Longitudinal characterisation of function and structure of Bietti crystalline dystrophy: report on a novel homozygous mutation in CYP4V2

Abstract

Background: Bietti crystalline dystrophy (BCD) is a rare inherited disorder characterised by fine crystalline deposits in the corneal limbus and retinal posterior pole. In 2004, mutations in the CYP4V2 gene were identified as the cause of BCD. Here, we describe the report of a homozygous point mutation in a patient with BCD and provide detailed characterisation of functional and structural changes over 20 years.

Methods: At regular intervals, the patient underwent repeat ophthalmic evaluations. DNA was extracted from buccal swabs, amplified by standard PCR and analysed for homology to the CYP4V2 sequence. Homology modelling was conducted using Iterative Threading ASSEmbly Refinement and molecular dynamics simulations using GROningen MAchine for Chemical Simulations.

Results: The proband, a 47-year-old woman of German ancestry was diagnosed with crystalline retinopathy at age 25. Over the next 20 years, visual acuity and function gradually declined with progression of retinal pigment epithelium and choroidal atrophy. When first tested at 39 years of age, the multifocal electroretinogram (ERG) was markedly abnormal, more so for the right eye, whereas the full-field ERG was more symmetrical and lagged other measures of visual function. Gene sequencing showed a single C>T point mutation in exon 9 encoding a R400C amino acid change. Computational modelling suggests the mutation impairs function due to loss of a hydrogen bonding interaction with the propionate side chains of the haeme prosthetic group.

Conclusion: This is the first report of a homozygous R400C mutation in CYP4V2 with protein modelling showing high likelihood of enzyme dysfunction. The comprehensive long-term clinical follow-up provides insight into disease progression and highlights possible anti-inflammatory modulation of disease severity.

Keywords: Bietti crystalline dystrophy; CYP4V2; macular edema; protein modeling.

Figure 1

(A) BCVA over time with best-fit line depicting progressive decline with slope in each eye that was significantly different than zero but not significantly different than each other. The last two BCVA measurements were made within months following the surgery. Improvement from 20/500 to 20/50 occurred for the right eye, whereas the BCVA for the left eye was essentially unchanged from the previous measurements prior to the surgery. (B) Rod b-wave amplitude response to dim white light and cone b-wave amplitude response to photopic bright flash stimuli. Both rod and cone photoreceptor-dependent full-field ERG responses declined and were significantly below normal at 39 years of age in each eye. Progression for each photoreceptor-mediated amplitude is presented as the percentage of the patient’s amplitude compared with the normal mean value. (C) Multifocal ERG, performed at 39 years of age, showed diffuse attenuation of amplitude densities that was greater for the right eye. BCVA, best-corrected visual acuity; ERG, electroretinogram; OD, right eye; OS, left eye; y/o, years old; yrs, years.

Figure 2

(A) Kinetic perimetry shows rapid development of worsening central and pericentral scotomas over the course of 2 years. (B) The area of the III4e isopter plotted over time showed a progressive decline in both eyes. deg², squared degrees; OD, right eye; OS, left eye; y/o, years old; yrs, years.

Figure 3

(A) Colour fundus photography show posterior pole crystalline deposits and confluent retinal atrophy, which progresses over time. (B) Montage of infrared images highlights the crystalline deposits and areas of retinal atrophy. (C) Wide-field autofluorescence shows confluent heterogeneous hypoautofluorescence throughout the posterior pole, which is surrounded by lacey hyperfluorescence throughout the midperipheral transition zone. (D) Early-phase wide-field fluorescein angiography shows confluent hyperfluorescent window defect of the posterior pole with patchy hypofluorescent choroidal atrophy. OD, right eye; OS, left eye; y/o, years old.

Figure 4

(A) Optical coherence tomography. Horizontal linear B scans through the fovea show cystoid macular oedema in both eyes at baseline and response to treatment. Retinal pigment epithelial atrophy and outer retinal attenuation with outer retinal tubulations are also observed. (B) Fluorescein angiography show late leakage mainly in the right eye. Hyperfluorescent window defect and hypofluroescent choroidal atrophy are also evident. OD, right eye; OS, left eye; OU, both eyes; mg/d, milligrams per day.

Figure 5

Computational comparison of wild-type CYP4V2 with the R400C mutant. (A) RMSD of each trajectory from its starting structure serves to demonstrate that our homology models can feasibly be studied by computational means. The RMSD rapidly achieves a plateau, indicating that the protein as a whole has assumed a locally stable conformation in silico. (B) Histogram of shortest distances between residue 400 and the haeme for each frame of our wild-type and mutant simulations. The wild-type R400 interacts much more closely with the haeme group and spends the majority of its time within hydrogen-bonding distance of the haeme propionates. In contrast, the R400C mutant has lost these interactions and is much more distant from the haeme. (C) Initial states of our simulated system, with wild-type (white) on the left and R400C (pale orange) on the right. Both enzymes are in highly similar conformations, with the haeme facing the active site and apparently available for catalysis. The peptide backbone is shown as a loop, while residue 400, the haeme prosthetic group and the thiolate ligand (C467) are depicted with ball-and-stick representation. (D) An illustration of the dramatic changes that can result from the loss of R400. After 50 ns of simulation, the haeme orientation is essentially unchanged in wtCYP4V2, but has everted by ~70° in the R400C mutant, presumably contributing to diminished stability or catalytic activity. RMSD, root mean square deviation.