Multimodal Imaging observation of Bietti crystalline dystrophy progression

Shengjuan Zhang 1. Beijing Institute of Ophthalmology, Beijing Ophthalmolgy and Visual Science Key Laboratory, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University. 2. Hebei Provincial Key Laboratory of Ophthalmology, Hebei Provincial Eye Institute, Hebei Provincial Eye Hospital. https://orcid.org/0000-0003-4988-0953 Qian Li Beijing Institute of Ophthalmology, Beijing OPhthalmolgy and Visual Science Key Laboratory, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University Lifei Wang Hebei Provincial Key Laboratory of Ophthalmology, Hebei Provincial Eye Institute, Hebei Provincial Eye Hospital XiaoYan Peng (  74000041@ccmu.edu.cn ) Hebei Provincial Key Laboratory of Ophthalmology, Hebei Provincial Eye Institute, Hebei Provincial Eye Hospital https://orcid.org/0000-0002-3946-5724

encodes the CYP4V2 protein and is relatively common among Asian people, and especially among Chinese and Japanese persons, but it is rare among European and American people [2,3]. BCD is characterized by the accrual of large amounts of yellowish crystals deposits at the posterior pole of the retina, retinal pigment epithelium (RPE) atrophy, and choroid sclerosis [4]. BCD has been receiving more attention in recent years, and reports on BCD have increased. However, most articles have focused on gene diagnosis [5,6], cross-sectional imaging studies [7,8], and case reports [9,10]. Few studies have conducted follow-up observations, and most of these are case reports [11,12].
Our aim in the present study was to use color fundus photography, fundus auto uorescence (FAF), infrared auto uorescence (IRAF), fundus uorescein angiography (FFA), and spectral domain optical coherence tomography (SD-OCT) to observe the progression of retinal and choroidal changes in 8 eyes of 4 patients with BCD. Our overall goal was to gain a better understanding of BCD progression and changes.

Subjects
The implementation of all research methods in this study followed the provisions of the Declaration of Helsinki, the Ethics Committee of Beijing Tongren Hospital, Capital Medical University, and the Ethics Committee of Hebei Provincial Eye Hospital. This study included 4 unrelated Chinese patients who visited our hospital twice between January 2017 and October 2018 and were diagnosed with BCD after clinical examination. The clinical characteristics and imaging data of these patients were retrospectively analyzed. For convenience, cases 1, 2, 3, and 4 included in this study are referred to as P1, P2, P3, and P4 respectively. The intervals of P1, P2, P3, and P4 were 10, 16, 20, and 20 months respectively. According to the staging criteria proposed by Yuzawa et al [13], the stage of the fundus lesions at the rst visit to our hospital were as follows: P1 and P2 were stage 2, with RPE atrophy extending to the mid-peripheral area, with choroidal capillary atrophy. P3 and P4 were stage 3, with extensive RPE and choroidal capillary atrophy. The rst examination of the 4 patients was referred to as P1F, P2F, P3F, and P4F, and the last examination was referred to as P1L, P2L, P3L, and P4L. All 4 patients included in this study agreed to undergo a genetic diagnosis at our hospital. Peripheral venous blood was collected upon receiving signed informed consent. The genomic DNA was extracted from the blood and the BCD pathogenic gene CYP4V2 was sequenced. Further sequence alignments and mutation screenings were also performed.
The FAF, IRAF, and FFA images over a 55×55° eld were obtained with a confocal scanning laser ophthalmoscope (Heidelberg Spectralis, Heidelberg Engineering, Heidelberg, Germany). The results of the two examinations were compared and changes were documented.
SD-OCT images were adopted by a Spectralis Optical coherence tomography (OCT) (Heidelberg Engineering, Heidelberg, Germany). The Spectralis SD-OCT scanning mode for horizontal linear scans selects and enhances the depth imaging (EDI) mode. Each scan was centered on the macular fovea (because the fovea OCT images of the P1 left eye and P2 left eye were not scanned during the rst examination, and the scanning image was slightly deviated from fovea. In the last examination, we followed the scans to track changes at this slightly deviated level. For convenience, in this study, the lowest point of the fovea shown at this level is also described as a fovea). Measurement data included the central macular thickness (CMT) and subfoveal choroidal thickness (SCT) [14]. The measurements were made on choroidal macrovascularities selected from the nasal side and the temporal side of the subfoveal choroid. The diameter of the vessel was measured to calculate the difference in blood vessel diameter between the rst and the last examination and the changes were recorded. The ellipsoid zone (EZ) at the fovea of the P1 right eye is missing, but the residual EZ was seen at the temporal side of the fovea. The distance between the residual EZ at the proximal foveal end and the Bruch membrane below the fovea was measured and the difference calculated between the rst and the last measurements. Little residual EZ was seen at the central fovea of the P1 left eye, the P2 and P4 both eyes. The EZ width was used to calculate the difference between the rst and the last measurements; the EZ of P3 both eyes macular area is missing.

Results
The general conditions and genetic diagnosis results for P1-P4 are shown in Table 1 and Table 2.
The mean patient age was 34.75 ± 5.75 years. The BCVA (ETDRS) of the 8 eyes was signi cantly decreased, with a mean decrease of 14.5±17.5.

Fundus photography
The yellowish crystalline deposits in the posterior pole of the retina were reduced at the last examination.
A greater atrophy of the retinal pigment epithelium (RPE) choriocapillaris complex was associated with fewer yellowish crystals. The numbers of pigmentation plaques scattered in the posterior retina increased (Fig. 1C), and part of the original pigment plaque was larger at the last examination (Fig. 1D).

Fundus auto uorescence (FAF) and infrared auto uorescence (IRAF)
The lesions area shown by FAF and IRAF were consistent in the P1 and P2 both eyes. Signi cant expansion of the hypo-AF and hypo-IRAF area at the macular area and around optic papilla area was observed, and the partial patchy hypo-AF areas merged together. IRAF revealed the choroid macrovascular morphology in the posterior hypo-IRAF area. The hypo-IRAF area was larger than the hypo-AF area. In P1, the hyper-AF spots and hyper-IRAF spots were observed at the junction of the hypo-AF and normal retina and the hypo-IRAF and normal retina. The number of hyper-AF spots was signi cantly greater than the number of hyper-IRAF spots. Because the lesion area was extended, this changed the position of the hyper-AF spots and hyper-IRAF spots compared with P1F, but the quantity of spots showed no obvious change. Comparison of P2L and P2F revealed a reduction in the number of hyper-AF spots. No obvious hyper-IRAF was found in P2F or P2L ( Fig. 2A-H).
Comparison of P3L versus P3F and of P4L versus P4F revealed signi cant expansion of the hypo-AF area at the macular area and around the optic papilla area, with partial patchy hypo-AF merged together.
IRAF showed the hypo-IRAF morphology of optic disc, retinal macrovascularities, and choroidal macrovascularities. P3 and P4 showed hyper-AF spots, but no hyper-IRAF spots. P3 and P4 also showed uneven auto uorescence intensity of the hypo-AF areas, but the corresponding IRAF spots showed uniform hypo-IRAF, revealing hypo-IRAF of the choroidal macrovascularities ( Fig. 2I-P).

Fundus uorescein angiography (FFA)
The posterior region of the P1F both eyes showed choroidal macrovascular uorescence in hypouorescence area, with surrounding mottled uorescence. The peripheral retina showed mottled uorescence. Annular normal retinal uorescence was seen in the mid-peripheral area. The lesions of the posterior and peripheral parts of the P1L both eyes extended to the mid-peripheral parts, and the atrophic hypo-uorescence of the RPE choriocapillaris complex in the posterior and superior peripheral parts was larger than that of P1F ( Fig. 3A-D).
In the macular area and around the optic papilla area of the P2F both eyes, choroidal macrovascular uorescence was observed in hypo-uorescence area, with surrounding mottled uorescence. Partial normal retinal uorescence was evident between the temporal peripheral lesion area and the macular lesion area. The hypo-uorescence area in the posterior of the P2L both eyes was enlarged, the mottled uorescence was more disordered than in P2F, and the normal retinal uorescence of the macular area temporal side was reduced because of the progression of the surrounding lesions ( Fig. 3E-H).
The posterior patchy hypo-uorescence areas of the P3F both eyes reached the mid-peripheral area and the rest of the retina outside the hypo-uorescence area, including part of the macular area, which showed mottled uorescence. The posterior patchy hypo-uorescence was enlarged and partially merged together. The hypo-uorescence was mainly expanded to the periphery, and no obvious changes were noted in the mottled uorescence in macular area ( Fig. 3I-L).
In addition to the mottled uorescence of the superior temporal, temporal, and inferior peripheral retina, choroidal macrovascular uorescence was observed in other regions of the P4F both eyes. The mottled uorescence of the superior temporal, temporal, and inferior peripheral retina was smaller in P4L than in P4F, and the choroidal macrovascular uorescence in the other area was clearer than in P4F ( Fig. 3M-P).
Spectral domain optical coherence tomography (SD-OCT) The outer nuclear layer, EZ, interdigitation zone, and RPE in the macular area of P1-P4 showed atrophy to different degrees, and a partial re ection was missing. The outer retinal tubulation (ORT) could be seen in some layers. OCT-related data from the P1-P4 rst and last examinations were recorded. Retinal thickness in the fovea was thinner in all eyes except the P1 left eye and P3 left eye, with an average thickness of 24.428±48.572 µm. The macular edema of the P1L left eye was increased over that of P1F, so the CMT was increased by 12 microns. The CMT of the P3 left eye showed no obvious change. The SCT of all 8 eyes were thinner, with an average thickness of 42.125±50.875 µm. The diameter of the temporal side choroidal macrovascular was reduced in all 8 eyes by an average of 21.125±18.875 µm. The diameter of the P1, P2, and P4 nasal side choroidal macrovascularities were reduced by an average of 22.5±15.5 µm. The diameter of the P3 nasal side choroidal macrovascular showed no obvious change; The residual EZ length of the P1 left eye, P2 both eyes, and P4 both eyes was shortened by an average of 269.4±206.6µm. No residual EZ was detected in the fovea of the P1 right eye, so we measured the distance between the temporal residual EZ proximal foveal end and the Bruch membrane below the fovea, the difference between P1F and P1L was 165 µm. The EZ and interdigitation zone were absent in the P3 both eyes macular area. The residual EZ only existed at the fovea of P2 and P4 and was absent at the nasal and temporal fovea. P1F had a small number of intraretinal cystoid cavities in the inner nuclear layer of the right eye and no intraretinal cystoid cavities in the left eye. The number of intraretinal cystoid cavities in the inner nuclear layer of the right eye was greater in P1L than in P1F, inner nuclear layer intraretinal cystoid cavities were present in the left eye of P1L, and the outer nuclear layer was thicker in P1L than in P1F. No intraretinal cystoid cavities were found in the macular area in the two examinations of P2 and P3. No intraretinal cystoid cavities were detected in the macular area of P4F but they were detected in countable numbers in P4L. (Fig. 4A-P).

Discussion
Our examination of multimodal images of 8 eyes of 4 BCD patients (P1, P2, P3, and P4) at different time intervals of 10, 16, 20, and 20 months, respectively, revealed expansion of the areas of hypo-AF and hypo-IRAF but a decrease in the hyper-AF spots and hyper-IRAF spots. FFA showed that the lesion area expanded and the hypo-uorescence area with severe atrophy also expanded. OCT showed thinning of both the retina and choroid and progressive atrophy of the outer nuclear layer, EZ, and RPE, as well as signi cant thinning of diameter of the choroidal macrovascularities.
The four patients ranged in age from 29 to 38 years, which is the common onset age of BCD [15,16]. The results of gene examinations were all showed BCD-related gene alleles [17,18]. The BCVA (ETDRS) of all 8 eyes signi cantly decreased. Color fundus photography showed yellowish crystalline deposits in the posterior fundus. The number of yellowish crystalline deposits declined by the last examination, which was consistent with previous studies [12,15,19,20]. The deposition of yellowish crystalline lipids is caused by abnormal expression of the CYP4V2 gene in the human retina and RPE [16,21,22], and it is reduced with atrophy of the retina and RPE.
Comparison of P1F, P2F, P3F, and P4F versus P1L, P2L, P3L, and P4L indicated a wider range of hypo-AF and hypo-IRAF at the posterior fundus, and a partial merging together of the patchy hypo-AF. Choroidal macrovascular morphology was observed in the hypo-IRAF. The junction of hypo-AF and the normal retina and hypo-IRAF and the normal retina of P1 showed the presence of hyper-AF spots and hyper-IRAF spots. The number of hyper-AF spots was signi cantly greater than the number of hyper-IRAF spots. The position of the hyper-IRAF spots corresponds to the hyper-AF spots, but the hyper-IRAF spots is smaller than hyper-AF spots.
FAF is known to represent mainly uorescence of lipofuscin [23], while IRAF is mainly melanin uorescence [24]. Some studies had also observed hyper-AF and hyper-IRAF in AMD. The degradation products of melanin may also be added to lipofuscin with increasing age [25]; therefore, we considered that this phenomenon might also be caused by the accumulation of lipofuscin and melanin degradation products in damaged RPE cells.
The FAF of P2, P3, and P4 showed hyper-AF spots, while the IRAF of P2, P3, and P4 showed choroidal macrovascular morphology in the hypo-IRAF. No hyper-IRAF spots were observed. Considering that the dysfunction of the RPE produced more lipofuscin than melanin degradation products. In P1, hyper-AF spots and hyper-IRAF spots appeared in the hypo-AF and hypo-IRAF peripheral areas. Some hyper-AF spots were observed between the patchy hypo-AF of P2, P3, and P4, which was consistent with the ndings of Kojima et al [26], and was considered due to retention of some functions by the impaired RPE.
We had the 4 patients do puzzles for the two FFA examinations to provide a more intuitive and comprehensive view of the progression of the disease. The retinal choroid atrophy was signi cantly worse in all 8 eyes of the 4 patients in the last than in the rst examination, in agreement with previous reports [11,12]. The left eye and right eye of each patient were essentially the same, but differed among the 4 patients. Normal retinal uorescence was observed in the mid-peripheral part in P1, and expansion of the lesions was observed from the posterior pole and peripheral parts to the normal retina in the midperipheral part. Partial normal retinal uorescence was observed at the temporal side of the P2 both eyes macular area, and P2L showed a reduction in the area of the normal retina, with expansion of the peripheral lesions to the normal retina. The P3 macular area retina showed no obvious changes, but the surrounding lesions were obviously atrophy aggravations. The P4 both eyes showed less atrophy in the superior temporal area and in the temporal side of the macular area and the inferior peripheral area. The rest of the region was severely atrophied and showed choroidal vascular uorescence. P4L showed further shrinkage of the less atrophy areas, and the areas with severer atrophy were further extended, with clearer of choroidal macrovascularities. This situation differed from previous research indicating that BCD is an eccentric extension starting from the posterior pole [27,28,29,30]. BCD may not develop exclusively by posterior polar eccentricity as, at least in some cases, the lesion develops from the posterior and peripheral parts and extends to the mid-peripheral part, and the temporal retinal atrophy develops relatively later. This may occur because the temporal retina is farther away from the posterior pole of the BCD initiation site.
In this study, OCT was applied for the rst time to quantify the changes in the retina and choroid. The P1 left eye showed increased retinal thickness due to macular edema, an aggravated atrophy of the EZ and RPE, and a thinner choroid. The P1 right eye, P2 both eyes, and P4 both eyes showed thinner retinas, with increased atrophy of the outer retinal nuclear layer, EZ, and RPE and a thinner choroid. The diameter of choroidal macrovascularities was shortened in P1, P2, and P4, consistent with the degenerative properties of the BCD lesions [31]. Our view is that the decrease in the diameter of the choroidal vessels is only one of the reasons for choroidal thinning. We observed that choroidal capillaries also become thinner; however, because the di culty in de ning the boundary of the capillary layer made obtaining speci c data impossible. Previous studies have reported reductions in choroidal capillary blood ow, as determined by OCTA [32].
No change, or a change of only a few micrometers, was observed in the P3 macular area measurements, except that the retinal thickness of the right eye fovea thinned to 17 µm, which we did not consider statistically signi cant. The combination of FAF and FFA results indicated a progression in the retinal choroidal lesions other than in the macular area, but no signi cant FAF and FFA changes were noted in the macular area. The re ection of the EZ in the P3 macular area was missing. Determining whether this is related to the P3 gene phenotype will require further study on more cases. It was not reported in previous cases [11,12].
The ORT was rst reported in 2009 [33], and it appears in OCT as a low-re ective oval space with a highly re ective boundary in the outer nuclear layer. Since the rst report, ORTs have been associated with poor visual prognosis [34,35,36]. In our study, ORTs were observed in the two examinations of all 8 eyes, con rming their high incidence in BCD and in agreement with previous results [26,37]. Two patients in our case showed intraretinal cystoid cavities in the inner nuclear layer in the macular area, suggesting that macular edema is common in BCD. The macular edema in P1 and P4 starts from a zero baseline and progresses from less to more, with adverse effects on the patients' visual acuity. Many previous reports have documented BCD macular cystoid edema [6,8,31,38,39], but the pathogenesis of the macular cystoid edema is still unclear. The consensus is that it may be the same as the pathogenesis of cystic macular disease in retinal dystrophy, which is caused mainly by damage to the blood-retinal barrier [8,37,38]. The exact pathogenesis needs further study.
This study had several limitations, including the small number of included eyes and the lack of primary patient observation. However, considering the rarity of the disease and reviewing the previous literature, our study on the progression of BCD using multimodal images provides one of the largest numbers of images and the largest number of patients. Another limitation is that the large retinal vessels of the P2L right eye and the P4F left eye showed only slight uorescence in the FAF due to the intravenous injection of the uorescein sodium test uid, and this had an impact on the accuracy of our FAF inspections. A third limitation is that the FFA evaluation was not well planned, so the puzzles were inconsistent and some fundus area pictures were missing. A fourth limitation was that the rst examination of the 4 patients was retrospective. Therefore, the horizontal B-scan OCT of the P1F left eye and P2F left eye did not cross the fovea, but slightly deviated from the fovea. Consequently, in the last examination, we used this slightly off-fovea layer for comparison.

Conclusions
Color fundus photography, FAF, IRAF, FFA, and SD-OCT observations of patients with BCD revealed a signi cant progression in the depth and width of the lesions in a short period of time. FAF, IRAF, and OCT-EDI are all non-invasive examination models, so they have great advantages in detecting disease development and in assessing disease progression.