In December 2019, an initial cluster of interstitial bilateral pneumonia emerged in Wuhan, China. A human-to-human transmission was assumed and a previously unrecognized entity, termed coronavirus disease-19 (COVID-19) due to a novel coronavirus (SARS-CoV-2) was described. The infection has rapidly spread out all over the world and Italy has been the first European country experiencing the endemic wave with unexpected clinical severity in comparison with Asian countries. It has been shown that SARS-CoV-2 utilizes angiotensin converting enzyme 2 (ACE2) as host receptor and host proteases for cell surface binding and internalization. Thus, a predisposing genetic background can give reason for interindividual disease susceptibility and/or severity. Taking advantage of the Network of Italian Genomes (NIG), here we mined whole-exome sequencing data of 6930 Italian control individuals from five different centers looking for ACE2 variants. A number of variants with a potential impact on protein stability were identified. Among these, three more common missense changes, p.(Asn720Asp), p.(Lys26Arg), and p.(Gly211Arg) were predicted to interfere with protein structure and stabilization. Rare variants likely interfering with the internalization process, namely p.(Leu351Val) and p.(Pro389His), predicted to interfere with SARS-CoV-2 spike protein binding, were also observed. Comparison of ACE2 WES data between a cohort of 131 patients and 258 controls allowed identifying a statistically significant (P value < 0.029) higher allelic variability in controls compared with patients. These findings suggest that a predisposing genetic background may contribute to the observed interindividual clinical variability associated with COVID-19, allowing an evidence-based risk assessment leading to personalized preventive measures and therapeutic options.

Toll-like receptors (TLR) are crucial components in the initiation of innate immune responses to a variety of pathogens, triggering the production of pro-inflammatory cytokines and type I and II interferons, which are responsible for innate antiviral responses. Among the different TLRs, TLR7 recognizes several single-stranded RNA viruses including SARS-CoV-2. We and others identified rare loss-of-function variants in X-chromosomal TLR7 in young men with severe COVID-19 and with no prior history of major chronic diseases, that were associated with impaired TLR7 signaling as well as type I and II IFN responses. Here, we performed RNA sequencing to investigate transcriptome variations following imiquimod stimulation of peripheral blood mononuclear cells isolated from patients carrying previously identified hypomorphic, hypofunctional, and loss-of-function TLR7 variants. Our investigation revealed a profound impairment of the TLR7 pathway in patients carrying loss-of-function variants. Of note, a failure in IFNγ upregulation following stimulation was also observed in cells harboring the hypofunctional and hypomorphic variants. We also identified new TLR7 variants in severely affected male patients for which a functional characterization of the TLR7 pathway was performed demonstrating a decrease in mRNA levels in the IFNα, IFNγ, RSAD2, ACOD1, IFIT2, and CXCL10 genes.

Objective Somatic mosaicism of KRAS gene is currently recognized as the only established molecular basis of arteriovenous malformations (AVM). However, given the limitations of the current technologies, KRAS somatic mutations are detected only in a limited proportion of AVMs and tissue biopsy remains an invasive high risky, sometimes life-threatening, diagnostic procedure. Next-generation sequencing liquid biopsy using cell-free DNA (cfDNA) has emerged as an innovative noninvasive approach for early detection and monitoring of cancer. This approach overcomes the space-time profile constraint of tissue biopsies opens a new scenario for vascular malformations owing to somatic mosaicism. Here, we propose a new approach as a fast noninvasive reliable tool in order to investigate the cfDNA coming from the AVMs. Methods A group of five patients suffering from AVM were selected. Blood samples from peripheral vein and efferent vein from vascular malformation were collected and cfDNA was extracted. The cfDNA libraries were performed using Oncomine Pan-Cancer Cell-Free Assay. We used Ion Proton for sequencing and Ion Reporter Software for analysis (Life Technologies, Carlsbad, Calif). Results In all cases, either G12D or G12V mutations in KRAS were identified. The mutational load was higher in the efferent vein than in peripheral blood, confirming the causative role of the identified mutation at a somatic level. Conclusions We demonstrate that cfDNA next-generation sequencing liquid biopsy is able to identify the KRAS mutation detected in affected tissues. Moreover, we have shown that blood sample withdrawal at the lesion site increases variant allele frequency with an order of magnitude above the limit of detection (usually 0.05%), decreasing the risk of a false negative. Finally, the noninvasiveness of the method avoids any risk of bleeding, being easily performed also in children. We propose this technique as the method of choice to better investigate AVMs and consequently to identify the therapy tailored to the genetic defect. Clinical Relevance This article highlights the importance of using liquid biopsy as a new method to investigate the molecular profile of AVMs. In view of the frequent inaccessibility of vascular tissues owing to the invasiveness of solid biopsy and the relative high incidence of biopsies with low diagnostic power, here we evaluated the efficacy of detecting cfDNA fragments released into the bloodstream from the affected tissue cells. Through a simple blood draw from the efferent vein at the vascular malformation site, the liquid biopsy allowed us to identify KRAS pathogenic mutations piloting a personalized therapeutic approach and opening a new scenario for new therapeutic strategies.