Mutations in CYP1B1 cause primary congenital glaucoma by reduction of either activity or abundance of the enzyme

Abstract

Primary congenital glaucoma (PCG) is an autosomal recessive disorder caused predominantly by mutations in the CYP1B1 gene. A total of five frequent single nucleotide polymorphisms (SNPs) have been identified in the coding sequence of CYP1B1: rs10012C>G (p.R48G), rs1056827G>T (p.A119S), rs1056836C>G (p.V432L), rs1056837C>T (p.D449D), and rs1800440A>G (p.N453S). We performed a functional characterization of four common CYP1B1 variants presenting different coding SNP haplotypes (RAVDN, GSLDN, RALDS, and RALDN) and five CYP1B1 mutations reported for PCG patients: c.182G>A (p.G61E), c.608A>G (p.N203S), c.1033_1035del (p.L343del), c.241 T>A (p.Y81N), and c.685G>A (p.E229 K). Each mutation was embedded in its corresponding background SNP haplotype. The common variants revealed variation in enzymatic activity; among them, RAVDN showed the highest activity. Mutants p.G61E, p.N203S, and p.L343del each revealed a residual activity (<10%) of their respective haplotype. The microsomal CYP1B1 abundance relative to total protein also showed variation in common variants and a significant reduction in p.L343del, p.Y81N, and p.E229 K. The free energy of folding (DeltaDeltaG) values suggest that the lower stability of the mutants is one key property leading to the experimentally observed lower protein abundance. Our new measure of relative enzymatic activity (U/mg total protein), which combines activity and abundance values, was significantly lower for all five mutations compared to the corresponding background haplotype. We classified p.Y81N and p.E229 K not as mutations but as hypomorphic alleles, since their relative activity values are intermediate between bona fide mutations and the common variant with the lowest activity (RALDS). We propose that CYP1B1 mutations can act by either reducing enzymatic activity (p.G61E and p.N203S), reducing the abundance of the enzyme (p.Y81N and p.E229 K), or both (p.L343del).

Molecular complexity of primary open angle glaucoma: current concepts

“…Direct evidence of CYP1B1 mutation dependent alteration of retinoic acid catabolism activity is still lacking. But studies from our lab (M. Acharya, S. Mookherjee and K. Ray, unpublished data) and others suggest that mutations in CYP1B1 alters its steroid metabolism either by perturbing the structure of the protein (Achary et al 2006;Achary and Nagarajam 2008) or by decreasing its stability (Jansson et al 2001;Bagiyeva et al 2007;Chavarria-Soley et al 2008;Choudhary et al 2008). We observed that Leu432Val polymorphism is associated with POAG with Val432 acting as a risk factor for the disease.…”

Exome sequencing in developmental eye disease leads to identification of causal variants in GJA8, CRYGC, PAX6 and CYP1B1

“…Enzymatic assays indicate c.1103G4A, p.(Arg368His) has markedly reduced activity while c.685G4A, p.(Glu229Lys) is a hypomorphic allele. 33,34 There was no evidence of glaucoma or Peters anomaly in Patient 9 (Family 9, II.1) or Patient 11 (Family 11, II.1). CYP1B1 can contribute to retinoic acid synthesis in embryonic development, 35 and retinoic Table 3 Other variants lacking appropriate segregation-allele frequencies in public databases, conservation and prediction categories acid receptor signalling regulates choroid fissure closure.…”

“…This missense mutation is in a conserved residue in the P450 domain of this protein (http://smart.embl.de), and previous enzymatic studies have indicated that it is a hypomorphic allele. 33 Patient 11 (Family 11, II.1) who had a heterozygous change in GDF3, was also a compound heterozygote for two missense mutations in CYP1B1, c.1103G4A, p.(Arg368His), and c.685G4A, p.(Glu229Lys) ( Table 3, Supplementary Figure S3). Both of these variants have been found to have higher frequencies in individuals with primary congenital glaucoma than in control populations, including in controls with this patient's ethnicity.…”

Genetic Testing for Eye Diseases: A Comprehensive Guide and Review of Ocular Genetic Manifestations from Anterior Segment Malformation to Retinal Dystrophy

“…Primary congenital glaucoma generally presents from birth to the first few years of life with excessive tearing, photophobia, corneal clouding, increased intraocular pressure, Haab striae (breaks in Descemet's membrane of the cornea), and buphthalmos. The majority of patients have deleterious variants in the CYP1B1 gene [17]. A few patients have been reported with truncating variants in the LTBP2 gene [18,19].…”