title: Screening of common CYP1B1 mutations in Iranian POAG patients using a microarray-based PrASE protocol.
abstract: OBJECTIVE
  The gene coding cytochrome P4501B1 (CYP1B1) has been shown to be a major cause of primary congenital glaucoma in the Iranian population. More recently it was shown to also be important in juvenile-onset open angle glaucoma (JOAG). We aimed to further investigate the role of CYP1B1 in a larger cohort of primary open angle glaucoma (POAG) patients which included late-onset patients. We also aimed to set up a microarray based protocol for mutation screening with an intent of using the protocol in a future population level screening program.
  
  
  METHODS
  Sixty three POAG patients, nine affected family members, and thirty three previously genotyped primary congenital glaucoma (PCG) patients were included in the study. Clinical examination included slit lamp biomicroscopy, IOP measurement, gonioscopic evaluation, fundus examination, and measurement of perimetry. G61E, R368H, R390H, and R469W were screened by a protocol that included multiplexed allele specific amplification in the presence of a protease (PrASE), use of sequence tagged primers, and hybridization to generic arrays on microarray slides. The entire coding sequences of CYP1B1 and myocilin (MYOC) genes were sequenced in all individuals assessed by the microarray assay to carry a mutation. Intragenic single nucleotide polymorphism (SNP) haplotpes were determined for mutated alleles.
  
  
  RESULTS
  Genotypes assessed by the array-based PrASE methodology were in 100% concordance with sequencing results. Seven mutation carrying POAG patients (11.1%) were identified, and their distribution was quite skewed between the juvenile-onset individuals (5/21) as compared to late-onset cases (2/42). Four of the seven mutation carrying Iranian patients harbored two mutated alleles. CYP1B1 mutated alleles in Iranian PCG and POAG patients shared common haplotypes. MYOC mutations were not observed in any of the patients.
  
  
  CONCLUSIONS
  The PrASE approach allowed reliable simultaneous genotyping of many individuals. It can be an appropriate tool for screening common mutations in large sample sizes. The results suggest that CYP1B1 is implicated in POAG among Iranians, notably in the juvenile-onset form. Contrary to POAG patients studied in other populations, many mutation harboring Iranian patients carry two mutated alleles. We propose an explanation for this observation.
authors.affiliation: School of Biology, University College of Science, University of Tehran, Tehran, Iran.
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Suri
authors.forename: Reza
authors.initials: R
authors.lastname: Kalhor
authors.forename: Seyed Jalal
authors.initials: SJ
authors.lastname: Zargar
authors.forename: Navid
authors.initials: N
authors.lastname: Nilforooshan
authors.forename: Shahin
authors.initials: S
authors.lastname: Yazdani
authors.forename: Hossein
authors.initials: H
authors.lastname: Nezari
authors.forename: Seyed Hassan
authors.initials: SH
authors.lastname: Paylakhi
authors.forename: Mehrnaz
authors.initials: M
authors.lastname: Narooie-Nejhad
authors.forename: Behnaz
authors.initials: B
authors.lastname: Bayat
authors.forename: Tina
authors.initials: T
authors.lastname: Sedaghati
authors.forename: Afshin
authors.initials: A
authors.lastname: Ahmadian
authors.forename: Elahe
authors.initials: E
authors.lastname: Elahi
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D001189:Aryl Hydrocarbon Hydroxylases; D044466:Asian People / Q000235:genetics; D001483:Base Sequence; D002648:Child; D065633:Cytochrome P-450 CYP1B1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004252:DNA Mutational Analysis; D005260:Female; D005820:Genetic Testing; D005838:Genotype; D005902:Glaucoma, Open-Angle / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D007492:Iran; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010375:Pedigree; D010447:Peptide Hydrolases / Q000378:metabolism*; D010641:Phenotype
chemical_list: D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D065633:Cytochrome P-450 CYP1B1; D010447:Peptide Hydrolases