HLA-DPB1*13:01 associates with enhanced, and KIR2DS4*001 with diminished protection from developing severe COVID-19.

Extreme polymorphism of HLA and killer-cell immunoglobulin-like receptors (KIR) differentiates immune responses across individuals. Additional to T cell receptor interactions, subsets of HLA class I act as ligands for inhibitory and activating KIR, allowing natural killer (NK) cells to detect and kill infected cells. We investigated the impact of HLA and KIR polymorphism on the severity of COVID-19. High resolution HLA class I and II and KIR genotypes were determined from 403 non-hospitalized and 1575 hospitalized SARS-CoV-2 infected patients from Italy collected in 2020. We observed that possession of the activating KIR2DS4*001 allotype is associated with severe disease, requiring hospitalization (OR = 1.48, 95% CI 1.20-1.85, pc = 0.017), and this effect is greater in individuals homozygous for KIR2DS4*001 (OR = 3.74, 95% CI 1.75-9.29, pc = 0.003). We also observed the HLA class II allotype, HLA-DPB1*13:01 protects SARS-CoV-2 infected patients from severe disease (OR = 0.49, 95% CI 0.33-0.74, pc = 0.019). These association analyses were replicated using logistic regression with sex and age as covariates. Autoantibodies against IFN-α associated with COVID-19 severity were detected in 26% of 156 hospitalized patients tested. HLA-C*08:02 was more frequent in patients with IFN-α autoantibodies than those without, and KIR3DL1*01502 was only present in patients lacking IFN-α antibodies. These findings suggest that KIR and HLA polymorphism is integral in determining the clinical outcome following SARS-CoV-2 infection, by influencing the course both of innate and adaptive immunity.

The landscape of the immunoglobulin repertoire in endemic pemphigus foliaceus.

Introduction: Primarily driven by autoreactive B cells, pemphigus foliaceus (PF) is an uncommon autoimmune blistering skin disease of sporadic occurrence worldwide. However, PF reaches a prevalence of 3% in the endemic areas of Brazil, the highest ever registered for any autoimmune disease, which indicates environmental factors influencing the immune response in susceptible individuals. We aimed to provide insights into the immune repertoire of patients with PF living in the endemic region of the disease, compared to healthy individuals from the endemic region and a non-endemic area. Methods: We characterized the B-cell repertoire in i) nontreated patients (n=5); ii) patients under immunosuppressive treatment (n=5); iii) patients in remission without treatment (n=6); and two control groups iv) from the endemic (n=6) and v) non-endemic areas in Brazil (n=4). We used total RNA extracted from peripheral blood mononuclear cells and performed a comprehensive characterization of the variable region of immunoglobulin heavy chain (IGH) in IgG and IgM using next-generation sequencing. Results: Compared to individuals from a different area, we observed remarkably lower clonotype diversity in the B-cell immune repertoire of patients and controls from the endemic area (p < 0.02), suggesting that the immune repertoire in the endemic area is under geographically specific and intense environmental pressure. Moreover, we observed longer CDR3 sequences in patients, and we identified differential disease-specific usage of IGHV segments, including increased IGHV3-30 and decreased IGHV3-23 in patients with active disease (p < 0.04). Finally, our robust network analysis discovered clusters of CDR3 sequences uniquely observed in patients with PF. Discussion: Our results indicate that environmental factors, in addition to disease state, impact the characteristics of the repertoire. Our findings can be applied to further investigation of the environmental factors that trigger pemphigus and expand the knowledge for identifying new targeted and more effective therapies.

Selecting Genetic Variants and Interactions Associated with Amyotrophic Lateral Sclerosis: A Group LASSO Approach.

Amyotrophic lateral sclerosis (ALS) is a multi-system neurodegenerative disease that affects both upper and lower motor neurons, resulting from a combination of genetic, environmental, and lifestyle factors. Usually, the association between single-nucleotide polymorphisms (SNPs) and this disease is tested individually, which leads to the testing of multiple hypotheses. In addition, this classical approach does not support the detection of interaction-dependent SNPs. We applied a two-step procedure to select SNPs and pairwise interactions associated with ALS. SNP data from 276 ALS patients and 268 controls were analyzed by a two-step group LASSO in 2000 iterations. In the first step, we fitted a group LASSO model to a bootstrap sample and a random subset of predictors (25%) from the original data set aiming to screen for important SNPs and, in the second step, we fitted a hierarchical group LASSO model to evaluate pairwise interactions. An in silico analysis was performed on a set of variables, which were prioritized according to their bootstrap selection frequency. We identified seven SNPs (rs16984239, rs10459680, rs1436918, rs1037666, rs4552942, rs10773543, and rs2241493) and two pairwise interactions (rs16984239:rs2118657 and rs16984239:rs3172469) potentially involved in nervous system conservation and function. These results may contribute to the understanding of ALS pathogenesis, its diagnosis, and therapeutic strategy improvement.

The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males.

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3L412F-encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways.Abbreviations: AP: autophagosome; AUC: area under the curve; BafA1: bafilomycin A1; COVID-19: coronavirus disease-2019; HCQ: hydroxychloroquine; RAP: rapamycin; ROC: receiver operating characteristic; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor.

Genetic association and differential expression of HLAComplexGroup lncRNAs in pemphigus.

BACKGROUND: Pemphigus is a group of bullous diseases characterized by acantholysis and skin blisters. As for other autoimmune diseases, the strongest genetic associations found so far for pemphigus foliaceus (PF) and vulgaris (PV) are with alleles of HLA genes. However, apart from protein-coding genes, the MHC region includes a set of poorly explored long non-coding RNA (lncRNA) genes, the HLA complex group (HCG). OBJECTIVES: To investigate if HCG lncRNA alleles are associated with pemphigus susceptibility. METHODS AND RESULTS: We analyzed SNPs in 13 HCG lncRNA genes, both in PV (Germany: 241 patients; 1,188 controls) and endemic PF (Brazil: 227 patients; 194 controls), applying multivariate logistic regression. We found 55 associations with PV (pcorr < 0.01) and nine with endemic PF (pcorr < 0.05), the majority located in TSBP1-AS1 (which includes HCG23) and HCG27 lncRNA genes, independently of HLA alleles previously associated with pemphigus. The association of TSBP1-AS1 rs3129949*A allele was further replicated in sporadic PF (p = 0.027, OR = 0.054; 75 patients and 150 controls, all from Germany). Next, we evaluated the expression levels of TSBP1-AS1, TSBP1, HCG23, and HCG27 in blood mononuclear cells of Brazilian patients and controls. HCG27 was upregulated in endemic PF (p = 0.035, log2 FC = 1.3), while TSBP1-AS1 was downregulated in PV (p = 0.029, log2 FC = -1.29). The same expression patterns were also seen in cultured keratinocytes stimulated with IgG antibodies from patients and controls from Germany. TSBP1 mRNA levels were also decreased in endemic PF blood cells (p = 0.042, log2 FC = -2.14). TSBP1-AS1 and HCG27 were also observed downregulated in CD19+ cells of endemic PF (p < 0.01, log2 FC = -0.226 and -0.46 respectively). CONCLUSIONS: HCG lncRNAs are associated with susceptibility to pemphigus, being TSBP1-AS1 and HCG27 also differentially expressed in distinct cell populations. These results suggest a role for HCG lncRNAs in pemphigus autoimmunity.

Dysregulation of Transcription Factor Networks Unveils Different Pathways in Human Papillomavirus 16-Positive Squamous Cell Carcinoma and Adenocarcinoma of the Uterine Cervix.

Squamous cell carcinoma (SCC) and adenocarcinoma (ADC) are the most common histological types of cervical cancer (CC). The worse prognosis of ADC cases highlights the need for better molecular characterization regarding differences between these CC types. RNA-Seq analysis of seven SCC and three ADC human papillomavirus 16-positive samples and the comparison with public data from non-tumoral human papillomavirus-negative cervical tissue samples revealed pathways exclusive to each histological type, such as the epithelial maintenance in SCC and the maturity-onset diabetes of the young (MODY) pathway in ADC. The transcriptional regulatory network analysis of cervical SCC samples unveiled a set of six transcription factor (TF) genes with the potential to positively regulate long non-coding RNA genes DSG1-AS1, CALML3-AS1, IGFL2-AS1, and TINCR. Additional analysis revealed a set of MODY TFs regulated in the sequence predicted to be repressed by miR-96-5p or miR-28-3p in ADC. These microRNAs were previously described to target LINC02381, which was predicted to be positively regulated by two MODY TFs upregulated in cervical ADC. Therefore, we hypothesize LINC02381 might act by decreasing the levels of miR-96-5p and miR-28-3p, promoting the MODY activation in cervical ADC. The novel TF networks here described should be explored for the development of more efficient diagnostic tools.

Variation in genes implicated in B-cell development and antibody production affects susceptibility to pemphigus.

Pemphigus foliaceus (PF) is an autoimmune blistering skin disease characterized by the presence of pathogenic autoantibodies against desmoglein 1, a component of intercellular desmosome junctions. PF occurs sporadically across the globe and is endemic in some Brazilian regions. Because PF is a B-cell-mediated disease, we aimed to study the impact of variants within genes encoding molecules involved in the different steps of B-cell development and antibody production on the susceptibility of endemic PF. We analysed 3,336 single nucleotide polymorphisms (SNPs) from 167 candidate genes genotyped with Illumina microarray in a cohort of 227 PF patients and 193 controls. After quality control and exclusion of non-informative and redundant SNPs, 607 variants in 149 genes remained in the logistic regression analysis, in which sex and ancestry were included as covariates. Our results revealed 10 SNPs within or nearby 11 genes that were associated with susceptibility to endemic PF (OR >1.56; p < 0.005): rs6657275*G (TGFB2); rs1818545*A (RAG1/RAG2/IFTAP);rs10781530*A (PAXX), rs10870140*G and rs10781522*A (TRAF2); rs535068*A (TNFRSF1B); rs324011*A (STAT6);rs6432018*C (YWHAQ); rs17149161*C (YWHAG); and rs2070729*C (IRF1). Interestingly, these SNPs have been previously associated with differential gene expression, mostly in peripheral blood, in publicly available databases. For the first time, we show that polymorphisms in genes involved in B-cell development and antibody production confer differential susceptibility to endemic PF, and therefore are candidates for possible functional studies to understand immunoglobulin gene rearrangement and its impact on diseases.

A 3'UTR polymorphism marks differential KLRG1 mRNA levels through disruption of a miR-584-5p binding site and associates with pemphigus foliaceus susceptibility.

Genetic variations mapping to 3' untranslated regions (3'UTRs) may overlap with microRNA (miRNA) binding sites, therefore potentially interfering with translation inhibition or messenger RNA (mRNA) degradation. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) located within the 3'UTRs of six candidate genes and predicted to interfere with miRNA ligation could account for disease-relevant differential mRNA levels. Focusing on pemphigus foliaceus (PF) - an autoimmune blistering skin condition with unique endemic patterns - we investigated whether nine 3'UTR SNPs from the CD1D, CTLA4, KLRD1, KLRG1, NKG7, and TNFSF13B genes differentially expressed in PF were disease-associated. The heterozygous genotype of the KLRG1 rs1805672 polymorphism was associated with increased predisposition to PF (A/G vs. A/A: P=0.038; OR=1.60), and a trend for augmented susceptibility was observed for carriers of the G allele (P=0.094; OR=1.44). In silico analyses suggested that rs1805672 G allele could disrupt binding of miR-584-5p, and indicated rs1805672 as an expression Quantitative Trait Locus (eQTL), with an effect on KLRG1 gene expression. Dual-luciferase assay showed that miR-584-5p mediated approximately 50% downregulation of the reporter gene's activity through the 3'UTR of KLRG1 harboring rs1805672 A allele (vs. miRNA-negative condition, P=0.006). This silencing relationship was lost after site-directed mutation to G allele (vs. miRNA-negative condition, P=0.391; vs. rs1805672 A allele, P=0.005). Collectively, these results suggest that a disease-associated SNP located within the 3'UTR of KLRG1 directly interferes with miR-584-5p binding, allowing for KLRG1 mRNA differential accumulation, which in turn may contribute to pathogenesis of autoimmune diseases, such as pemphigus.

KIR and HLA genotyping of Japanese descendants from Curitiba, a city of predominantly European ancestry from Southern Brazil.

We hereby report the KIR gene frequencies and the frequencies of HLA ligands of KIR for Brazilians of Japanese ancestry. A total of 51 individuals were genotyped for presence/absence of KIR (killer cell immunoglobulin-like receptors) genes and presence of HLA (human leukocyte antigens) ligands. KIR was genotyped using two pairs of sequence-specific primers (PCR-SSP) and HLA ligands were typed by LABType® SSO reagent kits (One Lambda, USA). These data are fully available in Allele Frequencies Net Database, under the population name "Brazil Curitiba Japanese KIR".