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Inherited retinal dystrophies comprise a broad group of genetic eye diseases without effective treatment. Among them, Stargardt disease is the second most prevalent pathology. This pathology triggers progressive retinal degeneration and vision loss in children and adults. In recent years, the evolution of several genome editing technologies, such as the CRISPR-Cas9 system, has revolutionized disease modeling and personalized medicine. Human induced pluripotent stem cells also provide a valuable tool for in vitro disease studies and therapeutic applications. Here, we show precise correction of two ABCA4 pathogenic variants in human induced pluripotent stem cells from two unrelated patients affected with Stargardt disease. Gene editing was achieved with no detectable off-target genomic alterations, demonstrating efficient ABCA4 gene correction without deleterious effects. These results will contribute to the development of emerging gene and cell therapies for inherited retinal dystrophies.

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Best Vitelliform Macular dystrophy (BVMD) is the most prevalent of the distinctive retinal dystrophies caused by mutations in the BEST1 gene. This gene, which encodes for a homopentameric calcium-activated ion channel, is crucial for the homeostasis and function of the retinal pigment epithelia (RPE), the cell type responsible for recycling the visual pigments generated by photoreceptor cells. In BVMD patients, mutations in this gene induce functional problems in the RPE cell layer with an accumulation of lipofucsin that evolves into cell death and loss of sight. In this work, we employ iPSC-RPE cells derived from a patient with the p.Pro77Ser dominant mutation to determine the correlation between this variant and the ocular phenotype. To this purpose, gene and protein expression and localization are evaluated in iPSC-RPE cells along with functional assays like phagocytosis and anion channel activity. Our cell model shows no differences in gene expression, protein expression/localization, or phagocytosis capacity, but presents an increased chloride entrance, indicating that the p.Pro77Ser variant might be a gain-of-function mutation. We hypothesize that this variant disturbs the neck region of the BEST1 channel, affecting channel function but maintaining cell homeostasis in the short term. This data shed new light on the different phenotypes of dominant mutations in BEST1, and emphasize the importance of understanding its molecular mechanisms. Furthermore, the data widen the knowledge of this pathology and open the door for a better diagnosis and prognosis of the disease.

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Familial hypercholesterolemia (FH) is associated with mutations in the low-density lipoprotein (LDL) receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin 9 (PCSK9) genes. A pathological variant has not been identified in 30-70% of clinically diagnosed FH patients, and a burden of LDL cholesterol (LDL-c)-raising alleles has been hypothesized as a potential cause of hypercholesterolemia in these patients. Our aim was to study the distribution of weighted LDL-c-raising single-nucleotide polymorphism (SNP) scores (weighted gene scores or wGS) in a population recruited in a clinical setting in Catalonia. The study included 670 consecutive patients with a clinical diagnosis of FH and a prior genetic study involving 250 mutation-positive (FH/M+) and 420 mutation-negative (FH/M-) patients. Three wGSs based on LDL-c-raising variants were calculated to evaluate their distribution among FH patients and compared with 503 European samples from the 1000 Genomes Project. The FH/M- patients had significantly higher wGSs than the FH/M+ and control populations, with sensitivities ranging from 42% to 47%. A wGS based only on the SNPs significantly associated with FH (wGS8) showed a higher area under the receiver operating characteristic curve, and higher diagnostic specificity and sensitivity, with 46.4% of the subjects in the top quartile. wGS8 would allow for the assignment of a genetic cause to 66.4% of the patients if those with polygenic FH are added to the 37.3% of patients with monogenic FH. Our data indicate that a score based on 8 SNPs and the75th percentile cutoff point may identify patients with polygenic FH in Catalonia, although with limited diagnostic sensitivity and specificity.

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PURPOSE: This study aimed to identify the underlying genetic cause(s) of inherited retinal dystrophy (IRD) in 12 families of Kuwaiti origin affected by macular dystrophy and four Spanish patients affected by retinitis pigmentosa (RP). METHODS: Clinical diagnoses were based on standard ophthalmic evaluations (best-corrected visual acuity, retinography, fundus autofluorescence imaging, optical coherence tomography, electroretinography and visual field tests). Panel-based whole exome sequencing was used to simultaneously analyse 224 IRD genes in one affected member of each family. The putative causative variants were confirmed by Sanger sequencing and cosegregation analyses. Haplotype analysis was performed using single nucleotide polymorphisms. RESULTS: A homozygous missense mutation c.606C>A (p.Asp202Glu) in RP1 was found to be the molecular cause of IRD in all 12 families from Kuwait. These patients exhibited comparable symptoms, including progressive decline in visual acuity since adolescence. Fundus autofluorescence images revealed bilateral macular retinal pigment epithelium disturbances, with neither perimacular flecks nor peripheral alterations. A shared haplotype spanning at least 1.1 Mb was identified in all families, suggesting a founder effect. Furthermore, RP1 variants involving nonsense and/or frameshifting mutations (three of them novel) were identified in three Spanish autosomal-recessive RP families and one dominant RP pedigree. CONCLUSION: This study describes, for the first time, a macular dystrophy phenotype caused by an RP1 mutation; establishing a new genotype-phenotype correlation in this gene, expanding its mutation spectrum and further highlighting the clinical heterogeneity associated with IRD.

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Best disease, also known as Best vitelliform macular dystrophy, is an autosomal dominant form of macular degeneration. Here, we have generated an induced pluripotent stem cell (iPSC) line derived from a Best disease patient carrying a new dominant mutation in the BEST1 gene. Skin fibroblasts were reprogrammed to iPSCs by the non-integrative Sendai-virus method. The iPSC line has been characterized preserving the BEST1 mutation, expressing the pluripotency markers and being capable to differentiate to endoderm, mesoderm and ectoderm in vitro.

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Cone-rod dystrophies (CRD) are a group of Inherited Retinal Dystrophies (IRD) characterized by the primary involvement of cone photoreceptors, resulting in the degeneration of the central retina, or macula. Although there are more than 55 CRD genes, a considerable percentage of cases remain unsolved. In this context, the present study aimed to describe and characterize the phenoptype and the genetic cause of 3 CRD families from a cohort of IRD cases. Clinical evaluation in each patient was supported by a complete ophthalmological examination, including visual acuity measurement, fundus retinography, fundus autofluorescence imaging, optical coherence tomography and full-field electroretinography. Molecular diagnoses were performed by whole exome sequencing analyzing a group of 279 IRD genes, and cosegregation of the identified pathogenic variants was confirmed by Sanger sequencing. Three novel homozygous mutations in the autophagy gene DRAM2 were identified as the molecular cause of disease in the three families: c.518-1G>A, c.628_629insAG and c.693+2T>A. Clinical data revealed that the 3 patients presented a shared CRD phenotype with adult-onset macular involvement and later peripheral degeneration, although the age of onset, evolution and severity were variable. In order to characterize the transcription effects of these variants, mRNA expression studies were performed. The results showed alterations in the DRAM2 transcription, including alternative splicing forms and lower levels of mRNA, which correlated with the phenotypic variability observed between patients. For instance, frameshift mutations were related to a less severe phenotype, with circumscribed mid-peripheral involvement, and lower levels of mRNA, suggesting an activation of the nonsense-mediated decay (NMD) pathway; while a more severe and widespread retinal degeneration was associated to the inframe alternative splicing variant reported, possibly due to a malfunctioning or toxicity of the resulting protein. Following these findings, DRAM2 expression was assessed in several human tissues by semi-quantitative RT-PCR and two isoforms were detected ubiquitously, yet with a singular tissue-specific pattern in retina and brain. Altogether, although the unique retinal phenotype described did not correlate with the ubiquitous expression, the retinal-specific expression and the essential role of autophagy in the photoreceptor survival could be key arguments to explain this particular DRAM2 phenotype.

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The antinociceptive effects of the carbon monoxide-releasing molecule tricarbonyldichlororuthenium (II) dimer (CORM-2) during chronic pain are well documented, but most of its possible side-effects remain poorly understood. In this work, we examine the impact of CORM-2 treatment on the lipoprotein profile and two main atheroprotective functions attributed to high-density lipoprotein (HDL) in a mouse model of type 1 diabetes while analyzing the effect of this drug on diabetic neuropathy. Streptozotocin (Stz)-induced diabetic mice treated with CORM-2 (Stz-CORM-2) or vehicle (Stz-vehicle) were used to evaluate the effect of this drug on the modulation of painful diabetic neuropathy using nociceptive behavioral tests. Plasma and tissue samples were used for chemical and functional analyses, as appropriate. Two main antiatherogenic properties of HDL, i.e., the ability of HDL to protect low-density lipoprotein (LDL) from oxidation and to promote reverse cholesterol transport from macrophages to the liver and feces in vivo (m-RCT), were also assessed. Stz-induced diabetic mice displayed hyperglycemia, dyslipidemia and pain hypersensitivity. The administration of 10 mg/kg CORM-2 during five consecutive days inhibited allodynia and hyperalgesia and significantly ameliorated spinal cord markers (Cybb and Bdkrb1expression) of neuropathic pain in Stz mice, but it did not reduce the combined dyslipidemia shown in Stz-treated mice. Its administration to Stz-treated mice led to a significant increase in the plasma levels of cholesterol (∼ 1.4-fold vs. Ctrl, ∼ 1.3- fold vs. Stz-vehicle; p < 0.05) and was attributed to significant elevations in both non-HDL (∼ 1.8-fold vs. Ctrl; ∼ 1.6-fold vs. Stz-vehicle; p < 0.05) and HDL cholesterol (∼ 1.3-fold vs. Ctrl, ∼ 1.2-fold vs. Stz-vehicle; p < 0.05). The increased HDL in plasma was not accompanied by a commensurate elevation in m-RCT in Stz-CORM-2 compared to Stz-vehicle mice; instead, it was worsened as revealed by decreased [3H]-tracer trafficking into the feces in vivo. Furthermore, the HDL-mediated protection against LDL oxidation ex vivo shown by the HDL isolated from Stz-CORM-2 mice did not differ from that obtained in Stz-vehicle mice. In conclusion, the antinociceptive effects produced by a high dose of CORM-2 were accompanied by antioxidative effects but were without favorable effects on the dyslipidemia manifested in diabetic mice.

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Inherited retinal dystrophies (IRD) comprise a wide group of clinically and genetically complex diseases that progressively affect the retina. Over recent years, the development of next-generation sequencing (NGS) methods has transformed our ability to diagnose heterogeneous diseases. In this work, we have evaluated the implementation of whole exome sequencing (WES) for the molecular diagnosis of IRD. Using Ion ProtonTM system, we simultaneously analyzed 212 genes that are responsible for more than 25 syndromic and non-syndromic IRD. This approach was used to evaluate 59 unrelated families, with the pathogenic variant(s) successfully identified in 71.18% of cases. Interestingly, the mutation detection rate varied substantially depending on the IRD subtype. Overall, we found 63 different mutations (21 novel) in 29 distinct genes, and performed in vivo functional studies to determine the deleterious impact of variants identified in MERTK, CDH23, and RPGRIP1. In addition, we provide evidences that support CDHR1 as a gene responsible for autosomal recessive retinitis pigmentosa with early macular affectation, and present data regarding the disease mechanism of this gene. Altogether, these results demonstrate that targeted WES of all IRD genes is a reliable, hypothesis-free approach, and a cost- and time-effective strategy for the routine genetic diagnosis of retinal dystrophies.

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PURPOSE: To characterize the molecular basis of two novel BEST1 mutations causing autosomal recessive bestrophinopathy (ARB). Strong evidence argues in favor of the dominant negative effects of most autosomal dominantly inherited mutations, whereas there is only weak support for the molecular basis of the ARB phenotype. METHODS: Patients underwent ophthalmic examination, color and autofluorescence fundus imaging, optical coherence tomography (OCT), electrooculogram, and full-field electroretinogram (ERG). BEST1 was directly screened for mutations in two ARB unrelated patients. The pathogenicity of the new BEST1 variants was assessed in silico and in vivo. RESULTS: Two unrelated patients with diagnoses of ARB showed retinal pigment epithelial disturbances and abnormal ERGs. Each patient was homozygous for a novel BEST1 mutation, c.521_522del and c.1100+1G>A. A carrier sibling (WT/c.521_522del) was unaffected. Both mutations generate a frameshift and a premature stop codon that, if translated, would seriously compromise bestrophin-1 function. However, the in vivo quantitative RT-PCR assays showed that most of the mutated transcripts were eliminated before translation because the mRNA-BEST1 levels were dramatically diminished the controls. CONCLUSIONS: In truncating BEST1 mutations, the null phenotype associated with ARB is attributed to a substantial decrease of BEST1 expression promoted by the nonsense-mediated decay (NMD) surveillance mechanism. Moreover, the severity of the phenotype increases with the preserved amount of altered transcript, suggesting that the clinical outcome reflects the combined null and dominant negative effects of the two mutations over the patient's genetic background.

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