Genotype And Phenotype Analysis Of Chinese Patients With Two Kinds Of Inherited Retinal Degenerative Disease

Purpose:Bietti crystalline corneoretinal dystrophy （BCD） is a rare monogenic autosomal recessively inherited retinal degenerative disease characterized by the presence of crystals in the retina and retinal pigment epithelium atrophy. Mutations in the CYP4V2 gene have been found to be causative for BCD. The purpose of this study was to analyze the CYP4V2 mutations in five unrelated Chinese pedigrees with BCD and to provide clinical features of the patients.Methods:Ophthalmic examinations were carried out in the affected individuals. Peripheral blood samples were collected and genomic DNA was extracted. All exons and flanking intronic regions of the CYP4V2 gene were amplified with polymerase chain reaction and screened for mutations by direct DNA sequencing. One hundred control chromosomes were also screened to exclude nonpathogenic polymorphisms.Results:Fundus examination revealed the presence of tiny yellowish-sparkling crystals at the posterior pole of the fundus and atrophy of the retinal pigment epithelium in all patients. Choroid neovascularization was noted in one patient. Five different CYP4V2 mutations were identified, including two missense mutations （p.F73L, p.R400H）, two splice site mutations （c.802-8₈10de117insGC, c.1091-2A>G）, and one single base deletion （p.T479TfsX7 or c.1437de1C）. The two splice site mutations were identified in three of the patients with BCD. Mutation p.T479TfsX7 was a novel mutation not observed in any of 100 ethnically matched control chromosomes.Conclusions:Mutation c.802-8 810del17insGC and c.1091-2A>G are common mutations in Chinese patients with BCD. Our results expand the allelic heterogeneity of Bietti’s crystalline corneoretinal dystrophy.Purpose:Human BEST1 gene mutations are associated with at least four clinically distinguishable retinal degenerative diseases, collectively referred to as bestrophinopathies:Best vitelliform macular dystrophy （BVMD）, autosomal recessive bestrophinopathy （ARB）, adult-onset vitelliform macular degeneration, and autosomal dominant vitreoretinochoroidopathy. The purpose of this study was to analyze BEST1 gene mutations in Chinese patients with bestrophinopathy and to describe the clinical features of these patients.Methods:Thirteen patients from 12 unrelated Chinese families affected by bestrophinopathy were recruited and clinically evaluated with best-corrected visual acuity examination, slit-lamp biomicroscopy, fundus examination and photography, optical coherence tomography, fundus autofluorescence, electro-oculography, and electroretinography. Blood samples were collected for DNA extraction. Mutation analysis was performed by direct sequencing of the BEST1 gene. One hundred control chromosomes were also screened to exclude nonpathogenic polymorphisms.Results:Seven patients showed clinical pictures of BVMD and were found to harbor heterozygous mutations compatible with autosomal dominant inheritance. Two novel mutations （p.T4I and p.A291 V） and three reported mutations （p.R218C, p.Q293H, and p.D301G） were identified among them. Six patients were found to carry BEST1 mutations on both alleles compatible with autosomal recessive inheritance. Compound heterozygous mutations were detected in four patients who presented a BVMD phenotype, while homozygous mutations were detected in two patients with ARB. Mutation analysis revealed eight mutations among them. Four （p.Y33H, p.R130L, p.M163R, and c.519delA） were novel and four （p.R13H, p.A195V, p.R255W, and p.W287*） had previously been reported.Conclusions:Patients with bi-allelic BEST1 mutations were common among Chinese bestrophinopathy patients and they displayed variability in phenotypes. The features and combinations of different BEST] mutations as well as epistatic effects may influence phenotype expression. Our results expand the BEST1 mutation spectrum.Purpose:Autosomal recessive bestrophinopathy （ARB） is a retinal dystrophy resulting from homozygous or compound heterozygous mutations in the BEST1 gene. It is a distinct disease characterized by clinical features different with typical Best vitelliform macular dystrophy. To elucidate the pathogenic effects of novel homozygous missense mutations p.R130L and p.M163R of the BEST] gene identified in the previous part, we investigated the cellular localization and stability of the mutant protein.Methods:Polymerase chain reaction （PCR） based site-specific mutagenesis using BEST1-pCMV6 （wild type） as a template was performed to construct BEST1-pCMV6 （p.R130L and p.M163R mutant）. MDCKII （Madin-Darby canine kidney II） cells were cultured and transiently transfected with BEST1-pCMV6 （wild type and ARB mutant）. The effect of ARB mutations on the cellluar localization of the protein was determined by confocal immunofluorescence on transiently transfected MDCK Ⅱ cells after immunofluorescent staining. Protein stability of wild-type and ARB mutant forms of hBestl was determined by the addition of lysosomal inhibitors or proteasomal inhibitors to transiently transfected MDCK II cells. Lysates were then analyzed by Western blot analysis.Results:BESTl-pCMV6 （p.R130L and p.M163R mutant） was successfully constructed in vitro. Wild type hBestl and hBestlR130L localized predominantly to the plasma membrane in transiently transfected MDCK Ⅱ cells. By contrast, hBest1M13R exhibited a primarily cytoplasmic localization and was rapidly degraded by the proteasome. The addition of lysosomal inhibitors did not notably alter the degradation of wild type hBestl or ARB-causing mutants.Conclusions:The molecular pathogenic mechanism associated with hBest1M163R is a consequence of both protein misfolding and mislocalization. Differences in pathogenic mechanisms for different ARB associated mutants lead to the same disease phenotype. Our study of disease-associated BEST1 variants has helped to elucidate pathogenic mechanisms underlying ARB.