Under non-stressed conditions, the redox status of the different subcellular compartments is tightly controlled . Hence, the cytoplasm has a reducing environment that favours cysteine protein residues in their dithiol form while the endoplasmic reticulum environment is oxidizing to facilitate the formation of disulfide bonds for protein folding . This situation is reversed in the presence of aggregation-prone proteins, as both com partments undergo a dramatic shift in their respective redox status, with the cytoplasm becoming more oxidized and the endoplasmic reticulum more reducing [1]. However, whether changes in the cellular redox status affect protein aggregation has not yet been addressed .We approached this hypothesis by using a C. elegans mutant strain lacking the gsr-1 gene, encoding glutathione reductase, the enzyme responsible for recycling oxidized glutathione (GSSG) and thus maintenance of glutathione redox homeostasis (2]. We found that gsr-1 deficiency enhances the deleterious phenotypes of worm di sea se models ca u sed by aggregating proteins like human b-amyloid peptide, a-synuclein or polyglutamine repeats containing proteins. lmportantly, gsr-1 dependent proteostatic disruption is also found in C. elegans strains expressing endogenous UNC-52 and LET-60 aggregate-prone metastable proteins. This deleterious effect is largely phenocopied by the GSH depleting agent diethyl ma leate [3].Protein aggregates can be disposed by autophagy and consistent with a role of GSR-1 in this process, gsr-1 mutants abolish nuclear translocation of the TFEB/HLH-30 transcription factor (a key mediator of autophagy induction) and strongly impair the degradation of the autophagy substrate p62/SQST -1 ::GFP. In agreement, genetic disruption of autophagy in gsr-1 mutants expressing aggregation prone proteins resulted in strong synthetic developmental phenotypes and in sorne cases lethality. Downregulation of glutathione reductase and GSH levels in both yeast and mammalian cell models also caused phenotypes associated to protein aggregation and impaired TFEB nuclear translocation [3]. Together, this study demonstrates a novel, evolutionarily conserved role of glutathione redox homeostasis in proteostasis maintenance through autophagy regulation . {1] Kirstein J, et al. (2015) Proteotoxtc stress and aqeinq triqqers the loss of redox homeostasis across ce/fular compartments. EMBO J. 34: 2334-49. {2] Mora-Larca JA et al. (20 16) Glutathione reductase qsr-1 is an essenttal gene required for Caenorhabditis eleqans earlv embryomc development. Free Radie Biol Med. 96:446-61 . {3] Guerrero-Gómez O, et al. (2019) Loss of qlutathtone redox homeostasis impairs proteostasis bv inhibitinq autophaqy-dependent protein deqradation. Ce// Oeath Oiffer. m press. 9

Background Retinitis Pigmentosa (RP) is a clinically and genetically heterogeneous disorder that results in inherited blindness. Despite the large number of genes identified, only ~ 60% of cases receive a genetic diagnosis using targeted-sequencing. The aim of this study was to design a whole genome sequencing (WGS) based approach to increase the diagnostic yield of complex Retinitis Pigmentosa cases. Methods WGS was conducted in three family members, belonging to one large apparent autosomal dominant RP family that remained unsolved by previous studies, using Illumina TruSeq library preparation kit and Illumina HiSeq X platform. Variant annotation, filtering and prioritization were performed using a number of open-access tools and public databases. Sanger sequencing of candidate variants was conducted in the extended family members. Results We have developed and optimized an algorithm, based on the combination of different open-access tools, for variant prioritization of WGS data which allowed us to reduce significantly the number of likely causative variants pending to be manually assessed and segregated. Following this algorithm, four heterozygous variants in one autosomal recessive gene (USH2A) were identified, segregating in pairs in the affected members. Additionally, two pathogenic alleles in ADGRV1 and PDZD7 could be contributing to the phenotype in one patient. Conclusions The optimization of a diagnostic algorithm for WGS data analysis, accompanied by a hypothesis-free approach, have allowed us to unmask the genetic cause of the disease in one large RP family, as well as to reassign its inheritance pattern which implies differences in the clinical management of these cases. These results contribute to increasing the number of cases with apparently dominant inheritance that carry causal mutations in recessive genes, as well as the possible involvement of various genes in the pathogenesis of RP in one patient. Moreover, our WGS-analysis approach, based on open-access tools, can easily be implemented by other researchers and clinicians to improve the diagnostic yield of additional patients with inherited retinal dystrophies.

To enhance the use of Whole Genome Sequencing (WGS) in clinical practice, it is still necessary to standardize data analysis pipelines. Herein, we aimed to define a WGS-based algorithm for the accurate interpretation of variants in inherited retinal dystrophies (IRD). This study comprised 429 phenotyped individuals divided into three cohorts. A comparison of 14 pathogenicity predictors, and the re-definition of its cutoffs, were performed using panel-sequencing curated data from 209 genetically diagnosed individuals with IRD (training cohort). The optimal tool combinations, previously validated in 50 additional IRD individuals, were also tested in patients with hereditary cancer (n = 109), and with neurological diseases (n = 47) to evaluate the translational value of this approach (validation cohort). Then, our workflow was applied for the WGS-data analysis of 14 individuals from genetically undiagnosed IRD families (discovery cohort). The statistical analysis showed that the optimal filtering combination included CADDv1.6, MAPP, Grantham, and SIFT tools. Our pipeline allowed the identification of one homozygous variant in the candidate gene CFAP20 (c.337 C > T; p.Arg113Trp), a conserved ciliary gene, which was abundantly expressed in human retina and was located in the photoreceptors layer. Although further studies are needed, we propose CFAP20 as a candidate gene for autosomal recessive retinitis pigmentosa. Moreover, we offer a translational strategy for accurate WGS-data prioritization, which is essential for the advancement of personalized medicine.