Identification Of Pathogenic Mutationsin Chinese Patients With Cone Dystrophy Diseases And Gene Therapy Of A Mouse Model Of Achromatopsia

Purpose:Alstrom syndrome （AS） is a rare monogenic autosomal recessively inherited disorder characterized by cone rod dystrophy and multiple organs dysfunction. Mutations in the ALMS1have been found to be causative for AS. The purpose of this study was to identify ALMS1mutations and to assess the clinical features of Chinese patients with AS.Methods:Detailed ocular and laboratory examinations were performed. Peripheral blood samples were collected from patients and their parents. Genomic DNA was extracted. Exons and exon/intron junctions of ALMS1were amplified by polymerase chain reaction （PCR） and screened for mutations with Sanger sequencing. The results were compared with ALMS1transcript （NM₀15120.4） to exclude polymorphism and confirm pathogenic mutations.Results:Seven patients from five unrelated non-consanguineous families were diagnosed as AS. All patients had cone rod dystrophy with impaired visual acuity, photophobia and nystagmus. Other clinical features, including sensorineural hearing loss, truncal obesity, insulin resistance, type2diabetes mellitus, renal and hepatic dysfunction, hyperlipidemia, hypothyroidism, mental retardation, acanthosis nigricans and scoliosis were present. Sequencing revealed two novel frameshift mutations p.N3150Kfs2X and p.V3154Xfs in patient1, one novel frameshift mutation p.N3672Ifs11X and one previously reported nonsense mutation p.R3703X in patient2, novel nonsense mutation p.S2479X and frameshift mutation p.R3611Efs7X in patient3, one novel homozygous nonsense mutationp.S695X in patient4and5, two novel frmeshift mutations p.H688HfsX and p.Q3147Qfs2X in patient6and7. These mutations were not present in100unrelated healthy Chinese control subjects. Parents were heterozygous carriers of the mutant allele respectively.Conclusions:Seven Chinese patients affected with AS showed typical ophthalmic features and multiple organs dysfunction. Novel loss of function mutations in the ALMS1are the underlying genetic defects. PurposeTo study the clinical features and identify the pathogenic mutationsin Chinese achromats.Methods15patients from10unrelated familieswere included in this study.Detailed ocular examinations including best corrected visual acuity （BCVA）, color vision, slit lamp, fundus, electroretinography, perimetry and optical coherent topography （SD-OCT） were performed for affected subjects. Peripheral blood samples were obtained from all patients and their family members for genomic DNA extraction. All exons of CNGA3,CNGB3,GNAT2,PDE6C and PDE6H were amplified by polymerase chain reaction and screened for mutation by direct Sanger sequencing. The sequences were analyzed using Blat tool compared with the gene transcript. Segregation test was confirmed in patients’ parents if they were available. The variants were compared with the database of1000genomes to exclude polymorphism.There were2patients who found none mutation of5known pathogenic mutations.ResultsNystagmus, photophobia and impaired color discrimination were occurred in all patients. The BCVA of the affected subjects ranged from0.05to0.2. Severely depressed and non-recordable cone electroretinograms were observed. Noticeable structural changes including disruption or loss of the macular inner/outer segments （IS/OS） junction of the photoreceptors under SD-OCT. CNG A3mutations were identified in13patientsfrom8families. Sequencing revealed7novel missense mutations,3novel deletion mutations and4previously reported mutations among those patients.ConclusionsCNGA3mutation is the most frequent cause of Chinese patients with achromatopsia.10novel mutations were identified in CNGA3. Genetic characterization of patients with achromatopsia is important for genetic counseling and future gene therapies. To the best of our knowledge this is the first report of genetic study related to Chinese achromats. Purpose:To elucidate the exact pathogenic effects of novel homozygous missense mutations D211E of CNGA3identified in the previous study.Methods:To functionally characterize one novel homozygous missense mutationD211E of CNGA3, site-directed mutagenesis, using CNGA3-pCMV6（wildtype） as a template, polymerase chain reaction（PCR） based site-specific mutagenesis was performed to construct CNGA3-pCMV6（D211E mutant）.HEK293cells were cultured and transfected with CNGA3-pCMV6（wildtype） and CNGA3-pCMV6（D211E mutant）. Localization of the wild and mutant protein in HEK293cells after immunofluorescent staining.Thesubcellular distribution ofCNGA3（D211E） in HEK293cells wascompared to wild-type by visualizing with fluorescence microscopy.Results:CNGA3-pCMV6（D211E mutant） was successfully constructed in vitro.Functional analysis of mutant protein HEK293cells were transfectedwith CNGA3-pCMV6（wildtype）, CNGA3-pCMV6（D211E mutant） encoding wild-type （WT） CNGA3protein and mutational CNGA3proteinrespectively.WT localized predominantly to the plasmamembraneindicating the normal biogenesis and transportation of theprotein to the membrane. By contrast, the D211E mutant was nottrafficked to the plasma membrane butaccumulatedintracellularly.Conclusions:Expression analyses of mutant CNGA3protein confirmed that the missense mutation D211Eled to abnormalprotein accumulation in the cytoplasm and the correct intracellular formation. PurposeTo clarify whetherthe AAV5vector-mediated gene therapy could restore cone function in the cnga3cpf15mouse model of achromatopsia.MethodsAAV5-IRBP/GNAT2-hCNGA3were injected subretinally into one eye ofmice at postnatal day21. The mice that had more than80%retinal detachment and minimal complications after surgery were kept for follow-up experiments. Contralateral eyes were uninjected as controls. Electroretinographic were recorded to detect cone response at170days after treating. Then eyes were enucleated after sacrifice and prepared for cryosections. Experimentwhich were designed to detect the opsinand CNGA3expression of retina were conducted with immunofluorescenceusing S-opsinand CNGA3antibodies as primary antibodies.ResultsCompared with the untreated eyes, AAV5mediated gene therapy could rescuecone response evidently. Both M-and S-opsins were expressedin outer segment of the retina. The curative effect of gene therapy for cnga3cpfl5lasted for at least5～6months after injection.ConclusionsIn this study,the time of gene therapy a week later usual postnatal14days, but we show that therapeutic effect is also distinct in a naturally occurring mouse model of CNGA3achromatopsia. The results provide us theillumination for future AAV5-based gene therapy for human CNGA3achromatopsia.