Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction.

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.

Optical genome mapping and revisiting short-read genome sequencing data reveal previously overlooked structural variants disrupting retinal disease-associated genes.

PURPOSE: Structural variants (SVs) play an important role in inherited retinal diseases (IRD). Although the identification of SVs significantly improved upon the availability of genome sequencing, it is expected that involvement of SVs in IRDs is higher than anticipated. We revisited short-read genome sequencing data to enhance the identification of gene-disruptive SVs. METHODS: Optical genome mapping was performed to improve SV detection in short-read genome sequencing-negative cases. In addition, reanalysis of short-read genome sequencing data was performed to improve the interpretation of SVs and to re-establish SV prioritization criteria. RESULTS: In a monoallelic USH2A case, optical genome mapping identified a pericentric inversion (173 megabase), with 1 breakpoint disrupting USH2A. Retrospectively, the variant could be observed in genome sequencing data but was previously deemed false positive. Reanalysis of short-read genome sequencing data (427 IRD cases) was performed which yielded 30 pathogenic SVs affecting, among other genes, USH2A (n = 15), PRPF31 (n = 3), and EYS (n = 2). Eight of these (>25%) were overlooked during previous analyses. CONCLUSION: Critical evaluation of our findings allowed us to re-establish and improve our SV prioritization and interpretation guidelines, which will prevent missing pathogenic events in future analyses. Our data suggest that more attention should be paid to SV interpretation and the current contribution of SVs in IRDs is still underestimated.

Potential Molecular Mechanisms of Rare Anti-Tumor Immune Response by SARS-CoV-2 in Isolated Cases of Lymphomas.

Recently, two cases of complete remission of classical Hodgkin lymphoma (cHL) and follicular lymphoma (FL) after SARS-CoV-2 infection were reported. However, the precise molecular mechanism of this rare event is yet to be understood. Here, we hypothesize a potential anti-tumor immune response of SARS-CoV-2 and based on a computational approach show that: (i) SARS-CoV-2 Spike-RBD may bind to the extracellular domains of CD15, CD27, CD45, and CD152 receptors of cHL or FL and may directly inhibit cell proliferation. (ii) Alternately, upon internalization after binding to these CD molecules, the SARS-CoV-2 membrane (M) protein and ORF3a may bind to gamma-tubulin complex component 3 (GCP3) at its tubulin gamma-1 chain (TUBG1) binding site. (iii) The M protein may also interact with TUBG1, blocking its binding to GCP3. (iv) Both the M and ORF3a proteins may render the GCP2-GCP3 lateral binding where the M protein possibly interacts with GCP2 at its GCP3 binding site and the ORF3a protein to GCP3 at its GCP2 interacting residues. (v) Interactions of the M and ORF3a proteins with these gamma-tubulin ring complex components potentially block the initial process of microtubule nucleation, leading to cell-cycle arrest and apoptosis. (vi) The Spike-RBD may also interact with and block PD-1 signaling similar to pembrolizumab and nivolumab- like monoclonal antibodies and may induce B-cell apoptosis and remission. (vii) Finally, the TRADD interacting "PVQLSY" motif of Epstein-Barr virus LMP-1, that is responsible for NF-kB mediated oncogenesis, potentially interacts with SARS-CoV-2 Mpro, NSP7, NSP10, and spike (S) proteins, and may inhibit the LMP-1 mediated cell proliferation. Taken together, our results suggest a possible therapeutic potential of SARS-CoV-2 in lymphoproliferative disorders.

The Spike of SARS-CoV-2: Uniqueness and Applications.

The Spike (S) protein of the SARS-CoV-2 virus is critical for its ability to attach and fuse into the host cells, leading to infection, and transmission. In this review, we have initially performed a meta-analysis of keywords associated with the S protein to frame the outline of important research findings and directions related to it. Based on this outline, we have reviewed the structure, uniqueness, and origin of the S protein of SARS-CoV-2. Furthermore, the interactions of the Spike protein with host and its implications in COVID-19 pathogenesis, as well as drug and vaccine development, are discussed. We have also summarized the recent advances in detection methods using S protein-based RT-PCR, ELISA, point-of-care lateral flow immunoassay, and graphene-based field-effect transistor (FET) biosensors. Finally, we have also discussed the emerging Spike mutants and the efficacy of the Spike-based vaccines against those strains. Overall, we have covered most of the recent advances on the SARS-CoV-2 Spike protein and its possible implications in countering this virus.

A novel multi-omics-based highly accurate prediction of symptoms, comorbid conditions, and possible long-term complications of COVID-19.

Comprehensive clinical pictures, comorbid conditions, and long-term complications of COVID-19 are still unknown. Recently, using a multi-omics-based strategy, we predicted potential drugs for COVID-19 with ∼70% accuracy. Herein, using a novel multi-omics-based bioinformatic approach and three ways of analysis, we identified the symptoms, comorbid conditions, and short-, mid-, and possible long-term complications of COVID-19 with >90% precision including 27 parent, 170 child, and 403 specific conditions. Among the specific conditions, 36 viral, 53 short-term, 62 short-mid-long-term, 194 mid-long-term, and 57 congenital conditions are identified. At a threshold "count of occurrence" of 4, we found that 83-100% (average 92.67%) of enriched conditions are associated with COVID-19. Except for dry cough and loss of taste, all the other COVID-19-associated mild and severe symptoms are enriched. CVDs, and pulmonary, metabolic, musculoskeletal, neuropsychiatric, kidney, liver, and immune system disorders are top comorbid conditions. Specific diseases like myocardial infarction, hypertension, COPD, lung injury, diabetes, cirrhosis, mood disorders, dementia, macular degeneration, chronic kidney disease, lupus, arthritis, etc. along with several other NCDs were found to be top candidates. Interestingly, many cancers and congenital disorders associated with COVID-19 severity are also identified. Arthritis, gliomas, diabetes, psychiatric disorders, and CVDs having a bidirectional relationship with COVID-19 are also identified as top conditions. Based on our accuracy (>90%), the long-term presence of SARS-CoV-2 RNA in human, and our "genetic remittance" assumption, we hypothesize that all the identified top-ranked conditions could be potential long-term consequences in COVID-19 survivors, warranting long-term observational studies.

Multi-omics-based identification of SARS-CoV-2 infection biology and candidate drugs against COVID-19.

SARS-CoV-2 has ushered a global pandemic with no effective drug being available at present. Although several FDA-approved drugs are currently under clinical trials for drug repositioning, there is an on-going global effort for new drug identification. In this paper, using multi-omics (interactome, proteome, transcriptome, and bibliome) data and subsequent integrated analysis, we present the biological events associated with SARS-CoV-2 infection and identify several candidate drugs against this viral disease. We found that: (i) Interactome-based infection pathways differ from the other three omics-based profiles. (ii) Viral process, mRNA splicing, cytokine and interferon signaling, and ubiquitin mediated proteolysis are important pathways in SARS-CoV-2 infection. (iii) SARS-CoV-2 infection also shares pathways with Influenza A, Epstein-Barr virus, HTLV-I, Measles, and Hepatitis virus. (iv) Further, bacterial, parasitic, and protozoan infection pathways such as Tuberculosis, Malaria, and Leishmaniasis are also shared by this virus. (v) A total of 50 candidate drugs, including the prophylaxis agents and pathway specific inhibitors are identified against COVID-19. (vi) Betamethasone, Estrogen, Simvastatin, Hydrocortisone, Tositumomab, Cyclosporin A etc. are among the important drugs. (vii) Ozone, Nitric oxide, plasma components, and photosensitizer drugs are also identified as possible therapeutic candidates. (viii) Curcumin, Retinoic acids, Vitamin D, Arsenic, Copper, and Zinc may be the candidate prophylaxis agents. Nearly 70% of our identified agents are previously suggested to have anti-COVID-19 effects or under clinical trials. Among our identified drugs, the ones that are not yet tested, need validation with caution while an appropriate drug combination from these candidate drugs along with a SARS-CoV-2 specific antiviral agent is needed for effective COVID-19 management.