Two patients in our cohort presented with BCD and CNV (). The first patient, a 31-year-old male, stated that he had experienced a decrease in central vision in both eyes during the past year, with associated difficulty in night vision. There was no history of similar eye problems in the family. Upon examination, he had a best-corrected Snellen visual acuity of 6/9 in both eyes. The anterior segment examination showed paralimbal crystals in both eyes. Fundus examination revealed diffuse RPE alterations, with the numerous intraretinal crystalline deposits in both eyes suggestive of BCD. A scarred subfoveal CNV was noted in the right eye (). Subretinal hemorrhage with active CNV was noted in the subfoveal region of the left eye (). Spectral domain optical-adherence tomography (SD OCT; Cirrus, Carl Zeiss Meditec, Inc., Dublin, CA) revealed the presence of high-reflective intraretinal deposits in both eyes; the left eye had subfoveal CNV with increased retinal thickness and moderately reflective echoes suggestive of hemorrhage (). Full-field ERG showed waveforms of normal amplitude and implicit time for both photopic and scotopic responses, while multifocal ERG revealed a reduced central, paracentral, and peripheral ring response for both amplitude and implicit time in both eyes that was different than expected (based on normal reference points) given the individual’s age (). Fundus fluorescein angiography of right eye and left eye shows areas of window defects corresponding to areas of RPE and choriocapillaris atrophy with blocked fluorescence along the areas of crystalline deposits and subretinal fluid in the left eye. Right eye also showed staining suggestive of scarred CNV and leakage in the left eye suggestive of active CNV (). The patient’s serum triglyceride level was found to be 171 mg/dl (normal range, 60 to 165 mg/dl). The nature of BCD with CNV was explained to the patient and he was offered a treatment option of anti-vascular endothelial growth factor (anti-VEGF) injection after its roles and limitations were explained. The patient consented to this treatment and was administered three injections of 0.05 mg ranibizumab (Lucentis®; Novartis Pharma AG, Basel, Switzerland; Genentech Inc., South San Francisco, CA), intravitreally at monthly intervals. At the last follow up, six months after the final injection, the patient maintained the same visual acuity. Fundus examination showed scarring from CNV in both eyes () that was confirmed on OCT ().
Clinical Presentation of Bietti crystalline dystrophy (BCD) patients.
Figure 1 Clinical and investigation pictures of patient 1. A: Color fundus photograph of right eye showing scarred choroidal neovascularization (CNV). B: Pretreatment color fundus photograph of the left eye showing retinal crystals with active CNV (arrow) and (more ...)
The second patient was a 26-year-old male with diminished vision, of six months duration, in both eyes. His best-corrected visual acuity was 6/48 and 6/7.5 in his right and left eyes, respectively. The rest of the findings in this patient were similar to those in the first patient, except that the patient did not have crystal deposits in the cornea and had no sign of CNV. At the time of the patient’s one-year follow up visit, his visual acuity had significantly dropped to 6/60 in the right eye and 6/18 in the left eye. OCT revealed foveal scarring in the right eye with RPE pigmentation. The left eye revealed scarring of the choroidal neovascular membrane with a slight increase in retinal thickness.
In addition to these two patients, we included a third patient in the study, a female with BCD, but not CNV. The patient had diminished vision at night of 5 years duration. Her sister had the same problem in her medical history, but was not examined. The best-corrected visual acuity was 6/9 in both eyes. There were typical crystalline deposits in the posterior pole of the retina and in the supranasal portion of the cornea. Full-field ERG waveforms revealed a reduction in both amplitude and in implicit time for both photopic and scotopic responses in comparison to normal reference points for the woman’s age.
CYP4V2 and TIMP3 analysis
Screening for CYP4V2 in the patient with BCD without CNV revealed a novel mutation, a single base-pair duplication c.1062_1063dupA. None of the controls screened showed this variation. The variation resulted in the change of a non-polar hydrophobic amino acid valine to a neutral hydrophilic serine. For this base-pair duplication, two additional amino acids with different charges are replaced before undergoing truncation to produce an aberrant peptide (negatively charged hydrophilic aspartic acid is replaced by neutral hydrophilic glycine, and positively charged hydrophilic histidine is replaced by neutral hydrophilic serine). Genotyping CYP4V2 in both patients with CNV identified three novel benign variations in the coding region without any functional significance. One of the novel benign variations c.775C>A resulted in the replacement of aminoacid glutamine with lysine. This variation was found in a homozygous or heterozygous state in 65% of the controls screened. No causative pathogenic variations were observed in CYP4V2 in these two patients (). Screening exon 5 of TIMP3 did not show a variation for any of the three patients.
Mutation profile of CYP4V2 in Bietti crystalline dystrophy (BCD) patient cohort.
The templates chosen for modeling CYP2C5 and CYPBM3 (with heme as a cofactor) sequences showed an overall identity of 25% and 29%, respectively, with WT-CYP4V2. Both CYP2C5 and CYPBM3 had a 44% similarity with WT-CYP4V2 and were modeled using Modeller9v7 (Laboratory of Dr. Andrej Sali). The predicted structure of WT was used as a template for modeling the structures of MT1 and MT2. MT1 and MT2 shared a 99% and 97% sequence identity with the WT, respectively, and were further refined using similar optimization protocols to those implemented for WT. No residues were found in the disallowed region of the Ramachandran plot for the generated structures. This confirms the plausibility of the generated models WT (), MT1 (superimposed view of WT with MT1; ), and MT2 (superimposed view of WT with MT2; ) of CYP4V2. The subtle conformational variation with the conversion of a few secondary structural elements in MT1 could confer only mild variation in the heme binding regions () and may not affect the function of the protein. The truncation of an axial ligand-binding residue, cys467 in MT2 (), affects protein function by altering substrate binding. The normalized conservation scores for the mutant position in MT1 and MT2 were found to be 4 and 5, respectively. The blosum70 score generated using a substitution matrix for MT1 variant was found to be +1. This emphasizes the conservative nature, whereas in the case of MT2, a negative blosum score of −2 was observed, and thus predicts a defective mutant protein.
Figure 2 A structural analysis of CYP4V2 WT, MT1, and MT2. A: Homology model of WT- CYP4V2. B: Backbone superimposition of WT (cyan) with MT1 (Red), the altered secondary structures are shown in zoom view. C: Backbone superimposition of WT (cyan) with MT2 (Blue), (more ...)
The predicted folding rate for WT was found to be 2.43/s, while for MT1 and MT2 it was found to be 3.65/s and −3.24/s, respectively, demonstrating that the MT2 mutation results in an unstable protein. The atomic contact energy of MT2 mutation was found to be highest with −243.405 J/mol, whereas for the WT and MT1 mutations the atomic contact energies were −510.510 J/mol and −534.050 J/mol, respectively. At the structural level, the backbone superimposition of MT1 with WT shows subtle conformational variation with the change in secondary structure elements at regions 406 to 408 and 411 to 414 in MT1 (). WT with MT2 superimposition shows a loss, due to truncation, of extended secondary structural elements from amino acid position 357 (). A significant variation was observed in the electrostatic potential at the heme-binding region of MT2 (), which may modulate the molecular interactions with its binding partners. In the case of MT1, it was observed that the variation at 259 with K is predicted to be tolerated with a SIFT score of 1.00, and is also benign as per polymorphism phenotyping, with a difference in position-specific independent counts (PSIC) of 0.442. Conversely, the phenotypic variation using SIFT with MT2 was found to be intolerant, with a score of 0.00. Polymorphism phenotyping also predicted that MT2 mutation would lead to impairment of protein function, with a PSIC difference of 2.095.