Underlying this study is the hypothesis that different combinations of CYP1B1 mutations may produce a range of structural alterations in the tissues of the conventional aqueous outflow pathway, including the trabecular meshwork and Schlemm’s canal. A corollary to this idea is that the degree of angle dysgenesis is congruous with the severity of the disease process in congenital glaucoma. The significant percentage of cases with genetic mutations and phenotypic abnormalities identified in this cohort perhaps compensates for the limited number of patients in this study. Detecting CYP1B1 mutations in two thirds of the cohort supports the concept that CYP1B1 mutations are responsible for the development of glaucoma in many children with congenital glaucoma. In addition, the goniodysgenesis in each of the six cases was readily apparent histologically and could account for the obstruction of aqueous outflow and the development of the glaucomatous process.
The phenotypes could be separated into three distinct categories based on histological features. Furthermore, the degree of dysgenesis observed histologically closely correlated with disease severity and the difficulty in achieving IOP control. In the type I category, with the absence of Schlemm’s canal, both patients presented at birth with congenital glaucoma. In addition, multiple surgeries and topical glaucoma medications were necessary to achieve and maintain IOP control. The distention of the uveoscleral outflow pathway in both cases with this phenotype might be expected, as this is the only route for aqueous outflow in patients lacking Schlemm’s canal. In the type II phenotype, in which a connective-tissue band is present within either the juxtacanalicular tissues or the outer trabecular meshwork, patients presented with congenital glaucoma between 6 and 8 months of age. IOP control was achieved with medications alone or a single operation in five of six (83.3%) of the eyes of the three patients in this group, and several years later four (66.7%) of six eyes are no longer using any topical medications. In the type III category, a single patient had a relatively normal outflow pathway with the exception of a mucopolysaccharide-type substance deposited in the juxtacanalicular tissue. The original trabeculectomy flap remained tightly sutured, and both eyes were treated for 2 years with topical medications alone. For the last several years, the patient has maintained normal IOP in both eyes despite being taken off all glaucoma medications. This patient provides a histological explanation for a previously described group of congenital glaucoma patients who experience spontaneous resolution of the glaucomatous process, potentially secondary to similar minor anomalies.23,24
The histological phenotypes identified here are consistent with those previously reported, though never systematically categorized. In trabeculectomy specimens from three patients, Anderson19
observed a “broad layer of collagen and amorphous material in the juxtacanalicular connective tissue,” consistent with the type II phenotype described here. Similarly, the majority of specimens from eight patients examined by Tawara and Inomata25
also had a deposition of fibrillar material containing bundles of collagen fibers within the outer trabecular meshwork. In their study, Schlemm’s canal was absent in one patient who developed early-onset glaucoma bilaterally, characteristic of type I cases. In addition, the electron micrograph of another case in this study of late-onset congenital glaucoma demonstrated a “homogeneous ground substance” in the outer meshwork, which closely resembles the type III phenotype.25
Of note, Barkan’s membrane was not present in any of the six cases described here, similar to the studies of both Anderson and Tawara. However, we have had the opportunity to correlate the gonioscopic appearance with the histological phenotype in patients with Barkan’s membrane (J.A.A., unpublished observations).
This study has demonstrated that multiple CYP1B1
mutations can be found in a heterogeneous group of patients with primary congenital glaucoma. The majority of the detected CYP1B1
mutations impacted highly conserved regions of the gene, and CYP1B1
mutations were identified in both of the cases of severe goniodysgenesis (type I) and two of the three cases of moderate dysgenesis (type II). The R390H mutation detected in case 1 lies within the k-helix core and makes up part of the Glu-X-X-Arg (387–390) motif, a highly conserved sequence among all members of the cytochrome P-450 family.11
Similarly, the frameshift mutation (4340 delG) in case 2 results in a premature stop codon upstream from the critical heme-binding region.26
The novel mutations identified in cases 1 and 2 were located in exon II and may possibly lead to changes in three-dimensional folding based on the different chemical properties of the substituted amino acids. The G61E mutation and R368H mutations in case 3 are also both within highly conserved regions, affecting the hinge region11,27
respectively. The E229K mutation found in patient 5 is located in exon II and, on the basis of prior reports, may also disrupt the three-dimensional helical structure.28,29
Several interesting observations emerged from the genetic analysis of this cohort. Incomplete penetrance was observed in the family of case 2 as each of the three children was compound heterozygous for both mutations (ie, each had identical CYP1B1 mutations), yet only two of the three siblings were affected (). This incomplete penetrance, previously observed in Saudi Arabian and Indian populations, suggests the existence of a modifier locus, which may be an autosomal dominant gene separately inherited.12,16
The screening in this study was limited to the coding regions of the gene (exons II and III), and mutations of the promoter region may not have been identified. Also of note, the presumed second mutant allele in case 5, based on the autosomal recessive mode of inheritance, was not detected and may be located in either the promoter region of the CYP1B1
gene or in a separate gene, as suggested in other reports.26,29,30
Pedigree of case 2. Corresponding DNA sequencing demonstrating the same two CYP1B1 mutations present in each of three offspring, only two of which developed congenital glaucoma.
The major weakness of this study is the small number of patients in this cohort, which makes direct genotype to phenotype correlations somewhat tenuous. Nevertheless, the observed phenotypic angle anomalies corresponding to particular CYP1B1
mutations were highly predictive of disease severity. Furthermore, the clinical course of some patients in this cohort corresponded to that described in prior studies of patients with the same mutations. For example, 11 (91.7%) of 12 patients previously reported with the 4340delG frameshift mutation (case 2) presented in the first month of life, each with severe, recalcitrant glaucoma.26
Additional studies examining different combinations of CYP1B1
mutations are ultimately necessary to provide an even better prediction of phenotype.
The cases in the series reported here demonstrate an anatomic basis for the elevation of IOP, indicating that in congenital glaucoma, it is the obstruction of the outflow pathway that leads to a pressure elevation and a secondary optic neuropathy. Similar obstruction of the outflow pathway has yet to be demonstrated for primary open-angle glaucoma. For this and other reasons, the prevailing notion among glaucoma specialists is that primary open-angle glaucoma represents a primary optic neuropathy in which IOP elevation serves as a major risk factor.31,32
An understanding of phenotype may provide a guide to glaucoma management in the future. For example, type I congenital glaucoma patients lacking Schlemm’s canal may require a filtration procedure at the outset of management. In contrast, in type II cases with a connective tissue band, a goniotomy knife may successfully disrupt this barrier and improve aqueous outflow. From a clinical point of view, the glaucoma specialist may be best able to determine a surgical intervention if the status of Schlemm’s canal is known. Currently, high-resolution ultrasound devices and spectral ocular coherence tomography devices are being tested to determine if they will allow one to image the lumen of Schlemm’s canal. Should these imaging devices prove to be both sensitive and specific, the demonstration of the presence or absence of Schlemm’s canal could be critical to surgical planning. In the future, genetic analysis may provide the glaucoma specialist with a useful tool to aid in the diagnosis of congenital glaucoma, the prediction of clinical course, and the development of tailored treatments.