ACE2 and TMPRSS2 variants and expression as candidates to sex and country differences in COVID-19 severity in Italy

This article is a preprint and has not been peer-reviewed. It reports new medical research that has yet to be evaluated and so should not be used to guide clinical practice.medRxiv 2020.03.30.20047878. This article is a preprint and has not been peer-reviewed. It reports new medical research that has yet to be evaluated and so should not be used to guide clinical practice.Background: As the outbreak of coronavirus disease 2019 (COVID-19) progresses, prognostic markers for early identification of high-risk individuals are an urgent medical need. Italy has the highest rate of SARS-CoV-2 infection, the highest number of deaths, and the highest mortality rate among large countries. Worldwide, a more severe course of COVID-19 is associated with older age, comorbidities, and male sex. Hence, we searched for possible genetic components of the peculiar severity of COVID-19 among Italians, by looking at expression levels and variants in ACE2 and TMPRSS2 genes, which are crucial for viral infection. Methods: Exome and SNP array data from a large Italian cohort representative of the country's population were used to compare the burden of rare variants and the frequency of polymorphisms with European and East Asian populations. Moreover, we looked into gene expression databases to check for sex-unbalanced expression. Results: While we found no significant evidence that ACE2 is associated with disease severity/sex bias in the Italian population, TMPRSS2 levels and genetic variants proved to be possible candidate disease modulators, contributing to the observed epidemiological data among Italian patients. Conclusions: Our analysis suggests a role for TMPRSS2 variants and expression levels in modulating COVID-19 severity, a hypothesis that fosters a rapid experimental validation on large cohorts of patients with different clinical manifestations.Preprin

Identification of a glucocorticoid response element in the human gamma chain fibrinogen promoter

The effect of the synthetic glucocorticoid hormone dexamethasone on human gamma chain fibrinogen gene expression was examined. The whole promoter region of 3.8 kb of this gene and progressive 5'-deletions were inserted into a promoterless expression vector, upstream of the luciferase gene and transiently transfected into the human hepatoma HepG2 cells, in the presence or in the absence of dexamethasone stimulation. Deletion analysis allowed to identify a region located between -1359 and -954 bp upstream from the transcription start site, involved in hormone inducibility. On the basis of a computer-assisted analysis, a putative GRE was found in this region at bases -1116 to -1102. Specific point mutations eliminating this putative GRE led to complete loss of glucocorticoid inducibility, thus indicating its functional role. Binding of the rat glucocorticoid receptor to this site was demonstrated by mobility-shift assays

POS0361 DNA METHYLATION SIGNATURES CHARACTERIZE PSORIASIS AND PSORIATIC ARTHRITIS IN MONOZYGOTIC TWINS DISCORDANT FOR THE DISEASE

Background:Psoriatic disease is a chronic inflammatory disorder spanning from skin disease (psoriasis) to psoriatic arthritis (PsA). The genetic background is insufficient to explain disease onset as illustrated by not very informative Genome Wide Association Studies and monozygotic (MZ) twin studies recently performed. It is strongly assumed that epigenetics may contribute to disease susceptibility modulating gene expression. DNA methylation has been found involved in several autoimmune inflammatory rheumatic diseases. Here we have analysed the DNA methylation profile of a selected cohort of MZ twins discordant for psoriasis/PsA.Objectives:To identify the methylome associated with psoriasis and PsA in the peripheral blood of MZ twins discordant for these conditions.Methods:Peripheral blood from 7 couples of MZ twins discordant for psoriatic disease was collected and DNA extracted for a genome-wide evaluation of the DNA methylation profile, with the Infinium MethylationEPIC BeadChip. Minfi and the packages of the Bioconductor were used to analyse the data obtained. Quality control and exclusion criteria were applied to the raw data having a final number of 762.451 probes, which accounts for 88% of the total.Results:The approach first identified 2564 differentially methylated positions (DMPs; *p<0.005) with 19 genes potentially affected (with at least two DMPs within 1 kb of distance), including SMAD3 and SMARCA4/BRG1 involved in the Interferon and TGFβ pathways. Gene Ontology (GO) analysis of DMP-associated genes showed a significative enrichment (*p<0.005) in transcription factor binding, transcription corepressor and transcription coactivator activity, SMAD binding and histone -lysine-N-methyltransferase activity. To further validate the results, 5'-methylcytosine immunoprecipitation (MedIP) followed by Real Time PCR was performed to assess the methylation level of SMAD3 and SMARCA4/BRG1 promoters in the same cohort of MZ twins. We found significantly DNA methylation enrichment in SMARCA4/BRG1 promoter in psoriatic disease twins (p<0.05). SMAD3 and SMARCA4/BRG1 mRNA expression was also assessed to evaluate any inverse correlation with promoter methylation level, on the MZ cohort used for the EPIC array (n=4) and on a cohort of PsA/Ps patients (n=8) and appropriate healthy controls (n=3). Reduced mRNA expression (p<0.05) was demonstrated for SMARCA4/BRG1 (n=4). Conversely, no changes were found for SMAD3.Conclusion:We report the first DNA methylation approach in MZ twins discordant for psoriatic disease. We believe that the observed changes in SMAD3 and SMARCA/BRG1 genes may suggest an epigenetic imbalance of chromatin remodelling factors involved in inflammation pathways with a potential role in PsA/psoriasis immunopathogenesis.Disclosure of Interests:None declare

Analysis of factor V in zebrafish demonstrates minimal levels needed for early hemostasis

In humans, coagulation factor V (FV) deficiency is a rare, clinically heterogeneous bleeding disorder, suggesting that genetic modifiers may contribute to disease expressivity. Zebrafish possess many distinct advantages including high fecundity, optical clarity, external development, and homology with the mammalian hemostatic system, features that make it ideal for genetic studies. Our aim was to study the role of FV in zebrafish through targeted mutagenesis and apply the model to the study of human F5 variants. CRISPR-mediated genome editing of the zebrafish f5 locus was performed, generating mutants homozygous for a 49 base pair deletion in exon 4. Thrombus formation secondary to vascular endothelial injury was absent in f52/2 mutant embryos and larvae. Despite this severe hemostatic defect, homozygous mutants survived before succumbing to severe hemorrhage in adulthood. Human F5 variants of uncertain significance from patients with FV deficiency were evaluated, and the causative mutations identified and stratified by their ability to restore thrombus formation in larvae. Analysis of these novel mutations demonstrates variable residual FV function, with minimal activity being required to restore hemostasis in response to laser-induced endothelial injury. This in vivo evaluation may be beneficial for patients whose factor activity levels lack correlation with bleeding symptomatology, although limitations exist. Furthermore, homozygous mutant embryos tolerate what is a severe and lethal defect in mammals, suggesting the possibility of species-specific factors enabling survival, and allowing further study not possible in the mouse. Identification of these factors or other genetic modifiers could lead to novel therapeutic modalities

Molecular genetic analysis of severe coagulation factor XI deficiency in six Italian patients

BACKGROUND AND OBJECTIVES: Factor XI (FXI) deficiency is a rare autosomal recessive coagulopathy which is, however, frequent among Ashkenazi Jews. Two mutations, type II (Glu117stop) and type III (Phe283Leu), account for the majority of abnormal alleles in this population. The aim of this study was to analyze the molecular basis of FXI deficiency in six unrelated Italian probands with severe deficiency, a population hitherto largely unexplored. DESIGN AND METHODS: All patients showed unmeasurable functional FXI levels in plasma. Mutational screening was performed by sequencing. Haplotype analysis was performed using intragenic polymorphisms. Expression studies were performed by transient transfection in COS-1 cells. RESULTS: Sequencing identified two novel mutations: a nonsense mutation (Cys118stop) in exon 5 in two probands, and a 6-bp deletion (643-648delATCGAC) in exon 7 in one proband. The Cys118stop is predicted to cause FXI deficiency by a secretion defect and/or by increased mRNA degradation. The 6-bp deletion causes the loss of residues Ile197 and Asp198. There was a remarkable secretion impairment of the deleted FXI protein. In four of the six probands, the type II mutation was found. Haplotype analysis in patients carrying the type II mutation revealed that they share a common haplotype, perhaps derived from a Jewish ancestor. INTERPRETATION AND CONCLUSIONS: The identification and characterization of two novel mutations widen the mutational spectrum of FXI deficiency. Haplotype analysis is compatible with a Jewish origin of the type II mutation. The high occurrence of the type II mutation among Italian patients will be helpful to direct future genetic screenings

Functional and clinical implications of genetic structure in 1686 Italian exomes

To reconstruct the phenotypical and clinical implications of the Italian genetic structure, we thoroughly analyzed a whole-exome sequencing data set comprised of 1686 healthy Italian individuals. We found six previously unreported variants with remarkable frequency differences between Northern and Southern Italy in the HERC2, OR52R1, ADH1B, and THBS4 genes. We reported 36 clinically relevant variants (submitted as pathogenic, risk factors, or drug response in ClinVar) with significant frequency differences between Italy and Europe. We then explored putatively pathogenic variants in the Italian exome. On average, our Italian individuals carried 16.6 protein-truncating variants (PTVs), with 2.5% of the population having a PTV in one of the 59 American College of Medical Genetics (ACMG) actionable genes. Lastly, we looked for PTVs that are likely to cause Mendelian diseases. We found four heterozygous PTVs in haploinsufficient genes (KAT6A, PTCH1, and STXBP1) and three homozygous PTVs in genes causing recessive diseases (DPYD, FLG, and PYGM). Comparing frequencies from our data set to other public databases, like gnomAD, we showed the importance of population-specific databases for a more accurate assessment of variant pathogenicity. For this reason, we made aggregated frequencies from our data set publicly available as a tool for both clinicians and researchers (http://nigdb.cineca.it; NIG-ExIT)

Protein-Truncating Variants at the Cholesteryl Ester Transfer Protein Gene and Risk for Coronary Heart Disease.

RATIONALE: Therapies that inhibit CETP (cholesteryl ester transfer protein) have failed to demonstrate a reduction in risk for coronary heart disease (CHD). Human DNA sequence variants that truncate the CETP gene may provide insight into the efficacy of CETP inhibition. OBJECTIVE: To test whether protein-truncating variants (PTVs) at the CETP gene were associated with plasma lipid levels and CHD. METHODS AND RESULTS: We sequenced the exons of the CETP gene in 58 469 participants from 12 case-control studies (18 817 CHD cases, 39 652 CHD-free controls). We defined PTV as those that lead to a premature stop, disrupt canonical splice sites, or lead to insertions/deletions that shift frame. We also genotyped 1 Japanese-specific PTV in 27561 participants from 3 case-control studies (14 286 CHD cases, 13 275 CHD-free controls). We tested association of CETP PTV carrier status with both plasma lipids and CHD. Among 58 469 participants with CETP gene-sequencing data available, average age was 51.5 years and 43% were women; 1 in 975 participants carried a PTV at the CETP gene. Compared with noncarriers, carriers of PTV at CETP had higher high-density lipoprotein cholesterol (effect size, 22.6 mg/dL; 95% confidence interval, 18-27; P<1.0×10-4), lower low-density lipoprotein cholesterol (-12.2 mg/dL; 95% confidence interval, -23 to -0.98; P=0.033), and lower triglycerides (-6.3%; 95% confidence interval, -12 to -0.22; P=0.043). CETP PTV carrier status was associated with reduced risk for CHD (summary odds ratio, 0.70; 95% confidence interval, 0.54-0.90; P=5.1×10-3). CONCLUSIONS: Compared with noncarriers, carriers of PTV at CETP displayed higher high-density lipoprotein cholesterol, lower low-density lipoprotein cholesterol, lower triglycerides, and lower risk for CHD