Alejandro Mendoza-Alvarez

Hereditary angioedema is a rare, autosomal dominant, genetic disorder characterized by recurrent episodes of angioedema. Over 800 SERPING1 gene variants have been reported, and their clinical profiles and causal genetic variants are highly heterogeneous. We report two cases of hereditary angioedema (HAE) in a Portuguese family: a 27-year-old male, under lanadelumab, and his 57-year-old father, kept on on-demand treatment. Genetic testing was performed following international guidelines. The Hereditary Angioedema Database Annotation highlighted a heterozygous insertion at exon 3 (c.336_337insC). This variant predicts a frameshift of the transcript, with the introduction of a premature STOP codon in C1-INH protein (p.Ser113LeufsTer20). We report the identification of a novel pathogenic SERPING1 variant in a family with HAE type 1, assisted by the Hereditary Angioedema Database Annotation variant prioritization tool. Our results may contribute to the identification of additional families with the same variant and can further enhance the knowledge about this condition.

Background and Objectives: One of the most common causes of drug-induced angioedema (AE-DI) is related to reduced bradykinin breakdown after the use of certain medications. This is the case for forms of AE-DI due to the use of angiotensin-converting enzyme inhibitors (ACEi), which are used for the treatment of cardiovascular conditions. The causes of AE are not clear in these patients. Given the limited number of AE-ACEi genetic loci identified by genome-wide association studies, we opted to assess the utility of NGS of a panel of relevant genes to identify candidate genetic risk factors in severely affected patients. Methods: Five hypertensive patients from unrelated families with clinical AE-ACEi were included in the study. Whole-exome sequencing, variant calling, and annotation techniques were used. ANNOVAR v18.04.16 was used to annotate the variant calls. The resulting variants for each patient were assessed using the Hereditary Angioedema Database Annotation tool and Franklin genomic platform for variant prioritization and clinical impact interpretation. Results: The genetic variant rs6025 in the F5 gene was identified in all recruited samples, which has been associated with an increase in blood clotting in AE-ACEi patients. In two patients, a common synonymous genetic variant of the ACE gene was found (rs4343). Finally, we identified the ACE genetic variant rs142947404 in only one patient. This variant has not been assessed in AE-ACEi. Conclusions: More studies will be needed to clarify the genetics involved in AE-DI. In this way, we will be able to try to predict future episodes of angioedema due to the use of ACEi.

Background/Objectives One of the most common causes of bradykinergic angioedema (Bk-AE) is related to reduced Bk breakdown after the use of certain medications. This is the case of forms of Acquired Angioedema (AAE) due to the use of angiotensin-converting enzyme inhibitors (ACEi), which are used for the treatment of cardiovascular conditions. The AE causes is not clear in these patients. Given the limited number of AAE-ACEi genetic loci identified by genome-wide association studies, here we opted for assessing the utility of NGS of a panel of relevant genes to aim to identify candidate genetic risk factors in severely affected patients. Methods Five Hypertensive patients from unrelated families with clinical AAE-ACEi were included in the study. Whole-exome sequencing, variant calling, and annotation techniques were used. ANNOVAR was used to annotate the variant calls . These variant were processed for each patient by Hereditary Angioedema Database Annotation tool and Franklin genomic platform for variant prioritization and clinical impact interpretation. Results The genetic variant rs6025 in F5 gene was identified in all recruited samples, which has been associated with an increase in blood clotting in AAE-ACEi patients. In two patients, a common synonymous genetic variant of ACE gene was found (rs4343). Finally, we identified the ACE genetic variant rs142947404 in only one patient. This variant has not been assessed in AAE-ACEi. Conclusions More studies will be needed to clarify the genetics involved in acquired forms of Bk-AE. In this way, we will be able to try to predict future episodes of angioedema due to use of ACEi.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major human pathogens. It could carry numerous resistance genes and virulence factors in its genome, some of which are related to the severity of the infection. An observational, descriptive, cross-sectional study was designed to molecularly analyze MRSA isolates that cause invasive infections in Paraguayan children from 2009 to 2013. Ten representative MRSA isolates of the main clonal complex identified were analyzed with short-read paired-end sequencing and assessed for the virulome, resistome, and phylogenetic relationships. All the genetically linked MRSA isolates were recovered from diverse clinical sources, patients, and hospitals at broad gap periods. The pan-genomic analysis of these clones revealed three major and different clonal complexes (CC30, CC5, and CC8), each composed of clones closely related to each other. The CC30 genomes prove to be a successful clone, strongly installed and disseminated throughout our country, and closely related to other CC30 public genomes from the region and the world. The CC5 shows the highest genetic variability, and the CC8 carried the complete arginine catabolic mobile element (ACME), closely related to the USA300-NAE-ACME+, identified as the major cause of CA-MRSA infections in North America. Multiple virulence and resistance genes were identified for the first time in this study, highlighting the complex virulence profiles of MRSA circulating in the country. This study opens a wide range of new possibilities for future projects and trials to improve the existing knowledge on the epidemiology of MRSA circulating in Paraguay. IMPORTANCE: The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) is a public health problem worldwide. The most frequent MRSA clones identified in Paraguay in previous studies (including community and hospital acquired) were the Pediatric (CC5-ST5-IV), the Cordobes-Chilean (CC5-ST5-I), the SouthWest Pacific (CC30-ST30-IV), and the Brazilian (CC8-ST239-III) clones. In this study, the pan-genomic analysis of the most representative MRSA clones circulating in invasive infection in Paraguayan children over the years 2009–2013, such as the CC30-ST30-IV, CC5-ST5-IV, and CC8-ST8-IV, was carried out to evaluate their genetic diversity, their repertoire of virulence factors, and antimicrobial resistance determinants. This revealed multiple virulence and resistance genes, highlighting the complex virulence profiles of MRSA circulating in Paraguay. Our work is the first genomic study of MRSA in Paraguay and will contribute to the development of genomic surveillance in the region and our understanding of the global epidemiology of this pathogen.

2. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major human pathogens, causing a wide range of infections from food poisoning to necrotizing pneumonia, endocarditis, or septicemia. It could carry numerous resistance genes and virulence factors, some of which are related to the severity of the infection, being also regarded as a potential “Super Bug”. In Paraguay, the prevalence of CC30-ST30-IV clones is the leading cause of S. aureus infections both at the regional level and in pediatric population. Here we aimed to deeply analyze the genomic features of MRSA isolates that cause invasive infections in Paraguayan children. An observational, descriptive, cross-sectional study was designed to analyze representative MRSA isolates of the main clones identified between 2009 to 2013 in Paraguayan children. All the genetically linked MRSA isolates were recovered from diverse clinical sources, patients, and hospitals at broad gap periods. Cases were primarily community-acquired, which excludes in-hospital transmission or outbreaks. MRSA isolates were analyzed with short-read paired-end sequencing and assessed for the virulome, resistome, and phylogenetic relationships. The pan-genomic analysis of these clones revealed three major and different clonal complexes (CC8, CC30, and CC5), each composed of clones closely related to each other, despite having different spa types. Furthermore, multiple virulence and resistance genes were identified for the first time in this study, pointing out the complex virulence profiles of MRSA circulating in the country. This study opens a wide range of new possibilities for future projects and trials to improve the existing knowledge on the epidemiology of MRSA circulating in Paraguay. 3. Impact statement: The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) is a public health problem worldwide. The most frequent MRSA clones identified in Paraguay in previous studies were (including community and hospital-acquired) the Pediatric (CC5-ST5-IV), the Cordobes-Chilean (CC5-ST5-I), the SouthWest Pacific (CC30-ST30-IV) and the Brazilian (CC8-ST239-III) clones. In this study, the pan-genomic analysis of the most representative MRSA clones circulating in invasive infection in Paraguayan children over ten years (2009-2019), such as the CC30-ST30-IV, CC5-ST5-IV, and CC8-ST8-IV, were carried out to evaluate their genetic diversity, their virulence factors repertoire, and antimicrobial resistance mechanisms revealed multiple virulences and resistance genes pointing out the complex virulence profiles of MRSA circulating in Paraguay. Our work is the first genomic study of MRSA in Paraguay and will contribute to the development of genomic surveillance in the region and our understanding of this pathogen’s global epidemiology. 4. Data summary: The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

Most causal variants of Mendelian diseases are exonic. Whole-exome sequencing (WES) has become the diagnostic gold standard, but causative variant prioritization constitutes a bottleneck. Here we assessed an in-house sample-to-sequence pipeline and benchmarked free prioritization tools for germline causal variants from WES data. WES of 61 unselected patients with a known genetic disease cause was obtained. Variant prioritizations were performed by diverse tools and recorded to obtain a diagnostic yield when the causal variant was present in the first, fifth, and 10th top rankings. A fraction of causal variants was not captured by WES (8.2%) or did not pass the quality control criteria (13.1%). Most of the applications inspected were unavailable or had technical limitations, leaving nine tools for complete evaluation. Exomiser performed best in the top first rankings, while LIRICAL led in the top fifth rankings. Based on the more conservative top 10th rankings, Xrare had the highest diagnostic yield, followed by a three-way tie among Exomiser, LIRICAL, and PhenIX, then followed by AMELIE, TAPES, Phen-Gen, AIVar, and VarNote-PAT. Xrare, Exomiser, LIRICAL, and PhenIX are the most efficient options for variant prioritization in real patient WES data.

Hereditary angioedema (HAE) is a rare genetic condition whose main symptoms are recurrent swelling in the skin, mucosa, and internal organs. Recent studies suggested that the regulation of the inflammatory response and the complement cascade are two of the pathways significantly enriched in the Canary Islands, Spain. Here, we describe the first HAE patient series in this region. Forty-one patients (33 F, 8 M) and nine healthy relatives belonging to twenty-nine families were recruited for this study, obtaining their clinical and demographic features using a data collection form, as well as blood samples for biochemical analysis. The mean age of patients was 36.8 years (ranging from 4 to 72 years). Positive family history of HAE was reported in 13 patients (32.5%), and a mean diagnosis delay of 7.9 (±12.5) years was estimated, ranging from months to 50 years. Cutaneous edema was the most common symptom (53.6%), while airway symptoms was present in 11 patients. Prophylactic treatment was indicated for 23 patients, while 14 also require on-demand rescue treatment. We estimate a minimum prevalence of 1.25:100,000 for HAE due to C1-INH deficiency or dysfunction in the Canary Islands, which is higher than the estimates for mainland Spanish populations. HAE continues to be a disease poorly recognized by health care professionals due to its confusing symptoms, leading to longer diagnosis delay. Altogether, the evidence reinforces the need for a rapid and accurate diagnosis and precision medicine-based studies to improve the patient’s quality of life.

Whole-exome sequencing has become a popular technique in research and clinical settings, assisting in disease diagnosis and increasing the understanding of disease pathogenesis. In this study, we aimed to compare common enrichment capture solutions available in the market. Peripheral blood-purified DNA samples were enriched with SureSelect^QXT V6 (Agilent) and various Illumina solutions: TruSeq DNA Nano, TruSeq DNA Exome, Nextera DNA Exome, and Illumina DNA Prep with Enrichment, and sequenced on a HiSeq 4000. We found that their percentage of duplicate reads was as much as 2 times higher than previously reported values for the previous HiSeq series. SureSelect^QXT and Illumina DNA Prep with Enrichment showed the best average on-target coverage, which improved when off-target regions were included. At high coverage levels and in shared bases, these two solutions and TruSeq DNA Exome provided three of the best performances. With respect to the number of small variants detected, SureSelect^QXT presented the lowest number of detected variants in target regions. When off-target regions were considered, its ability equalized to other solutions. Our results show SureSelect^QXT and Illumina DNA Prep with Enrichment to be the best enrichment capture solutions.

Background: Hereditary angioedema is a rare genetic condition caused by C1 esterase inhibitor deficiency, dysfunction, or kinin cascade dysregulation, leading to an increased bradykinin plasma concentration. Hereditary angioedema is a poorly recognized clinical entity and is very often misdiagnosed as a histaminergic angioedema. Despite its genetic nature, first-line genetic screening is not integrated in routine diagnosis. Consequently, a delay in the diagnosis, and inaccurate or incomplete diagnosis and treatment of hereditary angioedema are common. Objective: In agreement with recent recommendations from the International Consensus on the Use of Genetics in the Management of Hereditary Angioedema, to facilitate the clinical diagnosis and adapt it to the paradigm of precision medicine and next-generation sequencing–based genetic tests, we aimed to develop a genetic annotation tool, termed Hereditary Angioedema Database Annotation (HADA). Methods: HADA is built on top of a database of known variants affecting function, including precomputed pathogenic assessment of each variant and a ranked classification according to the current guidelines from the American College of Medical Genetics and Genomics. Results: HADA is provided as a freely accessible, user-friendly web-based interface with versatility for the entry of genetic information. The underlying database can also be incorporated into automated command-line stand-alone annotation tools. Conclusions: HADA can achieve the rapid detection of variants affecting function for different hereditary angioedema types, and further integrates useful information to reduce the diagnosis odyssey and improve its delay.

Corrigendum: Bradykinin-Mediated Angioedema: An Update of the Genetic Causes and the Impact of Genomics