Clinical And Molecular Genetic Studies Of OPA1 Gene-related Hereditary Optic Atrophy And Mechanism Studies Based On Patient-derived Cell Models

Purpose:To summarize the clinical characteristics,molecular genetic characteristics and genotype-phenotype correlation of OPA1 gene-related inherited optic atrophy(OPA1-OA).To explore the possible pathogenesis of the disease based on patients' skin fibroblasts.To establish and identify the iPS cell lines derived from patients and to explore the method of differentiation from iPSC to RGC.Methods:Patients who were diagnosed with inherited optic atrophy in Peking Union Medical College Hospital from 2010 to 2020 and who were variated to carry OPA1 variants were enrolled in this study.(1)Clinical research methods:The patient's medical history and family history were recorded,and the visual acuity,color vision,fundus,visual field,OCT,PERG and VEP were performed.(2)Molecular genetics methods:the venous blood of patients and family members were drawn to extract genomic DNA.The pathogenic variants of the OPA1 gene were detected by direct Sanger sequencing or next generation sequencing,and all patients with pathogenic variants of OPA1 were screened and their molecular genetic characteristics were summarized.(3)Primary culture of skin fibroblasts,mitochondrial morphology,CCK-8 cell proliferation test,Seahorse mitochondrial oxygen consumption test,apoptosis detection and reactive oxygen species detection by flow cytometry were performed.(4)IPSC of OPA1-OA patients was established by Sendai reprogramming kit,and iPSC was differentiated into RGC by dual SMAD inhibition combined with Wnt inhibition.Results:A total of 33 patients from 19 OPA1-OA families were included in this study.There were 22 males and 11 females.The age ranged from 1 to 62 years old.The average age was 24.4 years old,and the median age was 26 years old.(1)Clinical features:Among the 33 patients,30 cases suffered only eye disease,2 cases were complicated with sensorineural deafness,1 case was complicated with sensorineural deafness and motor abnormality.The visual acuity was from LP from 0.6,and the median visual acuity was 0.15.75%of the patients had abnormal color vision.All patients suffered optic nerve atrophy,presenting with temporal disc pallor(65.2%)or entire disc pallor(34.8%).85.7%of the affected eyes showed different range of central or central-temporal scotoma on the visual field examination.OCT showed different ranges and degrees of thinning of the retinal nerve fiber layer(RNFL),of which 60.4%of the eyes presenting with temporal RNFL thinning,and 39.6%of the eyes showed RNFL thinning in all quadrants.(2)Molecular genetics features:A total of 19 OPA1 pathogenic variants were found,of which 9 have been reported and 10 have not been reported.Among them,there were 10 missense variants,5 small fragment deletions,1 small fragment insertion,2 nonsense variants and 1 splice site variation.Seven were located in the GTP domain of OPA1 protein and seven in the C-terminal helix domain.Most of the missense variants are located in the GTP enzyme domain(6/10)or in the unknown functional sequence in front of GTP enzyme domain(3/10).Most of the variants which lead to the premature termination of the amino acid sequence are located in the C-terminal helix domain(5/6).The phenomenon of incomplete penetrance were found in 4 families(21.1%).(3)Genotype-phenotype correlation:Patients were divided into two groups according to different genotypes.The first group was missense variant group,including 19 patients from 11 families.The second group was non-missense variant group,including 14 patients from 8 families.The visual acuity of the missense mutation group was worse than that of the non-missense variant group(1.28 LogMAR vs 0.66 LogMAR).All the patients in the missense variant group had abnormal color vision,while 54.5%of the patients in the non-missense mutation group had normal color vision.Most of the eyes in the missense variant group showed a pale disc in all quadrants(58.3%),while most of the eyes in the non-missense mutation group showed a pale disc in temporal side(93.3%).All eyes in the non-missense mutation group suffered just optic nerve atrophy,while 36.8%of the missense mutation group were associated with other ocular signs,including nystagmus and strabismus.There were two patients with sensorineural deafness in addition to eye involvement,F5-2 with sensorineural deafness and F16-1 with sensorineural deafness and motor abnormality,both of which were from missense variant group.(4)Fibroblast experiment:The percentage of fragmented mitochondria of OPA1 mutated skin fibroblasts was significantly higher than that of normal controls.The proliferative activity of OPA1 mutated skin fibroblasts decreased when cell density was high or undernourished.The basic respiratory function of OPA1 mutated skin fibroblasts was nearly normal,but the max respiratory function was significantly decreased.Skin fibroblasts of asymptomatic OPA1 carriers were more resistant to apoptosis stimulation.(5)IPSC induction and RGC differentiation:IPSCs of two patients with OPA1-OA were established.They carry the c.2681delT(p.L894*)and c.805T>C(p.S269P)variant,respectively.The alkaline phosphatase staining and stem cell marker staining were positive.The karyotype is normal.The iPSCs had the ability to differentiate into three germ layers,and can differentiate into RGC.Conclusions:(1)Patients with OPA1-OA have various severity of clinical manifestations and may share different mutation spectra from Caucasian people.(2)Patients with non-missense variants are less severe than those with missense variants,showing better visual acuity,color vision and lighter degree of optic nerve atrophy.(3)Insufficient reserve respiratory capacity may be one of the pathogenic factors in patients with OPA1-OA,and the different response to apoptosis stimulation of patients from the same family may be one of the reason for incomplete penetrance.(4)OPA1 mutated cells can be induced into iPSC and have the ability of multi-directional differentiation.