|Molecular Vision 2005;
Received 1 June 2004 | Accepted 13 December 2005 | Published 21 January 2005
A compound heterozygous change found in Peters' anomaly
Amanda Jane Churchill,
Department of Ophthalmology, University of Bristol, Bristol, United Kingdom
Correspondence to: Amanda Jane Churchill, Bristol Eye Hospital, Lower Maudlin Street, Bristol, BS1 2LX, UK; Phone: (+44) 117-928-4949, FAX: (+44) 117-925-1421; email: email@example.com
Purpose: To determine whether sequence variations in the congenital glaucoma gene, CYP1B1, are present in individuals with Peters' anomaly, a developmental eye anomaly frequently associated with glaucoma.
Methods: The CYP1B1 coding region was screened in 26 individuals with Peters' anomaly (9 familial and 17 simplex cases) by heteroduplex analysis using the Transgenomic Wave® nucleic acid fragment analysis system. Deviations from the wild type pattern were determined by sequencing.
Results: Six nucleotide positions varied from the wild type. Four of these have previously been observed in clinically normal individuals: -13 in intervening sequence 1 (IVS1), codons 48, 432, and 449. We found a novel sequence variation at -16 in IVS1 in one affected individual. A novel compound heterozygote pattern was observed at codon 432 in 6 of our 26 unrelated cases with Peters' anomaly.
Conclusions: This is the first report of 2 novel CYP1B1 sequence variations seen in Peters' anomaly. The -16 IVS1 change is outside the coding region and likely to be a rare polymorphism. The compound heterozygous change at codon 432 is within a conserved part of the coding region and substitutes valine with either leucine or arginine. This change has not been observed in 100 normal controls. Furthermore, we propose how this finding may affect protein function.
CYP1B1 is a member of the cytochrome P450 enzyme family concerned with Phase I metabolism of a wide variety of compounds. Although generally involved with detoxification, CYP1B1 sequence variations have been associated with a wide variety of human tumors including colonic adenocarcinoma, endometrial, and prostrate cancer, and ocular disease. In 1997 Stoilov et al.  reported mutations in CYP1B1 as the cause of primary congenital glaucoma (PCG) in families linked to the GLC3A locus on chromosome 2p21. Since then there have been a number of publications recounting individuals with PCG and mutations in the CYP1B1 gene with no apparent genotype-phenotype correlation [2-4]. Gene expression studies have shown that CYP1B1 transcripts are abundant in human trabecular meshwork, along with transcripts from other glaucoma genes such as myocilin, PITX2 and optineurin . The exact mechanism of action of CYP1B1 in the eye is not understood. In 2001 Vincent et al.  described a child with features of Peters' anomaly and congenital glaucoma with a compound heterozygous mutation in CYP1B1, the effect of which was predicted to cause premature truncation of the protein. This is the only case to date of an alternate ocular phenotype being caused by a CYP1B1 mutation. We undertook the present study to determine whether sequence changes in CYP1B1 are likely to be a major cause of Peters' anomaly.
Twenty-six individuals (9 familial and 17 simplex cases) with Peters' anomaly had undergone a thorough ophthalmologic examination by one of the authors (AJC) and formed part of a panel for screening novel genes implicated in anterior segment development. The diagnostic clinical criteria  used for Peters' anomaly included the presence of a central or peripheral corneal leukoma with or without iris adhesions, with an anatomically normal retina (where ultrasound examination or direct visualization was possible). Lens opacities were present in some individuals. No individual had raised intraocular pressure documented at birth, nor did any show signs of congenital glaucoma such as Haab's striae or buphthalmos. Approximately 50% developed raised pressure later in life. The genetic background of the individuals was mixed but the majority were from the UK (2 Asian, 2 Arabian, 5 USA, 2 Ireland, 15 UK). Most individuals had previously been screened for mutations in PAX6 and no disease causing mutations were identified . Ethics approval for the study was obtained from the United Bristol Healthcare Trust and was carried out in accordance with the Declaration of Helsinki. Peripheral blood samples were obtained from all participants after informed consent.
DNA was extracted using either standard phenol/chloroform extraction, or by the Genomic DNA Purification System (Whatman BioScience, UK).
Six overlapping primer sets were used to amplify the coding region of the CYP1B1 gene (Table 1). PCR reactions were carried out in a DNA engine DYAD Peltier Thermal Cycler (MJ Research, UK) using Amplitaq Gold DNA Polymerase (Applied Biosystems, Warrington, UK). DMSO 10% was added to reactions for primer sets 1A, 1B, and 2A. Cycling parameters were 10 min at 94 °C, followed by 30 cycles of denaturation at 94 °C for 45 s, annealing at 52-60 °C for 30 s and extension at 72 °C for 1 min. Final extension was at 72 °C for 10 min. PCR products were checked on a 1.5% agarose gel containing ethidium bromide.
Denaturing high performance liquid chromatography (dHPLC) on the Transgenomic Wave® nucleic acid fragment analysis system (Transgenomic, Crewe, UK) was used to screen samples for sequence variations. Since CYP1B1 mutations may be homozygous and dHPLC Wave® technology works on the basis of heteroduplex formation, all samples were mixed with an equal volume of wild type sample prior to hybridization. Samples were heated to 95 °C for 5 min, and then cooled to 34 °C over a 40 min period to induce heteroduplex formation. CYP1B1 analysis was performed using an 8 μl injection volume, the standard protocol for the Transgenomic Wave®heteroduplex detection method at melting temperatures 59.1-66.0 °C and a slope of 2.0% increase in buffer B (0.1 M TEAA, 25% acetonitrile) per min. The resulting chromatograms were studied for deviation from the wild type homozygous pattern. Two clinically normal individuals were analysed alongside the samples as negative controls.
PCR products were purified using QIAquick columns (Qiagen, Crawley, UK) before being sequenced bidirectionally on a LICOR 4200 DNA sequencer. Sequencing reactions were carried out using the Thermo Sequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech, Chalfont St. Giles, UK). M13 sequencing primers were synthesized by MWG-Biotech AG (Ebersberg, Germany). Sequencing reactions were loaded onto a polyacrylamide Sequagel XR gel (National Diagnostics, Hessle, UK) and run at 2000 V, 50 mA and 50 °C for 2-3 h.
The coding region of the CYP1B1 gene was analysed in 6 overlapping fragments: 1A, 1B, 2A, 2B, 3A, and 3B. Twenty-eight sequence changes were detected in individuals with Peters' anomaly. Sequence variations were found in Fragments 1A, 3A, and 3B; the positive results are summarized in Table 2.
Nine subjects exhibited a pattern that deviated from the wild type (Figure 1) but was present in one of the negative control individuals. Sequencing showed the negative control to have a heterozygous single base change in codon 48 (CGG>GGG) resulting in arginine being replaced by glycine and a heterozygous C>T at the -13 position of Intron 1. Seven of the subjects had the same two heterozygous changes and one had the heterozygous change in codon 48 and an additional heterozygous C>T change in the -16 position upstream of the initiation codon. There was one individual with Peters' anomaly (P5) who had a homozygous C>G change at codon 48 and a homozygous C>T change in the -13 nucleotide. This would result in glycine replacing arginine in both copies of the CYP1B1 product.
Twelve subjects exhibited a pattern that deviated from the wild type (Figure 2) but was present in one of the negative control individuals. Sequencing showed the negative control to have a heterozygous single base change in codon 432 (GTG>CTG) resulting in valine being replaced by leucine. Five of the subjects had the same change but it was homozygous in nature. Four of these were familial with all affecteds having the same genotype; one was a simplex case. Seven further individuals with Peters' anomaly showed not only the homozygous change in the first base of codon 432 but also a heterozygous change in the second base of the same codon resulting in the substitution of the amino acid arginine. For these seven individuals, instead of a valine in each copy of the CYP1B1 protein they would have a leucine residue in one copy and an arginine residue in the other.
Three subjects exhibited a pattern that deviated from the wild type but was present in both negative control samples and six subjects gave poor chromatograms and so were directly sequenced. Sequencing showed the negative controls to have a conservative heterozygous single base change in codon 449 (GAT>GAC) resulting in no amino acid change. Four of the subjects had the same heterozygous change but in a further five it was homozygous in nature.
Peters' anomaly has been observed in families but the vast majority are simplex cases with no family history nor parental consanguinity. A few cases have been associated with mutations in PAX6, PITX2, and FOXE3 [9-11]. In 2001, Vincent et al.  reported a simplex case of Peters' anomaly and congenital glaucoma with a compound heterozygous mutation (M1T and W57X) in exon 2 of CYP1B1. The combined mutations in this patient are predicted to disrupt initiation of translation and truncate the remaining polypeptide, respectively, thereby reducing the amount of functional CYP1B1. Ten additional unrelated individuals with Peters' anomaly were screened by SSCP for sequence changes in CYP1B1 but none were identified. Both of these mutations have since been reported individually in association with PCG (homozygote M1T and W57X in combination with either R368H or 7901-7913 delGAGTGCAGGCAGA ). These findings suggest that it is not possible to predict the ocular phenotype from the genotype.
In 1998, Stoilov et al.  reported six polymorphisms in the CYP1B1 gene after screening normal controls from the United Kingdom (n=50) and Turkey (n=50). These include the variations we detected in fragment 1A in the -13 nucleotide of Intron 1, codon 48, the first base of codon 432 in fragment 3A, and codon 449 in fragment 3B. Since the population base is similar (50 normals from UK), we have used the data from the Stoilov study as a basis for comparison with the data from individuals with Peters' anomaly in this study.
When the percentage of the codon 48 and -13 polymorphisms found in the Peters' anomaly patients from this study are compared to those found in the Stoilov normal controls, the trends are similar. Formal statistical analysis is unhelpful because the sample sizes are so different (Table 3). The -16 nucleotide change in Intron 1 is novel and was found in one individual with Peters' anomaly. We have only sequenced two normal controls so we cannot exclude this as a polymorphism but it was not detected in 100 normal controls screened by direct sequencing in the Stoilov study.
Stoilov et al.  detected the homozygous/heterozygous codon 449 sequence variation in fragment 3B in 51% and 39% normal controls, respectively. The change is silent with no alteration of the amino acid. Our percentages are lower in Peters' anomaly patients at 20% and 15%, respectively. While it is tempting to speculate that the wild type should in fact be GAC at this position, and that tissue specific codon usage may be relevant here, the differences in percentages between the two studies may just reflect the smaller numbers of patients in our study.
Eleven percent of normal controls and 54% individuals with Peters' anomaly had the wild type valine at codon 432. In 2002, Stoilov et al.  reported that 42% Brazilians with PCG had valine at this position compared to 23% Brazilian controls. They comment that this is the only CYP1B1 polymorphism for which there is a reversal in the allele frequency between PCG and control groups. Valine in this position has been shown to increase the production of the carcinogen 4-hydroxyestradiol and is thought to increase susceptibility to ovarian, endometrial, prostate, and smoking related head and neck squamous cell carcinomas [13-15]. The high numbers of normal controls with leucine in place of valine (54% UK) might be evolutionary in reducing the risk of cancer. Five individuals with Peters' anomaly (19%) were heterozygous valine/leucine compared to 35% normal controls. What is more interesting is that seven individuals with Peters' anomaly were compound heterozygotes at codon 432 with leucine on one allele and arginine on the other. Two of these individuals are related (P3A and P3B) so the true percentage of unrelated compound heterozygote cases is 23% (6/26). This sequence variation was not detected in 100 normal controls in the Stoilov study . Since the individuals in our study herald from genetically diverse origins this finding cannot be attributed to a founder effect. If it were purely of evolutionary benefit to rid oneself of the valine residues, one would expect normal controls to have the same frequency of compound heterozygotes. As this is not the case, one might speculate that this genotype may be responsible for the Peters' anomaly phenotype. Valine and leucine are fairly compact amino acids, nonpolar and hydrophobic so this change alone may not be significant. Rat and mouse cyp1b1 have alanine at position 432, another small, nonpolar amino acid. Arginine, however, is relatively bulky in comparison, is basic, positively charged and hydrophilic. Codon 432 lies within a run of 13 amino acids (HDPVKWPNPENFD) where just three residues differ between human, mouse, and rat cyp1b1, just prior to the "meander" region of the protein . This region may influence correct folding of the protein, and it is possible that the presence of arginine/leucine could disrupt this and interfere with the function of CYP1B1. It has not yet been determined what level of functional CYP1B1 is required for normal ocular development. Functional studies of two other CYP1B1 mutations have yielded complex biochemical phenotypes making simple genotype-phenotype comparisons impossible . Since the true significance of these changes will not be fully understood until functional studies can be undertaken, we suggest that further investigation of this compound heterozygote is merited to determine whether this is the likely genetic cause of Peters' anomaly in these individuals.
In summary we have detected a novel sequence variation at the -16 position of Intron 1 in one individual with Peters' anomaly, and we have identified a compound heterozygous change, V432L/R, in 23% of unrelated individuals with Peters' anomaly.
We thank Dr. Brenda Powell for helping to supervise Anna Yeung, Dr. Linda Tyfield for the use of DNA sequencing facilities, and all the individuals who took part in this study. We acknowledge the generous financial support from the National Eye Research Center, UK and the Medical Research Committee of the Charitable Trusts for the United Bristol Hospitals, UK.
1. Stoilov I, Akarsu AN, Sarfarazi M. 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; 6:641-7.
2. Stoilov IR, Costa VP, Vasconcellos JP, Melo MB, Betinjane AJ, Carani JC, Oltrogge EV, Sarfarazi M. Molecular genetics of primary congenital glaucoma in Brazil. Invest Ophthalmol Vis Sci 2002; 43:1820-7.
3. Panicker SG, Reddy AB, Mandal AK, Ahmed N, Nagarajaram HA, Hasnain SE, Balasubramanian D. Identification of novel mutations causing familial primary congenital glaucoma in Indian pedigrees. Invest Ophthalmol Vis Sci 2002; 43:1358-66.
4. Soley GC, Bosse KA, Flikier D, Flikier P, Azofeifa J, Mardin CY, Reis A, Michels-Rautenstrauss KG, Rautenstrauss BW. Primary congenital glaucoma: a novel single-nucleotide deletion and varying phenotypic expression for the 1,546-1,555dup mutation in the GLC3A (CYP1B1) gene in 2 families of different ethnic origin. J Glaucoma 2003; 12:27-30.
5. Tomarev SI, Wistow G, Raymond V, Dubois S, Malyukova I. Gene expression profile of the human trabecular meshwork: NEIBank sequence tag analysis. Invest Ophthalmol Vis Sci 2003; 44:2588-96.
6. Vincent A, Billingsley G, Priston M, Williams-Lyn D, Sutherland J, Glaser T, Oliver E, Walter MA, Heathcote G, Levin A, Heon E. Phenotypic heterogeneity of CYP1B1: mutations in a patient with Peters' anomaly. J Med Genet 2001; 38:324-6.
7. Peters A. Uber angeboren defektbildung der descementschen membran. Klin Monatsbl Augenheilkd 1906; 44:27-40.
8. Churchill AJ, Booth AP, Anwar R, Markham AF. PAX 6 is normal in most cases of Peters' anomaly. Eye 1998; 12:299-303.
9. Hanson IM, Fletcher JM, Jordan T, Brown A, Taylor D, Adams RJ, Punnett HH, van Heyningen V. Mutations at the PAX6 locus are found in heterogeneous anterior segment malformations including Peters' anomaly. Nat Genet 1994; 6:168-73.
10. Doward W, Perveen R, Lloyd IC, Ridgway AE, Wilson L, Black GC. A mutation in the RIEG1 gene associated with Peters' anomaly. J Med Genet 1999; 36:152-5.
11. Ormestad M, Blixt A, Churchill A, Martinsson T, Enerback S, Carlsson P. Foxe3 haploinsufficiency in mice: a model for Peters' anomaly. Invest Ophthalmol Vis Sci 2002; 43:1350-7.
12. Stoilov I, Akarsu AN, Alozie I, Child A, Barsoum-Homsy M, Turacli ME, Or M, Lewis RA, Ozdemir N, Brice G, Aktan SG, Chevrette L, Coca-Prados M, Sarfarazi M. Sequence analysis and homology modeling suggest that primary congenital glaucoma on 2p21 results from mutations disrupting either the hinge region or the conserved core structures of cytochrome P4501B1. Am J Hum Genet 1998; 62:573-84.
13. Sasaki M, Kaneuchi M, Fujimoto S, Tanaka Y, Dahiya R. CYP1B1 gene in endometrial cancer. Mol Cell Endocrinol 2003; 202:171-6.
14. Chang B, Zheng SL, Isaacs SD, Wiley KE, Turner AR, Hawkins GA, Bleecker ER, Walsh PC, Meyers DA, Isaacs WB, Xu J. A common haplotype of CYP1B1 is associated with prostrate cancer risk in Caucasians. Am J Hum Genet 2002; 71:S418.
15. Ko Y, Abel J, Harth V, Brode P, Antony C, Donat S, Fischer HP, Ortiz-Pallardo ME, Thier R, Sachinidis A, Vetter H, Bolt HM, Herberhold C, Bruning T. Association of CYP1B1 codon 432 mutant allele in head and neck squamous cell cancer is reflected by somatic mutations of p53 in tumor tissue. Cancer Res 2001; 61:4398-404.
16. Graham-Lorence SE, Peterson JA. Structural alignments of P450s and extrapolations to the unknown. Methods Enzymol 1996; 272:315-26.
17. Jansson I, Stoilov I, Sarfarazi M, Schenkman JB. Effect of two mutations of human CYP1B1, G61E and R469W, on stability and endogenous steroid substrate metabolism. Pharmacogenetics 2001; 11:793-801.