Impact of placental malaria on maternal, placental and fetal cord responses and its role in pregnancy outcomes in women from Blue Nile State, Sudan

Pes al naixement; Citocines; Malària placentàriaPeso al nacer; Citoquinas; Paludismo placentarioBirth weight; Cytokines; Placental malariaBackground The sequestration of Plasmodium falciparum infected cells in the placenta results in placental malaria (PM). It activates the mother's immune cells and induces secretion of inflammatory cytokines, which might influence pregnancy outcomes. This study aims to investigate the cytokines (levels IL-4, IL-6, IL-10, IL-17A, and INF γ) in maternal peripheral, placental, and umbilical cord blood in response to PM and the extent to which this may influence maternal haemoglobin levels and birth weight. Methods A total of 185 consenting Sudanese women from Blue Nile State were enrolled at delivery time in a cross-sectional study conducted between Jan 2012-Dec 2015. Malaria infection in the collected maternal peripheral, placental, umbilical cord samples was determined microscopically, and ELISA was used to measure the plasma levels IL-4, IL-6, IL-10, IL-17A, and INF γ in the collected positive and negative malaria samples. Results Elevated levels of IL-4 and IL-10 and reduced levels of IL-6 were detected in the malaria positive samples in comparison to the negative ones in the three types of the samples investigated. Maternal, IL-4 and IL-10 were significantly higher in the samples collected from the PM infected group compared to the non-infected control (P < 0.001). While the absence of PM was significantly associated with the IL-6 and maternal IFN-γ levels, maternal IL-17A, placental and umbilical cord IFN-γ levels showed no significant difference (P = 0.214, P = 0.065, P = 0.536, respectively) due to infection. Haemoglobin level and birth weight were increased in the group with high levels of IL-6 and IL-17A, but not in the group with IL-4 and IL-10 levels. While significantly negative correlation was found between IFN-γ levels and birth weight for all three types of samples, only maternal peripheral IFN-γ level was significantly positively correlated with maternal haemoglobin (r = 0.171, P = 0.020). Conclusion These results suggest that PM induces mother’s immune response and impairs her cytokine profile, which might alter maternal haemoglobin levels and the baby's birth weight.This work was supported by Vall d´Hebron Research Institute as part of Samia Omer fellowship of the 4th edition of Science by Women, Foundation Mujeres por África (FMxA), Madrid –Spain

Common Variable Immunodeficiency and Neurodevelopmental Delay Due to a 13Mb Deletion on Chromosome 4 Including the NFKB1 Gene: A Case Report

Chromosomal rearrangements; Primary immunodeficiencies; Syndromic immunodeficienciesReordenacions cromosòmiques; Immunodeficiències primàries; Immunodeficiències sindròmiquesReordenamientos cromosómicos; Inmunodeficiencias primarias; Inmunodeficiencias sindrómicasSyndromic immunodeficiencies are a heterogeneous group of inborn errors of immunity that can affect the development of non-immune organs and systems. The genetic basis of these immunodeficiencies is highly diverse, ranging from monogenic defects to large chromosomal aberrations. Antibody deficiency is the most prevalent immunological abnormality in patients with syndromic immunodeficiencies caused by chromosomal rearrangements, and usually manifests as a common variable immunodeficiency (CVID)-like phenotype. Here we describe a patient with a complex phenotype, including neurodevelopmental delay, dysmorphic features, malformations, and CVID (hypogammaglobulinemia, reduced pre-switch and switch memory B cells, and impaired vaccine response). Microarray-based comparative genomic hybridization (aCGH) revealed a 13-Mb deletion on chromosome 4q22.2-q24 involving 53 genes, some of which were related to the developmental manifestations in our patient. Although initially none of the affected genes could be linked to his CVID phenotype, subsequent reanalysis identified NFKB1 haploinsufficiency as the cause. This study underscores the value of periodic reanalysis of unsolved genetic studies performed with high-throughput technologies (eg, next-generation sequencing and aCGH). This is important because of the ongoing incorporation of new data establishing the relationship between genes and diseases. In the present case, NFKB1 had not been associated with human disease at the time aCGH was performed. Eight years later, reanalysis of the genes included in the chromosome 4 deletion enabled us to identify NFKB1 haploinsufficiency as the genetic cause of our patient’s CVID. In the future, other genes included in the deletion may be linked to human disease, allowing us to better define the molecular basis of our patient’s complex clinical phenotype.This study was funded by Instituto de Salud Carlos III, grants PI17/00660 and PI20/00761, cofinanced by the European Regional Development Fund (ERDF). This study was also funded by the Jeffrey Modell Foundation. This work is supported by the European Reference Network for Rare Immunodeficiency, Autoinflammatory and Autoimmune Diseases Network (ERN-RITA)

Case report: Cytokine hemoadsorption in a case of hemophagocytic lymphohistiocytosis secondary to extranodal NK/T-cell lymphoma

Cytokine hemoadsorption; Hemophagocytic lymphohistiocytosis; Multiorgan dysfunctionHemoadsorció de citocines; Limfohistiocitosi hemofagocítica; Disfunció multiorgànicaHemoadsorción de citoquinas; Linfohistiocitosis hemofagocítica; Disfunción multiorgánicaWe discuss a single case of Hemophagocytic lymphohistiocytosis (HLH) due to NK-type non-Hodgkin lymphoma and Epstein-Barr virus reactivation with multiorgan dysfunction and distributive shock in which we performed cytokine hemoadsorption with Cytosorb ®. A full microbiological panel was carried out, including screening for imported disease, standard serologies and cultures for bacterial and fungal infection. A liver biopsy and bone marrow aspirate were performed, confirming the diagnosis. The patients fulfilled the HLH-2004 diagnostic criteria, and according to the 2018 Consensus Statements by the HLH Steering Committee of the Histiocyte Society, dexamethasone and etoposide were started. There was an associated hypercytokinemia and, due to refractory distributive shock, rescue therapy with cytokine hemoadsorption was performed during 24 h (within day 2 and 3 from ICU admission). After starting this procedure, rapid hemodynamic control was achieved with a significant reduction in vasopressor support requirements. This case report highlights that cytokine hemoadsorption can be an effective since rapid decrease in IL-10 levels and a significant hemodynamic improvement was achieved

Expanding the clinical and genetic spectra of primary immunodeficiency-related disorders with clinical exome sequencing: expected and unexpected findings

Inmunodeficiencias primarias; Secuenciación de próxima generación; Secuenciación clínica del exomaImmunodeficiències primàries; Seqüenciació de propera generació; Seqüenciació clínica d’exomesPrimary immunodeficiencies; Next generation sequencing; Clinical exome sequencingPrimary immunodeficiencies (PIDs) refer to a clinically, immunologically, and genetically heterogeneous group of over 350 disorders affecting development or function of the immune system. The increasing use of next-generation sequencing (NGS) technology has greatly facilitated identification of genetic defects in PID patients in daily clinical practice. Several NGS approaches are available, from the unbiased whole exome sequencing (WES) to specific gene panels. Here, we report on a 3-year experience with clinical exome sequencing (CES) for genetic diagnosis of PIDs. We used the TruSight One sequencing panel, which includes 4,813 disease-associated genes, in 61 unrelated patients (pediatric and adults). The analysis was done in 2 steps: first, we focused on a virtual PID panel and then, we expanded the analysis to the remaining genes. A molecular diagnosis was achieved in 19 (31%) patients: 12 (20%) with mutations in genes included in the virtual PID panel and 7 (11%) with mutations in other genes. These latter cases provided interesting and somewhat unexpected findings that expand the clinical and genetic spectra of PID-related disorders, and are useful to consider in the differential diagnosis. We also discuss 5 patients (8%) with incomplete genotypes or variants of uncertain significance. Finally, we address the limitations of CES exemplified by 7 patients (11%) with negative results on CES who were later diagnosed by other approaches (more specific PID panels, WES, and comparative genomic hybridization array). In summary, the genetic diagnosis rate using CES was 31% (including a description of 12 novel mutations), which rose to 42% after including diagnoses achieved by later use of other techniques. The description of patients with mutations in genes not included in the PID classification illustrates the heterogeneity and complexity of PID-related disorders.This study was funded by Instituto de Salud Carlos III, grants PI14/00405 and PI17/00660, cofinanced by the European Regional Development Fund (ERDF)

Reference values for interleukin-6 in the amniotic fluid of asymptomatic pregnant women

Amniocentesis; Interleukin-6; Intra-amniotic inflammationAmniocentesi; Interleucina-6; Inflamació intraamniòticaAmniocentesis; Interleucina-6; Inflamación intraamnióticaIntroduction Nowadays, proinflammatory factors are considered to play an important role in the pathophysiology of threatened preterm labor or chorioamnionitis. The aim of this study was to establish the normal reference range for interleukin-6 (IL-6) levels in the amniotic fluid and to identify factors which may alter this value. Material and methods Prospective study in a tertiary-level center including asymptomatic pregnant women undergoing amniocentesis for genetic studies from October 2016 to September 2019. IL-6 measurements in amniotic fluid were performed using a fluorescence immunoassay with microfluidic technology (ELLA Proteinsimple, Bio Techne). Maternal history and pregnancy data were also recorded. Results This study included 140 pregnant women. Of those, women who underwent termination of pregnancy were excluded. Therefore, a total of 98 pregnancies were included in the final statistical analysis. The mean gestational age was 21.86 weeks (range: 15–38.7) at the time of amniocentesis, and 38.6 weeks (range: 30.9–41.4) at delivery. No cases of chorioamnionitis were reported. The log10 IL-6 values follow a normal distribution (W = 0.990, p = 0.692). The median, and the 5th, 10th, 90th, and 95th percentiles for IL-6 levels were 573, 105, 130, 1645, and 2260 pg/mL, respectively. The log10 IL-6 values were not affected by gestational age (p = 0.395), maternal age (p = 0.376), body mass index (p = 0.551), ethnicity (p = 0.467), smoking status (p = 0.933), parity (p = 0.557), method of conception (p = 0.322), or diabetes mellitus (p = 0.381). Conclusions The log10 IL-6 values follow a normal distribution. IL-6 values are independent of gestational age, maternal age, body mass index, ethnicity, smoking status, parity and method of conception. Our study provides a normal reference range for IL-6 levels in the amniotic fluid that can be used in future studies. We also observed that normal IL-6 values were higher in the amniotic fluid than in serum.Some of the authors are members of the European Reference Network on Rare Congenital Malformations and Rare Intellectual Disability ERN-ITHACA (EU Framework Partnership Agreement ID: 3HP-HP-FPA ERN-01-2016/739516)

Immunological and genetic kinetics from diagnosis to clinical progression in chronic lymphocytic leukemia

Progressió clínica; Evasió immuneProgresión clínica; Evasión inmuneClinical progression; Immune evasionBackground Mechanisms driving the progression of chronic lymphocytic leukemia (CLL) from its early stages are not fully understood. The acquisition of molecular changes at the time of progression has been observed in a small fraction of patients, suggesting that CLL progression is not mainly driven by dynamic clonal evolution. In order to shed light on mechanisms that lead to CLL progression, we investigated longitudinal changes in both the genetic and immunological scenarios. Methods We performed genetic and immunological longitudinal analysis using paired primary samples from untreated CLL patients that underwent clinical progression (sampling at diagnosis and progression) and from patients with stable disease (sampling at diagnosis and at long-term asymptomatic follow-up). Results Molecular analysis showed limited and non-recurrent molecular changes at progression, indicating that clonal evolution is not the main driver of clinical progression. Our analysis of the immune kinetics found an increasingly dysfunctional CD8+ T cell compartment in progressing patients that was not observed in those patients that remained asymptomatic. Specifically, terminally exhausted effector CD8+ T cells (T-betdim/−EomeshiPD1hi) accumulated, while the the co-expression of inhibitory receptors (PD1, CD244 and CD160) increased, along with an altered gene expression profile in T cells only in those patients that progressed. In addition, malignant cells from patients at clinical progression showed enhanced capacity to induce exhaustion-related markers in CD8+ T cells ex vivo mainly through a mechanism dependent on soluble factors including IL-10. Conclusions Altogether, we demonstrate that the interaction with the immune microenvironment plays a key role in clinical progression in CLL, thereby providing a rationale for the use of early immunotherapeutic intervention.This work was supported by the Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias (PI17/00950, M.C., PI18/01392, P.A. and PI17/00943, F.B.) and co-financed by the European Regional Development Fund (ERDF) and Fundación Asociación Española Contra el Cáncer (M.C. and P.A.), Gilead Fellowships (GLD16/00144, GLD18/00047, F.B.) and Fundació la Marató de TV3 (201905–30-31 F.B). S.B. is the recipient of a postdoctoral fellowship from Fundación Alfonso Martin Escudero. R.V-M. is supported by a Torres Quevedo fellowship from the Spanish Ministry of Science and Innovation (PTQ-16-08623). A.E-C. is funded by ISCIII/MINECO (PT17/0009/0019) which is co-funded by FEDER. M.C. holds a contract from Ministerio de Ciencia, Innovación y Universidades (RYC-2012-2018)

Newborn screening for presymptomatic diagnosis of complement and phagocyte deficiencies

The clinical outcomes of primary immunodeficiencies (PIDs) are greatly improved by accurate diagnosis early in life. However, it is not common to consider PIDs before the manifestation of severe clinical symptoms. Including PIDs in the nation-wide newborn screening programs will potentially improve survival and provide better disease management and preventive care in PID patients. This calls for the detection of disease biomarkers in blood and the use of dried blood spot samples, which is a part of routine newborn screening programs worldwide. Here, we developed a newborn screening method based on multiplex protein profiling for parallel diagnosis of 22 innate immunodeficiencies affecting the complement system and respiratory burst function in phagocytosis. The proposed method uses a small fraction of eluted blood from dried blood spots and is applicable for population-scale performance. The diagnosis method is validated through a retrospective screening of immunodeficient patient samples. This diagnostic approach can pave the way for an earlier, more comprehensive and accurate diagnosis of complement and phagocytic disorders, which ultimately lead to a healthy and active life for the PID patientsThis work was supported by the Swedish Research Council (VR) and grants provided by the Stockholm County Council (ALF)

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Clinical and molecular characterization of Factor I and C5 complement deficiencies: from diagnosis to population studies

El Sistema de Complemento es una parte de la respuesta inmune humoral que, entre otras funciones, se encarga de la defensa frente a patógenos y eliminación de inmunocomplejos. Está compuesto por más de treinta proteínas solubles y unidas a membrana, que se activan en forma de cascada proteolítica para poder ejercer su función. Los defectos congénitos en proteinas del sistema de complemento incrementan la susceptibilidad a infecciones por patógenos encapsulados y aumentan el riesgo de recurrencia de infecciones por bacterias del género Neisseria meningitidis. A pesar de considerarse enfermedades raras, la hipótesis del trabajo es que se están infradiagnosticando por la falta de conocimiento y de técnicas de laboratorio para el estudio de esta parte del sistema inmune. Además, consideramos que un diagnóstico temprano de este tipo de defectos permite adoptar medidas terapéuticas preventivas que mejoran la calidad de vida de los pacientes. En este trabajo de tesis se implementan 10 nuevas técnicas para el estudio del sistema de complemento en la rutina asistencial del Servicio de Inmunología del Hospital Universitario Vall d’Hebron. Este hecho ha permitido el diagnóstico y caracterización molecular de nueve casos de defectos de complemento (tres familias con defectos de C5 y tres familias con defectos de Factor I). Dos de los casos diagnosticados fueron en recién nacidos, hermanos de pacientes índice. Este hecho permitió la vacunación temprana y la indicación de profilaxis antibiótica para evitar futuras infecciones. Debido a la variabilidad geográfica descrita en la frecuencia de los defectos en moléculas de la vía terminal del complemento (C5-C9), estudiamos la presencia de alelos que presentaran la mutación p.A252T en 2710 muestras de poblaciones representativas de las diferentes regiones continentales. De acuerdo con nuestra hipótesis, observamos que existe una sobrerrepresentación de esta mutación en paises de África Sub-sahariana, coincidiendo en parte, con los paises englobados en el cinturón de la meningitis africano. Por contra, también identificamos dos muestras que eran portadoras del alelo mutado en regiones fuera de África (Israel y Pakistán). De cara a responder la pregunta de si es necesario estudiar el sistema de complemento en los casos de enfermedad meningocócica invasiva, en esta tesis recogemos un nuevo algoritmo en el que se suman a la presencia de recurrencias, el hecho de que haya consanguineidad, que la infección venga determinada por un serotipo poco frecuente o que el paciente sea originario de África o de Oriente Medio.The Complement System is a part of the humoral immune response that, among other functions, is responsible for the defense against pathogens and elimination of immune complexes. It is composed of more than thirty soluble and membrane-bound proteins, which are activated as a proteolytic cascade to be able to exert their function. Congenital defects in complement proteins increase susceptibility to infections by encapsulated pathogens and increase the risk of recurrence of infections by bacteria of the genus Neisseria meningitidis. Despite being considered rare diseases, the hypothesis of the work is that they are underdiagnosed by the lack of awareness and laboratory techniques for the study of this part of the immune system. In addition, we consider that an early diagnosis of this type of defects allows adopting preventive therapeutic measures that improve the quality of life of patients. In this work, 10 new techniques are implemented for the study of the complement system in the routine of the Immunology Department of the Hospital Universitario Vall d'Hebron. This fact allowed the diagnosis and molecular characterization of nine cases of complement defects (three families with defects of C5 and three families with defects of Factor I). Two of the diagnosed cases were in newborns, siblings of index patients. This fact allowed the early vaccination and the indication of antibiotic prophylaxis to avoid future infections. Due to the geographic variability described in the frequency of defects in molecules of the complement (C5-C9) terminal pathway, we studied the presence of alleles that presented the p.A252T mutation in 2710 samples from representative populations of the different continental regions. According to our hypothesis, we observe that there is an over-representation of this mutation in countries of Sub-Saharan Africa, coinciding in part with the countries included in the African meningitis belt. In contrast, we also identified two samples that were carriers of the mutated allele in regions outside of Africa (Israel and Pakistan). In order to answer the question of whether it is necessary to study the complement system in the cases of invasive meningococcal disease, in this thesis we present a new algorithm in which they are added to the presence of recurrences, the fact that there is consanguinity, that Infection is determined by a rare serotype or the patient is from Africa or the Middle East