely conform to the classical diagnostic criteria for either CDB1 or CDB2, and therefore represents a novel phenotype-genotype correlation.