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Purpose: The aim of this work was to assess the possible effects of CYP1B1 mutations on the extracellular matrix (ECM) in glaucoma patients. CYP1B1 mutations are the cause of disease in a notable fraction of primary congenital glaucoma (PCG) patients and in a smaller fraction of primary open angle glaucoma (POAG) patients., Methods: The study was performed on a glaucoma family with the common homozygous p.Gly61Glu CYP1B1 mutation. The father was affected with POAG and three siblings had PCG. Microscopy was performed on the skin of the father and one son, as well as controls. Immunohistochemical studies were done using anti-CYP1B1 and anti-fibrillin-1 antibodies. Fibrillin-1 served as a marker for the ECM, and electron microscopy was also performed., Results: CYP1B1 expression patterns were the same in the patients and controls. However, microfibrils that are associated with fibrillin-1 were less abundant and more fragmented in both patients. Electron microscopy showed disturbed collagen fibers only in the PCG patient., Conclusions: The p.Gly61Glu mutation in CYP1B1 affects the ECM structure. This implies that the ECM of the trabecular meshwork may also be disrupted in a manner that affects aqueous humor flow resulting in increased intraocular pressure and contributing to the glaucoma phenotype., (© 2016 S. Karger AG, Basel.)

Purpose: To assess for the first time the possible contribution of latent transforming growth factor (TGF)-beta binding protein 2 (LTBP2), an extracellular matrix (ECM) protein that associates with fibrillin-1-containing microfibrils, to the etiology of primary open angle glaucoma (POAG) and pseudoexfoliation (PEX) syndrome. Mutations in LTBP2 have previously been shown to be the cause of primary congenital glaucoma (PCG) and other disorders that often manifest as secondary glaucoma., Methods: All exons of LTBP2 were sequenced in the DNA of 42 unrelated patients with POAG and 48 unrelated patients with PEX syndrome. Contribution of candidate variations to disease was assessed by screening in control individuals and use of biochemical, bioinformatics, and evolutionary criteria, and in one case by segregation analysis within the family of a proband with POAG. Microscopy was performed on the skin of a patient with PEX syndrome whose condition developed into PEX glaucoma during the course of the study and on the skin of her son previously identified with PCG who harbored the same LTBP2 mutation., Results: Among the 30 sequence variations observed in LTBP2, five found in five patients with POAG and two found in two patients with PEX glaucoma syndrome may contribute to their diseases. One of the mutations was observed in a patient with POAG and in a patient with PEX glaucoma syndrome. Light, fluorescent, and electron microscopy showed that a mutation present in one of the individuals affected with PEX glaucoma syndrome and in her son affected with PCG causes disruptions in the ECM., Conclusions: Some LTBP2 sequence variations can contribute to the etiology of POAG and PEX glaucoma syndrome. It is not expected that in these diseases LTBP2 mutations behave in a strictly Mendelian fashion with complete penetrance. In conjunction with recent findings, the results suggest that anomalies in the ECM are among the factors that can contribute to POAG and PEX glaucoma syndrome. LTBP2 and other related ECM protein coding genes should be screened in larger cohorts with these diseases, which are common disorders and important to the public health.

Objective: To investigate penetrance and expressivity of CYP1B1 genotypes associated with primary congenital glaucoma (PCG)., Design: Observational case series, systematic review, and comparative analysis of the literature., Participants: Forty probands affected with PCG, 16 siblings affected with PCG, and 103 siblings and 75 parents of the probands reported not to be affected by history. The participants were members of 40 unrelated families., Methods: Mutations were screened by restriction fragment length polymorphism, allele-specific polymerase chain reaction amplification, and direct sequencing. Ophthalmologic examination included slit-lamp biomicroscopy, intraocular pressure (IOP) measurement, gonioscopy, and high magnification stereoscopic fundus examination, followed by standard achromatic perimetry., Main Outcome Measures: Identification of subjects carrying CYP1B1 mutations. Glaucoma diagnosis based on slit-lamp examination, IOP measurement, gonioscopic findings, optic nerve appearance, and perimetry., Results: Fifteen different homozygous or compound heterozygous mutant CYP1B1 genotypes were identified. Most probands and previously diagnosed subjects harbored G61E, R368H, R390H, and R469W mutations. Among the 178 apparently unaffected family members, 20 subjects from 12 families were observed to harbor 2 CYP1B1 mutations, suggesting an average penetrance of 73% for all the mutations. These 20 subjects ranged in age from 14 to 54 years. R390H appeared to have a notably high penetrance. Penetrance was 50% in the subset of families with incomplete penetrance. Ophthalmologic examination on 14 of the 20 apparently nonpenetrant individuals showed that 8 subjects were affected with juvenile open-angle glaucoma (JOAG) or primary open-angle glaucoma (POAG), and that 3 subjects were glaucoma suspect. One of the individuals with a JOAG diagnosis was the identical twin sibling of a proband affected with PCG., Conclusions: At least 57% of the PCG nonpenetrant individuals examined clinically were affected with JOAG or POAG to varying degrees, and overall penetrance of "affected CYP1B1 genotypes" with respect to glaucoma may be more than 90%. These findings suggest that "affected CYP1B1 genotypes" exhibit variable expressivity rather than nonpenetrance. The clinical implication of this observation is that seemingly unaffected relatives of patients with PCG, particularly those known to harbor CYP1B1 mutations, should undergo regular ophthalmologic examination to allow early diagnosis.

Purpose: To investigate the role of MYOC and CYP1B1 in Iranian juvenile open angle glaucoma (JOAG) patients., Methods: Twenty-three JOAG probands, their available affected and unaffected family members, and 100 ethnically matched control individuals without history of ocular disease were recruited. Clinical examinations of the probands included slit lamp biomicroscopy, intraocular pressure (IOP) measurement, gonioscopic evaluation, fundus examination, and perimetry measurement. Familial cases were classified according to the mode of inheritance. Exons of MYOC and CYP1B1 were sequenced, and novel variations assessed in the control individuals. Potential disease-associated variations were tested for segregation with disease status in available family members., Results: The mode of inheritance of the disease in the families of four probands (17.4%) appeared to be autosomal dominant and in at least eight (34.8%) to be autosomal recessive. Four patients carried MYOC mutations, and an equal number carried CYP1B1 mutations. The MYOC mutations were heterozygous; two of them (p.C8X and p.L334P) are novel, and one codes for the shortest truncated protein so far reported. Autosomal recessive inheritance was consistent with inheritance observed in families of patients carrying CYP1B1 mutations. All these patients carried homozygous mutations., Conclusions: MYOC and CYP1B1 contributed equally to the disease status of the Iranian JOAG patients studied. The contribution of the two genes appeared to be independent in that no patient carried mutations in both genes. The fraction of Iranian patients carrying MYOC mutations was comparable to previously reported populations.

Purpose: The gene coding cytochrome P4501B1 (CYP1B1) has been shown to be a major cause of primary congenital glaucoma in the Iranian population. More recently it was shown to also be important in juvenile-onset open angle glaucoma (JOAG). We aimed to further investigate the role of CYP1B1 in a larger cohort of primary open angle glaucoma (POAG) patients which included late-onset patients. We also aimed to set up a microarray based protocol for mutation screening with an intent of using the protocol in a future population level screening program., Methods: Sixty three POAG patients, nine affected family members, and thirty three previously genotyped primary congenital glaucoma (PCG) patients were included in the study. Clinical examination included slit lamp biomicroscopy, IOP measurement, gonioscopic evaluation, fundus examination, and measurement of perimetry. G61E, R368H, R390H, and R469W were screened by a protocol that included multiplexed allele specific amplification in the presence of a protease (PrASE), use of sequence tagged primers, and hybridization to generic arrays on microarray slides. The entire coding sequences of CYP1B1 and myocilin (MYOC) genes were sequenced in all individuals assessed by the microarray assay to carry a mutation. Intragenic single nucleotide polymorphism (SNP) haplotpes were determined for mutated alleles., Results: Genotypes assessed by the array-based PrASE methodology were in 100% concordance with sequencing results. Seven mutation carrying POAG patients (11.1%) were identified, and their distribution was quite skewed between the juvenile-onset individuals (5/21) as compared to late-onset cases (2/42). Four of the seven mutation carrying Iranian patients harbored two mutated alleles. CYP1B1 mutated alleles in Iranian PCG and POAG patients shared common haplotypes. MYOC mutations were not observed in any of the patients., Conclusions: The PrASE approach allowed reliable simultaneous genotyping of many individuals. It can be an appropriate tool for screening common mutations in large sample sizes. The results suggest that CYP1B1 is implicated in POAG among Iranians, notably in the juvenile-onset form. Contrary to POAG patients studied in other populations, many mutation harboring Iranian patients carry two mutated alleles. We propose an explanation for this observation.