Compound heterozygous mutations in CYP1B1 gene leads to severe primary congenital glaucoma phenotype

Abstract

Aim: To identify the novel mutation alleles in the CYP1B1 gene of primary congenital glaucoma (PCG) patients at Shandong Province of China, and investigate their correlation with glaucomatous features.

Methods: The DNA from the peripheral blood of 13 congenital glaucoma patients and 50 ethnically matched healthy controls from the affiliated hospital of Qingdao University were extracted. The coding region of the CYP1B1 gene was amplified by PCR and direct DNA sequencing was performed. Disease causing-variants were analyzed by comparing the sequences and the structures of wild type and mutant CYP1B1 proteins by PyMOL software.

Results: Two missense mutations, including A330F caused by c.988G>T&c.989C>T, and R390H caused by c.1169G>A, were identified in one of the 13 PCG patients analyzed in our study. A330F mutation was observed to be novel in the Chinese Han population, which dramatically altered the protein structure of CYP1B1 gene, including the changes in the ligand-binding pocket. Furthermore, R390H mutation caused the changes in heme-protein binding site of this gene. In addition, the clinical phenotype displayed by PCG patient with these mutations was more pronounced than other PCG patients without these mutations. Multiple surgeries and combined drug treatment were not effective in reducing the elevated intraocular pressure in this patient.

Conclusion: A novel A330F mutation is identified in the CYP1B1 gene of Chinese PCG patient. Moreover, in combination with other mutation R390H, this PCG patient shows significant difference in the CYP1B1 protein structure, which may specifically contribute to severe glaucomatous phenotype.

Keywords: CYP1B1 gene; missense mutation; primary congenital glaucoma; protein structure.

Figure 1. Electropherograms of two mutations in the CYP1B1 gene

The normal and the mutated nucleic acids from region 983 to 992 (A); and region 1164 to 1173 (B) of CYP1B1 gene have been listed. The mutated nucleic acids are labeled with arrows.

Figure 2. The structural analysis of wild-type and mutated CYP1B1 protein

A: The normal CYP1B1 protein structure including amino acid, alanine (labeled as red) at position 330 and arginine (labeled as purple) at position 390. The ANF ligand and HEM (Protoporphyrin Ix Containing Fe) ligand have been labeled as blue and yellow, respectively. B: The interaction between alanine and ANF ligand under high magnification. C, D: The influence of the mutations on the interactions between amino acid and ligands.

Figure 3. The clinical characteristics of PCG patient with A330F and R390F mutations

A: The appearance of the eyes at 23 years of age; B: Information about the changes in IOPs after different treatments and ages.