Inherited retinal diseases (IRDs) are the major cause of refractory blinding eye diseases, and gene replacement therapy has already made preliminary progress in the treatment of IRDs. For IRDs that cannot be treated by gene replacement therapy, gene editing provides an alternative therapeutic method. Strategies like disruption of pathogenic variants with or without gene augmentation therapy and precise repair of pathogenic variants can be applied for IRDs with various inheritance patterns and pathogenic variants. In animal models of retinitis pigmentosa, Usher syndrome, Leber congenital amaurosis, cone rod cell dystrophy, and other disorders, CRISPR/Cas9, base editing, and prime editing showed the potential to edit pathogenic variations in vivo, indicating a promising future for gene editing therapy of IRDs.
Inherited retinal diseases (IRD) are a group of genetic disorders with high genetic and clinical heterogeneity. Genetic diagnosis has become one essential method for patients with IRD in their clinical management. So far, about 30% of the patients with IRD cannot get molecular diagnosis (no pathogenic variant detected or only mono-allele variant identified in AR genes) using target or whole exome sequencing. Most missing heritability or variants for these patients were variants located in no-coding regions (deep intron or promoter regions) and structure variants of the known IRD genes. It is more challenge to reveal this kind of missing variants, which need using whole genome sequencing combined with other cellular or molecular assays.
RCBTB1 gene associated hereditary retinopathy is an extremely rare inherited retinal disease (IRD) discovered recently. The mutation of RCBTB1 gene can lead to a variety of IRD clinical phenotypes, such as early retinitis pigmentosa and delayed chorioretinal atrophy. The hereditary mode of RCBTB1 gene associated retinopathy is autosomal recessive. RCBTB1 gene plays an important role in maintaining mitochondrial function and anti-oxidative stress defense mechanism of retinal pigment epithelium cells. In the future, it is necessary to further determine whether there is a genotypic and phenotypic correlation in the age of onset of RCBTB1 gene associated retinopathy or multi-organ involvement, and evaluate the safety and efficacy of adeno-associated virus-mediated RCBTB1 gene replacement therapy in animal models, to explore the feasibility of gene replacement therapy and stem cell therapy.
Objective To observe the expression of genes related to hereditary retinal diseases (IRD) in human microglia (hMG). MethodsA experimental study. Efficient differentiation of human induced pluripotent stem cells (iPSC) into hMG. Identification of octamer-binding transcription factor 4 (OCT4), sex-determining transcription factor 2 (SOX2), Nanog homeobox (NANOG), stage-specific embryonic antigen-4 (SSEA4), alpha-fetoprotein (AFP), α-smooth muscle actin (α-SMA) as markers associated with iPSC dryness and pluripotency by immunofluorescence staining Glial fibrillary acidic protein (GFAP); hMG associated marker transmembrane protein 119 (TMEM119), purinergic receptor P2Y12 (P2RY12), and allograft inflammatory factor 1 (IBA1). The proportion of CD11b+ and CD45+ cells was detected by flow cytometry. Mature hMG was collected and stimulated with lipopolysaccharide for 0, 4, 8 and 12 h, and were divided into groups 0 h, 4 h, 8 h and 12 h, respectively. Total RNA samples from the 4 groups were extracted for transcriptome sequencing, and the persistently significant differentially expressed genes (DEG) were screened. Real-time quantitative polymerase chain reaction (qPCR) was used to verify and analyze the expression of DEG mRNA. The two-tailed Student t test was used for comparison between the two groups. ResultsiPSC expressed the dry related markers OCT4, SOX2, NANOG and SSEA4, and differentiated into endoderm, mesoderm and ectoderm, expressing the corresponding markers AFP, α-SMA and GFAP, respectively. iPSC formed embryoid bodies under specific culture conditions, and then differentiated into hMG, and hMG expressed related markers TMEM119, P2RY12 and IBA1 by immunofluorescence staining. The double positive ratio of CD11b+ and CD45+ was > 95%. Transcriptomic analysis showed that the expression of 18 DEG in hMG stimulated by LPS was changed. qPCR test results showed that compared with group 0 h, mRNA expressions of Toll-like receptor 4 (TLR4), phosphoglycerate kinase 1, disintegrin and metallopeptidase domain 9 (ADAM9) in LPS stimulated group 4 h were significantly increased (t=25.43, 15.54, 6.26; P＜0.01). The mRNA expression levels of MER proto-oncogene tyrosine kinase (MERTK), non-hydrolase domain containing lysophospholipase 12 (ABHD12), retinal dehydrogenase 11 (RDH11), DNA damage autophagic regulator 2 (DRAM2) decreased (t=5.94, 14.14, 8.21, 6.97; P＜0.01), and the differences were statistically significant. Compared with group 0 h, mRNA expressions of RDH11, MERTK, ABHD12, DRAM2 and ADAM9 in group 8 h stimulated by LPS were significantly decreased, with statistical significance (t=25.97, 5.47, 43.97, 38.40, 3.84; P＜0.05). Compared with the group 0 h, the mRNA expressions of TLR4, ADAM9, MERTK, ABHD12, RDH11 and DRAM2 in the 12 h stimulated group were significantly decreased, and the differences were statistically significant (t=6.39, 46.11, 5.34, 14.14, 25.97, 25.65; P＜0.05). ConclusionIRD-related genes may be involved in the occurrence and development of IRD by regulating the function of hMG.
ObjectiveTo investigate and analyze the relationship between genotype and phenotype in patients with early-onset high myopia (eoHM) associated with hereditary eye diseases. MethodsA family-based study was conducted among 30 families diagnosed with eoHM at Department of Ophthalmology of People's Hospital of Ningxia Hui Autonomous Region from January 2022 to June 2023. Seven families (23.3%, 7/30), all probands, and their 14 parents were included. These seven families were unrelated. Detailed patient and family histories were collected. All participants underwent comprehensive ophthalmic examinations, including best-corrected visual acuity, color vision testing, fundus color photography, optical coherence tomography, fluorescein fundus angiography, and fundus autofluorescence imaging. Full-field electroretinography was performed in four cases. Peripheral venous blood samples were collected from patients and their parents for whole-genome DNA extraction and whole-exome sequencing. Potential pathogenic variants were identified, and their pathogenicity was analyzed and confirmed by Sanger sequencing. The pathogenicity of newly discovered gene variants was evaluated according to the guidelines of the American College of Medical Genetics and Genomics (ACMG). Literature on previously reported eoHM associated with hereditary eye diseases was reviewed to analyze the relationship between variant genes and clinical phenotypes. ResultsAmong the seven families, three exhibited X-linked inheritance, two showed autosomal recessive inheritance, and two demonstrated autosomal dominant inheritance. All the patients were male. Among the seven patients, one case each was identified with congenital stationary night blindness (CSNB), Bornholm eye disease, X-linked retinitis pigmentosa (XL-RP), cone-rod dystrophy, Knobloch syndrome, familial exudative vitreoretinopathy (FEVR), and Stickler syndrome. Genetic testing revealed nine gene variants highly correlated with the observed phenotypes. The genetic testing results revealed that all patients were found to carry nine gene variants highly associated with the phenotype, including: a hemizygous missense variant NYX c.647A>T (p.N216I) (M1), an OPN1LW LIAVA haplotype variant (M2), a hemizygous frameshift variant RPGR c.3096_3097del (p.E1033RfsTer45) (M3), compound heterozygous variants TTLL5 c.1588_1589del (p.L531EfsTer24) and c.850G>C (p.D284H) (M4, M5), compound heterozygous variants COL18A1 c.2118dup (p.G707RfsTer23) and c.3523_3524del (p.L1175VfsTer72) (M6, M7), a heterozygous missense mutation FZD4 c.1499C>T (p.T500I) (M8), and a heterozygous frameshift variant COL11A2 c.966dup (p.T323HfsTer19) (M9). Among them, M2, M4, M5, M8 and M9 were newly discovered mutation sites, and M1, M3, M6 and M7 were known mutation sites. According to the classification standards and guidelines of genetic variation issued by ACMG, M2, M3, M4, M6, M7, and M9 were judged to be pathogenic variants, while M1, M5, and M8 were of unknown clinical significance. Through literature review, it was found that eoHM was more common among the clinical phenotypes of 4 types of hereditary retinal diseases, including CSNB, Stickler syndrome, FEVR and XL-RP. ConclusioneoHM is intricately associated with inherited eye diseases and may serve as the earliest indicator of such conditions.