New Compound Heterozygous CYP4V2 Mutations in Bietti Crystalline Corneoretinal Dystrophy.

Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive retinal dystrophy which is caused by the mutations of CYP4V2, usually progressing to legal blindness by the 5th or 6th decade of life. Here we identified CYP4V2 compound heterozygous mutations in two female siblings with BCD without subjective symptoms. After 381 pathogenic genes related to retinal diseases were screened by targeted sequence capture array techniques and confirmed by Sanger sequencing, two compound heterozygous mutations in CYP4V2 were found. One was missense mutation c.1198C>T (p.R400C) and the other was frameshift mutation c.802-8_810delinsGC (p.V268_E329del). Optical coherence tomography (OCT) showed that the ellipsoid zone was absent in the macular regions and electroretinogram (ERG) revealed poor cone and rod responses. Compound heterozygous mutations in CYP4V2 are related to the BCD. Our study expands our knowledge of heterogenic phenotypes and genotypes through genetic diagnosis of the BCD patients.


Background
Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive retinal dystrophy which is characterized by numerous tiny glistening yellow-white crystals at posterior pole of the retina. Some cases also have similar crystals at the corneoscleral limbus. Clinically, this disease can be divided into three stages: Stage 1: RPE atrophy with uniform ne white crystalline deposits at the macular area. Stage 2: RPE atrophy extends beyond the posterior pole and the atrophy of choriocapillaris in the posterior pole.
Stage 3: RPE-choriocapillaris complex atrophy throughout the fundus. Crystalline deposits at the corneal limbus are probably prominent at the advanced stage of atrophy of the RPE-choriocapillaris complex. 1 Although BCD has been reported worldwide, it seems more frequent in East Asia, especially in China and Japan. The prevalence of BCD in China was 1 per 24 000 for the total population, but in Europe, BCD only accounted for 3% of all nonsyndromic retinitis pigmentosa. 2 BCD is caused by the mutations of CYP4V2, which encodes a 525 amino acid protein and plays an important role in fatty acid and steroid metabolism. 3 Over 100 cases of mutations in CYP4V2 gene have been reported. 4 Here we aim to perform genetic screening for BCD based on targeted next generation sequencing. A largescale mutation screening of 381 known retinal degenerative-related genes was performed and identi ed two compound heterozygous mutations in the CYP4V2 gene.

Methods
A case of 35-year-old lady came to our clinic for myopia laser correction without any other ocular complains or any general diseases. However, we found crystalline lesions in the posterior pole of both eyes during fundus examination. After obtaining medical and ophthalmic histories, we gave the patient and her family a complete ophthalmological examination including central visual acuity with EDTRS charts, slit lamp biomicroscopy of the anterior segment, fundus exam, fundus photography, visual eld, Optical coherence tomography (OCT), Fluorescence angiography (FFA), standard ERG and multifocal ERG (mfERG). The approval of the Ethics Committee of Shenzhen Eye Hospital and the informed consent from the patients were obtained. We extracted genomic DNA from peripheral blood by standard protocols and collected the protein coding regions of targeted genes by targeted sequence capture array technique.
This technique could capture 381 pathogenic genes related to retinal diseases, which included CYP4V2 gene associated with BCD. Polymerase chain reaction(PCR) and Sanger sequencing were used to con rm the screening results.

Results And Discussion
The best corrected visual acuity (BCVA) of the proband was 0.9 (nearly 20/20) in both eyes. The intraocular pressure was normal. The sclera, conjunctiva, cornea and lens were normal. The physiological blind spot was enlarged by visual eld tests. Optical coherence tomography (OCT) showed that the ellipsoid zone was absent in the macular regions ( Fig. 1). Fundus uorescein angiography (FFA) showed that the surrounding granules in the posterior pole were stained with uorescence and the omental vessels were thin. Photopic and scotopic electroretinogram (ERG) revealed that the amplitudes of a-and b-waves were severely decreased, indicating poor cone and rod responses (Fig. 1). We found that her elder sister had the same sign. For her elder sister, we also found that the retinal phenotypes were almost identical (Fig. 1). We found two compound heterozygous mutations in CYP4V2 of the proband. One was missense mutation c.1198C > T (p.R400C) and the other was frameshift mutation c.802-8_810delinsGC (p.V268_E329del). Similarly, her sister also carried the same mutations (Fig. 2). The missense mutation c.1198C > T (p.R400C) resulted in a change from arginine to cysteine at amino acid codon 400. The other frameshift mutation c.802-8_810delinsGC (p.V268_E329del) leads to the deletion of all 62 amino acids in exon 7. Missense mutation c.1198C > T(p.R400C) has been reported twice before, which was compound heterozygous mutation with R400H in one case and a homozygous point mutation in the other case. 5,6 Frameshift mutation c.802-8_810delinsGC (p.V268_E329del) is common in BCD patients in East Asia. 7 Although most patients with BCD develop decreased vision, nyctalopia, and paracentral scotomata, few patients complain no symptoms. In our case, the patients have no subjective symptoms, which may be due to the speci c genetic defects (new compound heterozygous mutations). In addition, most patients of BCD usually present in the 2nd or 3rd decade of life and clinical features do not seem to correlate well with the age of debut. We can't exclude the possibility that the disease is still in its early stages. Patients are often asymptomatic during the earliest stage of BCD, which increase di culty in diagnosis. Some reports suggested that phenotype associated with genetic disease is determined by genotype and environment commonly and different diseases depend on the degree of in uence of these two factors. 8 Studies of genotype and phenotype analysis based on large sample are necessary to explore the pathogenesis of BCD and the determinant of disease progression.
In this study we found new compound heterozygous mutations in CYP4V2 in BCD, which were a new pathogenic cause. Our study expands our knowledge of heterogenic phenotypes and genotypes through genetic diagnosis of the BCD patients.  Figure 1 A family with Bietti crystalline corneoretinal dystrophy. A.Pedigree of the family with Bietti crystalline corneoretinal dystrophy; B. The proband patient: scanning laser ophtalmoscope showed crystalline points in the posterior pole of both eyes and fundus auto uorescence imaging showed a butter y-shaped hypo uorescence with rim increased auto uorescence in both eyes;C. The sister of the proband: the retinal phenotype is identi cal to the proband. D. The amplitudes of the a-and b-waves were signi cantly reduced in the photopic (which re ects cone response) and scotopic ERG (which re ects rod response). E.Visional eld tests showed that the blind spot of both eyes was enlarged. F. OCT indicated that ellipsoid zonewas absentin both eyes; G. Fluorescence angiography showed that crystalline lesions were stained with uorescence in the posterior pole and the omental vessels were thin in both eyes.

Figure 2
Mutations in CYP4V2 gene in the proband and her sister.Sequence shows the heterozygous missense mutation c.1198C>T(p.R400C) and frameshift mutation c.802-8_810delinsGC(p.V268_E329del) found in both patients.