BACKGROUNDThere is considerable variation in disease behavior among patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 . Genomewide association analysis may allow for the identification of potential genetic factors involved in the development of Covid-19. METHODSWe conducted a genomewide association study involving 1980 patients with Covid-19 and severe disease (defined as respiratory failure) at seven hospitals in the Italian and Spanish epicenters of the SARS-CoV-2 pandemic in Europe. After quality control and the exclusion of population outliers, 835 patients and 1255 control participants from Italy and 775 patients and 950 control participants from Spain were included in the final analysis. In total, we analyzed 8,582,968 single-nucleotide polymorphisms and conducted a meta-analysis of the two case-control panels. RESULTSWe detected cross-replicating associations with rs11385942 at locus 3p21.31 and with rs657152 at locus 9q34.2, which were significant at the genomewide level (P<5×10 −8 ) in the meta-analysis of the two case-control panels (odds ratio, 1.77; 95% confidence interval [CI], 1.48 to 2.11; P = 1.15×10 −10 ; and odds ratio, 1.32; 95% CI, 1.20 to 1.47; P = 4.95×10 −8 , respectively). At locus 3p21.31, the association signal spanned the genes SLC6A20, LZTFL1, CCR9, FYCO1, CXCR6 and XCR1. The association signal at locus 9q34.2 coincided with the ABO blood group locus; in this cohort, a blood-group-specific analysis showed a higher risk in blood group A than in other blood groups (odds ratio, 1.45; 95% CI, 1.20 to 1.75; P = 1.48×10 −4 ) and a protective effect in blood group O as compared with other blood groups (odds ratio, 0.65; 95% CI, 0.53 to 0.79; P = 1.06×10 −5 ). CONCLUSIONSWe identified a 3p21.31 gene cluster as a genetic susceptibility locus in patients with Covid-19 with respiratory failure and confirmed a potential involvement of the ABO blood-group system. (Funded by Stein Erik Hagen and others.

Background. Respiratory failure is a key feature of severe Covid-19 and a critical driver of mortality, but for reasons poorly defined affects less than 10% of SARS-CoV-2 infected patients. Methods. We included 1,980 patients with Covid-19 respiratory failure at seven centers in the Italian and Spanish epicenters of the SARS-CoV-2 pandemic in Europe (Milan, Monza, Madrid, San Sebastian and Barcelona) for a genome-wide association analysis. After quality control and exclusion of population outliers, 835 patients and 1,255 population-derived controls from Italy, and 775 patients and 950 controls from Spain were included in the final analysis. In total we analyzed 8,582,968 single-nucleotide polymorphisms (SNPs) and conducted a meta-analysis of both case-control panels. Results. We detected cross-replicating associations with rs11385942 at chromosome 3p21.31 and rs657152 at 9q34, which were genome-wide significant (P<5x10-8) in the meta-analysis of both study panels, odds ratio [OR], 1.77; 95% confidence interval [CI], 1.48 to 2.11; P=1.14x10-10 and OR 1.32 (95% CI, 1.20 to 1.47; P=4.95x10-8), respectively. Among six genes at 3p21.31, SLC6A20 encodes a known interaction partner with angiotensin converting enzyme 2 (ACE2). The association signal at 9q34 was located at the ABO blood group locus and a blood-group-specific analysis showed higher risk for A-positive individuals (OR=1.45, 95% CI, 1.20 to 1.75, P=1.48x10-4) and a protective effect for blood group O (OR=0.65, 95% CI, 0.53 to 0.79, P=1.06x10-5). Conclusions. We herein report the first robust genetic susceptibility loci for the development of respiratory failure in Covid-19. Identified variants may help guide targeted exploration of severe Covid-19 pathophysiology.

Abnormalities in alternative splicing (AS) are emerging as recurrent features in autoimmune diseases (AIDs). In particular, a growing body of evidence suggests the existence of a pathogenic association between a generalized defect in splicing regulatory genes and multiple sclerosis (MS). Moreover, several studies have documented an unbalance in alternatively-spliced isoforms in MS patients possibly contributing to the disease etiology. In this work, using a combination of PCR-based techniques (reverse-transcription (RT)-PCR, fluorescent-competitive, real-time, and digital RT-PCR assays), we investigated the alternatively-spliced gene encoding Gasdermin B, GSDMB, which was repeatedly associated with susceptibility to asthma and AIDs. The in-depth characterization of GSDMB AS and backsplicing profiles led us to the identification of an exonic circular RNA (ecircRNA) as well as of novel GSDMB in-frame and out-of-frame isoforms. The non-productive splicing variants were shown to be downregulated by the nonsense-mediated mRNA decay (NMD) in human cell lines, suggesting that GSDMB levels are significantly modulated by NMD. Importantly, both AS isoforms and the identified ecircRNA were significantly dysregulated in peripheral blood mononuclear cells of relapsing-remitting MS patients compared to controls, further supporting the notion that aberrant RNA metabolism is a characteristic feature of the disease.

Long non-coding RNAs (lncRNAs) are post-transcriptional and epigenetic regulators, whose implication in neurodegenerative and autoimmune diseases remains poorly understood. We analyzed publicly available microarray data sets to identify dysregulated lncRNAs in multiple sclerosis (MS), a neuroinflammatory autoimmune disease. We found a consistent upregulation in MS of the lncRNA MALAT1 (2.7-fold increase; meta-analysis, P = 1.3 × 10−8; 190 cases, 182 controls), known to regulate alternative splicing (AS). We confirmed MALAT1 upregulation in two independent MS cohorts (1.5-fold increase; P &lt; 0.01; 59 cases, 50 controls). We hence performed MALAT1 overexpression/knockdown in cell lines, demonstrating that its modulation impacts on endogenous expression of splicing factors (HNRNPF and HNRNPH1) and on AS of MS-associated genes (IL7R and SP140). Minigene-based splicing assays upon MALAT1 modulation recapitulated IL7R and SP140 isoform unbalances observed in patients. RNA-sequencing of MALAT1-knockdown Jurkat cells further highlighted MALAT1 role in splicing (approximately 1100 significantly-modulated AS events) and revealed its contribution to backsplicing (approximately 50 differentially expressed circular RNAs). Our study proposes a possible novel role for MALAT1 dysregulation and the consequent AS alteration in MS pathogenesis, based on anomalous splicing/backsplicing profiles of MS-relevant genes.

Multiple sclerosis (MS) is the most common neurological disorder in young adults. Despite extensive studies, only a fraction of MS heritability has been explained, with association studies focusing primarily on protein-coding genes, essentially for the difficulty of interpreting non-coding features. However, non-coding RNAs (ncRNAs) and functional elements, such as super-enhancers (SE), are crucial regulators of many pathways and cellular mechanisms, and they have been implicated in a growing number of diseases. In this work, we searched for possible enrichments in non-coding elements at MS genome-wide associated loci, with the aim to highlight their possible involvement in the susceptibility to the disease. We first reconstructed the linkage disequilibrium (LD) structure of the Italian population using data of 727,478 single-nucleotide polymorphisms (SNPs) from 1,668 healthy individuals. The genomic coordinates of the obtained LD blocks were intersected with those of the top hits identified in previously published MS genome-wide association studies (GWAS). By a bootstrapping approach, we hence demonstrated a striking enrichment of non-coding elements, especially of circular RNAs (circRNAs) mapping in the 73 LD blocks harboring MS-associated SNPs. In particular, we found a total of 482 circRNAs (annotated in publicly available databases) vs. a mean of 194 ± 65 in the random sets of LD blocks, using 1,000 iterations. As a proof of concept of a possible functional relevance of this observation, we experimentally verified that the expression levels of a circRNA derived from an MS-associated locus, i.e., hsa_circ_0043813 from the STAT3 gene, can be modulated by the three genotypes at the disease-associated SNP. Finally, by evaluating RNA-seq data of two cell lines, SH-SY5Y and Jurkat cells, representing tissues relevant for MS, we identified 18 (two novel) circRNAs derived from MS-associated genes. In conclusion, this work showed for the first time that MS-GWAS top hits map in LD blocks enriched in circRNAs, suggesting circRNAs as possible novel contributors to the disease pathogenesis.