CYP1B1 mutations in Spanish patients with primary congenital glaucoma: phenotypic and functional variability.

Purpose: To analyze the contributions of cytochrome P4501B1 (CYP1B1) mutations to primary congenital glaucoma (PCG) in Spanish patients.
Methods: We analyzed, by polymerase chain reaction (PCR) DNA sequencing, the presence of promoter (-1 to -867) and exon CYP1B1 mutations in 38 unrelated Spanish probands affected by PCG. Functional analysis of nine identified mutations was performed measuring ethoxyresorufin O-deethylation activity and CYP1B1 stability in transiently transfected human embryonic kidney 293T (HEK-293-T) cells.
Results: We found a total of 16 different mutations in 13 (34.2%) index cases. The identified mutations included nine missense and three nonsense nucleotide changes, three small deletions, and a short duplication. Eleven probands were compound heterozygotes and two were heterozygotes. Six of the identified mutations were novel (A106D, E173X, F261L, E262X, W341X, and P513_K514del). Mutations T404fsX30 and R355fsX69 were the most prevalent among index cases and were detected in six (23.0%) and three (11.5%) patients, respectively. Functional analysis showed that the three nonsense mutants assayed (E173X, E262X, and W341X) and F261L were null alleles. Of the remaining mutants, four (P52L, G61E, Y81N, and E229K) showed catalytic activities ranging from 20% to 40% of wild-type CYP1B1 and high protein instability. Mutation P400S showed normal catalytic activity and moderate instability. These five mutants were classified as hypomorphic alleles. Patients carrying two null alleles showed severe phenotypes featured by very early PCG onset usually at birth or in the first month of life (0.6+/-0.9 months). Incomplete penetrance was detected in patients carrying hypomorphic alleles.
Conclusions: Our data indicate that approximately one-third of Spanish patients with PCG carry loss-of-function CYP1B1 and show that null alleles are associated with the most severe phenotypes. Hypomorphic alleles may contribute to some cases of incomplete penetrance.