Molecular Vision 2007; 13:1458-1468 <>
Received 30 April 2007 | Accepted 22 August 2007 | Published 27 August 2007

Mutational screening of CYP1B1 in Turkish PCG families and functional analyses of newly detected mutations

Sefayet Bagiyeva,1 Gemma Marfany,1,2,3 Olga Gonzalez-Angulo,1,2,3 Roser González-Duarte1,2,3

1Departament de Genética, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain; 2Centre for Biomedical Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Barcelona, Spain; 3Institut de Biomedicina de la Universitat de Barcelona (IBUB), Barcelona, Spain

Correspondence to: Sefayet Bagiyeva, Hacettepe University, Teknokent, GENAR Biotechnology and Molecular Genetics, Research and Application Labotarories, 06800 Beytepe, Ankara, Turkiye; Phone: +90 312 299 23 26; FAX: +90 312 299 23 09; email: or


Purpose: To investigate the genetic basis of primary congenital glaucoma (PCG) in a collection of Turkish patients and to assess the pathogenicity of two novel alleles

Methods: Intragenic single nucleotide polymorphisms (SNPs) genotyping and mutational screening of CYP1B1, the major PCG causing gene, were performed by PCR amplification and sequencing. PCG cases with either none or a single heterozygous mutation in CYP1B1 were further screened for mutations in myocilin (MYOC), claimed to be a minor contributor to the PCG disease through a digenic mode of inheritance. The subcellular localization and enzymatic activity of the two novel mutant proteins were assessed by immunofluorescent confocal techniques, and by an easy, user-friendly method that we have adapted from toxicity tests that use modified-luciferine substrates.

Results: CYP1B1 mutations were found in 15 out of 35 PCG patients either in the homozygous or heterozygous state. Two novel (p.R117W and p.G329V), as well as six previously reported mutations were identified. No mutation in the MYOC gene was found in any of the PCG cases analyzed. The two new mutant proteins showed considerably reduced enzyme activity as well as a diminished localization in the mitochondria, probably due to a slower traffic rate through the ER compared to the wild-type form.

Conclusions: The present work provides a mutation and intragenic SNP haplotype profile of the CYP1B1 gene in Turkish PCG families and suggests a modest contribution at best of the MYOC gene to PCG in Turkey. In addition, it describes two new missense mutations and and reports a simple enzymatic assay to assess the pathogenicity of novel variants.


Glaucoma, a group of eye diseases characterized by the abnormal appearance of the optic nerve head, affects approximately 67 million people worldwide [1]. Data from population based studies of OAG (open angle glaucoma) and ACG (angle closure glaucoma) revealed that there will be 60.5 million people with OAG and ACG by 2010, increasing to 79.6 million by 2020 [2]. Primary open angle glaucoma (POAG; OMIM 137760) is the most common type and affects 37 million people [3]. POAG comprises juvenile and adult onset forms, which, respectively follow monogenic and complex patterns of inheritance. MYOC (myocilin; OMIM 601652), OPTN (optineurin; OMIM 602432), the WDR36 gene (WD repeat-containing protein 36; OMIM 609669) and recently, CYP1B1 (OMIM 601771), have been already shown to cause POAG, although at least 15 genetic loci have been reported to contribute to the POAG phenotype [4-8].

Compared to juvenile and adult onset forms, the primary congenital form of glaucoma (PCG; OMIM 231300) is rare. Usually diagnosed at birth or during the first 3 years of life, PCG is characterized by aberrant development of the eye drainage channels and is frequently associated with increased intraocular pressure (IOP). The PCG prevalence varies across populations, being higher in the middle East (1/2500 newborns) than in western countries (1/10 000) [9,10].

CYP1B1, located on chromosome 2p21 in the GLC3A locus, is the main known gene responsible for PCG [11]. Although the precise role of CYP1B1 in the eye is still unknown, it appears to be crucial in ocular development. CYP1B1 is the major cause of the disease in Saudi Arabia (85%), whereas in other populations its contribution ranges from 50% in Brazil to 12% in Ecuador [12-16]. In addition, there is another reported PCG locus at 1p36, GL3B (OMIM 600975) [15-17], whose gene has yet to be identified, and another putative locus at 14q36, GLC3C, which still awaits confirmation [18].

Besides the heterogeneous genetic basis of PCG, incomplete penetrance has been reported, supporting the action of modifier genes, as yet unknown, which would modulate the phenotypic clinical severity of the CYP1B1 mutations [12]. Recently, tyrosinase (Tyr) deficiency was also found to worsen the drainage structure/ocular dysgenesis phenotype of Cyp1b1-/- knockout mice [19]. In addition, CYP1B1 has been involved in other eye pathologies, such as juvenile open-angle glaucoma (JOAG; OMIM 137750, OMIM 608695, OMIM 608696), and Peter's anomaly (PA; OMIM 604229) [20-22].

CYP1B1 is a mixed-function monoxygenase [23,24] that belongs to the cytochrome P450 1B subfamily, whose substrate(s) and their relevance for ocular development and differentiation are as yet unknown. In spite of their differences in substrate recognition and enzymatic activity, all P450s share a highly conserved COOH-terminal core (CCS), a segment that includes four helix bundles (D, E, I, and L), helices J and K, β-sheets 1 and 2, the heme-binding and the "meander" regions [23]. The NH2-terminal CYP1B1 segment contains a transmembrane domain and a hinge region. Between these two fragments, several SRS-substrate recognition sites are located [24,25]. Several mutant forms of CYP1B1 have been studied and differ in their enzymatic properties. G61E has been related to a less stable protein complex, whereas R469W showed the opposite effect [26]. In a parallel report, G61E, G365W, D374N, P437L, and R469W missense mutations were shown to reduce CYP1B1 activity [27].

Here we report the mutational analysis of CYP1B1 in 35 Turkish patients to assess its contribution to PCG. A recent report relating MYOC gene mutations to PCG [28,29] and the digenic inheritance reported for some MYOC and CYP1B1 mutations in different types of glaucoma [28,30] led us to evaluate the pressumptive contribution of MYOC to the disease as well. In addition, we aimed to investigate the effect of any novel mutation on CYP1B1 protein activity as a means to provide solid grounds to establish their pathogenicity and when enough data has been gathered, to draw genotype-phenotype correlations.


Patient clinical assessment

Affected individuals were patients from both, the Ankara Numune Research and Training Hospital and The Hospital of the Medical Faculty of Ankara University. All subjects were clinically evaluated by glaucoma specialists and diagnosed with trabeculodisgenesis (PCG). Age of onset was in most cases unknown, as many patients were sent to the hospital years after the onset of the disease. There was no evidence of differences in ethnic origin nor of any genetic relationship among patients from different families. In all the recruited cases, the disease showed autosomal recessive inheritance. After obtaining informed consent, thirty-five PCG cases were selected. All the procedures used in this study conformed to the tenets of the Declaration of Helsinki and received approval from the Ethical Review Board of Hacettepe University, Ankara, Turkey.

Mutational screening of the CYP1B1 and MYOC genes and SNP haplotype analysis

Genomic DNAs of PCG patients and family members when available were isolated from blood samples using the Gentra Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN). All exons of the MYOC and CYP1B1 genes were amplified using intronic primers flanking the exons (Table 1) in order to confirm the integrity of the intron-exon boundaries. The amplification conditions were as follows: an initial denaturation step at 94 °C for 1 min, followed by 35 cycles of denaturation, annealing and extension at 94 °C (30 s), 55-59 °C (30 s), 72 °C (40 s), respectively, with a final extension step at 72 °C for 3 min. The PCR products were purified using spin-columns (QIAQuick PCR Purification Kit; Qiagen, Hilden, Germany). Samples were then sequenced, at least twice, using the BigDye Terminator Kit version 3.1 (Applied Biosystems, Foster City, CA) and analyzed in an automated DNA sequencer (ABIPrism3730). All nucleotide mutations and SNP variations were carefully assessed by resequencing. The location of the mutations along the gene is shown in Figure 1, and summarized in Table 2. The SNP nomenclature was according to dbSNP. Five of the SNPs are in the coding region whereas the first is 12 bp upstream of the first coding exon (Table 2). Multiple sequence alignments of proteins of the CYP enzyme family were performed using ClustalW1.8.

Cosegregation analysis of PCG families

Restriction fragment length polymorphism (RFLP) analysis was performed to show cosegregation in families with homozygous or compound heterozygous missense mutations (Table 3). One hundred control matched chromosomes from the same population were screened to assess new mutations. Amplifications were performed under the same reaction conditions as described above. The fragments were resolved either on 6% polyacrylamide or 1.5% agarose gels and stained by ethidium bromide.

Microsatellite haplotype analysis

Three chromosomal markers, D2S177, D2S1346, and D2S2974, closely located to CYP1B1 (respective approximate distance to CYP1B1 is -268 kb, -28 kb, +125 kb) were used to assess chromosomal segregation in family PCG9 (Figure 2C). The amplification conditions consisted of an initial denaturing step at 94 °C for 1 min, followed by 35 cycles of denaturation, annealing and extension steps at 94 °C (30 s), 53-55 °C (30 s), 72 °C (30 s), respectively, with a final extension at 72 °C for 3 min.

RNA Extraction and RT-PCR

Total RNA was extracted from the HEK293T (human embryonic kidney) cell line grown in DMEM (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, and 100 μg/ml streptomycin at 37 °C, in a fully humidified 5% CO2 atmosphere. cDNA synthesis was performed using the Cells-to-cDNAII kit (Ambion, Foster City, CA). AccuPrime Taq DNA Polimerase (Invitrogen) and 4% DMSO were used for the PCR reaction: an initial denaturation step at 94 °C for 2 min, followed by 40 cycles of denaturation, annealing and extension at 94 °C (30 s), 52 °C (30 s), and 68 °C (90 s), respectively. For amplification of the CYP1B1 cDNA, four different primers: NF- GAG AAG CTT CATG GG CAC CAG CCT CAG C, NR-GAT AAA GGC GTC CAT CAT GTC for the NH2-terminal encoding region, and CF-AGT GCC GTG TGT TTC GGC T, CR-CGG GAT CCC TTG GCA AGT TTC CTT GGC T for the COOH-terminal encoding segment (the position of the first and last CYP1B1 amino acids are in red), were designed so that the PCR product was flanked by suitable cloning restriction enzyme sites (HindIII for the NH2-terminus and BamHI for the COOH-terminus, in italics). The NH2-terminal and COOH-terminal fragments thus amplified overlapped a small stretch of sequence containing a unique internal EcoRI site that was used to reconstitute the full coding sequence in frame (see below).

Eukaryotic expression constructs and site-directed mutagenesis

The amplification products of the CYP1B1 cDNA were sequentially cloned into the HindIII/EcoRI and EcoRI/BamHI sites of the same pBluescript +SK vector (Stratagene, La Jolla, CA) to reconstitute the full-length CYP1B1 coding sequence and the construct was resequenced. We generated a CYP1B1-EGFP fusion protein in the vector pEGFP-N2 (BD-Clontech, San Jose, CA). The mutations, c.4154C >T and c.4791G >T (p.R117W and p.G329V, respectively) were generated by oligonucleotide-directed PCR mutagenesis, using the following primers: 5'-CCT TCG CCG ACT GGC CGG CCT TC-3' and 5'-CAC TGA CAT CTT CGT CGC CAG CCA GGA C-3' (mutations in red) and the QuickChange Site-directed Mutagenesis kit (Stratagene). The wild-type and mutant CYP1B1 coding sequences were cloned into the HindIII/BamHI sites of the pcDNA3.1 vector (Invitrogen Life Technologies) in which the HA-epitope had already been introduced at the NH2-terminus. All these cDNA-derived constructs were verified by sequencing (BigDye Terminator kit version 3.1-Applied Biosystems).

Immunolocalization of wild-type and mutant CYP1B1 proteins in transiently transfected COS-7 cells

COS-7 (African green monkey kidney) cells were grown in DMEM containing 10% fetal bovine serum, 4 mM L-glutamine, 100 U/ml of penicillin and 100 μg/ml streptomycin (Invitrogen Life Technologies). Cells grown on coverslips in 24 well plates were transiently transfected with 0.8 μg of the different constructs using LipofectamineTM 2000 (Invitrogen Life Technologies). Forty-eight h post-transfection cells were rinsed with 100 mM PBS and fixed in 3% paraformaldehyde and 2% sucrose in 0.1 M phosphate buffer at 4 °C for 30 min, then washed and permeabilized with 0.1% Triton X-100/20 mM glycine/10 mM PBS for 10 min. Cells were rinsed, blocked in 1% BSA/20 mM glycine/10 mM PBS and incubated with anti-calnexin (Endoplasmic reticulum; 1:25) monoclonal primary antibody (BD Biosciences, Bedford, MA) at 37 °C for 1 h. Upon washing, cells were incubated with AlexaFluor 546-conjugated anti-mouse (1:300; Molecular Probes, Invitrogen, Carlsbad, CA) secondary antibody. To detect the Golgi apparatus, cells were rinsed and blocked with goat serum (1:50 in 1X PBS) for 1 h at RT, rinsed in 0.5% Triton X-100/0.25% gelatin/0.9% NaCl/0.05 M Tris-HCl (pH 7.4), incubated with monoclonal antibody anti-GM130 (1:250; BD Biosciences), and a final incubation with Cy5-conjugated anti-mouse secondary antibodies (1:100). To label mitochondria, reduced MitoTracker Orange (Molecular Probes, Invitrogen) was added to the cell culture medium at a final concentration of 500 nM for 45 min before the fixation procedure. All preparations were mounted in Vectashield medium for fluorescence (Vector Laboratories, Burlingame, CA) and analyzed by confocal laser scanning microscopy with Olympus Fluoview 500, Leica TCS NT and Leica SP2 laser scanning microscopes.

Wild-type and mutant CYP1B1 enzyme activity in transiently transfected HEK293T cells

The wild-type as well as the R117W and G329V mutant CYP1B1 coding sequences were cloned into the pcDNA3.1 vector. HEK293T cells were grown in DMEM containing 10% fetal bovine serum, 4 mM L-glutamine, 100 U/ml of penicillin, and 100 μg/ml streptomycin (Invitrogen Life Technologies) and seeded (75,000 cells/well) in 24 well plates. After 24 h, cells were transiently transfected with 0.8 μg of either empty pcDNA 3.1 vector, wild-type, R117W, or G329V CYP1B1 constructs using LipofectamineTM 2000 (Invitrogen Life Technologies). In all cases, 0.3 μg of pEGFP (BD-Clontech) was also co-transfected for normalization of the transfection efficiency. Forty-eight h post-transfection, total CYP1B1 activity was measured in vivo using the P450-GLO CYP1B1 kit (catalog number V8762; Promega, Madison, WI) with a modified protocol. In brief, the media was changed and the luciferin-CEE substrate (0.1 mM final concentration) was added to each well. After 4 h of incubation, most of the luciferin-CEE has been converted to luciferin by the activity of CYP1B1 and released to the media. The medium from each well was collected and processed following the protocol of the aforementioned luciferase-based kit and the final amount of luciferin determined using a luminometer (FB12-Luminometer; Berthold, Bundoova, Australia). The cells from each well were trypsinized and washed, and 1/10 of the volume was used to assess the efficiency of transfection (EGFP positive cells are counted using a FACS and obtained as a percentage of the total amount of cells) and the rest is lysed and the total protein concentration used for normalization of the total number of cells. The endogenous CYP1B1 activity (assessed from HEK293T cells transfected with the empty pcDNA vector and normalized against protein concentration) is substracted from the values obtained in the CYP1B1 transfected cells. The final enzyme activity values were corrected for transfection efficiency (per each construct and well). More than 5 replicates in 2 different experiments were used for each DNA construct. Statistical significance was verified by the Mann-Whitney test (p<0.01).

Protein electrophoresis and western blotting

Protein samples were resolved in 10% SDS-polyacrylamide (MiniPROTEAN II; BioRad, Herucles, CA) and transferred to a PVDF membrane (Hybond-P; GE Healthcare, Bedford, UK) by electroblotting (1 h) using the Mini Trans-Blot system (BioRad). Membranes were then blocked in 0.1% Tween-20, 5% non-fat milk in PBS and incubated with anti-HA monoclonal antibodies (1:1000) overnight at 4 °C. Anti-tubulin (1:10000) was also used as a loading control. The corresponding secondary antibody was added and the ECL-western blotting detection kit (GE-Healthcare) was used for chemiluminiscent detection.


Mutational screening of CYP1B1

To assess the contribution of CYP1B1 in a panel of 35 Turkish patients affected with PCG, we performed direct amplification and sequencing of the 3 exons (the first is noncoding). We detected 8 different mutations in CYP1B1, scattered all along the coding sequence, in 15 samples (Figure 1, Table 2). Six of these mutations had already been reported (c.8037dup10, c.4668insC, G61E, E229K, R368H, and R469W). The mutations c.8037dup10 and c.4668insC were observed, respectively, in homozygosity in two patients, PCG1 and PCG10, and both caused a frameshift that introduced a premature STOP codon, thus generating a truncated CYP1B1 protein. The G61E segregated with the disease phenotype in 5 PCG families (14% of the total families) and was the most frequent mutation in the Turkish gene pool (detected in 8 alleles), followed by the R469W mutation, found in four PCG families (11% of the total families, detected in 6 alleles). Overall, the G61E and R469W alleles represented about 58% of the mutated CYP1B1 sequences (9 out of 15 patients bore one of these alleles, either in homozygous or heterozygous combination; Table 2). Mutations E229K and R368H were much less frequent and found in heterozygous state in individuals where the other mutated allele could not be identified (Table 2).

Notably, we identified two novel missense mutations, R117W and G329V (Figure 2A, Table 2). The R117W mutation appeared as a compound heterozygote with the already reported R469W, and the patient PCG21 presented the G329V variant in homozygosity. For these two novel mutations, G329V and R117W, one hundred control chromosomes were screened using RFLP analyses. None of these changes were detected in healthy controls, thus supporting their pathogenicity. Taking all these results together, CYP1B1 mutations in both alleles were involved in 9 out of 35 PCG families (26%), and in one allele in 6 PCG families (17%), demonstrating a 26% prevalence, which most probably would add up to 43% (if the second mutation is found in those PCG families with a single mutated allele).

An intragenic SNP profile analysis of all the patients was also performed to evaluate consanguinity and/or a presumptive founder effect for mutations shared by different families (Table 2). We genotyped 6 SNPs (rs2617266, rs10012, rs1056827, rs1056836, rs1056837, and rs1800440), which had been widely used when tracing CYP1B1 mutations, 5 of them in the coding sequence and one located 12 bp upstream of the first coding exon. A systematic haplotypic analysis had already revealed that reported mutations were associated to 5 different haplotypes [31]. In fact, most mutations were embedded in haplotypes CYP1B1-A and CYP1B1-B (identical for the 6 analyzed SNPs), also the most common haplotypes in the healthy population [31]. In our case, patients homozygous for a mutation were also homozygous for the intragenic SNP haplotype, thus suggesting homozygosity by descent. Moreover, the mutation-haplotype associations found in our patients were in agreement to those reported [31]. Besides, all the patients with the G61E allele, as it also happened with the R469W, shared the same intragenic haplotype (Table 2).

Cosegregation analysis of the mutant alleles was performed in a consanguineous family (PCG9) with affected siblings in two generations. Notably, all patients were compound heterozygotes for R117W and R469W, as confirmed by an RFLP analysis devised to directly detect each mutant (Table 3, Figure 2B,C). As homozygosity is usually assumed to explain the disease in consanguineous families, three CYP1B1 closely located markers were used to ascertain chromosomal segregation in this family. The haplotypes revealed that all the members of the nuclear family (generation II) had inherited the same allele from I.2, also shared by II.6, III.1, and III.2, whereas inherited alleles from I.1 differed between affected and unaffected individuals. Moreover, one nonpenetrant phenotype was observed in II.5, who bore the mutation R117W in a compound heterozygous state with R469W, while the mother (II.6) was heterozygous for R117W (Figure 2C). Given that incomplete penentrance is a recurrent trait of PCG cases, these cosegregation results agree with the pathogenicity of these two mutations.

Sequence analysis of the first (noncoding) exon did not reveal any mutation, although two polymorphisms that did not cosegregate with the disease were identified: c.226C >A in homozygous state, and c.370G >A in heterozygosity (the latter already reported as a rare variant, Human Genome Browser 2004).

Mutational screening of MYOC coding regions

As aforementioned, digenic inheritance for PCG has been claimed when only one mutant CYP1B1 allele has been found. Myocilin (MYOC), one of the major genes involved in POAG, has been shown to be a minor contributor in PCG. Hence, we analyzed the MYOC exons and intron-exon boundaries in all those patients harboring none or only one CYP1B1 mutant allele in heterozygosis. According to our results, MYOC was not involved in any of these patients, as no pathogenic mutations were found and only a silent substitution (p.I71I) was identified in exon 1. Therefore, the contribution of MYOC to the PCG in Turkey would be very modest (<1/35) in any case.

The new missense mutants alter conserved CYP1 residues

To evaluate the pathogenicity of the new mutations inferred after the cosegregation analysis, we aligned several protein sequences of the cytochrome P450 CYP1 subfamily from different species. Positions R117 and G329 (residue number according to the human CYP1B1) were remarkably conserved among orthologues and paralogues of different vertebrate species (CYP1B1, CYP1A1, and CYP1A2; Figure 3). Moreover, the in silico SIFT (Sorting Intolerant from Tolerant) analysis [32] predicted that the two substitutions would affect the protein function with a high score, thus further supporting their pathogenicity.

Subcellular localization of the wild-type and mutant CYP1B1 proteins

CYP1B1 is a transmembrane protein and, as such, subject to intracellular traffic. In order to assess the putative effect of the R117W and G329V missense mutations on the proper localization of the CYP1B1 protein, we transfected COS-7 cells with constructs bearing the wild-type and mutant coding cDNAs fused at the NH2-terminus of EGFP. Several markers from different subcellular compartments were used (such as calnexin V for the ER, GM130 for the Golgi, and MitoTracker Orange to label mitochondria) for counterstaining and subcellular compartment assignment.

According to our results, the wild-type CYP1B1 first localized in the ER and then translocated to mitochondria, where most of the protein was detected 48 h post-transfection (Figure 4, first row). No clear colocalization with Golgi markers was observed. As for the mutants and in comparison to the wild-type, increased amounts of the protein were detected in the ER, whereas the localization in the mitochondria was decreased (Figure 4, note the differences in the colocalization intensities in the third and sixth columns), pointing to a partial ER-retention or decreased trafficking through the ER. Besides, some of the G329V mutant protein mislocalized to the Golgi (Figure 4, third row). Note that the GFP protein moiety alone (pEFGP-N2 empty vector) diffuses throughout the cell and localizes to most organelles without any obvious pattern.

Decreased enzymatic activity in R117W and G329V mutants compared to the wild-type CYP1B1

We then explored whether the missense mutations caused any reduction in the enzyme activity. To this end, we adapted an existent kit, the P450-Glo kit assay (Promega), which is devised to measure the in vivo conversion of a modified Luciferin-CEE (Luciferin 6' chloroethyl ether) into a luciferin substrate, which is then secreted to the medium and quantifiable by standard luciferase assays. We modified the protocol as to measure the activity of exogenous CYP1B1 in transiently transfected HEK293T cells with constructs bearing the wild-type or the R117W and G329V mutant CYP1B1 proteins. As shown in Figure 5A, the expression levels of the recombinant proteins were comparable in all the transfected cells. For the final activity values, we considered: (1) normalization against total cell protein, (2) substraction of the cellular endogenous CYP1B1 activity, and (3) transfection efficiency per construct and well (as measured by cotransfection with a pEGFP vector).

Remarkably, the enzymatic activity of the R117W and G329V CYP1B1 mutants was decreased, respectively, down to 60% and 30% of the wild-type protein (p<0.01, Mann-Whitney test), providing solid grounds for the pathogenicity of these mutations (Figure 5B).


Of the eight CYP1B1 mutations found in Turkish patients, our work has unveiled two novel missense substitutions, R117W and G329V, whose pathogenicity is based on the following grounds: (1) the two segregate with the disease phenotype; (2) both are absent in 100 control chromosomes; (3) they are located in evolutionarily conserved positions among different species and across various members of the cytochrome P450 family (Figure 3), and are also predicted to affect protein function by the SIFT program and finally, (d) they produce a decrease in CYP1B1 enzymatic activity (Figure 5).

Residue R117 is conserved in the CYP families of different species (Figure 3) and it is embedded in the putative active site of CYP1B1 [33]. The replacement of the basic residue arginine with a neutral aromatic tryptophan may impair the native protein structure in this site and consequently, affect proper folding and substrate accommodation. Interestingly, the substitution of the adjacent and also conserved P118 residue (P118T) has been already involved in Peter's anomaly [34] (a developmental eye anomaly, frequently associated with glaucoma), thus giving credence to the relevance of this domain for CYP1B1 function. The R117W substitution was found in a compound heterozygous state with mutation R469W and notably, one of the obligate carriers showed a nonpenetrant phenotype. Incomplete penetrance of some mutations in PCG had already been observed in families from Saudi Arabia, this was one of the reasons why the contribution of as yet unknown modifier genes in PCG had been suggested [12]. In this previous report, 40 individuals carrying the corresponding causative mutations were not affected compared to 108 affected siblings, rendering a penetrance of 0.73 [12], which is a similar number to that found in our pedigree (0.8). Interestingly, one of the previously reported CYP1B1 alleles with incomplete penetrance was R469W (either in homozygous or compound heterozygous state), as it is also the case in our study. We could not detect any other case of non-penetrance.

The mutation at position G329 occurred in SRS4, well within helix I of the heme-binding four-helix bundles. The primary structure and relative position of these four helices is highly conserved among P450s [24]. Comparative sequence alignment of closely related CYP protein families in different species indicate that this position is evolutionarily conserved (Figure 3). Although the G329V substitution involves two neutral amino acids, the bulkier lateral chain of valine might alter the integrity of the helix and the correct orientation of the substrate. It is worth noting that this substitution caused the highest decrease in CYP1B1 activity (reduced down to 30% of the wild-type enzyme), thus clearly supporting the pathogenicity of the mutation. The other six mutations found in this study had already been reported to be pathogenic [12,35,36].

We also sequenced the first (noncoding) exon of CYP1B1 as well as the coding regions of MYOC in the patients where none or only one CYP1B1 mutant allele in heterozygosis was detected, but we could not detect any additional mutations. However, we identified two polymorphisms in the first non-coding exon of CYP1B1: one new c.226C>A (in homozygosity), and one previously reported as a rare variant, c.370G>A (in heterozygous state). Although variant c.226C>A was not detected in 50 healthy control subjects, we did not consider this substitution as pathogenic, since the patient was already homozygous for the pathogenic c.4668insC, which causes a frameshift and a premature truncation of the protein. For all these unassigned patients, we cannot rule out a second mutation in unscreened promotor regions or in other genes involved in other types of glaucoma.

CYP1B1 mutations had been claimed to be responsible for 90%-100% of Saudi Arabian, Romany Slovakians, and Turkey PCG families [15]. However, other reports support that CYP1B1 contribution to PCG is lower in other populations, from roughly 12% in native americans from Ecuador, to 20% in Japan, 30% in Morocco, and India, or 50% in Brazil [13-16]. Considering this wide range, our results are much closer to the latter, as the mutational screening of the CYP1B1 gene in these 35 Turkish families indicate that this gene may be involved in 43% of the PCG phenotype. These apparent discordances between, or even within, similar populations had also been noticed in most previous reports, although no straightforward explanation could be offered. In addition, the MYOC gene, which is involved in juvenile-onset POAG and has been associated with the PCG phenotype, did not show any mutation in this study. Therefore, we could not find any evidence of the MYOC contribution to PCG in our subjects, even though coexistence of PCG and POAG was observed in 3 families (PCG11, PCG26, and PCG27). Because of the high genetic heterogeneity of POAG and PCG, we concluded that the etiology of diseases in these families may be linked to other loci. In some populations, a common haplotype for 6 intragenic CYP1B1 SNPs (5'-CCGGTA-3' SNP haplotype) was found associated with several CYP1B1 mutations [12,15,26]. In the families included in this study, this same haplotype is shared for all the G61E (as well as all the R469W) alleles, in accordance to other previous reports [31,37].

In this study, we investigated the effect of two novel missense mutations, R117W and G329V, on CYP1B1 subcellular localization and enzymatic activity. To our knowledge, little data has been gathered on CYP1B1 subcellular localization and none relies on immunofluorescence techniques. Previous reports on human tissues using an immunohistochemistry approach detected CYP1B1 protein in the nuclei, although it was also found in the cytoplasm of some cell types [38]. Our results using confocal microscopy for detailed subcellular localization clearly showed endoplasmic reticulum (ER) and mitochondrial localization. No CYP1B1 protein could be detected in nuclei. A recent report on quite a different member of the cytochrome P450 protein super-family, CYP11B1, also showed a similar subcellular localization in the mitochondria and ER [39]. Moreover, according to our results, the two new missense mutants carrying R117W and G329V were partially retained in the ER and showed decreased colocalization with the mitochondrial marker. In the case of G329V, some protein was even mislocalized to Golgi. It is thus conceivable that a slower CYP1B1 traffic through the ER contributes to the eventual lower enzyme activity and hence, the pathogenesis of PCG in those patients.

The role of P4501B1 in ocular development is as yet undefined. CYP1B1 is capable of metabolizing ligands for nuclear receptor families such as steroids and retinoids. Any alteration in the CYP1B1 expression pattern, as a result of DNA mutations or environmental influences, may alter the normal distribution of CYP metabolites and interfere with the normal eye development [40]. Given the CYP1B1 expression in the pigmented ciliary epithelium and the trabecular meshwork, some authors have hypothesized that the molecular basis of PCG could be related to an unknown endogenous substrate, metabolized by the CYP1B1 enzyme in the fetal eye [41,42]. Either the concentration of the biologically active parental molecule or the metabolites generated by the enzymatic CYP1B1 activity are secreted to the aqueous humor, thereby diffusing to distant sites and playing a relevant developmental role in anterior segment structures.

There is still much to be done to understand the etiology of glaucoma. The mutational screening of CYP1B1 and other candidates and the identification of new genes will be helpful in decreasing the risk of congenital glaucoma and of blindness and will provide further understanding of the genetic basis of PCG by establishing genotype-phenotype correlations, and will also contribute to our understanding of the role of CYP1B1 in development and differentiation.


We thank Dr. Oya Tekeli and Dr. Sinan Saricaoglu for providing the samples. We are indebted to the patients and their families by their cooperation in this work. This research was funded by BMC2003-05211 (MEC) to R.G-D. S. B. was in receipt of a grant by Bidons Egara, Additional funding was provided by ONCE and FUNDALUCE.


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