Phylogenetic evaluation of the CYP1B1 gene in patients with primary congenital glaucoma

Abstract

Editor, Primary congenital glaucoma is a rare genetic disorder that usually manifests itself at birth or within the first year of life. Tearing, photophobia and enlargement of the eyeball resulting from elevated intraocular pressure characterize the disease. The disorder is autosomally recessively inherited and has been assumed to comprise a group of heterogeneous diseases. Candidate gene studies of the 2p21 locus have identified disease-causative mutations in the cytochrome P4501B1 (CYP1B1) gene, a member of the P-450 superfamily, and a variety of mutations have been demonstrated to be causes of primary congenital glaucoma (Sarfarazi & Stoilov 2000). The CYP1B1 gene has two long-coding exons, which translate 543 amino acids. Besides pathological mutations, the CYP1B1 gene has innocent polymorphic nucleotide changes presenting genetic haplotypes. The majority of pathological mutations have been identified as amino acids conserved throughout distinct species and analogous genes, such as in Best's disease (Marquardt et al. 1998). To investigate the anthropological background of the CYP1B1 gene, the nucleotide sequence in the entire coding region was phylogenetically assessed in Japanese congenital glaucoma patients with or without the relevant gene mutation. Eleven patients (GLC1−GLC11) were diagnosed with primary congenital glaucoma and followed up at Kagoshima University Medical Centre. All the patients belonged to unrelated families who resided in Kagoshima Prefecture in southwestern Japan. The nucleotide sequence in the entire coding exons of the CYP1B1 gene has been evaluated elsewhere (Kakiuchi-Matsumoto et al. 2001). The patients had two independent pathological mutations and five innocent nucleotide variations. GLC1 had Arg48Gly, Ala119Ser, Ala330Val and Asp449Asp (gac to gat). GLC2 had 1620insG mutation, Leu432Val and Asp449Asp. GLC3 had Arg48Gly and Asp449Asp. GLC4 had Ala119Ser. GLC5 had Ala119Ser. GLC6 had Arg48Gly and Ala119Ser. GLC7 had Asp449Asp. GLC8 had Arg444Gln mutation and Asp449Asp. GLC9 had Ala119Ser and Asp449Asp. GLC10 had Arg48Gly and Ala119Ser. GLC11 had Arg48Gly. Polymorphic alleles did not co-segregate with the disease and were found in healthy individuals as well. The estimated value of nucleotide diversity within the examined patients was 0.13%. The obtained CYP1B1 gene sequence was edited into a contiguous alignment with software YooEdit. The datasets of nucleotide alignments of all individuals were loaded into the multiple-sequence alignment interface clustal x and the phylogenetic tree was constructed by the neighbour-joining method (Thompson et al. 1997). This study was carried out in adherence with the tenets of the Declaration of Helsinki. Based on the branching patterns, no specific cluster(s) were found in the examined patients. On the phylogenetic tree, GLC2 and GLC8 were located on the upper stream far from the other GLC patients examined. Given the pathological CYP1B1 gene mutations in both GLC2 and GLC8, the phylogenetic assessment suggests that mutational events happened in the early stage of CYP1B1 gene evolution. In the case of an alternate genetic ocular disease, Leber's hereditary optic neuropathy, the mutational event of mtDNA G11778A appears to be retroactive from a considerably ancient time (Isashiki et al. 2003). From the anthropological point of view, major genetic mutations significant for pathology appear to happen in the earlier phase of genetic evolution and to be conserved for long periods.