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.
Conclusion: 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.