|Molecular Vision 2006;
Received 22 February 2006 | Accepted 20 April 2006 | Published 20 April 2006
Primary role of CYP1B1 in Indian juvenile-onset POAG patients
Moulinath Acharya,1 Suddhasil Mookherjee,1 Ashima
Bhattacharjee,1 Arun Kumar Bandyopadhyay,2 Sanjay Kumar
Daulat Thakur,2 Gautam Bhaduri,2 Abhijit Sen,3
1Human Genetics & Genomics Division, Indian Institute of Chemical Biology, Kolkata, India; 2Regional Institute of Ophthalmology, Medical College, Kolkata, India; 3Dristi Pradip, Jodhpur Park, Kolkata, India
Correspondence to: Dr. Kunal Ray, Human Genetics & Genomics Division, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata - 700 032, India; Phone: 91-33-2473-3491 or 0492 or 6793; FAX: 91-33-2473-5197 or 91-33-2472-3967; email: firstname.lastname@example.org or email@example.com
Purpose: CYP1B1, a member of the cytochrome P450 superfamily of enzymes, has been implicated in primary congenital glaucoma (PCG). Recent studies suggest a role of CYP1B1 in primary open-angle glaucoma (POAG) as a modifier locus. The purpose of the study was to further investigate the potential role of CYP1B1 in POAG patients.
Methods: Two hundred unrelated Indian POAG patients and 100 unrelated ethnically matched controls were enrolled in this study. The coding sequence of CYP1B1 was amplified by polymerase chain reaction (PCR) from genomic DNA, followed by direct DNA sequencing to identify the allelic variants.
Results: Six mutations were identified in nine patients and none of the controls examined. One novel mutation (R523T) was detected in the homozygous condition while three reported (W57C, E229K, and R368H) and two novel mutations (S515L and D530G) were found in the heterozygous state. The homozygous mutation of a conserved residue, detected in a familial juvenile onset POAG (JOAG) patient (lacking MYOC or OPTN mutations), cosegregated with the disease locus in an autosomal recessive mode of transmission. All the novel mutations (R523T, S515L and D530G) were detected in a region of CYP1B1 that did not harbor any of the 34 point mutations implicated in PCG. In addition, six previously reported (p.R48G, p.A119S, p.V432L, p.D449D, p.N453S, and 372-12C>T in intron 1) and four novel (p.V395V, p.P400P, p.V518A, and c.2016C>G in the 3'-UTR) single nucleotide polymorphism (SNPs) were also observed in POAG patients and controls.
Conclusions: Our observation suggests that on rare occasions CYP1B1 may be primarily responsible for JOAG by possible monogenic association, and this observation emphasizes the importance of screening for mutation in this gene of JOAG patients that are determined not to harbor mutations in previously characterized candidate genes and loci for POAG.
Glaucoma is part of a heterogeneous group of neurodegenerative disorders of the eye affecting about 67 million people worldwide . It is the second largest blinding disorder after cataract . Untreated glaucoma is a leading cause of irreversible blindness. In glaucoma, vision loss is caused by damage to the optic nerve. Different forms of glaucoma share some common clinical manifestations that usually include specific abnormal appearance of the optic nerve head, characteristic loss of visual fields, and chronic painless progression.
Primary open angle glaucoma (POAG; OMIM 137760) is the most common form of the disease. Vision is lost progressively, often silently, and can lead to bilateral blindness. The onset of the disease is not obvious to the patient until there is appreciable and irreversible loss of the field of vision. POAG mainly affects older people and the incidence increases with age, but it may occur in middle age or younger. So far, three different genes, Myocilin (MYOC), Optineurin (OPTN), and WDR36 [3-5] have been implicated in POAG. On the other hand, primary congenital glaucoma (PCG; OMIM 231300) or infantile glaucoma, a rare genetic disorder that usually manifests itself at birth or within the first year of life but may emerge up to the age of three [6-8], is caused by CYP1B1 (a member of the cytochrome P450 superfamily of enzymes) when the disease is transmitted in a family in an autosomal recessive mode . Later, CYP1B1 was demonstrated to be a modifier locus for POAG that, together with MYOC mutations, expedite the disease progression from adult onset to a juvenile form in a digenic mode of inheritance . However, the underlying molecular mechanism involving these two genes in glaucoma pathogenesis is not yet understood. CYP1B1 mutations have been identified with a heterozygous genotype in both familial and sporadic POAG patients. It has been reported that in familial cases, the disease does not cosegregate with all the individuals in the family harboring the CYP1B1 mutations. Based on these observations, it has been suggested that CYP1B1 mutations pose a significant risk for JOAG and might also modify glaucoma phenotype in patients who do not carry a MYOC mutation .
Selection of study subjects
Indian patients affected with POAG with or without a positive family history were recruited in this study from the glaucoma departments in the Regional Institute of Ophthalmology and Drishti Pradip, Kolkata. Diagnosis was based on clinical ocular and systemic examinations. Ocular examinations involved measurement of intraocular pressure by applanation tonometry (Goldmann). Gonioscopy by Goldmann 3-mirror gonioscope (Shaffer's grading), which revealed the angles of the anterior chamber, was also used for optic disc evaluation and fundoscopy. Optic disc was further evaluated with +78D lens and visual field was assessed by Humphrey's automated perimeter.
An increased intraocular pressure above 21 mm Hg, significant cupping of optic disc with or without peripapillary changes, and presence of an open angle of the anterior chamber raised the suspicion of POAG, which was confirmed by typical reproducible visual field changes in automated perimetry test. Also included were individuals with an IOP less than 21 mm Hg and who had cupping of the optic disc and visual field changes characteristic of POAG. Individuals with any history of inflammation or ocular trauma (past and present) were excluded from this study.
Controls selected from the general population had no personal or family history of ocular disease. They were determined to be negative for POAG based on a routine eye examination for glaucoma including direct ophthalmoscopy, thorough examination of the optic disc, intraocular tension, gonioscopy, automated visual field analysis, and retinal nerve fiber layer (RNFL) analysis with the help of scanning laser polarimetry (SLP) with variable corneal compensation.
Collection of blood samples and genomic DNA preparation
A 10 ml peripheral blood sample was collected alone with informed consent from each POAG patient and controls. EDTA was used as an anticoagulant. The internal review committee on research using human subject cleared the project, and the research was conducted adhering to the tenets of the Declaration of Helsinki.
Genomic DNA was prepared from fresh whole blood using the conventional phenol-chloroform method, followed by ethanol precipitation. The DNA was then dissolved in TE (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) .
Polymerase chain reaction
Polymerase chain reaction (PCR) was carried out in a total volume of 25.0 μl containing 50-100 ng genomic DNA, 0.4 μM of each primer, 0.2 mM of each dNTP, an appropriate concentration of MgCl2, and 0.5 unit of Taq polymerase (Invitrogen, Carlsbad, CA) in a thermocycler (GeneAmp-9700, PE Applied Biosystems, Foster City, CA). Exon 2, with adjoining splice junctions was amplified using a primer pair PCG 17F (5'-TCT CCA GAG AGT CAG CTC CG-3') and PCG 18R (5'-CTA CTC CGC CTT TTT CAG A-3'). Coding regions of exon 3 with adjoining splice junction and 3' UTR was amplified by PCG15F (5'-GTC ACT GAG CTA GAT AGC CT-3') and PCG20R (5'-GGA CAG TTG ATT TAT GCT CAC C-3').
The PCR products were column-purified using a Qiagen PCR-purification kit (Qiagen, Hilden, Germany), and bidirectional sequencing was performed in an ABI 3130XL capillary DNA sequencer using the dye-termination chemistry. Nucleotide sequences were analyzed using pairwise BLAST  to examine if there were any changes from the normal sequence available in the database.
We screened CYP1B1 in 200 unrelated POAG patients comprising 156 sporadic and 44 familial cases by direct sequencing of the coding exons and splice junctions. Among these patients, 34 had JOAG (<30 years of age) and 166 had adult-onset POAG. The mean age of diagnosis of the patients was 52.43±19.33 years with a range varying between 7-84 years.
Altogether, nine patients (4.5%) carried a mutation in CYP1B1 (Table 1), similar to a French report (4.6%) . Six mutations were identified in CYP1B1, half of which were previously reported (W57C, E229K, and R368H) in PCG and the rest (S515L, R523T, and D530G) were novel mutations (Table 1). In addition, six SNPs, one in intron 1 and others in the coding sequence previously reported (Table 2), were detected in POAG patients and controls. Consistent with a previous report on Indian patients, we observed that R48G and A119S are in complete linkage disequilibrium . Four novel SNPs were found including one in the 3' UTR region in the heterozygous state (Table 2).
Except for one novel mutation (R523T) discussed later, all other mutations (three previously reported and two novel changes) were found in the heterozygous state. Among these mutations, one (S515L) was detected in four sporadic patients (Table 3). Haplotype analysis was not possible in these patients for lack of DNA samples from the family members, but variable heterozygosity of intragenic SNP markers suggested that the chromosomal region harboring the mutation was not common among these patients. All of the novel missense changes identified only in patients are likely to be pathogenic because these alleles are absent in 100 ethnically matched controls selected from the general population without any personal or family history of ocular disease. The mean age of the controls was 53.86±8.92 years, varying between 40-80 years. In addition, the SIFT (Sorting Intolerant From Tolerant) homology tool, using 24 related sequences from plants and animals from a BLAST search of GenBank, was applied to predict whether a substitution was likely to have a phenotypic effect. SIFT is a software that predicts the potential of a substituted amino acid to be deleterious in a protein sequence. A score less than 0.05 is the benchmark of deleterious change . Although S515L, the most frequent novel mutation, shows an insignificant SIFT score (Table 1), its complete absence in 100 controls strongly suggests it to be a putative disease-causing alteration. All other mutations encountered in this group of POAG patients were previously observed in PCG patients, in India or elsewhere. The E229K and R368H mutations were previously associated with familial PCG in India  while W57C was associated with a familial Hispanic case . In the present study, the R368H mutation was found in a sporadic POAG patient while E229K and W57C were found to be associated with familial POAG cases (Table 3).
While we identified a few patients harboring CYP1B1 mutations in the heterozygous condition, we also observed a proband (25 years; III-2 in Figure 1) in a Muslim family who was affected with juvenile onset POAG (JOAG; OMIM 137750) at 18 years of age and homozygous for a novel mutation (c.1940G>C; R523T) in CYP1B1. The mutation was not detected in 100 ethnically matched controls, described previously. Following screening of MYOC and OPTN in this patient, no mutation was detected in either of the two genes. To check whether the patient represented a case of delayed presentation of PCG, we thoroughly examined the proband and found he did not have any of the characteristic features for PCG, such as buphthlamos, megalocornea, deep anterior chamber, etc. His corneas were transparent and normal in size and shape, and there were no "Haab's striae" present in either cornea. The proband had dimness of vision with visual acuity 2/60 in right eye and 1/60 in left eye (aided), intraocular pressure (IOP) of 16 mm Hg in both eyes controlled by 0.5% Timolol eye drops. He had undergone bilateral trabeculectomy. Gonioscopy revealed open angle (grade IV) glaucoma. The visual field study showed gross glaucomatous changes in the right eye but could not be done in the left eye due to severe visual impairment. Fundus oculi showed advanced glaucomatous cupping with thin neuroretinal rim and optic nerve atrophy.
Three additional members of the family (II-4, II-5, and III-1 in Figure 1) are blind and have been diagnosed with absolute glaucoma with total corneal opacity and bullous keratopathy. Their age of onset ranged from 17-19 years. All three patients are also homozygotes for the CYP1B1 mutation (c.1940G>C; Arg523Thr). Further, neither of the two heterozygotes in the family were observed to have glaucoma or other eye diseases. Therefore, the mutation completely cosegregated with JOAG within the family and showed an autosomal recessive mode of transmission. The arginine residue at codon 523 of CYP1B1 is conserved across many species (e.g., mouse, rat, striped dolphin, and zebrafish). In addition, the mutation was determined to have significantly lower SIFT value (0.00), suggesting threonine as a highly intolerant residue at codon 523. It was also interesting to note that the mutated residue is located in a region of CYP1B1 that does not harbor any of 34 missense and nonsense mutations that have been implicated in PCG.
To our knowledge, this is the first report that mutations in CYP1B1 may be primarily responsible for juvenile onset POAG by possible monogenic association. Thus it appears that CYP1B1 has a larger role to play in glaucoma pathogenesis that includes causation of PCG, modifying the pathogenesis of POAG, and, on rare occasion, being the primary cause of JOAG. A wider spectrum of clinical phenotypes, that would be distinguished as related but defined as different diseases, have been demonstrated earlier involving the RDS/peripherin gene. Historically, macular dystrophy and a form of autosomal dominant retinitis pigmentosa were independently mapped to chromosome 6p21.1-cen, finally revealing that in both cases RDS was the causal gene . The molecular bases for wide spectrum of clinical manifestation in glaucoma pathogenesis by mutation in CYP1B1 is not well understood and needs to be explored. Our observation provides evidence for a primary role of CYP1B1 in juvenile onset POAG and emphasizes the importance of screening for mutations in the gene of JOAG patients that are determined not to harbor mutations in already characterized candidate genes and loci for the disease.
The authors are thankful to the patients and their family members who participated in this study. Dr. Devdeep Aikath and Ms. Mahua Maulik deserve special thanks for their help in collection of blood samples from the donors and excellent technical assistance. The Council of Scientific and Industrial Research, Government of India provided the financial support (NMITLI and CMM 0016 grants) for the study as well as predoctoral fellowship to MA, SM, and AB.
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