Screening of CNVs using NGS data improves mutation detection yield and decreases costs in genetic testing for hereditary cancer.

INTRODUCTION: Germline CNVs are important contributors to hereditary cancer. In genetic diagnostics, multiplex ligation-dependent probe amplification (MLPA) is commonly used to identify them. However, MLPA is time-consuming and expensive if applied to many genes, hence many routine laboratories test only a subset of genes of interest. METHODS AND RESULTS: We evaluated a next-generation sequencing (NGS)-based CNV detection tool (DECoN) as first-tier screening to decrease costs and turnaround time and expand CNV analysis to all genes of clinical interest in our diagnostics routine. We used DECoN in a retrospective cohort of 1860 patients where a limited number of genes were previously analysed by MLPA, and in a prospective cohort of 2041 patients, without MLPA analysis. In the retrospective cohort, 6 new CNVs were identified and confirmed by MLPA. In the prospective cohort, 19 CNVs were identified and confirmed by MLPA, 8 of these would have been lost in our previous MLPA-restricted detection strategy. Also, the number of genes tested by MLPA across all samples decreased by 93.0% in the prospective cohort. CONCLUSION: Including an in silico germline NGS CNV detection tool improved our genetic diagnostics strategy in hereditary cancer, both increasing the number of CNVs detected and reducing turnaround time and costs.

BARD1 Pathogenic Variants are Associated with Triple-Negative Breast Cancer in a Spanish Hereditary Breast and Ovarian Cancer Cohort.

Only a small fraction of hereditary breast and/or ovarian cancer (HBOC) cases are caused by germline variants in the high-penetrance breast cancer 1 and 2 genes (BRCA1 and BRCA2). BRCA1-associated ring domain 1 (BARD1), nuclear partner of BRCA1, has been suggested as a potential HBOC risk gene, although its prevalence and penetrance are variable according to populations and type of tumor. We aimed to investigate the prevalence of BARD1 truncating variants in a cohort of patients with clinical suspicion of HBOC. A comprehensive BARD1 screening by multigene panel analysis was performed in 4015 unrelated patients according to our regional guidelines for genetic testing in hereditary cancer. In addition, 51,202 Genome Aggregation Database (gnomAD) non-Finnish, non-cancer European individuals were used as a control population. In our patient cohort, we identified 19 patients with heterozygous BARD1 truncating variants (0.47%), whereas the frequency observed in the gnomAD controls was 0.12%. We found a statistically significant association of truncating BARD1 variants with overall risk (odds ratio (OR) = 3.78; CI = 2.10-6.48; p = 1.16 × 10-5). This association remained significant in the hereditary breast cancer (HBC) group (OR = 4.18; CI = 2.10-7.70; p = 5.45 × 10-5). Furthermore, deleterious BARD1 variants were enriched among triple-negative BC patients (OR = 5.40; CI = 1.77-18.15; p = 0.001) compared to other BC subtypes. Our results support the role of BARD1 as a moderate penetrance BC predisposing gene and highlight a stronger association with triple-negative tumors.

Comprehensive analysis and ACMG-based classification of CHEK2 variants in hereditary cancer patients.

CHEK2 variants are associated with intermediate breast cancer risk, among other cancers. We aimed to comprehensively describe CHEK2 variants in a Spanish hereditary cancer (HC) cohort and adjust the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines for their classification. First, three CHEK2 frequent variants were screened in a retrospective Hereditary Breast and Ovarian Cancer cohort of 516 patients. After, the whole CHEK2 coding region was analyzed by next-generation sequencing in 1848 prospective patients with HC suspicion. We refined ACMG-AMP criteria and applied different combined rules to classify CHEK2 variants and define risk alleles. We identified 10 CHEK2 null variants, 6 missense variants with discordant interpretation in ClinVar database, and 35 additional variants of unknown significance. Twelve variants were classified as (likely)-pathogenic; two can also be considered "established risk-alleles" and one as "likely risk-allele." The prevalence of (likely)-pathogenic variants in the HC cohort was 0.8% (1.3% in breast cancer patients and 1.0% in hereditary nonpolyposis colorectal cancer patients). Here, we provide ACMG adjustment guidelines to classify CHEK2 variants. We hope that this study would be useful for variant classification of other genes with low effect variants.

CPAP vs oxigenoterapia convencional en lactantes trasladados por insuficiencia respiratoria / CPAP vs oxygen therapy in infants being transported due to acute respiratory failure

OBJETIVOS: Evaluar la efectividad y la seguridad de la presión continua en la vía aérea (CPAP) en lactantes trasladados por insuficiencia respiratoria aguda (IRA) y comparar el curso clínico y la evolución en la unidad de cuidados intensivos pediátricos (UCIP) de los pacientes trasladados con dicha terapia vs la oxigenoterapia convencional. MATERIAL Y MÉTODOS: Estudio observacional y analítico de una cohorte retrospectiva. Se revisaron las historias de los lactantes de 0 a 12meses con IRA que precisaron traslado interhospitalario a la UCIP. RESULTADOS: Se incluyeron 110 pacientes: 71 trasladados con CPAP y 39 con oxigenoterapia. La causa principal de IRA fue la bronquiolitis (81,8%). La mediana del nivel de CPAP fue de 7cmH2O (rango intercuartílico, 6-7). Controlando en análisis multivariables específicos por los valores previos, se obtuvo que la CPAP durante el traslado produjo una disminución significativa en el score de Wood-Downes (beta=−1,08; IC95%: −1,76 a 0,40; p = 0,002) y en la frecuencia cardiaca (beta=−19,64, IC95%: −28,46 a −10,81; p < 0,001). Ningún paciente precisó intubación endotraqueal durante el transporte. En la UCIP, la tasa de intubación fue similar en el grupo CPAP (7%) y en el de oxigenoterapia (5,1%) (p = 0,689). El porcentaje de pacientes que precisaron ventilación no invasiva con dos niveles de presión en las primeras 6h de ingreso en la UCIP fue mayor en el grupo de oxigenoterapia: 100% (11/11) vs 69,2% (18/26), p = 0,04. CONCLUSIONES: La administración precoz de CPAP en lactantes es segura durante el traslado interhospitalario. Durante el transporte, la CPAP comparada con la oxigenoterapia disminuye el score de Wood-Downes y la frecuencia cardiaca

OBJECTIVE: The aims of our study are to evaluate the effectiveness and security of CPAP (continuous positive airway pressure) in infants transferred with acute respiratory failure (ARF) and to compare their evolution in PICU between CPAP vs oxygen therapy. MATERIALS AND METHODS: We conducted a retrospective observational and analytical study by reviewing the health records of infants with ARF aged 0 to 12months that required interhospital transfer to the PICU. RESULTS: We included 110 patients: 71 transported with CPAP and 39 with oxygen therapy. The main cause of ARF was acute bronchiolitis (81.8%). The median level of CPAP was 7cmH2O (interquartile range, 6-7). Controlling by the previous values in specific multivariable models, CPAP produced a significant decrease in the Wood-Downes score (beta = −1.08; 95% CI = −1.76 to −0.40; P = .002) and the heart rate (beta = −19.64, 95% CI = −28.46 to −10.81; P < .001). No patients required endotracheal intubation during transport. During the PICU stay, the intubation rate was similar in the CPAP group (7%) and the oxygen therapy group (5.1%) (P=.689). The proportion of patients that required bilevel positive airway pressure within 6hours of admission to the PICU was higher in the oxygen therapy group: 100% (11/11) vs 69.2% (18/26), P=.04. CONCLUSIONS: Early administration of CPAP to infants with ARF was a safe respiratory support intervention during interhospital transport. During patient transport, the use of CPAP achieved greater decreases in the Wood-Downes score and heart rate compared to oxygen therapy

Exploring the Role of Mutations in Fanconi Anemia Genes in Hereditary Cancer Patients.

Fanconi anemia (FA) is caused by biallelic mutations in FA genes. Monoallelic mutations in five of these genes (BRCA1, BRCA2, PALB2, BRIP1 and RAD51C) increase the susceptibility to breast/ovarian cancer and are used in clinical diagnostics as bona-fide hereditary cancer genes. Increasing evidence suggests that monoallelic mutations in other FA genes could predispose to tumor development, especially breast cancer. The objective of this study is to assess the mutational spectrum of 14 additional FA genes (FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FANCP, FANCQ, FANCR and FANCU) in a cohort of hereditary cancer patients, to compare with local cancer-free controls as well as GnomAD. A total of 1021 hereditary cancer patients and 194 controls were analyzed using our next generation custom sequencing panel. We identified 35 pathogenic variants in eight genes. A significant association with the risk of breast cancer/breast and ovarian cancer was found for carriers of FANCA mutations (odds ratio (OR) = 3.14 95% confidence interval (CI) 1.4-6.17, p = 0.003). Two patients with early-onset cancer showed a pathogenic FA variant in addition to another germline mutation, suggesting a modifier role for FA variants. Our results encourage a comprehensive analysis of FA genes in larger studies to better assess their role in cancer risk.

[CPAP vs oxygen therapy in infants being transported due to acute respiratory failure]. / CPAP vs oxigenoterapia convencional en lactantes trasladados por insuficiencia respiratoria.

OBJECTIVE: The aims of our study are to evaluate the effectiveness and security of CPAP (continuous positive airway pressure) in infants transferred with acute respiratory failure (ARF) and to compare their evolution in PICU between CPAP vs oxygen therapy. MATERIALS AND METHODS: We conducted a retrospective observational and analytical study by reviewing the health records of infants with ARF aged 0 to 12months that required interhospital transfer to the PICU. RESULTS: We included 110 patients: 71 transported with CPAP and 39 with oxygen therapy. The main cause of ARF was acute bronchiolitis (81.8%). The median level of CPAP was 7cmH2O (interquartile range, 6-7). Controlling by the previous values in specific multivariable models, CPAP produced a significant decrease in the Wood-Downes score (beta = -1.08; 95% CI = -1.76 to -0.40; P = .002) and the heart rate (beta = -19.64, 95% CI = -28.46 to -10.81; P < .001). No patients required endotracheal intubation during transport. During the PICU stay, the intubation rate was similar in the CPAP group (7%) and the oxygen therapy group (5.1%) (P=.689). The proportion of patients that required bilevel positive airway pressure within 6hours of admission to the PICU was higher in the oxygen therapy group: 100% (11/11) vs 69.2% (18/26), P=.04. CONCLUSIONS: Early administration of CPAP to infants with ARF was a safe respiratory support intervention during interhospital transport. During patient transport, the use of CPAP achieved greater decreases in the Wood-Downes score and heart rate compared to oxygen therapy.

Physiological parameters for Prognosis in Abdominal Sepsis (PIPAS) Study: a WSES observational study.

Background: Timing and adequacy of peritoneal source control are the most important pillars in the management of patients with acute peritonitis. Therefore, early prognostic evaluation of acute peritonitis is paramount to assess the severity and establish a prompt and appropriate treatment. The objectives of this study were to identify clinical and laboratory predictors for in-hospital mortality in patients with acute peritonitis and to develop a warning score system, based on easily recognizable and assessable variables, globally accepted. Methods: This worldwide multicentre observational study included 153 surgical departments across 56 countries over a 4-month study period between February 1, 2018, and May 31, 2018. Results: A total of 3137 patients were included, with 1815 (57.9%) men and 1322 (42.1%) women, with a median age of 47 years (interquartile range [IQR] 28-66). The overall in-hospital mortality rate was 8.9%, with a median length of stay of 6 days (IQR 4-10). Using multivariable logistic regression, independent variables associated with in-hospital mortality were identified: age > 80 years, malignancy, severe cardiovascular disease, severe chronic kidney disease, respiratory rate ≥ 22 breaths/min, systolic blood pressure < 100 mmHg, AVPU responsiveness scale (voice and unresponsive), blood oxygen saturation level (SpO2) < 90% in air, platelet count < 50,000 cells/mm3, and lactate > 4 mmol/l. These variables were used to create the PIPAS Severity Score, a bedside early warning score for patients with acute peritonitis. The overall mortality was 2.9% for patients who had scores of 0-1, 22.7% for those who had scores of 2-3, 46.8% for those who had scores of 4-5, and 86.7% for those who have scores of 7-8. Conclusions: The simple PIPAS Severity Score can be used on a global level and can help clinicians to identify patients at high risk for treatment failure and mortality.

Dispositivo de presión positiva continua en la vía aérea de Boussignac durante el transporte interhospitalario en lactantes menores de tres meses / Boussignac continuous positive airway pressure device during inter-hospital transportation in infants aged less than three months

Fundamento: El objetivo del presente estudio es evaluar la seguridad y efectividad del dispositivo de generación de presión positiva continua en la vía aérea de Boussignac(R) (CPAPB) durante su uso en el transporte en lactantes menores de tres meses con bronquiolitis. Material y métodos: Estudio observacional analítico transversal de cuatro años de duración. Se recogieron datos de 25 lactantes que precisaron trasladado interhospitalario a la Unidad de Cuidados Intensivos Pediátrica (UCIP) de referencia con CPAPB e interfase tipo Helmet. Se registraron las caracteristicas epidemiológicas, del traslado y de la evolución en UCIP, además de los parámetros cardiorrespiratorios y gasométricos previos al traslado y a su llegada a UCIP. Resultados: La mediana del nivel de presión contínua en la vía aérea (CPAP) utilizada durante el traslado fue de 7 cm H2O (6-7,25). Ningún paciente precisó de intubación endotraqueal durante el traslado y un paciente la precisó durante las primeras seis horas de ingreso en UCIP. Los siguientes parámetros cardiorrespiratorios presentaron una mejoría estadisticamente significativa a su llegada a UCIP: score de Wood-Downes modificado [8,40 (2,1) vs 5,29 (1,68)], frecuencia respiratoria [60,72 (12,73) vs 47,28 (10,31)], frecuencia cardiaca [167,28 (22,60) vs 154,48 (24,83)] y saturación de oxígeno [92,08 (5,63) vs 97,64 (2,27)]. Conclusiones: La aplicación de CPAPB resultó ser un método de soporte respiratorio seguro en lactantes menores de tres meses. Su uso durante el transporte suposo una mejoría en los parámetros cardiorrespiratoriosl

Background: The present study aims to evaluate the safety and effectiveness of the Boussignac continuous positive airway pressure device (CPAPB) when used during the transportation of infants under three months of age with bronchiolitis. Methods: Transversal analytical observational study of four years duration. Data was collected on 25 infants who needed inter-hospital transportation to the reference Paediatric Intensive Care Unit (PICU), with CPAPB and Helmet interface. The epidemiological characteristics of the transportation and evolution in the PICU were registered, as well as the cardiorespiratory gastronomic parameters prior to transfer and on arrival at the PICU. Results: The median level of continuous airway pressure (CPAP) used during the transfer was 7 cm H2O (6-7.25). No patient required endotracheal intubation during transportation, while one patient required this during the first six hours of admission in the PICU. The following cardiorespiratory parameters presented a statistically significant improvement on arrival at the PICU: modified Wood-Downes score [8.40 (2.1) vs 5.29 (1.68)], respiratory frequency [60.72 (12.73) vs 47.28 (10.31)], cardiac frequency [167.28 (22.60) vs 154.48 (24.83)] and oxygen saturation [92.08 (5.63) vs 97.64 (2.27)]. Conclusions: Application of CPAPB proved to be a safe method of respiratory support in infants under three months of age. Its use during transportation brought an improvement in cardiorespiratory parameters

Seat Belt Syndrome and the Submarine Effect: A Case Report.

The seatbelt sign is indicative of severe internal lesions in as many as 30% of cases. In the "submarine effect" the body slides below the belt, acting like hinge. "Seatbelt syndrome" describes the presence of the seat belt sign plus an intra-abdominal or spinal injury. We present the case of a driver in a car accident in whom severe soft tissue and visceral lesions were caused by a two-point seat-belt reproducing a complete "seatbelt syndrome".

Elucidating the clinical significance of two PMS2 missense variants coexisting in a family fulfilling hereditary cancer criteria.

The clinical spectrum of germline mismatch repair (MMR) gene variants continues increasing, encompassing Lynch syndrome, Constitutional MMR Deficiency (CMMRD), and the recently reported MSH3-associated polyposis. Genetic diagnosis of these hereditary cancer syndromes is often hampered by the presence of variants of unknown significance (VUS) and overlapping phenotypes. Two PMS2 VUS, c.2149G>A (p.V717M) and c.2444C>T (p.S815L), were identified in trans in one individual diagnosed with early-onset colorectal cancer (CRC) who belonged to a family fulfilling clinical criteria for hereditary cancer. Clinico-pathological data, multifactorial likelihood calculations and functional analyses were used to refine their clinical significance. Likelihood analysis based on cosegregation and tumor data classified the c.2444C>T variant as pathogenic, which was supported by impaired MMR activity associated with diminished protein expression in functional assays. Conversely, the c.2149G>A variant displayed MMR proficiency and protein stability. These results, in addition to the conserved PMS2 expression in normal tissues and the absence of germline microsatellite instability (gMSI) in the biallelic carrier ruled out a CMMRD diagnosis. The use of comprehensive strategies, including functional and clinico-pathological information, is mandatory to improve the clinical interpretation of naturally occurring MMR variants. This is critical for appropriate clinical management of cancer syndromes associated to MMR gene mutations.

ICO amplicon NGS data analysis: a Web tool for variant detection in common high-risk hereditary cancer genes analyzed by amplicon GS Junior next-generation sequencing.

Next-generation sequencing (NGS) has revolutionized genomic research and is set to have a major impact on genetic diagnostics thanks to the advent of benchtop sequencers and flexible kits for targeted libraries. Among the main hurdles in NGS are the difficulty of performing bioinformatic analysis of the huge volume of data generated and the high number of false positive calls that could be obtained, depending on the NGS technology and the analysis pipeline. Here, we present the development of a free and user-friendly Web data analysis tool that detects and filters sequence variants, provides coverage information, and allows the user to customize some basic parameters. The tool has been developed to provide accurate genetic analysis of targeted sequencing of common high-risk hereditary cancer genes using amplicon libraries run in a GS Junior System. The Web resource is linked to our own mutation database, to assist in the clinical classification of identified variants. We believe that this tool will greatly facilitate the use of the NGS approach in routine laboratories.

Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes.

Next-generation sequencing (NGS) is changing genetic diagnosis due to its huge sequencing capacity and cost-effectiveness. The aim of this study was to develop an NGS-based workflow for routine diagnostics for hereditary breast and ovarian cancer syndrome (HBOCS), to improve genetic testing for BRCA1 and BRCA2. A NGS-based workflow was designed using BRCA MASTR kit amplicon libraries followed by GS Junior pyrosequencing. Data analysis combined Variant Identification Pipeline freely available software and ad hoc R scripts, including a cascade of filters to generate coverage and variant calling reports. A BRCA homopolymer assay was performed in parallel. A research scheme was designed in two parts. A Training Set of 28 DNA samples containing 23 unique pathogenic mutations and 213 other variants (33 unique) was used. The workflow was validated in a set of 14 samples from HBOCS families in parallel with the current diagnostic workflow (Validation Set). The NGS-based workflow developed permitted the identification of all pathogenic mutations and genetic variants, including those located in or close to homopolymers. The use of NGS for detecting copy-number alterations was also investigated. The workflow meets the sensitivity and specificity requirements for the genetic diagnosis of HBOCS and improves on the cost-effectiveness of current approaches.

Identification and comprehensive characterization of large genomic rearrangements in the BRCA1 and BRCA2 genes.

Large genomic rearrangements are estimated to account for approximately 5-10% of all disease-causing mutations in BRCA1 and BRCA2 genes in patients with hereditary breast and ovarian cancer syndrome (HBOC). We use MRC-Holland Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for such rearrangements in patients with HBOC and as a first step in our genetic testing workflow. The technique was applied to a set of 310 independent patients and detected eight different copy number alterations, corresponding to 2.6% of the studied samples. MLPA was also found to identify point mutations located in probe sequences. As commercial MLPA tests are not suitable for determining the specific breakpoints or for defining the exact extent of rearrangements, we applied a set of different complementary techniques to characterize these genetic alterations with greater precision. Long-range PCR amplification, RNA analysis, SNP-array chips, non-commercial MLPA probes, and FISH analysis were used to fully define the extent and mechanism of each alteration. In BRCA1, six rearrangements were characterized: deletion of E22, duplication of E9-E24, deletion of E16-E23, deletion of E1-E13, deletion of E1-E2 and duplication of E1-E2. In BRCA2, we studied a deletion of E15-E16 and a deletion of E1-E24. To the best of our knowledge, this is the most comprehensive study of the nature and underlying molecular causes of these mutational events in the BRCA1/2 genes.