December 2007
Volume 48, Issue 12
Free
Biochemistry and Molecular Biology  |   December 2007
Glaucoma-Associated CYP1B1 Mutations Share Similar Haplotype Backgrounds in POAG and PACG Phenotypes
Author Affiliations
  • Subhabrata Chakrabarti
    From the Kallam Anji Reddy Molecular Genetics Laboratory and the
  • Koilkonda R. Devi
    From the Kallam Anji Reddy Molecular Genetics Laboratory and the
  • Sreelatha Komatireddy
    From the Kallam Anji Reddy Molecular Genetics Laboratory and the
  • Kiranpreet Kaur
    From the Kallam Anji Reddy Molecular Genetics Laboratory and the
  • Rajul S. Parikh
    VST Centre for Glaucoma Care, L. V. Prasad Eye Institute, Hyderabad, India.
  • Anil K. Mandal
    VST Centre for Glaucoma Care, L. V. Prasad Eye Institute, Hyderabad, India.
  • Garudadri Chandrasekhar
    VST Centre for Glaucoma Care, L. V. Prasad Eye Institute, Hyderabad, India.
  • Ravi Thomas
    VST Centre for Glaucoma Care, L. V. Prasad Eye Institute, Hyderabad, India.
Investigative Ophthalmology & Visual Science December 2007, Vol.48, 5439-5444. doi:10.1167/iovs.07-0629
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Subhabrata Chakrabarti, Koilkonda R. Devi, Sreelatha Komatireddy, Kiranpreet Kaur, Rajul S. Parikh, Anil K. Mandal, Garudadri Chandrasekhar, Ravi Thomas; Glaucoma-Associated CYP1B1 Mutations Share Similar Haplotype Backgrounds in POAG and PACG Phenotypes. Invest. Ophthalmol. Vis. Sci. 2007;48(12):5439-5444. doi: 10.1167/iovs.07-0629.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

purpose. To understand the involvement of the CYP1B1 gene in cases of primary open-angle (POAG) and primary angle-closure (PACG) glaucomas and obtain the haplotype background of these mutations.

methods. The entire coding region of CYP1B1 was screened by resequencing in 224 unrelated cases of POAG (n = 134) and PACG (n = 90) and 200 ethnically matched normal control subjects from Indian populations. Six intragenic single nucleotide polymorphisms (SNPs) in CYP1B1 (−13T>C, R48G, A119S, V432L, D449D, and N453S) were used to generate haplotype data for the cases and controls and linkage disequilibrium (LD) and haplotype analysis were performed with Haploview software, which uses the EM (expectation-maximization) algorithm.

results. The frequency of CYP1B1 mutations was higher among POAG (18.6%; 95% CI, 12.9–26.1) than PACG (11.1%; 95% CI, 6.1–19.3) cases. There was a marked allelic heterogeneity, and the Arg368His was the most prevalent mutation across both the phenotypes. The spectrum of CYP1B1 mutations was largely similar across different POAG populations. Haplotypes generated with intragenic SNPs indicated the C-C-G-G-T-A to be a risk haplotype associated with CYP1B1 mutations in POAG (P = 0.006) and PACG (P = 0.043), similar to that observed in cases of primary congenital glaucoma worldwide.

conclusions. The results demonstrate an involvement of CYP1B1 in a proportion of POAG and PACG cases that should be explored further. The similar haplotype background of these mutations is indicative of their common origin across multiple glaucoma phenotypes.

Glaucoma comprises a group of clinically and genetically heterogeneous optic neuropathies characterized by a progressive loss of vision and is the second leading cause of irreversible blindness worldwide. 1 2 Based on gonioscopic findings, primary glaucomas are classified as primary open-angle glaucoma (POAG; OMIM 137750; Online Mendelian Inheritance in Man; http://www.ncbi.nlm.nih.gov/Omim/ provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD) and primary angle-closure glaucoma (PACG). Both have characteristic optic nerve head changes, degeneration of retinal ganglion cells, and visual field loss, but PACG also has a closed angle or peripheral anterior synechia (PAS) on gonioscopy. 3 Both of these conditions may be associated with elevated intraocular pressure (IOP) due to the obstruction of outflow. 4 POAG affects 33 million worldwide and is more common in the West, 2 5 6 whereas PACG is relatively more common among Asian populations. 7 8  
Genetic heterogeneity is well documented in POAG, and 11 chromosomal loci (GLC1A-GLC1K) have been mapped by linkage analysis. 9 Of these, only three genes harboring GLC1A (Myocilin; MYOC; OMIM 601652), 10 GLC1E (Optineurin; OPTN; OMIM 602432), 11 and GLC1G (WDR36; OMIM 609669) 12 have been characterized. In addition, approximately 15 candidate genes have been identified by association studies that require a thorough replication in different populations. 9 Glaucoma being a complex disease would be attributed to multiple gene variants with various magnitudes of effect. 13  
Although the human cytochrome P450 gene CYP1B1 (OMIM 601771) has been implicated in primary congenital glaucoma (PCG; OMIM 231300) worldwide, 14 15 16 it has been relatively less explored in POAG and not at all in PACG. An initial study implicated the involvement of CYP1B1 with MYOC through a digenic mechanism in a family with juvenile-onset open angle glaucoma (JOAG) and suggested that CYP1B1 is a modifier of MYOC expression. It was also observed that affected subjects harboring a mutant CYP1B1 allele in this family had an earlier age at onset than those with only a mutant MYOC allele. 17 These findings led to the screening of CYP1B1 as a candidate gene among the patients with POAG and largely among those with JOAG. The frequency of CYP1B1 mutations varied in patients from Canada (5.0%), 17 France (4.6%), 18 Spain (10.9%), 19 Eastern India (4.5%), 20 and Southern India (10.8%). 21 The differences in mutation frequency could be partly explained by the definition of disease used in these studies. The Canadian patients had JOAG 17 whereas the French patients had POAG, but elevated IOP was not an inclusion criterion, 18 similar to studies from Eastern 20 and Southern 21 India. The results from these studies indicate a minor involvement of CYP1B1 among JOAG and late-onset POAG cases and suggest a possible role of this gene in glaucoma pathogenesis. 
We have reported the extent of CYP1B1 mutations along with their structural properties in PCG. 22 23 We have also demonstrated a global clustering of these mutations on specific haplotype backgrounds, irrespective of geographic location, that could be useful in predictive testing. 16 Herein, we report an extensive screening of the CYP1B1 gene in a cohort of patients with POAG or PACG from India, to determine its mutation spectrum and understand the haplotype backgrounds of these mutations. 
Methods
Clinical Details of the Subjects
The study protocol adhered to the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board. The cohort comprised unrelated, consecutive patients with JOAG (n = 30), POAG (n = 104), PACG (n = 90), and 200 normal control subjects, who were seen at the L. V. Prasad Eye Institute (Hyderabad, India) between January 2002 and March 2007. The diagnoses of POAG and PACG were independently confirmed by two surgeons based on the following inclusion and exclusion criteria. 
POAG (Including JOAG)
The diagnosis of POAG was based on open angles on gonioscopy, an IOP >21 mm Hg, and characteristic optic disc changes and corresponding visual field defects in patients >35 years of age. Visual field defects were considered to be glaucomatous if they were consistent with optic disc damage and met at least two of the criteria laid out by Anderson and Patella. 24 The presence of a visual field defect required confirmation by a repeatable field performed within 2 weeks of the first reliable visual field result showing the defect. The field defects were further classified as mild, moderate, or severe. 25 Such findings in patients between 5 and 35 years of age were labeled as JOAG. As the presence of visual field defects was one of the inclusion criteria, only patients older than 10 years were included in the study. 
Primary Angle-Closure Glaucoma
PACG was defined as the presence of optic disc and visual field changes characteristic of glaucoma, along with appositional or synechial primary angle-closure (PAC) in patients older than 18 years. The visual field defects were as defined in POAG. PAC (appositional) was defined as increased IOP (>21 mm Hg) associated with nonvisibility of the filtering trabecular meshwork for more than 180°, in the absence of PAS, disc damage, or field changes. PAC (synechial) was defined as the presence of PAS with nonvisibility of the filtering trabecular meshwork for more than 180°, with or without increased IOP (>21 mm Hg), without disc damage or demonstrable field defects. The presence of even a single PAS in an angle with more than 180° of nonvisibility of trabecular meshwork was considered diagnostic of PAC. Other causes of synechiae were excluded. 
Ocular hypertension, normal-tension glaucoma, lens-induced glaucoma, neovascular and pseudoexfoliation glaucoma, and secondary open-angle glaucoma were excluded. Other ocular diseases that can lead to secondary glaucoma were also excluded. 
Normal adult individuals without any signs or symptoms of glaucoma and other systemic diseases served as control subjects. Their visual acuity ranged from 20/20 to 20/40, and their IOP was <21 mm Hg. Clinical examination on stereo biomicroscopy did not reveal any changes in the optic disc suggestive of glaucoma. The patients and controls were matched with respect to their ethnicity and geographical region of habitat. 
Molecular Analysis
Peripheral blood samples (5–10 mL) were collected from each subject by venipuncture, with prior informed consent. DNA was extracted by standard protocols 26 and the entire coding region of CYP1B1 was amplified using appropriate oligonucleotide primers and PCR protocols, as published earlier. 27 The amplicons were purified (SigmaSpin columns; Sigma-Aldrich, St. Louis, MO) and bidirectionally sequenced using dye termination chemistry (BigDye on a 3100 DNA Analyzer; Applied Biosystems, Inc. [ABI], Foster City, CA), according to the manufacturer’s protocol. Sequencing analysis software was used to read the individual sequences. Six mutations (G61E, Y81N, Q144R, P193L, E229K, and R368H) were further confirmed by restriction digestion of the amplicon with appropriate restriction enzymes as published earlier, 18 22 27 whereas the remaining five mutations were verified by resequencing. Multiple sequence alignment of the human CYP1B1 protein was performed along with other CYP1 protein across different families, to check for the conservation of the residues. The SIFT (sorting tolerant from intolerant) homology tool (http://blocks.fhcrc.org/sift/SIFT.html/ provided in the public domain by the Fred Hutchinson Cancer Research Center, Seattle, WA) was used to assess the effect of the substituted amino acid on the CYP1B1 protein, and a threshold score of less than 0.05 was considered to be deleterious to the protein. 28  
Statistical Analysis
The maximum-likelihood estimates of allele frequencies, Hardy-Weinberg equilibrium, and haplotype frequencies were estimated from the genotype data at six single-nucleotide polymorphism (SNP) loci using Haploview software, which uses the EM (expectation-maximization) algorithm. 29 Pair-wise linkage disequilibrium (LD) between the individual SNPs was calculated using the LD-plot function of this software. The odds ratios were calculated, to assess the risk of the individual genotypes at all six SNP loci. Clinical parameters, such as IOP at presentation, cup-to-disc ratio, and visual field defects for the worst eye were considered when correlating the genotype with the phenotype. All calculations were performed with commercial software (SPSS ver. 14; SPSS, Chicago, IL). 
Results
Mutation Screening of CYP1B1 in POAG and PACG
The study cohort conformed to Hardy-Weinberg equilibrium. A total of 11 CYP1B1 mutations were observed, of which 4 (Q144R, W434R, F445C, and g.8148-8152del5bp) were novel. The overall spectrum of CYP1B1 mutations observed in POAG and PACG is demonstrated in Figure 1(the electropherograms of all 11 mutations are provided in Supplementary Fig. S1). The frequency of mutations was higher in POAG (18.6%; 25/134) than in PACG (11.1%; 10/90). Arg368His was the most prevalent mutation across both the phenotypes, similar to earlier studies of PCG from India. 16 22 Further details are provided in Table 1 . Allelic heterogeneity was relatively more in POAG than in PACG. SIFT scores indicated a deleterious effect for all the mutations except E229K. 
The cosegregation of the heterozygous mutant allele was observed in only three families for the three mutations (G61E, Q144R, and P193L). DNA samples were unavailable from the relatives of the probands in the remaining cases harboring CYP1B1 mutations. Except for the Q144 residue, multiple sequence alignment indicated a strong conservation of the wild-type residues for all amino acids across multiple CYP1 families (Supplementary Fig. S2). 
Homozygosity of the mutant allele was noted in a JOAG case with G61E and in a POAG case with P193L mutations. There was only a single JOAG case with a compound heterozygous mutation (G61E and R368H). All other mutations were observed in the heterozygous state in JOAG (5/7), POAG (17/18), and all PACG. 
The CYP1B1 mutation frequencies were different across all the studies performed on POAG in Indian populations. 20 21 Of interest, the investigators in the study from Southern India found a carrier rate of 6.4% and 0.7% for the E229K and the R368H mutations, respectively, in their control populations 21 that was not observed in the cohorts from Eastern India 20 or in the present study. 
Table 2provides a comparison of JOAG, POAG, and PACG cases. As is evident from the table, JOAG cases had a higher prevalence of CYP1B1 mutations than did POAG cases. There was no significant difference in age at onset among JOAG cases with (20.1 ± 8.78 years) and without (20.9 ± 8.31 years) CYP1B1 mutations (P = 0.781). JOAG cases had a significantly higher mean IOP at presentation than did POAG cases, with and without CYP1B1 mutations (P < 0.001). The mean IOPs were similar among the JOAG and PACG cases with and without mutations. CYP1B1 mutations did not seem to be associated with disc changes (P = 0.192) and severe visual field defects (P = 0.417) in any of these phenotypes. 
Linkage Disequilibrium and Haplotype Analysis at the CYP1B1 Locus
Six intragenic SNPs were typed at the CYP1B1 locus, to generate haplotypes among the cases and controls. Pair-wise LD analysis indicated strong LD (D′ = 1) at two clusters, between three SNPs (−13T>C, R48G, and A119S) and between the two SNPs V432L and D449D (data not shown). The measure of LD between the other SNPs was similar to that in an earlier study. 35  
Four different haplotypes (with frequency >5%) were generated with these six SNPs in cases and controls. There were no significant differences in the haplotype frequencies when all POAG and PACG cases were compared with the controls (Tables 3 4) . Reanalysis of the cases with respect to their mutation status indicated a significantly higher frequency of the C-C-G-G-T-A haplotype in both POAG (P = 0.006) and PACG (P = 0.043) cases with CYP1B1 mutations (CYP1B1 +) than controls. However, there was no observable difference in frequencies of the other haplotypes among cases and controls. The significantly higher frequency of the C-C-G-G-T-A haplotype in POAG (P = 0.001) and PACG (P = 0.020) cases with CYP1B1 mutations was consistent, even when compared with cases without (CYP1B1 ) mutations (data not shown). 
Discussion
The present study provides a mutation spectrum of the CYP1B1 gene in POAG and PACG. The involvement of CYP1B1 highlights its role as a potential candidate in disease pathogenesis that should be explored further. We observed a higher proportion of mutations in POAG in the present cohort than in other populations (Table 5) . The spectrum of mutations observed in the present cohort was largely similar to that in the POAG populations in France, Spain, and India. 18 19 20 21 Except for the four novel mutations observed in this study, all other variants were observed earlier in patients with PCG in India and other countries (Table 1)
To the best of our knowledge, this is also the first study to report the involvement of CYP1B1 in PACG. Although there are differences in the mutation frequencies of CYP1B1 across JOAG, POAG, and PACG, the 95% CI of these frequencies overlap (Table 2) . It would also be interesting to investigate the role of CYP1B1 mutations in PAC, where there is no damage to the disc and visual fields, as opposed to PACG, where disc and fields are affected. Although that would be the subject for further study, we have screened 16 PAC cases, and none of them had CYP1B1 mutations (95% CI, 0–17.57). Although this was not significantly different from PACG cases with CYP1B1 mutations (as 95% CI overlaps; Table 2 ), the sample size was too small to draw any conclusion. Further studies on a larger sample are needed to determine CYP1B1 involvement in PAC. 
Although we observed a higher mutation frequency of CYP1B1 in POAG than in other populations, our results are not very different from those in a Spanish population, 19 when we look at the confidence intervals in these two studies (Table 5) . The frequency differs, however, from those in French and other Indian populations. These differences may be partially attributable to the definitions of POAG used in these studies. 18 20 21 In contrast to the French and other Indian studies, we used raised IOP (>21 mm Hg) in the definition of POAG and PACG, as it was our inclusion criterion. It is well known that CYP1B1 is a major candidate gene in PCG that is associated with increased IOP. 14 15 16 Hence, this could partially explain the higher frequency of CYP1B1 mutations in our patient cohort. The report on the Spanish patients with POAG 19 also included increased IOP (>21 mm Hg) as a major inclusion criterion, and, as just noted, their mutation rates are not very different from ours (Table 5)
It is interesting to note that the prevalent mutation was different across all previously reported POAG populations (Table 5) . Also, the frequency of heterozygous mutations was similar across these studies. Although the R368H mutation was common in patients in both the Indian and Canadian studies, it was noted in only 2 of the 60 patients with JOAG in the Canadian report. 17 One of the Canadian patients with the R368H mutation had an East Indian/Guyanese ancestry, 17 but we were not able to determine whether this patient shared a common haplotype background with the Indian patient due to unavailability of data. 
The median age at onset of the patients with POAG in the present cohort was similar to that of the French sample, 18 but was significantly lower than that of the Spanish 19 patients with POAG. The median age of the Canadian patients was significantly lower, as no cases older than 40 years were enrolled. 17 Another study on patients with POAG from Eastern India 20 reported a mutation frequency (4.5%) similar to that of the French population but a higher mean age (52.43 ± 19.33 years) than that of our cohort. Of interest, the prevalent mutation in the Eastern Indian (S515L) 20 and Southern Indian (E229K) 21 cohort was also different from that in the present study (R368H). 
Another interesting observation was the presence of CYP1B1 mutations on specific haplotypes that was earlier observed in PCG. 16 We noted that C-C-G-G-T-A was the risk haplotype in cases of POAG and PACG with CYP1B1 mutations. These results were consistent (even after reanalyzing the data set) based on a five-locus haplotype (i.e., C-G-G-T-A), similar to previous studies in different PCG populations worldwide. 16 32 33 35 37 On the other hand, the G-T-C-C-A haplotype that was largely associated with the unaffected controls and PCG cases without CYP1B1 mutations 16 was similar in frequency in the POAG and PACG cases with CYP1B1 mutations and the controls (Tables 3 4) . In tune with our previous study on PCG, 16 most of the mutations observed in the POAG and PACG clustered on the C-G-G-T-A haplotype. The R368H mutation, which was the prevalent mutation in POAG and PACG in the present study, similar to PCG in India, 16 22 was found on the background of the C-G-G-T-A haplotype across all these phenotypes. Of interest, this mutation was also found on the same haplotype in Saudi Arabian 32 and Brazilian 37 PCG patients. The G61E mutation in POAG was also found on the C-G-G-T-A haplotype, similar to that observed among the PCG patients from Ecuador, 31 Saudi Arabia, 32 and Morocco. 33 The E229K mutation that was observed on the G-T-C-C-A haplotype among patients with PCG in India 16 and Germany 35 was also seen to harbor the same mutation in POAG and PACG cases. Another striking similarity was the presence of the Y81N mutation in a case of POAG on the G-T-C-C-A haplotype. This mutation was also found on the same haplotype among German patients with PCG. 35  
Similar to our earlier hypothesis on the evolution of CYP1B1 mutations, we confirm that there is a strong clustering of these mutations on specific haplotype backgrounds, irrespective of geographical location. 16 A larger proportion of mutations were seen on the C-G-G-T-A haplotype and a smaller proportion on the G-T-C-C-A haplotype, further confirming the former to be an ancient haplotype and the latter to be a recent haplotype. 16 A formal haplotype comparison among other POAG cases with CYP1B1 mutations was not possible due to the unavailability of data from other populations. Based on the present analysis we speculate that the presence of specific CYP1B1 mutations on specific haplotype backgrounds in PCG worldwide and in patients with POAG and PACG in the present cohort is indicative of common founders. The mutations on these haplotypes would have migrated across different geographical regions due to population movements as reported in PCG in our previous study. 16 Thus, glaucoma-associated CYP1B1 mutations share a similar haplotype background across POAG, PACG, and PCG. 
The role of CYP1B1, particularly in retinoic acid synthesis is pivotal during embryonic development. Recent studies on chick embryogenesis have demonstrated its importance in the dorsoventral patterning of the neural tube that is consistent with its endogenous expression. 38 Several in vitro and in vivo studies in lower organisms have demonstrated the sites of expression of CYP1B1 at different stages of development in the anterior retina and anterior segment of the eye. 38 39 40 41 Although these studies have provided convincing evidence of the possible role of CYP1B1, its actual molecular mechanism leading to glaucoma in humans has to be deciphered. Although the functions of CYP1B1 mutations leading to POAG and PACG remain to be characterized, it is nevertheless an important candidate gene that should be screened in patients with glaucoma worldwide, to establish its involvement in the disease’s pathogenesis. 
 
Figure 1.
 
Schematic representation of the CYP1B1 mutations observed in POAG and PACG. Arrows: location of the mutations within the CYP1B1 gene. Shaded regions: coding regions in exon II and III of CYP1B1 gene.
Figure 1.
 
Schematic representation of the CYP1B1 mutations observed in POAG and PACG. Arrows: location of the mutations within the CYP1B1 gene. Shaded regions: coding regions in exon II and III of CYP1B1 gene.
Table 1.
 
Details of CYP1B1 Mutations Observed in Cases of POAG and PACG in the Present Study and Other Populations
Table 1.
 
Details of CYP1B1 Mutations Observed in Cases of POAG and PACG in the Present Study and Other Populations
Nucleotide Change Location Amino Acid Change Restriction Site Generated, † SIFT Score Number of Patients with the Mutation Controls with the Change Mutation Previously Observed in Other Populations
JOAG POAG PACG JOAG/POAG PCG
g.3987G>A Exon II G61E TaqI (+) 0.00 2/30 0/200 Spain 19 India, 22 Turkey, 30 Ecuador, 31 Saudi Arabia, 30 32 Morocco, 33 Kuwait 34
g.4046T>A Exon II Y81N HaeII (−) 0.00 1/30 0/200 Spain, 19 France 18 Germany 35
g.4236A>C Exon II Q144R* MspA1I (−) 0.01 1/30 1/104 1/90 0/200
g.4381C>T Exon II P193L Eco811 (+) 0.01 3/104 0/200 India 21 India 22
g.4491G>A Exon II E229K Eam1104I (−) 0.10 1/30 4/104 4/90 0/200 Spain, 19 France, 18 India 20 21 India, 22 France 36
g.4520A>C Exon II S239R 0.00 1/104 0/200 India 22
g.7940G>A Exon III R368H TaaI (−) 0.00 1/30 6/104 5/90 0/200 India 20 21 India, 22 Brazil, 37 Saudi Arabia, 30 32 Kuwait 34
g.8137T>C Exon III W434R* 0.00 1/104 0/200
g.8147C>T Exon III P437L 0.00 1/104 0/200 India, 22 Brazil, 37 Turkey 30
g.8171T>G Exon III F445C* 0.03 1/104 0/200
g.8148-8152del5bp Exon III Frame shift* 1/30 0/200
Total 7/30 18/104 10/90 0/200
Table 2.
 
Distribution of Mean Ages at Onset and IOPs at Presentation among JOAG, POAG, and PACG Groups
Table 2.
 
Distribution of Mean Ages at Onset and IOPs at Presentation among JOAG, POAG, and PACG Groups
Parameters JOAG Cases with CYP1B1 Mutations (n = 7) JOAG Cases without CYP1B1 Mutations (n = 23) POAG Cases with CYP1B1 Mutations (n = 18) POAG Cases without CYP1B1 Mutations (n = 86) PACG Cases with CYP1B1 Mutations (n = 10) PACG Cases without CYP1B1 Mutations (n = 80)
% Frequency of Cases (95% CI) 23.3 (11.8–40.9) 76.7 (59.1–88.2) 17.3 (11.2–25.7) 82.7 (74.3–88.7) 11.1 (6.1–19.2) 88.9 (80.7–93.8)
Age at Onset (mean years ± SD) 20.1 ± 8.78 20.9 ± 8.31 51.3 ± 12.22 54.1 ± 10.56 57.4 ± 12.43 54.4 ± 11.18
IOP at Presentation (mean mm Hg ± SD) 37.3 ± 8.43 34.1 ± 8.33 24.8 ± 3.04 27.1 ± 6.26 32.6 ± 16.73 32.9 ± 10.11
Table 3.
 
Distribution of Estimated CYP1B1 Haplotype Frequencies among POAG Cases and Controls
Table 3.
 
Distribution of Estimated CYP1B1 Haplotype Frequencies among POAG Cases and Controls
Haplotypes POAG (All Cases) Controls P POAG CYP1B1(+), † Controls P POAG CYP1B1(−), ‡ Controls P
T-G-T-C-C-A 36.7% 44.8% 0.079 40.1% 44.7% 0.557 36.0% 44.7% 0.072
C-C-G-G-T-A 21.2% 20.1% 0.809 38.4% 20.1% 0.006* 17.9% 20.1% 0.535
C-C-G-C-C-A 22.2% 18.2% 0.287 9.9% 18.3% 0.143 24.3% 18.3% 0.132
C-C-G-C-C-G 17.4% 13.5% 0.252 7.5% 13.3% 0.253 20.6% 13.5% 0.058
Table 4.
 
Distribution of Estimated CYP1B1 Haplotype Frequencies among PACG Cases and Controls
Table 4.
 
Distribution of Estimated CYP1B1 Haplotype Frequencies among PACG Cases and Controls
Haplotypes PACG (All Cases) Controls P PACG CYP1B1(+), † Controls P PACG CYP1B1(−), ‡ Controls P
T-G-T-C-C-A 36.8% 44.8% 0.099 44.9% 44.6% 0.978 37.3% 44.8% 0.130
C-C-G-G-T-A 20.8% 20.1% 0.861 39.9% 20.1% 0.043* 18.3% 20.1% 0.660
C-C-G-C-C-A 21.3% 18.5% 0.463 10.1% 18.4% 0.352 22.1% 18.5% 0.372
C-C-G-C-C-G 14.0% 13.3% 0.831 5.0% 13.3% 0.284 14.6% 13.3% 0.698
Table 5.
 
Characteristics of CYP1B1 Mutations across Different POAG Populations
Table 5.
 
Characteristics of CYP1B1 Mutations across Different POAG Populations
Populations (cases) Median Age at Onset in Years (Range) % Frequency of CYP1B1 Mutation (95% CI) % Frequency of Heterozygous CYP1B1 Mutation (95% CI) Prevalent CYP1B1 Mutation
Canada (n = 60) 17 23.6 (8–36) 5.0 (1.7–13.7) 66.6 (20.8–93.8) R368H
France (n = 236) 18 40 (13–52) 4.6 (2.6–8.2) 90.9 (62.3–98.4) A443G
Spain (n = 82) 19 59.9 (48–77) 11.0 (5.9–19.6) 100.0 (70.1–100.0) Y81N
Eastern India (n = 200) 20 NA* 4.5 (2.4–8.3) 88.9 (56.5–98.1) S515L
Southern India (n = 251) 21 NA* 10.7 (7.5–15.2) 92.5 (76.6–97.9) E229K
Present study; India (n = 134) 46 (10–80) 18.6 (12.9–26.1) 88.0 (80.2–93.0) R368H
Supplementary Materials
Multiple sequence alignment of the CYP protein across different species. The wild type residues for the different missense mutations are indicated by boxes. 
The authors thank all the patients and volunteers for their participation in this study. 
QuigleyHA. Number of people with glaucoma worldwide. Br J Ophthalmol. 1996;80:389–393. [CrossRef] [PubMed]
ResnikoffS, PascoloniD, Etya’aleD, et al. Global data on visual impairment in the year 2002. Bull World Health Org. 2004;82:844–851. [PubMed]
ThomasR, PaulP, MuliyilJ. Glaucoma in India. J Glaucoma. 2003;12:81–87. [CrossRef] [PubMed]
AlvaradoJA, MurphyCG. Outflow obstruction in pigmentary and primary open angle glaucoma. Arch Ophthalmol. 1992;110:1769–1778. [CrossRef] [PubMed]
TielschJM, SommerA, KatzZ, RoyallRM, QuigleyHA, JavittJ. Racial variations in prevalence of primary open angle glaucoma. JAMA. 1991;266:369–374. [CrossRef] [PubMed]
KleinBE, KleinR, SponselWE, et al. Prevalence of glaucoma: The Beaver Dam Eye Study. Ophthalmology. 1999;99:1499–1504.
FosterPJ, JohnsonGJ. Glaucoma in China: How big is the problem?. Br J Ophthalmol. 2001;85:1277–1282. [CrossRef] [PubMed]
FosterPJ, OenFT, MachinD, et al. The prevalence of glaucoma in Chinese residents of Singapore: a cross sectional population survey of the Tanjong Pagar district. Arch Ophthalmol. 2000;118:1105–1111. [CrossRef] [PubMed]
FanBJ, WangDY, LamDSC, PangCP. Gene mapping for primary open angle glaucoma. Clin Biochem. 2006;39:249–258. [CrossRef] [PubMed]
StoneEM, FingertJH, AlwardWL, et al. Identification of a gene that causes primary open angle glaucoma. Science. 1997;275:668–670. [CrossRef] [PubMed]
RezaieT, ChildA, HitchingsR, et al. Adult-onset primary open angle glaucoma caused by mutations in optineurin. Science. 2002;295:1077–1079. [CrossRef] [PubMed]
MonemiS, SpaethG, DaSilvaA, et al. Identification of a novel adult-onset primary open angle glaucoma (POAG) gene on 5q22.1. Hum Mol Genet. 2005;14:725–733. [CrossRef] [PubMed]
LibbyRT, GouldDB, AndersonMG, JohnSW. Complex genetics of glaucoma susceptibility. Annu Rev Genomics Hum Genet. 2005;6:15–44. [CrossRef] [PubMed]
StoilovI, AkarsuAN, SarfaraziM. Identification of three different truncating mutations in cytochrome P4501B1 (CYP1B1) as the principal cause of primary congenital glaucoma (buphthalmos) in families linked to the GLC3A locus on chromosome 2p21. Hum Mol Genet. 1997;6:641–647. [CrossRef] [PubMed]
SarfaraziM, StoilovI. Molecular genetics of primary congenital glaucoma. Eye. 2000;14:422–428. [CrossRef] [PubMed]
ChakrabartiS, KaurK, KaurI, et al. Globally, CYP1B1 mutations in primary congenital glaucoma are strongly structured by geographic and haplotype backgrounds. Invest Ophthalmol Vis Sci. 2006;47:43–47. [CrossRef] [PubMed]
VincentAL, BillingsleyG, BuysY, et al. Digenic inheritance of early onset glaucoma: CYP1B1, a potential modifier gene. Am J Hum Genet. 2002;70:448–460. [CrossRef] [PubMed]
MelkiR, ColombE, LefortN, BrezinAP, GarchonH-J. CYP1B1 mutations in French patients with early-onset primary open-angle glaucoma. J Med Genet. 2004;41:647–651. [CrossRef] [PubMed]
Lopez-GarridoMP, Sanchez-SanchezF, Lopez-MartinezF, et al. Heterozygous CYP1B1 gene mutations in Spanish patients with primary open-angle glaucoma. Mol Vis. 2006;12:748–755. [PubMed]
AcharyaM, MookherjeeS, BhattacharjeeA, et al. Primary role of CYP1B1 in Indian juvenile-onset POAG patients. Mol Vis. 2006;12:399–404. [PubMed]
KumarA, BasavarajMG, GuptaSK, et al. Role of CYP1B1, MYOC, OPTN and OPTC genes in adult-onset primary open-angle glaucoma: predominance of CYP1B1 mutations in Indian patients. Mol Vis. 2007;13:667–676. [PubMed]
ReddyABM, KaurK, MandalAK, et al. Mutation spectrum of the CYP1B1 gene in Indian primary congenital glaucoma patients. Mol Vis. 2004;10:696–702. [PubMed]
AcharyMS, ReddyABM, ChakrabartiS, et al. Disease-causing mutations in proteins: structural analysis of the CYP1b1 mutations causing primary congenital glaucoma in humans. Biophys J. 2006;91:4329–4339. [CrossRef] [PubMed]
AndersonDR, PatellaVM. Automated Static Perimetry. 1999; 2nd ed. 121–136.Mosby St. Louis, MO.
HodappE, ParrishRK, II, AndersonDR. Clinical Decisions in Glaucoma. 1993;63–92.Mosby St. Louis, MO.
SambrookJ, FritschEF, ManiatisT. Molecular Cloning: A Laboratory Manual. 1989; 2nd ed. 17–19.Cold Spring Harbor Press Cold Spring Harbor, NY.
PanickerSG, ReddyABM, MandalAK, et al. Identification of novel mutations causing familial primary congenital glaucoma in Indian pedigrees. Invest Ophthalmol Vis Sci. 2002;43:1358–1366. [PubMed]
NgPC, HenikoffS. SIFT: predicting amino acid changes that affect protein function. Nucleic Acids Res. 2003;31:3812–3814. [CrossRef] [PubMed]
BarrettJC, FryB, MallerJ, DalyMJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 2005;21:263–265. [CrossRef] [PubMed]
BejjaniBA, LewisRA, TomeyK, et al. Mutations in CYP1B1, the gene for cytochrome P4501B1, are the predominant causes of primary congenital glaucoma in Saudi Arabia. Am J Hum Genet. 1998;62:325–333. [CrossRef] [PubMed]
CurrySM, DaouAG, HermannsP, MolinariA, LewisRA, BejjaniBA. Cytochrome P450 1B1 mutations cause only part of primary congenital glaucoma in Ecuador. Ophthalmic Genet. 2004;25:3–9. [CrossRef] [PubMed]
BejjaniBA, StocktonDW, LewisRA, et al. Multiple CYP1B1 mutations and incomplete penetrance in an inbred population segregating primary congenital glaucoma suggest frequent de novo events and a dominant modifier locus. Hum Mol Genet. 2000;9:367–374. [CrossRef] [PubMed]
BelmoudenA, MelkiR, HamdaniM, et al. A novel frameshift founder mutation in the cytochrome P450 (CYP1B1) gene is associated with primary congenital glaucoma in Morocco. Invest Ophthalmol Vis Sci. 2002;62:334–339.
AlfadhliS, BehbehaniA, ElshafeyA, AbdelmoatyS, Al-AwadiS. Molecular and clinical evaluation of primary congenital glaucoma in Kuwait. Am J Ophthalmol. 2006;141:512–516. [CrossRef] [PubMed]
Chavarria-SoleyG, Michels-RautenstraussK, PasuttoF, et al. Primary congenital glaucoma and Rieger’s anomaly: extended haplotypes reveal founder effects for eight distinct CYP1B1 mutations. Mol Vis. 2006;12:523–531. [PubMed]
ColombE, KaplanJ, GarchonH-J. Novel cytochrome P4501B1 (CYP1B1) mutations in patients with primary congenital glaucoma in France. Hum Mutat. 2003;22:496.
StoilovIR, CostaVP, VasconcellosJPC, et al. Molecular genetics of primary congenital glaucoma in Brazil. Invest Ophthalmol Vis Sci. 2002;43:1820–1827. [PubMed]
ChambersD, WilsonL, MadenM, LumsdenA. RALDH-independent generation of retinoic acid during vertebrate embryogenesis by CYP1B1. Development. 2007;134:1369–1383. [CrossRef] [PubMed]
BejjaniBA, XuL, ArmstrongD, LupskiJR, RenekerLW. Expression patterns of cytochrome P4501B1 (Cyp1b1) in FVB/N mouse eyes. Exp Eye Res. 2002;75:249–257. [CrossRef] [PubMed]
StoilovI, RezaieT, JanssonI, SchenkmanJB, SarfaraziM. Expression of cytochrome P4501B1 (Cyp1b1) during early murine development. Mol Vis. 2004;10:629–636. [PubMed]
DoshiM, MarcusC, BejjaniBA, EdwardDP. Immunolocalization of CYP1B1 in normal, human, fetal and adult eyes. Exp Eye Res. 2006;82:24–32. [CrossRef] [PubMed]
Figure 1.
 
Schematic representation of the CYP1B1 mutations observed in POAG and PACG. Arrows: location of the mutations within the CYP1B1 gene. Shaded regions: coding regions in exon II and III of CYP1B1 gene.
Figure 1.
 
Schematic representation of the CYP1B1 mutations observed in POAG and PACG. Arrows: location of the mutations within the CYP1B1 gene. Shaded regions: coding regions in exon II and III of CYP1B1 gene.
Table 1.
 
Details of CYP1B1 Mutations Observed in Cases of POAG and PACG in the Present Study and Other Populations
Table 1.
 
Details of CYP1B1 Mutations Observed in Cases of POAG and PACG in the Present Study and Other Populations
Nucleotide Change Location Amino Acid Change Restriction Site Generated, † SIFT Score Number of Patients with the Mutation Controls with the Change Mutation Previously Observed in Other Populations
JOAG POAG PACG JOAG/POAG PCG
g.3987G>A Exon II G61E TaqI (+) 0.00 2/30 0/200 Spain 19 India, 22 Turkey, 30 Ecuador, 31 Saudi Arabia, 30 32 Morocco, 33 Kuwait 34
g.4046T>A Exon II Y81N HaeII (−) 0.00 1/30 0/200 Spain, 19 France 18 Germany 35
g.4236A>C Exon II Q144R* MspA1I (−) 0.01 1/30 1/104 1/90 0/200
g.4381C>T Exon II P193L Eco811 (+) 0.01 3/104 0/200 India 21 India 22
g.4491G>A Exon II E229K Eam1104I (−) 0.10 1/30 4/104 4/90 0/200 Spain, 19 France, 18 India 20 21 India, 22 France 36
g.4520A>C Exon II S239R 0.00 1/104 0/200 India 22
g.7940G>A Exon III R368H TaaI (−) 0.00 1/30 6/104 5/90 0/200 India 20 21 India, 22 Brazil, 37 Saudi Arabia, 30 32 Kuwait 34
g.8137T>C Exon III W434R* 0.00 1/104 0/200
g.8147C>T Exon III P437L 0.00 1/104 0/200 India, 22 Brazil, 37 Turkey 30
g.8171T>G Exon III F445C* 0.03 1/104 0/200
g.8148-8152del5bp Exon III Frame shift* 1/30 0/200
Total 7/30 18/104 10/90 0/200
Table 2.
 
Distribution of Mean Ages at Onset and IOPs at Presentation among JOAG, POAG, and PACG Groups
Table 2.
 
Distribution of Mean Ages at Onset and IOPs at Presentation among JOAG, POAG, and PACG Groups
Parameters JOAG Cases with CYP1B1 Mutations (n = 7) JOAG Cases without CYP1B1 Mutations (n = 23) POAG Cases with CYP1B1 Mutations (n = 18) POAG Cases without CYP1B1 Mutations (n = 86) PACG Cases with CYP1B1 Mutations (n = 10) PACG Cases without CYP1B1 Mutations (n = 80)
% Frequency of Cases (95% CI) 23.3 (11.8–40.9) 76.7 (59.1–88.2) 17.3 (11.2–25.7) 82.7 (74.3–88.7) 11.1 (6.1–19.2) 88.9 (80.7–93.8)
Age at Onset (mean years ± SD) 20.1 ± 8.78 20.9 ± 8.31 51.3 ± 12.22 54.1 ± 10.56 57.4 ± 12.43 54.4 ± 11.18
IOP at Presentation (mean mm Hg ± SD) 37.3 ± 8.43 34.1 ± 8.33 24.8 ± 3.04 27.1 ± 6.26 32.6 ± 16.73 32.9 ± 10.11
Table 3.
 
Distribution of Estimated CYP1B1 Haplotype Frequencies among POAG Cases and Controls
Table 3.
 
Distribution of Estimated CYP1B1 Haplotype Frequencies among POAG Cases and Controls
Haplotypes POAG (All Cases) Controls P POAG CYP1B1(+), † Controls P POAG CYP1B1(−), ‡ Controls P
T-G-T-C-C-A 36.7% 44.8% 0.079 40.1% 44.7% 0.557 36.0% 44.7% 0.072
C-C-G-G-T-A 21.2% 20.1% 0.809 38.4% 20.1% 0.006* 17.9% 20.1% 0.535
C-C-G-C-C-A 22.2% 18.2% 0.287 9.9% 18.3% 0.143 24.3% 18.3% 0.132
C-C-G-C-C-G 17.4% 13.5% 0.252 7.5% 13.3% 0.253 20.6% 13.5% 0.058
Table 4.
 
Distribution of Estimated CYP1B1 Haplotype Frequencies among PACG Cases and Controls
Table 4.
 
Distribution of Estimated CYP1B1 Haplotype Frequencies among PACG Cases and Controls
Haplotypes PACG (All Cases) Controls P PACG CYP1B1(+), † Controls P PACG CYP1B1(−), ‡ Controls P
T-G-T-C-C-A 36.8% 44.8% 0.099 44.9% 44.6% 0.978 37.3% 44.8% 0.130
C-C-G-G-T-A 20.8% 20.1% 0.861 39.9% 20.1% 0.043* 18.3% 20.1% 0.660
C-C-G-C-C-A 21.3% 18.5% 0.463 10.1% 18.4% 0.352 22.1% 18.5% 0.372
C-C-G-C-C-G 14.0% 13.3% 0.831 5.0% 13.3% 0.284 14.6% 13.3% 0.698
Table 5.
 
Characteristics of CYP1B1 Mutations across Different POAG Populations
Table 5.
 
Characteristics of CYP1B1 Mutations across Different POAG Populations
Populations (cases) Median Age at Onset in Years (Range) % Frequency of CYP1B1 Mutation (95% CI) % Frequency of Heterozygous CYP1B1 Mutation (95% CI) Prevalent CYP1B1 Mutation
Canada (n = 60) 17 23.6 (8–36) 5.0 (1.7–13.7) 66.6 (20.8–93.8) R368H
France (n = 236) 18 40 (13–52) 4.6 (2.6–8.2) 90.9 (62.3–98.4) A443G
Spain (n = 82) 19 59.9 (48–77) 11.0 (5.9–19.6) 100.0 (70.1–100.0) Y81N
Eastern India (n = 200) 20 NA* 4.5 (2.4–8.3) 88.9 (56.5–98.1) S515L
Southern India (n = 251) 21 NA* 10.7 (7.5–15.2) 92.5 (76.6–97.9) E229K
Present study; India (n = 134) 46 (10–80) 18.6 (12.9–26.1) 88.0 (80.2–93.0) R368H
Supplementary Figure S1
Supplementary Figure S2
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×