title: [Construction of CYP1B1 gene haplotypes predisposing to primary congenital glaucoma through allele-specific PCR/restriction fragment length polymorphism analysis].
abstract: OBJECTIVE
  To develop an allele-specific PCR (AS-PCR)/restriction fragment length polymorphism (RFLP) assay for CYP1B1 gene haplotypes predisposing to primary congenital glaucoma (PCG).
  
  
  METHODS
  Twenty Chinese PCG patients and 20 healthy controls were recruited. Peripheral blood sample was subjected to direct sequencing for common single nucleotide polymorphisms (SNPs) of the CYP1B1 gene. Based on the results, CYP1B1 gene haplotypes were constructed by PCR-RFLP and AS-PCR combined with RFLP.
  
  
  RESULTS
  Four SNPs loci were identified by sequencing, which included rs10012 G>C (S1 in exon 2), rs1056827 T/G (S2 in exon 2), rs1056836 C/G (S3 in exon 3) and rs1056837T>C (S4 in exon 3). The distribution of such loci showed different characteristics between the two groups. 50% of the PCG patients had rs10012 G>C and rs1056827 T>G, while 25% of PCG patients had rs1056836 C>G and rs1056837T>C. As for the controls, 25% had rs10012 G>C and rs1056827 T>G, 10% had rs1056836 C>G and rs1056837T>C. None of the SNP loci has presented alone. PCR-RFLP was carried out to confirm the results of SNPs typing, but could not confirm the linkage between the SNP loci. By contrast, AS-PCR combined with RFLP has achieved specific amplification for rs10012 G>C and thorough differentiation of 1056827 T>G polymorphism. Similar results have been obtained by the same method for rs1056836 C>G and rs1056837T>C typing and linkage disequilibrium analysis.
  
  
  CONCLUSIONS
  The AS-PCR/RFLP assay has successfully constructed the haplotypes of the CYP1B1 gene. For its accuracy, efficiency and specificity, the method may be used for constructing haplotypes for hereditary disease studies.
authors.affiliation: Department of Clinical Laboratory, Eye and Ear Nose Throat Hospital, Shanghai Medical School, Fudan University, Shanghai 200031, P.R. China. wgkjyk@aliyun.com; oryqun@163.com.
authors.forename: Aiping
authors.initials: A
authors.lastname: Zhang
authors.forename: Shengjie
authors.initials: S
authors.lastname: Li
authors.forename: Qi
authors.initials: Q
authors.lastname: Ouyang
authors.forename: Li
authors.initials: L
authors.lastname: Tang
authors.forename: Xiaolei
authors.initials: X
authors.lastname: Wang
authors.forename: Jian
authors.initials: J
authors.lastname: Ji
authors.forename: Wenjun
authors.initials: W
authors.lastname: Cao
mesh_terms: D000328:Adult; D000368:Aged; D000483:Alleles; D001483:Base Sequence; D065633:Cytochrome P-450 CYP1B1 / Q000235:genetics*; D005260:Female; D005787:Gene Frequency; D020022:Genetic Predisposition to Disease / Q000235:genetics*; D005838:Genotype; D005901:Glaucoma / Q000151:congenital / Q000235:genetics*; D006239:Haplotypes; D006801:Humans; D015810:Linkage Disequilibrium; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: C585496:CYP1B1 protein, human; D065633:Cytochrome P-450 CYP1B1