CYP1B1 Mutation Profile of Iranian Primary Congenital Glaucoma Patients and Associated Haplotypes
2007; Elsevier BV; Volume: 9; Issue: 3 Linguagem: Inglês
10.2353/jmoldx.2007.060157
ISSN1943-7811
AutoresFereshteh Chitsazian, Betsabeh Khoramian Tusi, Elahe Elahi, Heidar Amini Saroei, Mohammad Hossein Sanati, Shahin Yazdani, Mohammad Pakravan, Navid Nilforooshan, Yadollah Eslami, Mohammad Ali Zare Mehrjerdi, R Zareei, Mahmood Jabbarvand, Ali Abdolahi, Ali R. Lasheyee, Arash Etemadi, Behnaz Bayat, Mehdi Sadeghi, Mohammad Mehdi Banoei, Behnam Ghafarzadeh, Mohammad Reza Rohani, A Rismanchian, Yvonne R. Thorstenson, Mansoor Sarfarazi,
Tópico(s)Genetic Associations and Epidemiology
ResumoThe mutation spectrum of CYP1B1 among 104 primary congenital glaucoma patients of the genetically heterogeneous Iranian population was investigated by sequencing. We also determined intragenic single nucleotide polymorphism (SNP) haplotypes associated with the mutations and compared these with haplotypes of other populations. Finally, the frequency distribution of the haplotypes was compared among primary congenital glaucoma patients with and without CYP1B1 mutations and normal controls. Genotype classification of six high-frequency SNPs was performed using the PHASE 2.0 software. CYP1B1 mutations in the Iranian patients were very heterogeneous. Nineteen nonconservative mutations associated with disease, and 10 variations not associated with disease were identified. Ten mutations and three variations not associated with disease were novel. The 13 novel variations make a notable contribution to the ∼70 known variations in the gene. CYP1B1 mutations were identified in 70% of the patients. The four most common mutations were G61E, R368H, R390H, and R469W, which together constituted 76.2% of the CYP1B1 mutated alleles found. Six unique core SNP haplotypes were identified, four of which were common to the patients with and without CYP1B1 mutations and controls studied. Three SNP blocks determined the haplotypes. Comparison of haplotypes with those of other populations suggests a common origin for many of the mutations. The mutation spectrum of CYP1B1 among 104 primary congenital glaucoma patients of the genetically heterogeneous Iranian population was investigated by sequencing. We also determined intragenic single nucleotide polymorphism (SNP) haplotypes associated with the mutations and compared these with haplotypes of other populations. Finally, the frequency distribution of the haplotypes was compared among primary congenital glaucoma patients with and without CYP1B1 mutations and normal controls. Genotype classification of six high-frequency SNPs was performed using the PHASE 2.0 software. CYP1B1 mutations in the Iranian patients were very heterogeneous. Nineteen nonconservative mutations associated with disease, and 10 variations not associated with disease were identified. Ten mutations and three variations not associated with disease were novel. The 13 novel variations make a notable contribution to the ∼70 known variations in the gene. CYP1B1 mutations were identified in 70% of the patients. The four most common mutations were G61E, R368H, R390H, and R469W, which together constituted 76.2% of the CYP1B1 mutated alleles found. Six unique core SNP haplotypes were identified, four of which were common to the patients with and without CYP1B1 mutations and controls studied. Three SNP blocks determined the haplotypes. Comparison of haplotypes with those of other populations suggests a common origin for many of the mutations. Glaucoma is a heterogeneous group of optic neuropathies with common manifestations including a specific pattern of visual field loss and degeneration of the optic nerve resulting in a characteristic glaucomatous appearance.1Sarfarazi M Recent advances in molecular genetics of glaucomas.Hum Mol Genet. 1997; 6: 1667-1677Crossref PubMed Scopus (157) Google Scholar,2Ray K Mukhopadhyay A Acharya M Recent advances in molecular genetics of glaucoma.Mol Cell Biochem. 2003; 253: 223-231Crossref PubMed Scopus (60) Google Scholar Degeneration of the optic nerve may be caused by apoptosis of retinal ganglion cells.3Farkas RH Grosskreutz CL Apoptosis, neuroprotection, and retinal ganglion cell death: an overview.Int Ophthalmol Clin. 2001; 41: 111-130Crossref PubMed Scopus (65) Google Scholar Glaucoma leads to blindness if left untreated, and it is considered the second leading cause of blindness worldwide.4Thylefors B Negrel AD The global impact of glaucoma.Bull World Health Organ. 1994; 72: 323-326PubMed Google Scholar The disease is subclassified based on etiology, anatomy of the anterior chamber of the eye, and age of onset.2Ray K Mukhopadhyay A Acharya M Recent advances in molecular genetics of glaucoma.Mol Cell Biochem. 2003; 253: 223-231Crossref PubMed Scopus (60) Google Scholar The subgroup primary congenital glaucoma (PCG; Online Mendelian Inheritance of Man no. 231300) is a severe form of the disease. It is characterized by an anatomical defect of the trabecular meshwork (trabeculodysgenesis) and an age of onset in the neonatal or infantile period, generally before the age of 3 years.5Sarfarazi M Stoilov I Schenkman JB Genetics and biochemistry of primary congenital glaucoma.Ophthalmol Clin North Am. 2003; 16: 543-554Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar The developmental anomaly at the angle of the anterior chamber manifests itself by increased intraocular pressure (IOP), corneal edema, excessive tearing, photophobia, enlargement of the globe (buphthalmos), and opacity of the cornea. The details of the pathogenic pathways, including the relationship between elevated IOP and optic nerve damage, are not well understood. PCG occurs in both sporadic and familial patterns. In familial cases, inheritance is usually autosomal recessive, sometimes associated with incomplete penetrance.5Sarfarazi M Stoilov I Schenkman JB Genetics and biochemistry of primary congenital glaucoma.Ophthalmol Clin North Am. 2003; 16: 543-554Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar,6Bejjani BA Stockton DW Lewis RA Tomey KF Dueker DK Jabak M Astle WF Lupski JR 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: 367-374Crossref PubMed Google Scholar Pseudodominant transmission has also been reported.7Hewitt AW Mackinnon JR Elder JE Giubilato A Craig JE Mackey DA Familial transmission patterns of infantile glaucoma in Australia (abstract).Invest Ophthalmol Vis Sci. 2005; 46: E3207Google Scholar The incidence of PCG is geographically and ethnically variable, estimated at 1:10,000 in Western countries5Sarfarazi M Stoilov I Schenkman JB Genetics and biochemistry of primary congenital glaucoma.Ophthalmol Clin North Am. 2003; 16: 543-554Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar and higher in inbred populations such as those of Andhra Pradesh in India (1:3300),8Dandona L Williams JD Williams BC Rao GN Population-based assessment of childhood blindness in southern India.Arch Ophthalmol. 1998; 116: 545-546PubMed Google Scholar Saudi Arabia (1:2500),6Bejjani BA Stockton DW Lewis RA Tomey KF Dueker DK Jabak M Astle WF Lupski JR 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: 367-374Crossref PubMed Google Scholar Slovakia Roma (1:1250),9Plásilová M Stoilov I Sarfarazi M Kadasi L Ferakova E Ferak V Identification of a single ancestral CYP1B1 mutation in Slovak Gypsies (Roms) affected with primary congenital glaucoma.J Med Genet. 1999; 36: 290-294PubMed Google Scholar and Arab Bedouins of the Negro region in Israel (1 of 1200).10Levy J Tessler Z Tamir O Lifshitz T Primary congenital glaucoma.Harefuah. 2004; 143: 876-910PubMed Google ScholarSo far, three PCG loci have been identified by linkage analysis in multiply affected families, GLC3A,11Sarfarazi M Akarsu AN Hossain A Turacli ME Aktan SG Barsoum-Homsy M Chevrette L Sayli BS Assignment of a locus (GLC3A) for primary congenital glaucoma (Buphthalmos) to 2p21 and evidence for genetic heterogeneity.Genomics. 1995; 30: 171-177Crossref PubMed Scopus (209) Google Scholar GLC3B,12Akarsu AN Turacli ME Aktan SG Barsoum-Homsy M Chevrette L Sayli BS Sarfarazi M A second locus (GLC3B) for primary congenital glaucoma (Buphthalmos) maps to the 1p36 region.Hum Mol Genet. 1996; 5: 1199-1203Crossref PubMed Scopus (206) Google Scholar and GLC3C.5Sarfarazi M Stoilov I Schenkman JB Genetics and biochemistry of primary congenital glaucoma.Ophthalmol Clin North Am. 2003; 16: 543-554Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar,13Sarfarazi M, Stoilov I: The third genetic locus (GLC3C) for primary congenital glaucoma (PCG) maps to chromosome 14q24.3. Presented at the ARVO Annual Meeting, 2002 May 5–10, Fort Lauderdale, FLGoogle Scholar Only the gene associated with GLC3A, CYP1B1 (Online Mendelian Inheritance of Man no. 601771), has been identified.14Stoilov 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: 641-647Crossref PubMed Scopus (534) Google Scholar The CYP1B1 gene spans ∼12 kb on chromosome 2, has three exons, encodes cytochrome P4501B1, and is a member of the cytochrome P450 superfamily of genes.15Nebert DW Russell DW Clinical importance of the cytochromes P450.Lancet. 2002; 360: 1155-1162Abstract Full Text Full Text PDF PubMed Scopus (1086) Google Scholar Protein products of these genes catalyze oxidative, peroxidative, and reductive reactions and have roles in the metabolism of various substrates. Expression of the CYP1 gene family, which includes CYP1B1, is induced by the aryl hydrocarbon receptor. Although physiological studies have confirmed that mutations in CYP1B1 can cause disease, the pathway by which CYP1B1 affects development of the anterior chamber of the eye is unknown.15Nebert DW Russell DW Clinical importance of the cytochromes P450.Lancet. 2002; 360: 1155-1162Abstract Full Text Full Text PDF PubMed Scopus (1086) Google Scholar,16Stoilov I Jansson I Sarfarazi M Schenkman JB Roles of cytochrome P450 in development.Drug Metabol Drug Interact. 2001; 18: 33-55Crossref PubMed Scopus (77) Google Scholar Presumably, the metabolism of an endogenous substrate by the CYP1B1 protein is involved. Expression of CYP1B1 in the posterior segment of the eye, notably in the neuroretina, may be relevant to glaucoma pathogenesis.17Bejjani BA Xu L Armstrong D Lupski JR Reneker LW Expression patterns of cytochrome P4501B1 (Cyp1b1) in FVB/N mouse eyes.Exp Eye Res. 2002; 75: 249-257Crossref PubMed Scopus (38) Google Scholar In addition to glaucoma, CYP1B1 may have a role in carcinogenesis. Unusually high expression of the gene or increased frequencies of alleles coding more active isoforms have been reported in some cancers.18Bandiera S Weidlich S Harth V Broede P Ko Y Friedberg T Proteasomal degradation of human CYP1B1: effect of the Asn453Ser polymorphism on the post-translational regulation of CYP1B1 expression.Mol Pharmacol. 2005; 67: 435-443Crossref PubMed Scopus (79) Google Scholar19Guengerich PF Chun YJ Kim D Gillam EM Shimada T Cytochrome P450 1B1: a target for inhibition in anticarcinogenesis strategies.Mutat Res. 2003; 523–524: 173-182Crossref PubMed Scopus (138) Google Scholar20Ko Y Abel J Harth V Bröde P Antony C Donat S Fischer HP Ortiz-Pallardo ME Their R Sachinidis A Vetter H Bolt HM Herberhold C Brüning C 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: 4398-4404PubMed Google ScholarThe proportion of PCG patients whose disease is attributable to CYP1B1 mutations is generally high but varies among populations. Comparisons are not definitive, particularly because of differences in sample size, composition of samples with regard to familial and sporadic classifications, and detection protocols; nevertheless, the published figures clearly indicate that the variation exists. The numbers range from 100 to 20%: 100% in Slovakia Roma,9Plásilová M Stoilov I Sarfarazi M Kadasi L Ferakova E Ferak V Identification of a single ancestral CYP1B1 mutation in Slovak Gypsies (Roms) affected with primary congenital glaucoma.J Med Genet. 1999; 36: 290-294PubMed Google Scholar ∼90% in Saudi Arabia,6Bejjani BA Stockton DW Lewis RA Tomey KF Dueker DK Jabak M Astle WF Lupski JR 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: 367-374Crossref PubMed Google Scholar ∼50% in Brazil21Stoilov IR Costa VP Vasconcellose PC Melo MB Betinjane AJ Carani JCE Oltrogge EV Sarfarazi M Molecular genetics of primary congenital glaucoma in Brazil.Invest Ophthalmol Vis Sci. 2002; 43: 1820-1827PubMed Google Scholar and France,22Colomb E Kaplan J Garchon1 H-J Novel cytochrome P450 1B1 (CYP1B1) mutations in patients with primary congenital glaucoma in France.Hum Mutat. 2003; 22: 496Crossref PubMed Google Scholar ∼40% in India23Reddy ABM Kaur K Mandal AK Panicker SG Thomas R Hasnain SE Balasubramanian D Chakrabarti S Mutation spectrum of the CYP1B1 gene in Indian primary congenital glaucoma patients.Mol Vis. 2004; 10: 696-702PubMed Google Scholar and Morocco,24Belmouden A Melki R Hamdani M Zaghloul K Amraoui A Nadifi S Akhayat O Garchon HJR A novel frameshift founder mutation in the cytochrome P450 1B1 (CYP1B1) gene is associated with primary congenital glaucoma in Morocco.Clin Genet. 2002; 62: 334-339Crossref PubMed Scopus (54) Google Scholar and ∼20% in Japan.25Mashima Y Suzuki Y Sergeev Y Ohtake Y Tanino T Kimura I Miyata H Aihara M Tanihara H Inatani M Azuma N Iwata T Araie M Novel cytochrome P4501B1 (CYP1B1) gene mutations in Japanese patients with primary congenital glaucoma.Invest Ophthalmol Vis Sci. 2001; 42: 2211-2216PubMed Google Scholar CYP1B1 may have a lesser role in the disease status of African PCG patients as compared with Europeans.21Stoilov IR Costa VP Vasconcellose PC Melo MB Betinjane AJ Carani JCE Oltrogge EV Sarfarazi M Molecular genetics of primary congenital glaucoma in Brazil.Invest Ophthalmol Vis Sci. 2002; 43: 1820-1827PubMed Google ScholarThe worldwide profile of variations thus far reported is heterogeneous and includes ∼70 alterations (Human Genome Mutation Database; http://www.hgmd.cf.ac.uk/ac/index.php). The degree of heterogeneity within different populations, as well as the distribution of mutations, is quite variable.26Chakrabarti S Kaur K Kaur I Mandal AK Parikh RS Thomas R Majumder PP Globally, CYP1B1 mutations in primary congenital glaucoma are strongly structured by geographic and haplotype backgrounds.Invest Ophthalmol Vis Sci. 2006; 47: 43-47Crossref PubMed Scopus (49) Google Scholar A single allele, E387K, constitutes all CYP1B1 mutated alleles among the Slovak Roma patients.9Plásilová M Stoilov I Sarfarazi M Kadasi L Ferakova E Ferak V Identification of a single ancestral CYP1B1 mutation in Slovak Gypsies (Roms) affected with primary congenital glaucoma.J Med Genet. 1999; 36: 290-294PubMed Google Scholar Likewise, only the V364M mutation was found among PCG patients of Indonesian descent.27Sitorus R Ardjo SM Lorenz B Preising M CYP1B1 gene analysis in primary congenital glaucoma in Indonesian and European patients.J Med Genet. 2003; 40: e9Crossref PubMed Scopus (52) Google Scholar In Saudi Arabia, G61E constitutes ∼75% of the mutated alleles, and R469W and D374N account for almost all of the rest.6Bejjani BA Stockton DW Lewis RA Tomey KF Dueker DK Jabak M Astle WF Lupski JR 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: 367-374Crossref PubMed Google Scholar,28Bejjani BA Lewis RA Tomey KF Anderson KL Dueker DK Jabak M Astle WF Otterud B Leppert M Lupski JR Mutations in CYP1B1, the gene for cytochrome P4501B1, are the predominant cause of primary congenital glaucoma in Saudi Arabia.Am J Hum Genet. 1998; 62: 325-333Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar Mutation g.4339delG is the predominant mutation among patients from Morocco.24Belmouden A Melki R Hamdani M Zaghloul K Amraoui A Nadifi S Akhayat O Garchon HJR A novel frameshift founder mutation in the cytochrome P450 1B1 (CYP1B1) gene is associated with primary congenital glaucoma in Morocco.Clin Genet. 2002; 62: 334-339Crossref PubMed Scopus (54) Google Scholar Among less than 30 PCG patients with CYP1B1 mutations from India23Reddy ABM Kaur K Mandal AK Panicker SG Thomas R Hasnain SE Balasubramanian D Chakrabarti S Mutation spectrum of the CYP1B1 gene in Indian primary congenital glaucoma patients.Mol Vis. 2004; 10: 696-702PubMed Google Scholar and Brazil,21Stoilov IR Costa VP Vasconcellose PC Melo MB Betinjane AJ Carani JCE Oltrogge EV Sarfarazi M Molecular genetics of primary congenital glaucoma in Brazil.Invest Ophthalmol Vis Sci. 2002; 43: 1820-1827PubMed Google Scholar respectively, 16 and 11 different mutations were found. However, a single mutation, R368H in India and g.4340delG in Brazil, constituted ∼20% of the aberrant alleles in their respective populations. In contrast to these populations, 11 different mutations were found among only eight patients of French descent carrying CYP1B1 mutations.22Colomb E Kaplan J Garchon1 H-J Novel cytochrome P450 1B1 (CYP1B1) mutations in patients with primary congenital glaucoma in France.Hum Mutat. 2003; 22: 496Crossref PubMed Google Scholar The same number of mutations was identified among 13 Japanese patients.25Mashima Y Suzuki Y Sergeev Y Ohtake Y Tanino T Kimura I Miyata H Aihara M Tanihara H Inatani M Azuma N Iwata T Araie M Novel cytochrome P4501B1 (CYP1B1) gene mutations in Japanese patients with primary congenital glaucoma.Invest Ophthalmol Vis Sci. 2001; 42: 2211-2216PubMed Google Scholar These differences are likely attributable to variations in frequencies of consanguineous marriages and gene pools among the different populations.The genetic basis of PCG among Iranian patients has not been previously studied. Iran, having been a major gateway in human history, has encountered many populations and is expected to have a rich genetic legacy. Here, we report the frequency of Iranian PCG patients carrying mutations in the coding regions of the CYP1B1 gene. Mutations thought to be associated with PCG, including 10 novel ones, and variations thought not to be associated with PCG, three of which are novel, are described. Intragenic single nucleotide polymorphism (SNP) haplotypes associated with the mutations are presented and compared with those previously reported for other populations.Materials and MethodsThis research was performed in accordance with the Helsinki Declaration and with the approval of the ethics board of the International Institute for Genetic Engineering and Biotechnology in Iran. The families of patients all consented to participate after being informed of the nature of the research. One hundred four unrelated patients were recruited mostly from the ophthalmic divisions of the Farabi (associated with Tehran University of Medical Sciences), Labbafi-Nejhad (associated with Shaheed Beheshti University of Medical Sciences and Health Services), and Hazrat Rasoolakram (associated with Iran University of Medical Sciences) hospitals in Tehran. The hospitals are national reference centers, and patients from throughout the country are referred to them. All patients were diagnosed by glaucoma specialists. Slit lamp biomicroscopy, measurement of IPO, gonioscopy (if corneal clarity permitted), fundus examination, and measurement of perimetry were performed whenever possible. IOP measurements were obtained using Goldmann tonometry or the Tono-Pen (Medtronic, Minneapolis, MN) in cases with limited cooperation or central corneal scars. PCG manifested in the patients by IOP of ≥21-mm mercury (21 to 56 mm Hg) in at least one eye before treatment, corneal edema, Descemet membrane rupture, megalocornea (corneal diameter >12 mm), and optic nerve head changes suggestive of glaucomatous damage including high cup/disc ratio or neuronal rim thinning or notching. The cup/disc ratio of affected eyes when available ranged from 0.3 to total cupping (average, 5.8). Patients with other ocular or systemic anomalies were excluded. For example, patients diagnosed with Peters' anomaly or aphakic glaucoma after congenital cataract surgery were not included. Age of onset ranged from birth to 3 years. One hundred sixty ethnically matched but unrelated control individuals were recruited from those older than 60 years of age and without self-reported familial history of ocular diseases. Older individuals were recruited because mutations in CYP1B1 has been reported in some late onset glaucoma patients.22Colomb E Kaplan J Garchon1 H-J Novel cytochrome P450 1B1 (CYP1B1) mutations in patients with primary congenital glaucoma in France.Hum Mutat. 2003; 22: 496Crossref PubMed Google Scholar,29Melki R Colomb E Lefort N Brezin AP Garchon H-J CYP1B1 mutations in French patients with early-onset primary open-angle glaucoma.J Med Genet. 2004; 41: 647-651Crossref PubMed Scopus (118) Google ScholarThe patients were recruited consecutively, without regard to familial status of disease. Of the 104 PCG patients, 33 were sporadic in the sense that their parents indicated no consanguinity and no other incidence of disease in relatives of the patient. Fifty-eight patients were offspring of consanguineous parents. Of these, 46 had no other affected family member. Seventeen patients were recurrent cases in the sense that more than one family member was affected with PCG. Five of these were progeny of reported nonconsanguineous marriages. PCG in progeny of consanguineous marriages and in recurrent cases was considered familial; there were thus 63 familial PCG patients. The sporadic/familial status of eight patients could not be ascertained.Exon 1 of the CYP1B1 gene was amplified by polymerase chain reaction (PCR) in 50 patients. The primers corresponded to sequences adjacent to the exon (F, forward; R, reverse) (1F: 5′-GAAAGCCTGCTGGTAGAGCTCC-3′; 1R: 5′-CTGCAATCTGGGGACAACGCTG-3′). Exon 2, which contains the initiation codon, was amplified in all 104 patients in two overlapping PCR fragments (2Fa: 5′-ATTTCTCCAGAGAGTCAGCTCCG-3′; 2Ra: 5′-TGTAGCGGCAGCCGAAACACAC-3′; Fb: 5′-GCATGATGCGCAACTTCTTCACG-3′; 2Rb: 5′-TCACTGTGAGTCCCTTTACCGAC-3′). The coding region of exon 3 was also amplified in all of the patients (3F: 5′-AATTTAGTCACTGAGCTAGATAGCC-3′; 3R: 5′-TATGGAGCACACCTCACCTGATG-3′). The amplicon of exon 1 included 172 nucleotides upstream of the transcriptional initiation site and 136 nucleotides of intron 1. The amplicons of exon 2 included 133 and 161 nucleotides of introns 1 and 2, respectively. The amplicon of exon 3 included 129 nucleotides of intron 2 and 155 nucleotides downstream of its protein coding region. All PCR products were se-quenced in both forward and reverse directions with the same primers as used in the PCRs, using the ABI Big Dye terminator chemistry and an ABI Prism 3700 instrument (Applied Biosystems, Foster City, CA). The CYP1B1 amplicons of 10 control individuals were also fully sequenced. Sequences were analyzed by the Sequencher software (Gene Codes Corp., Ann Arbor, MI).Four of the novel single nucleotide variations deemed to be possibly associated with disease were assessed in 60 to 109 control individuals by restriction enzyme digestion and fragment length polymorphism (RFLP) as described below. All of the controls were Iranian, and at least 50 were from the same region of the country as the patients carrying the variations. Likewise, five SNPs contributing to unique core haplotypes were assessed in 100 control individuals from throughout Iran by RFLP. The enzymes used for g.3947C>G (R48G), g.4160G>T (A119S), g.8131G>C (V432L), g.8184T>C (D449D), and g.8195A>G (N453S) were BsaWI, NaeI, AleI, BseGI, and MwoI, respectively. The restriction enzymes were purchased from New England BioLabs (Boston, MA), Roche (Mannheim, Germany), or Cinnagen (Tehran, Iran). One SNP of the haplotypes of the controls, g.3793T>C, was assessed only in the 10 individuals sequenced because a restriction enzyme appropriate for its analysis by RFLP was not identified.Unique core haplotypes consisting of six SNPs were assessed in patients with CYP1B1 mutations, patients without CYP1B1 mutations, and the control group using the PHASE 2.0 software.30Stephens M Smith NJ Donnelly P A new statistical method for haplotype reconstruction from population data.Am J Hum Genet. 2001; 68: 978-989Abstract Full Text Full Text PDF PubMed Scopus (6367) Google Scholar,31Stephens M Donnelly P A comparison of Bayesian methods for haplotype reconstruction from population genotype data.Am J Hum Genet. 2003; 73: 1162-1169Abstract Full Text Full Text PDF PubMed Scopus (3012) Google Scholar This program of the software implements a Bayesian statistical method for reconstructing haplotypes. Use of the fastPHASE 1.0.1 software, which is based on a cluster model that includes an E-M algorithm, produced identical results.32Scheet P Stephens M A fast and flexible statistical model for large-scale population genotype data: applications to inferring missing genotypes and haplotypic phase.Am J Hum Genet. 2006; 78: 629-644Abstract Full Text Full Text PDF PubMed Scopus (1416) Google Scholar Statistical comparisons of haplotype frequencies between and among groups were done using χ2 contingency tables.33Armitage P Berry G Statistical Methods in Medical Research. Blackwell Scientific Publications, Boston1987: 371-378Google ScholarSequence variations and numbering were assessed by comparison with reference sequences associated with CYP1B1 available at National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov; genomic sequence, NT_022184.14; cDNA sequence, NM_000104.2, protein sequence, NP_000095.1). Predicted effects of variant sequences on splicing were determined by comparison with known canonical splice site motifs (http://www.fruitfly.org/seq_tools/splice.html). For determination of extent of conservation of amino acids altered because of nucleotide variations, the amino acid sequences of 34 cytochrome P450 proteins from 18 species were obtained from SwissProt (http://expasy.org./sprot/) and aligned using the ClustalW software (European Bioinformatics Institute, Hinxton, UK; http://www.ebi.ac.uk/clustalw).ResultsNovel VariationsTwenty-nine sequence variations were identified in the regions of the CYP1B1 gene sequenced in DNA of the Iranian PCG patients and controls (Tables 1 and 2). Ten of the variations have been previously reported to be mutations associated with disease in the literature and six others reported as variations thought not to cause PCG; most of these are listed in the Human Genome Mutation Database. Although CYP1B1 polymorphisms and mutations associated with PCG have now been reported in various ethnic groups, only limited information is available on genotype-phenotype correlations.21Stoilov IR Costa VP Vasconcellose PC Melo MB Betinjane AJ Carani JCE Oltrogge EV Sarfarazi M Molecular genetics of primary congenital glaucoma in Brazil.Invest Ophthalmol Vis Sci. 2002; 43: 1820-1827PubMed Google Scholar,34Panicker SG Reddy ABM 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: 1358-1366PubMed Google Scholar35Panicker SG Mandal AK Reddy ABM Gothwal VK Hasnain SE Correlations of genotype with phenotype in Indian patients with primary congenital glaucoma.Invest Ophthalmol Vis Sci. 2004; 45: 1149-1156Crossref PubMed Scopus (81) Google Scholar36Ohtake Y Tanino T Suzuki Y Miyata H Taomoto M Azuma N Tanihara H Araie M Mashima Y Phenotype of cytochrome P4501B1 gene (CYP1B1) mutations in Japanese patients with primary congenital glaucoma.Br J Ophthalmol. 2003; 87: 302-304Crossref PubMed Scopus (45) Google Scholar Among the previously reported disease-causing mutations also found among the Iranian patients, some phenotypic data for T404fs (g.8037-8096dup10) in the Brazilian population are available.21Stoilov IR Costa VP Vasconcellose PC Melo MB Betinjane AJ Carani JCE Oltrogge EV Sarfarazi M Molecular genetics of primary congenital glaucoma in Brazil.Invest Ophthalmol Vis Sci. 2002; 43: 1820-1827PubMed Google Scholar The phenotypic features of two Brazilian homozygotes carrying this mutation were similar to those of the single homozygous Iranian patient: the three were diagnosed before the age of 1 month, both eyes were affected in all, and their maximum recorded IOP ranged from 26 to 31.5 mm Hg. Phenotypic features associated with mutations found among a larger number of both Iranian and Indian patients are presented in Table 3.35Panicker SG Mandal AK Reddy ABM Gothwal VK Hasnain SE Correlations of genotype with phenotype in Indian patients with primary congenital glaucoma.Invest Ophthalmol Vis Sci. 2004; 45: 1149-1156Crossref PubMed Scopus (81) Google Scholar Only data on homozygous patients are presented so as to eliminate variations attributable to effects of differing second mutations. Data on R390H and R469W are included because these are common mutations among the Iranian patients. The data on R390H of the Iranians also allow comparison with R390C, which affects the same amino acid position and is found among the Indian patients. The remaining 13 novel variations were designated mutations associated with PCG or variations probably not associated with the disease on the basis of causing frameshifts or creating stop codons during translation, absence in control individuals, presence in more than one unrelated patient, occurring at same site as a previously reported mutation, nature of amino acid change caused, and/or degree of conservation during evolution.Table 1CYP1B1 Mutations Associated with PCG in Iranian PatientsGene location*The four most common mutations are shown in bold. Reference sequences used were NT_022184.14, NM_000104.2, and NP_000095.1.cDNA location*Th
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