|Molecular Vision 2004;
Received 27 July 2004 | Accepted 30 September 2004 | Published 30 September 2004
Mutation spectrum of the CYP1B1 gene in Indian primary congenital glaucoma patients
Aramati Bindu Madhava Reddy,1 Kiranpreet Kaur,1
Anil Kumar Mandal,2 Shirly George
Panicker,1 Ravi Thomas,2
Seyed Ehtesham Hasnain,3 Dorairajan
(The first two authors contributed equally to this publication)
1Kallam Anji Reddy Molecular Genetics Laboratory and 2Jasti V. Ramanamma Children's Eye Care Center, L.V. Prasad Eye Institute, Hyderabad, India; 3Center for DNA Fingerprinting and Diagnostics, Hyderabad, India
Correspondence to: Dr. Subhabrata Chakrabarti, Kallam Anji Reddy Molecular
Genetics Laboratory, L. V. Prasad Eye Institute, Road No. 2, Banjara
Hills, Hyderabad - 500034, India; Phone: +91-40-23543652; FAX:
+91-40-23548271; email: firstname.lastname@example.org
Dr. Reddy is now at the Department of Immunology, Childrens Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA.
Purpose: The human Cytochrome P450 gene CYP1B1 has been implicated in primary congenital glaucoma worldwide. The aim of this study was to understand the role of CYP1B1 mutations in causing primary congenital glaucoma in Indian populations.
Methods: The study included 64 new and unrelated cases of primary congenital glaucoma from different ethnic groups of India. Direct sequencing screened the coding and the promoter regions of CYP1B1.
Results: Sixteen pathogenic mutations were observed in 24 cases, of which 7 were novel. These included two frameshift mutations leading to deletions of 23 bp (g.3905del23bp) and 2 bp (g.7900-7901delCG) in exons II and III, respectively. Four novel missense mutations viz. A115P, M132R, Q144P, S239R were noted in exon II, and one in exon III (G466D), whose residue is a part of the "signature sequence" (NH2-FXXGXXXCXG-COOH) and is present in all heme binding cytochromes. Overall, CYP1B1 was involved in 37.50% (24/64) cases and homozygosity of the mutant allele was seen in 29.68% (19/64) and compound heterozygosity in 3.12% (2/64) of the cases, respectively. The frequency of CYP1B1 mutations was comparatively lower than Saudi Arabian, Slovakian Gypsys, and Turkish populations, largely due to genetic heterogeneity and ethnic diversities in Indian populations. Genotype-phenotype correlation indicated variable prognosis that could be due to the type of mutation, leading to alteration of CYP1B1 protein.
Conclusions: This study provides a mutation spectrum of CYP1B1 causing primary congenital glaucoma in Indian populations that has implications in devising molecular diagnostics for rapid screening.
Primary congenital glaucoma (PCG; OMIM 231300) is an inherited ocular congenital anomaly of the trabecular meshwork and anterior chamber angle that leads to the obstruction of aqueous outflow, increased intraocular pressure (IOP), and optic nerve damage resulting in childhood blindness. The disease manifests in the neonatal or early infantile period with symptoms of photophobia, epiphora, and signs of enlargement of the globe, edema, opacification of the cornea, breaks in the Descemet's membrane, and others . The prevalence of PCG varies across ethnic communities and geographical boundaries, ranging from 1 in 10,000-20,000 in the western populations  to 1 in 2,500 and 1 in 1,250 in the Saudi Arabian  and Gypsy populations of Slovakia , respectively. In the Indian state of Andhra Pradesh, prevalence is estimated to be around 1:3,300 and accounts for 4.2% of all childhood blindness . The mode of inheritance is largely autosomal recessive with variable penetrance but rare cases of pseudodominance are also seen in families with multiple consanguinity . Three chromosomal loci have been linked to PCG: GLC3A (2p21), GLC3B (1p36), and GLC3C (14q24.3) [5-7]. GLC3A, harboring the human cytochrome P450 gene CYP1B1 (OMIM 601771), has been characterized . More than 40 different mutations have been identified in CYP1B1 to be causal for PCG in different ethnic backgrounds and populations highlighting the allelic heterogeneity of the condition [8,9].
The involvement of CYP1B1 in other PCG populations varies from 20% in Indonesians and Japanese to 50% among the Brazilians and about 100% among the Saudi Arabians and Slovakian Gypsies [8,10-12]. Also it was shown that the Slovakian Gypsies and Saudi Arabian populations exhibited allelic homogeneity that was largely attributed to consanguinity and inbreeding [13,14]. Consanguinity and inbreeding are also prevalent in different ethnic groups of India  and thus provide an excellent opportunity to explore the role of CYP1B1 causing PCG in these populations.
Earlier we reported the involvement of CYP1B1 in 5 PCG families from India and devised PCR-based restriction fragment length polymorphism (RFLP) methods to detect these mutations . With this strategy we further screened 138 PCG cases and found that 30.8% of these cases were positive for one of the previously identified six mutations and that R368H happened to be the most prevalent mutation in the Indian population . In order to understand the spectrum of CYP1B1 mutations causing PCG in Indian populations, we screened CYP1B1 in 64 new and unrelated cases from different ethnic backgrounds.
Clinical examination and selection of cases
The study protocols adhered to the guidelines of the Declaration of Helsinki. After approval of the Institutional Review Board, 64 consecutively diagnosed PCG cases from different ethnic backgrounds, presenting at the L. V. Prasad Eye Institute from January 2001 to June 2003 were recruited. All patients underwent slit lamp biomicroscopy, applanation tonometry, and gonioscopy (where corneal clarity permitted). General anesthesia (Savoflorane) was used in the younger age groups and where required. The inclusion criteria were increased corneal diameter (>12.0 mm) along with raised intraocular pressure (>21 mm Hg) and/or presence of Haab's striae, or optic disc changes (where examination was possible). Symptoms of epiphora, photophobia, and rupture in the Descemet's membrane were the corroborating factors. The age of onset ranged from 0-1 years. Clinical diagnosis was confirmed independently by two glaucoma surgeons (AKM and RT) with expertise in congenital glaucoma. Seventeen cases had a positive family history of the disease, while the rest were sporadic. Parental consanguinity was seen in 55% of these cases. One hundred ethnically matched normal individuals without any ocular disorders served as controls. Peripheral blood samples were collected from the probands, their relatives, and controls by venipuncture with prior informed consent.
Screening of CYP1B1 gene
DNA was extracted from the leukocytes following standard protocols . The entire coding regions of CYP1B1 were amplified using three sets of pre-designed oligonucleotide primers with PCR conditions as described earlier . The 485 bp upstream region (2,748 bp to 3,233 bp) flanking four important regions for maximum promoter activity was screened using a set of forward (5'-AGC GGC CGG GGC AGG TTG TAC C-3') and reverse (5'-ATT GGG ATG GGG ACG GAG AA-3') primers. PCR was performed with 25 μl reaction mixtures containing 50 ng of genomic DNA, 1X PCR buffer (containing 100 mM Tris-HCl, 500 mM KCl and 0.8% Nonidet P40) with MgCl2, 200 mM of dNTPs, 0.5 μM of each primer, 10% DMSO, and 1 unit of Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania). The amplification conditions were an initial denaturation at 95 °C for 3 min followed by 30 cycles of denaturation, annealing and extensions at 95 °C (30 s), 62 °C (30 s), and 72 °C (45 s), respectively with a final extension at 72 °C for 7 min. The amplicons were purified using spin-columns (Sigma-Aldrich, St. Louis, MO) and subjected to bi-directional sequencing using BigDye terminator (version 3.1) chemistry according to the manufacturers protocol (Applied Biosystems, Foster city, CA). Sequencing was performed on an automated DNA sequencer (ABI 3100) and the data were analyzed with the SEQUENCING ANALYSIS software (Both from Applied Biosystems, Foster City, CA).
Identification of 7 novel mutations
Direct sequencing revealed 16 different mutations in 24 unrelated cases, of which 7 were novel. These included two frameshift mutations leading to deletions of 23 bp (g.3905del23bp, Figure 1) and 2 bp (g.7900-7901delCG, Figure 2) in the second and third exons, respectively. The deletions resulted in the creation of premature stop codons, thereby truncating the ORFs. The 23 bp deletion begins at nucleotide 3,905 and ends at 3,927 (reference sequence U56438) resulting in an inframe deletion of 8 amino acids i.e. the sequence his-val-gly-gln-arg-leu-leu-arg (or HVGQRLLR). Four novel missense mutations viz. g.4148G>C (A115P), g.4200T>G (M132R), g.4236A>C (Q144P), and g.4520A>C (S239R) were noted in the second exon, and one g.8234G>A (G466D) in the third exon (Figure 3). Except for S239R (that was present in 2 different cases), all other novel mutations were present in single cases only. All these mutations segregated with the affected phenotype and were absent in 200 normal chromosomes and their residues were mostly conserved across different species (Figure 4). Five of these mutations also resulted in the loss of restriction sites thereby facilitating their screening in the population (Table 1).
Other mutations in CYP1B1
Apart from these novel changes, 9 other mutations were noted in 16 PCG cases, which has already been reported in other ethnic groups [11,14,16,17,19-22]. These included a frameshift mutation leading to a 10 bp homozygous duplication g.8037-8046dupTCATGCCACC in PCG110 and 7 missense changes viz. L77P, R368H, R390H, R390C, P437L, E229K, and P193L (Table 1). The R368H was the most frequent mutant allele (7/25 cases) and also exhibited compound heterozygosity in association with R390C and G61E alleles in probands belonging to 2 non-consanguineous families. Three cases were heterozygous for the mutant alleles Q144P, R368H, and E229K. However no variation was observed in the non-coding regions or promoter of CYP1B1 in the heterozygous cases indicating the involvement of some other causal gene that is yet uncharacterized.
We also observed six benign intragenic variants at -13T/C, R48G, A119S, V432L, D449D, and N453S that were reported in other populations . But there was no common haplotype based on these single nucleotide polymorphisms that segregated with all these mutations, unlike in other populations [11,13,14].
Mutations in CYP1B1 have been associated with PCG with varying frequencies across different ethnic communities and geographical boundaries [8,10-12]. Populations with higher rate of inbreeding and consanguinity exhibit a higher frequency of CYP1B1 mutations in PCG as opposed to ethnically diverse populations [12,13]. Screening a cohort of 64 consecutive PCG cases from different geographical locales of India revealed 37.50% (24/64 cases) mutations in CYP1B1. Of these 24 cases, consanguinity was seen in 16 cases and 3 of them had a positive family history. Homozygosity of the mutant allele was seen in 29.68% (19/64) and compound heterozygosity in 3.12% (2/64) of the cases, respectively. However there was no association of any mutation with the gender or ethnicity of the individual.
The homozygous deletion at g.3905-3927delHVGQRLLR in the proband PCG050 (Figure 1) resulted in the truncation of the CYP1B1 protein to 51 aa, thereby missing 492 aa from the C-terminus. This occurred at the end of the membrane domain resulting in the elimination of all-important domains of CYP1B1 protein in exon II. It also led to the abolition of a restriction site for BbvCI. The other homozygous deletion at g.7900-7901delCG in the proband PCG081 (Figure 2) resulted in the truncation of CYP1B1 protein at 373 aa in the "J" helix (350 aa-363 aa). The resulting protein therefore lacked the "K" and "L" helices along with the "heme binding" regions and also caused the loss of a restriction site for TaqI in exon III. The PCG050 and PCG 081 probands presented at 7 years and 2 months, respectively. Although surgical intervention could lower their IOPs, visual recovery was poor in both of them (Table 2).
Among the missense mutations, the homozygous substitution at g.4148G->C resulted in a change from alanine to proline (A115P) in exon II in the proband PCG008. This mutation occurred in the N-terminal region of the cytosolic domain between the "B" (100 aa-107 aa) and "C" (141 aa-151 aa) helices causing a loss of a restriction site for BsgI. The importance of proline residues in the proline-rich region of microsomal cytochrome P450s has demonstrated the presence of typical carbon monoxide difference spectrum in the wild type P450 that was lacking in the mutant protein (due to the substitution of alanine by proline) . Taking this into consideration, we suspect that the A115P mutation might also cause a conformational change of the protein due to an extra proline residue. This patient underwent a surgical intervention at 6 days after birth and had a better IOP control and visual recovery (Table 2).
Another homozygous substitution at g.4200T->G caused a change from methionine to arginine (M132R) in exon II in the proband and his younger brother in PCG122 family. This mutation was located between the "B" and "C" helices of the CYP1B1 protein that resulted in a loss of a restriction site for NlaIII. A comparative modeling of CYP1B1 using CYP2C5 as a template showed the possibility of hydrogen bond formation with main chain amide in this residue (Unpublished). Thus this mutation could disrupt this hydrogen bond formation and affect the final conformation of the protein. Both the siblings in PCG122 family presented with raised IOP and visual acuity of "fixing and following of light" but the elder brother had to undergo Transcleralcyclophotocoagulation (TSCPC) due to his advanced nature of the glaucoma and late age of intervention (6 years).
A homozygous substitution at g.8234G->A resulted in the replacement of glycine by aspartic acid (G466D) in proband PCG009 (Figure 3). This residue is highly conserved across all cytochrome P450s and is a part of the "signature sequence" (NH2-FXX GXX XCX G-COOH) that is present in all heme binding cytochromes at the C-terminus of the protein (Figure 4). The cysteine residue in this sequence acts as the fifth ligand to heme. The other conserved residues and hydrophobic amino acids viz. phenylalanine and glycine next to the axial ligand (cysteine) are very important for the apoprotein to hold and/or incorporate the heme plane at the active site of P450 . In spite of early intervention, visual prognosis was poor in this proband (Table 2).
Another homozygous mutation at g.4520A->C resulted in the replacement of serine by arginine (S239R) between the "F" (219 aa-234 aa) and "G" (259 aa-281 aa) helices of CYP1B1 protein. This was the only mutation that was observed independently in the probands of 2 families (PCG021, PCG147). Both the probands with S239R mutation had advanced glaucoma at presentation. Although their IOPs could be controlled by surgical intervention, their visual acuity did not improve significantly (Table 2).
A heterozygous change g.4236A->C resulted in the substitution of glutamine by proline (Q144P) in exon II in the proband PCG086. Although this residue is conserved only in humans (Figure 4), it was not seen in either of his parents or controls. This de novo mutation lay in the "C" helix (141 aa-151 aa) of the protein and caused a loss of a restriction site for MspA1I. The insertion of proline in the middle of the "C" helix could induce a turn and further affect the final conformation of protein. This proband was intervened early and had a better prognosis with normal IOP and improved visual acuity (Table 2).
Among all the mutations, R368H was again found to be the most frequent change (29.16%), similar to our previous study that reported it to be the most predominant CYP1B1 allele in the Indian population . This mutation has rarely been reported from the other ethnic groups. So far only a few PCG families (from Saudi Arabia and Brazil) were found to have this mutation at a very low frequency [11,14]. Early intervention in 5 probands with the homozygous and heterozygous R368H mutant allele had a relatively better visual prognosis, as opposed to probands who were compound heterozygous for R368H in association with G61E and R390H mutant alleles and intervened at later ages (Table 2). The codon 390 in CYP1B1 happens to be a mutation hot spot in various populations [14,19,21]. However visual outcomes were relatively better in probands with the R390C mutation who underwent early surgical interventions than those with the R390H mutation, who were intervened late and also developed phthisis bulbi in one of their eyes (Table 2). Thus a variable prognosis was noted in probands with CYP1B1 mutations. Although our clinical experience suggests a better visual prognosis for early intervention in PCG , our current findings may suggest a larger role of the type of mutation, leading to structural and functional alteration of the CYP1B1 protein. However, functional studies of these mutations and analysis on a larger series would throw more light in understanding the protein alterations leading to the severity of phenotype in PCG.
The involvement of CYP1B1 in PCG was found to be lower (37.50%) in our population than those reported in Saudi Arabian (95.0%) and Slovakian Gypsy populations (100%). This could be due to a higher rate of inbreeding and allelic homogeneity in these populations [12,13]. Relatively lower frequencies of CYP1B1 mutations have been observed in Brazilian (50%) and south east Asian populations such as Japanese (20%) and Indonesians (about 34%) [8,10,11]. Interestingly, none of the CYP1B1 mutations in Japanese and Indonesian populations were found in Indian PCG populations. This could be attributed to the genetic heterogeneity and ethnic diversities in these populations. As there was no association of any specific intragenic haplotypes to these mutations, it could imply multiple independent origins of these mutations that warrants further investigations. In summary, this study provides a mutation spectrum of CYP1B1 causing PCG in Indian populations that has implications in devising molecular diagnostics for rapid screening in predisposed families  and would aid early intervention.
We thank the patients and their families for participating in this study and the clinical biochemistry staff for their help in collection of blood samples. ABMR and KK gratefully acknowledge the pre-doctoral research fellowship of the Council of Scientific and Industrial Research (CSIR), Government of India. This study was supported in parts by grants from the Department of Biotechnology and Indian Council of Medical Research, Government of India, to the Hyderabad Eye Research Foundation.
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