The Clinical And Basic Research Of Bietti Crystalline Dystrophy And Autosomal Dominant Optic Atrophy

Purposes:To characterize the clinical features of Chinese patients with Bietti crystalline dystrophy?BCD?,identify the disease-causing mutations and study the correlation between genotypes and phenotypes.To analyse blood plasma lipid profiles of BCD patients.Methods:Patients and families clinically diagnosed as BCD in Peking Union Medical College Hospital during 1998 to 2017 were collected.1.Clinical study:Detailed medical history and family history were recorded.The patients were given general ophthalmic examination,including visual acuity,optometry,anterior segment and fundus examination.Special ophthalmic examinations were conducted,including confocal microscopy of paralimbal cornea,fundus photography,optical coherence tomography?OCT?,fundus antofluorescence?AF?,visual field?VF?and electroretinogram?ERG?.2.Genetic study:The peripheral blood of the BCD patients and their family members were collected and extracted into genomic DNA.The exons and intron/exon junctions of CYP4V2 gene were amplified with polymerase chain reaction?PCR?and sequenced directly.For negative cases after Sanger sequencing,we performed multiplex ligation-dependent probe amplication?MLPA?to detect genomic rearragements.Targeted exome capture plus next generation sequencing?NGS?were applied for atypical patients.3.Lipodomics:Blood plasma were extracted from BCD patients and normal controls.Untargeted lipid profiling was performed with Ultra-performance liquid chromatography-mass spectrometry?UPLC-MS?.Results:A total of 168 BCD patients from 139 families were collected.Among the 139 index patiens,65 were males and 74 were females,with age between 17 to 71.11 consanguineous families?8.3%?,14 pseudo-dominant?10.6%?and 2 pseudo-X-linked families?1.5%?were identified.1.Clinical manifestations:The median age of onset was 27.62.1%of our BCD cohort presented initial symptom of night blindness,19%declined visual acuity,16.0%night blindness and declined visual acuity,2%were asymptomatic and 1 case complained of floaters.The best corrected visual acuity?BCVA?ranged from light perception to 1.5.40.4%complicated with mild myopia,35.1%moderate myopia,15.8%high myopia and approximately half of them presented mild to moderate astigmatism.Crystalline deposits at the limbus of cornea were observed among 17.8%of the patients under slit-lamp examination.All the 16 patients underwent confocal microscopy revealed crystal deposits at the limbus of cornea,including negative cases by slit-lamp or fundus examination.The confocal microscopy showed the needle-shaped or rod-shaped crytals located in the corneal epithelium and superficial layer of stroma.Retinal crystals,irregular pigment clamps,atrophy of retinal pigment epithelium?RPE?and choroid with various severity were the main fundus appearance of BCD.Fundus complications were identified in 14 patients?9.5%?,among which 8?57.1%?were choroidal neovascularization?CNV?and 4?28.6%?were macular hole?MH?with retinal detachment.OCT showed reduced retinal thickness,discontinued or disappeared ellipsoid layer,RPE and choroid atrophy.The average foveal thickness was 116.6 ?m.We categorized the BCD patients into the central type?18.2%?and peripheral type?66.9%?according to different progression patterns.15.9%were at the late stage of the disease and hard to be classified.The central type was characterized by retinal atrophy predominantly in the posterior hole,and gradually involve the periphery region.The peripheral type was characterized by retinal atrophy predominantly in the periphery region,and gradually involve the posterior hole,which resembles retinitis pigmentosa.The two types varied remarkably in the aspects of initial symptoms,average visual acuity,the risk of retinal complications,VF defects and retinal functions?P<0.001?.2.Molecular study:Sanger sequencing combined with MLPA identified the disease-causing mutations in 96.4%BCD patients.The most prevalent mutation was IVS6-8del17bp/insGC,with an allele frequency of 61.7%.A total of 33 causative mutations were identified,among which 51.5%were novel.There was no obvious correlation between different mutations and phenotypes.NGS revealed 2 families with multigenetic CYP4V2-related retinopathy.We also reported a typical BCD patient complicated with sclerodactyly.Genetic analysis revealed compound heterozygous IVS6-8del17bp/insGC and c.A761G mutations.3.Lipodomics:Using a combination of differential and correlation analysis,more than 10 lipid compounds were determined,consisted primarily of acylglycerols and free fatty acids.Conclusions:BCD patients presented typical ocular manifestations,but they varied in severity and morphology.Retinal complications were not rare in our BCD cohort.The new classification would facilitate in assessing phenotypes and prognosis of BCD patients.Confocal microscopy could be useful in diagnosing BCD in some cases.Sanger sequencing of CYP4V2 could identify causative mutations in most BCD patients.Screening of genomic rearrangements should be a routine for Chinese CYP4V2-negative cases.We suggest NGS as a useful tool to detect multigenetic mutations when the phenotype is atypical of BCD.Since the CYP4V2 mutant allele frequency is estimated to be relatively high in Chinese population,screening of CYP4V2 in the spouses of BCD patients is recommended.Lipodomic studies may show some enlightment in the diagnosis and treatment of BCD.Purpose:Autosomal dominant optic atrophy?ADOA?is one of the most common types of genetically inherited optic atrophy.OPA1 gene mutations have been found to account for the majority of ADOA.The purpose of this study was to identify OPA1 mutations and to assess the clinical features of a cohort of Chinese patients affected with ADOA.Methods:Detailed clinical evaluations were performed on 17 patients from 8 unrelated dominant optic atrophy families.Genomic DNA was extracted from peripheral blood.Sanger sequencing was used to analyze all exons and exon/intron junctions of OPA1 for 7 pedigrees.Target exome capture plus next generation sequencing?NGS?were applied for one atypical family.Reverse transcription polymerase chain reaction?RT-PCR?was carried out to further characterize the mRNA change of selected splice site mutation.Results:All 17 patients had impaired vision and optic disk pallor,however,the clinical severity varied markedly.Two patients complicated with hearing loss.Six novel and two reported mutations in OPA1?GenBank Accession No.NM₁30837?were identified including 4 missense mutations?c.T2235G,c.T2340G,c.T1468C and c.A1567G?,2 splice site mutations?c.2984-1₂986del and c.2983+5G>A?,1 small deletion?c.2960₂968delGCGTTCAAC?and 1 small insertion?c.3009₃010insA?.RNA analysis revealed the splice site mutation c.2984-1₂986del caused small deletion of mRNA?r.2819₂824del?.Conclusions:Six novel and 2 reported causative mutations in OPA1 were confirmed for 17 Chinese ADOA patients with variable clinical manifestations.Hearing loss was recorded in our ADOA cohort?2/17?.We suggest next generation sequencing as a useful diagnostic tool for genetic diagnosis when the phenotype is atypical of ADOA.