Genomic epidemiology of the primary methicillin-resistant Staphylococcus aureus clones causing invasive infections in Paraguayan children.

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.

A catalog of the genetic causes of hereditary angioedema in the Canary Islands (Spain).

Hereditary angioedema (HAE) is a rare disease where known causes involve C1 inhibitor dysfunction or dysregulation of the kinin cascade. The updated HAE management guidelines recommend performing genetic tests to reach a precise diagnosis. Unfortunately, genetic tests are still uncommon in the diagnosis routine. Here, we characterized for the first time the genetic causes of HAE in affected families from the Canary Islands (Spain). Whole-exome sequencing data was obtained from 41 affected patients and unaffected relatives from 29 unrelated families identified in the archipelago. The Hereditary Angioedema Database Annotation (HADA) tool was used for pathogenicity classification and causal variant prioritization among the genes known to cause HAE. Manual reclassification of prioritized variants was used in those families lacking known causal variants. We detected a total of eight different variants causing HAE in this patient series, affecting essentially SERPING1 and F12 genes, one of them being a novel SERPING1 variant (c.686-12A>G) with a predicted splicing effect which was reclassified as likely pathogenic in one family. Altogether, the diagnostic yield by assessing previously reported causal genes and considering variant reclassifications according to the American College of Medical Genetics guidelines reached 66.7% (95% Confidence Interval [CI]: 30.1-91.0) in families with more than one affected member and 10.0% (95% CI: 1.8-33.1) among cases without family information for the disease. Despite the genetic causes of many patients remain to be identified, our results reinforce the need of genetic tests as first-tier diagnostic tool in this disease, as recommended by the international WAO/EAACI guidelines for the management of HAE.

Evaluation of a whole-exome sequencing pipeline and benchmarking of causal germline variant prioritizers.

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.

First Census of Patients with Hereditary Angioedema in the Canary Islands.

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.

Evaluation of Whole-Exome Enrichment Solutions: Lessons from the High-End of the Short-Read Sequencing Scale.

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 SureSelectQXT 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. SureSelectQXT 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, SureSelectQXT 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 SureSelectQXT and Illumina DNA Prep with Enrichment to be the best enrichment capture solutions.

Interactive Web-Based Resource for Annotation of Genetic Variants Causing Hereditary Angioedema (HADA): Database Development, Implementation, and Validation.

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.

[This corrects the article DOI: 10.3389/fgene.2019.00900.].

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

Recurrent episodes of bradykinin-mediated angioedema (Bk-AE) can associate with acquired or hereditary conditions, the former most commonly developing secondarily to a pharmacological treatment. Despite successful genomic advances that have led to the identification of a large number of disease genes irrespective of disease prevalence, their application to Bk-AE has barely occurred. As a consequence, the genetic causes of Bk-AE remain poorly understood, obstructing the identification of patient subtypes and the development of precision medicine strategies. This review provides an update of the genetic studies completed to date on the acquired forms, which have almost exclusively focused on Bk-AE secondarily to the angiotensin-converting enzyme inhibitor treatment, and the blooming subdivision of the hereditary forms established by the identification of novel causal genes with next-generation sequencing (NGS) technology-based exome studies in genetically undiagnosed patients. Finally, based on the diverse benefits that are offered by the technology, we present arguments favoring the use of holistic NGS approaches as first-tier genetic tests as a promise to reduce the diagnostic odyssey of patients with suspected hereditary forms of Bk-AE.

Whole-Exome Sequencing Identifies Somatic Mutations Associated With Mortality in Metastatic Clear Cell Kidney Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is among the most aggressive histologic subtypes of kidney cancer, representing about 3% of all human cancers. Patients at stage IV have nearly 60% of mortality in 2-3 years after diagnosis. To date, most ccRCC studies have used DNA microarrays and targeted sequencing of a small set of well-established, commonly altered genes. An exception is the large multi-omics study of The Cancer Genome Atlas Kidney Renal Clear Cell Carcinoma (TCGA-KIRC), which identified new ccRCC genes based on whole exome-sequencing (WES) data, and molecular prognostic signatures based on transcriptomics, epigenetics and proteomics data. Applying WES to simultaneously interrogate virtually all exons in the human genome for somatic variation, here we analyzed the burden of coding somatic mutations in metastatic ccRCC primary tumors, and its association with patient mortality from cancer, in patients who received VEGF receptor-targeting drugs as the first-line therapy. To this end, we sequenced the exomes of ten tumor-normal pairs of ccRCC patient tissues from primary biopsies at >100× mean depth and called somatic coding variation. Mutation burden analysis prioritized 138 genes linked to patient mortality. A gene set enrichment analysis evidenced strong statistical support for the abundance of genes involved in the development of kidney cancer (p = 2.31 × 10-9) and carcinoma (p = 1.22 × 10-5), with 49 genes having direct links with kidney cancer according to the published records. Two of these genes, SIPA1L2 and EIF3A, demonstrated independent associations with mortality in TCGA-KIRC project data. Besides, three mutational signatures were found to be operative in the tumor exomes, one of which (COSMIC signature 12) has not been previously reported in ccRCC. Selection analysis yielded no detectable evidence of overall positive or negative selection, with the exome-wide number of nonsynonymous substitutions per synonymous site reflecting largely neutral tumor evolution. Despite the limited sample size, our results provide evidence for candidate genes where somatic mutation burden is tentatively associated with patient mortality in metastatic ccRCC, offering new potential pharmacological targets and a basis for further validation studies.