N-Glycosylation of the Ig Receptors Shapes the Antigen Reactivity in Chronic Lymphocytic Leukemia Subset #201.

Subset #201 is a clinically indolent subgroup of patients with chronic lymphocytic leukemia defined by the expression of stereotyped, mutated IGHV4-34/IGLV1-44 BCR Ig. Subset #201 is characterized by recurrent somatic hypermutations (SHMs) that frequently lead to the creation and/or disruption of N-glycosylation sites within the Ig H and L chain variable domains. To understand the relevance of this observation, using next-generation sequencing, we studied how SHM shapes the subclonal architecture of the BCR Ig repertoire in subset #201, particularly focusing on changes in N-glycosylation sites. Moreover, we profiled the Ag reactivity of the clonotypic BCR Ig expressed as rmAbs. We found that almost all analyzed cases from subset #201 carry SHMs potentially affecting N-glycosylation at the clonal and/or subclonal level and obtained evidence for N-glycan occupancy in SHM-induced novel N-glycosylation sites. These particular SHMs impact (auto)antigen recognition, as indicated by differences in Ag reactivity between the authentic rmAbs and germline revertants of SHMs introducing novel N-glycosylation sites in experiments entailing 1) flow cytometry for binding to viable cells, 2) immunohistochemistry against various human tissues, 3) ELISA against microbial Ags, and 4) protein microarrays testing reactivity against multiple autoantigens. On these grounds, N-glycosylation appears as relevant for the natural history of at least a fraction of Ig-mutated chronic lymphocytic leukemia. Moreover, subset #201 emerges as a paradigmatic case for the role of affinity maturation in the evolution of Ag reactivity of the clonotypic BCR Ig.

IgIDivA: immunoglobulin intraclonal diversification analysis.

Intraclonal diversification (ID) within the immunoglobulin (IG) genes expressed by B cell clones arises due to ongoing somatic hypermutation (SHM) in a context of continuous interactions with antigen(s). Defining the nature and order of appearance of SHMs in the IG genes can assist in improved understanding of the ID process, shedding light into the ontogeny and evolution of B cell clones in health and disease. Such endeavor is empowered thanks to the introduction of high-throughput sequencing in the study of IG gene repertoires. However, few existing tools allow the identification, quantification and characterization of SHMs related to ID, all of which have limitations in their analysis, highlighting the need for developing a purpose-built tool for the comprehensive analysis of the ID process. In this work, we present the immunoglobulin intraclonal diversification analysis (IgIDivA) tool, a novel methodology for the in-depth qualitative and quantitative analysis of the ID process from high-throughput sequencing data. IgIDivA identifies and characterizes SHMs that occur within the variable domain of the rearranged IG genes and studies in detail the connections between identified SHMs, establishing mutational pathways. Moreover, it combines established and new graph-based metrics for the objective determination of ID level, combined with statistical analysis for the comparison of ID level features for different groups of samples. Of importance, IgIDivA also provides detailed visualizations of ID through the generation of purpose-built graph networks. Beyond the method design, IgIDivA has been also implemented as an R Shiny web application. IgIDivA is freely available at https://bio.tools/igidiva.

Genetic Characterization of Kidney Failure of Unknown Etiology in Spain: Findings From the GENSEN Study.

RATIONALE & OBJECTIVE: Chronic kidney disease of unknown etiology (CKDUE) is one of the main global causes of kidney failure. Genetic studies may identify an etiology in these patients, but few studies have implemented genetic testing of CKDUE in a population-based series of patients, which was the focus of the GENSEN Study. STUDY DESIGN: Case series. SETTINGS & PARTICIPANTS: 818 patients aged≤45 years at 51 Spanish centers with CKDUE, and either an estimated glomerular filtration rate of<15mL/min/1.73m2 or treatment with maintenance dialysis or transplantation. OBSERVATIONS: Genetic testing for 529 genes associated with inherited nephropathies using high-throughput sequencing (HTS). Pathogenic and/or likely pathogenic (P/LP) gene variants concordant with the inheritance pattern were detected in 203 patients (24.8%). Variants in type IV collagen genes were the most frequent (COL4A5, COL4A4, COL4A3; 35% of total gene variants), followed by NPHP1, PAX2, UMOD, MUC1, and INF2 (7.3%, 5.9%, 2.5%, 2.5%, and 2.5%, respectively). Overall, 87 novel variants classified as P/LP were identified. The top 5 most common previously undiagnosed diseases were Alport syndrome spectrum (35% of total positive reports), genetic podocytopathies (19%), nephronophthisis (11%), autosomal dominant tubulointerstitial kidney disease (7%), and congenital anomalies of the kidney and urinary tract (CAKUT, 5%). A family history of kidney disease was reported by 191 participants (23.3%) and by 65 of 203 patients (32.0%) with P/LP variants. LIMITATIONS: Missing data, and selection bias resulting from voluntary enrollment. CONCLUSIONS: Genomic testing with HTS identified a genetic cause of kidney disease in approximately one quarter of young patients with CKDUE and advanced kidney disease. These findings suggest that genetic studies are a potentially useful tool for the evaluation of people with CKDUE. PLAIN-LANGUAGE SUMMARY: The cause of kidney disease is unknown for 1 in 5 patients requiring kidney replacement therapy, reflecting possible prior missed treatment opportunities. We assessed the diagnostic utility of genetic testing in children and adults aged≤45 years with either an estimated glomerular filtration rate of<15mL/min/1.73m2 or treatment with maintenance dialysis or transplantation. Genetic testing identified the cause of kidney disease in approximately 1 in 4 patients without a previously known cause of kidney disease, suggesting that genetic studies are a potentially useful tool for the evaluation of these patients.

Loss of Esr1 Does Not Affect Hearing and Balance.

Although estrogen affects the structure and function of the nervous system and brain and has a number of effects on cognition, its roles in the auditory and vestibular systems remain unclear. The actions of estrogen are mediated predominately through two classical nuclear estrogen receptors, estrogen receptor 1 (ESR1) and estrogen receptor 2 (ESR2). In the current study, we investigated the roles of ESR1 in normal auditory function and balance performance using 3-month-old wild-type (WT) and Esr1 knockout (KO) mice on a CBA/CaJ background, a normal-hearing strain. As expected, body weight of Esr1 KO females was lower than that of Esr1 KO males. Body weight of Esr1 KO females was higher than that of WT females, while there was no difference in body weight between WT and Esr1 KO males. Similarly, head diameter was higher in Esr1 KO vs. WT females. Contrary to our expectations, there were no differences in auditory brainstem response (ABR) thresholds, ABR waves I-V amplitudes and ABR waves I-V latencies at 8, 16, 32, and 48 kHz, distortion product otoacoustic emission (DPOAE) thresholds and amplitudes at 8, 16, and 32 kHz, and rotarod balance performance (latency to fall) between WT and Esr1 KO mice. Furthermore, there were no sex differences in ABRs, DPOAEs, and rotarod balance performance in Esr1 KO mice. Taken together, our findings show that Esr1 deficiency does not affect auditory function or balance performance in normal hearing mice, and suggest that loss of Esr1 is likely compensated by ESR2 or other estrogen receptors to maintain the structure and function of the auditory and vestibular systems under normal physiological conditions.

Purpose-Built Immunoinformatics for BcR IG/TR Repertoire Data Analysis.

The study of antigen receptor gene repertoires using next-generation sequencing (NGS) technologies has disclosed an unprecedented depth of complexity, requiring novel computational and analytical solutions. Several bioinformatics workflows have been developed to this end, including the T-cell receptor/immunoglobulin profiler (TRIP), a web application implemented in R shiny, specifically designed for the purposes of comprehensive repertoire analysis, which is the focus of this chapter. TRIP has the potential to perform robust immunoprofiling analysis through the extraction and processing of the IMGT/HighV-Quest output, via a series of functions, ensuring the analysis of high-quality, biologically relevant data through a multilevel process of data filtering. Subsequently, it provides in-depth analysis of antigen receptor gene rearrangements, including (a) clonality assessment; (b) extraction of variable (V), diversity (D), and joining (J) gene repertoires; (c) CDR3 characterization at both the nucleotide and amino acid level; and (d) somatic hypermutation analysis, in the case of immunoglobulin gene rearrangements. Relevant to mention, TRIP enables a high level of customization through the integration of various options in key aspects of the analysis, such as clonotype definition and computation, hence allowing for flexibility without compromising on accuracy.

Evidence of somatic hypermutation in the antigen binding sites of patients with CLL harboring IGHV genes with 100% germline identity.

Classification of patients with chronic lymphocytic leukemia (CLL) based on the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene has established predictive and prognostic relevance. The SHM status is assessed based on the number of mutations within the IG heavy variable domain sequence, albeit only over the rearranged IGHV gene excluding the variable heavy complementarity determining region 3 (VH CDR3). This may lead to an underestimation of the actual impact of SHM, in fact overlooking the most critical region for antigen-antibody interactions, i.e. the VH CDR3. Here we investigated whether SHM may be present within the VH CDR3 of cases bearing 'truly unmutated' IGHV genes (i.e. 100% germline identity across VH FR1-VH FR3) employing Next Generation Sequencing. We studied 16 patients bearing a 'truly unmutated' CLL clone assigned to stereotyped subsets #1 (n=12) and #6 (n=4). We report the existence of SHM within the germline-encoded 3'IGHV, IGHD, 5'IGHJ regions of the VH CDR3 in both the main IGHV-IGHD-IGHJ gene clonotype and its variants. Recurrent somatic mutations were identified between different patients of the same subset, supporting the notion that they represent true mutational events rather than technical artefacts; moreover, they were located adjacent to/within AID hotspots, pointing to SHM as the underlying mechanism. In conclusion, we provide immunogenetic evidence for intra-VH CDR3 variations, attributed to SHM, in CLL patients carrying 'truly unmutated' IGHV genes. Although the clinical implications of this observation remain to be defined, our findings offer a new perspective into the immunobiology of CLL, alluding to the operation of VH CDR3-restricted SHM in U-CLL.

Identifying port maritime communities: application to the Spanish case.

The aim of this paper is to detect port maritime communities sharing similar international trade patterns, by a modelisation of maritime traffic using a bipartite weighted network, providing decision-makers the tools to search for alliances or identify their competitors. Our bipartite weighted network considers two different types of nodes: one represents the ports, while the other represents the countries where there are major import/export activity from each port. The freight traffic among both types of nodes is modeled by weighting the volume of product transported. To illustrate the model, the Spanish case is considered, with the data segmented by each type of traffic for a fine tuning. A sort of link prediction is possible, finding for those communities with two or more ports, countries that are part of the same community but with which some ports do not have yet significant traffic. The evolution of the traffics is analyzed by comparing the communities in 2009 and 2019. The set of communities formed by the ports of the Spanish port system can be used to identify global similarities between them, comparing the membership of the different ports in communities for both periods and each type of traffic in particular.

Understanding Monoclonal B Cell Lymphocytosis: An Interplay of Genetic and Microenvironmental Factors.

The term monoclonal B-cell lymphocytosis (MBL) describes the presence of a clonal B cell population with a count of less than 5 × 109/L and no symptoms or signs of disease. Based on the B cell count, MBL is further classified into 2 distinct subtypes: 'low-count' and 'high-count' MBL. High-count MBL shares a series of biological and clinical features with chronic lymphocytic leukemia (CLL), at least of the indolent type, and evolves to CLL requiring treatment at a rate of 1-2% per year, whereas 'low-count' MBL seems to be distinct, likely representing an immunological rather than a pre-malignant condition. That notwithstanding, both subtypes of MBL can carry 'CLL-specific' genomic aberrations such as cytogenetic abnormalities and gene mutations, yet to a much lesser extent compared to CLL. These findings suggest that such aberrations are mostly relevant for disease progression rather than disease onset, indirectly pointing to microenvironmental drive as a key contributor to the emergence of MBL. Understanding microenvironmental interactions is therefore anticipated to elucidate MBL ontogeny and, most importantly, the relationship between MBL and CLL.

Accuracy of an amplicon-sequencing nanopore approach to identify variants in tuberculosis drug-resistance-associated genes.

A rapid and accurate diagnostic assay represents an important means to detect Mycobacterium tuberculosis, identify drug-resistant strains and ensure treatment success. Currently employed techniques to diagnose drug-resistant tuberculosis include slow phenotypic tests or more rapid molecular assays that evaluate a limited range of drugs. Whole-genome-sequencing-based approaches can detect known drug-resistance-conferring mutations and novel variations; however, the dependence on growing samples in culture, and the associated delays in achieving results, represents a significant limitation. As an alternative, targeted sequencing strategies can be directly performed on clinical samples at high throughput. This study proposes a targeted sequencing assay to rapidly detect drug-resistant strains of M. tuberculosis using the Nanopore MinION sequencing platform. We designed a single-tube assay that targets nine genes associated with drug resistance to seven drugs and two phylogenetic-determining regions to determine strain lineage and tested it in nine clinical isolates and six sputa. The study's main aim is to calibrate MinNION variant calling to detect drug-resistance-associated mutations with different frequencies to match the accuracy of Illumina (the current gold-standard sequencing technology) from both culture and sputum samples. After calibrating Nanopore MinION variant calling, we demonstrated 100% agreement between Illumina WGS and our MinION set up to detect known drug resistance and phylogenetic variants in our dataset. Importantly, other variants in the amplicons are also detected, decreasing the recall. We identify minority variants and insertions/deletions as crucial bioinformatics challenges to fully reproduce Illumina WGS results.

B Cell Receptor Immunogenetics in B Cell Lymphomas: Immunoglobulin Genes as Key to Ontogeny and Clinical Decision Making.

The clonotypic B cell receptor immunoglobulin (BcR IG) plays a seminal role in B cell lymphoma development and evolution. From a clinical perspective, this view is supported by the remarkable therapeutic efficacy of BcR signaling inhibitors, even among heavily pre-treated, relapsed/refractory patients. This clinical development complements immunogenetic evidence for antigen drive in the natural history of these tumors. Indeed, BcR IG gene repertoire biases have been documented in different B cell lymphoma subtypes, alluding to selection of B cell progenitors that express particular BcR IG. Moreover, distinct entities display imprints of somatic hypermutation within the clonotypic BcR IG gene following patterns that strengthen the argument for antigen selection. Of note, at least in certain B cell lymphomas, the BcR IG genes are intraclonally diversified, likely in a context of ongoing interactions with antigen(s). Moreover, BcR IG gene repertoire profiling suggests that unique immune pathways lead to distinct B cell lymphomas through targeting cells at different stages in the B cell differentiation trajectory (e.g., germinal center B cells in follicular lymphoma, FL). Regarding the implicated antigens, although their precise nature remains to be fully elucidated, immunogenetic analysis has offered important hints by revealing similarities between the BcR IG of particular lymphomas and B cell clones with known antigenic specificity: this has paved the way to functional studies that identified relevant antigenic determinants of classes of structurally similar epitopes. Finally, in certain tumors, most notably chronic lymphocytic leukemia (CLL), immunogenetic analysis has also proven instrumental in accurate patient risk stratification since cases with differing BcR IG gene sequence features follow distinct disease courses and respond differently to particular treatment modalities. Overall, delving into the BcR IG gene sequences emerges as key to understanding B cell lymphoma pathophysiology, refining prognostication and assisting in making educated treatment choices.

Dose dependent gene expression is dynamically modulated by the history, physiology and age of yeast cells.

Cells respond to external stimuli with transient gene expression changes in order to adapt to environmental alterations. However, the dose response profile of gene induction upon a given stress depends on many intrinsic and extrinsic factors. Here we show that the accurate quantification of dose dependent gene expression by live cell luciferase reporters reveals fundamental insights into stress signaling. We make the following discoveries applying this non-invasive reporter technology. (1) Signal transduction sensitivities can be compared and we apply this here to salt, oxidative and xenobiotic stress responsive transcription factors. (2) Stress signaling depends on where and how the damage is generated within the cell. Specifically we show that two ROS-generating agents, menadione and hydrogen peroxide, differ in their dependence on mitochondrial respiration. (3) Stress signaling is conditioned by the cells history. We demonstrate here that positive memory or an acquired resistance towards oxidative stress is induced dependent on the nature of the previous stress experience. (4) The metabolic state of the cell impinges on the sensitivity of stress signaling. This is shown here for the shift towards higher stress doses of the response profile for yeast cells moved from complex to synthetic medium. (5) The age of the cell conditions its transcriptional response capacity, which is demonstrated by the changes of the dose response to oxidative stress during both replicative and chronological aging. We conclude that capturing dose dependent gene expression in real time will be of invaluable help to understand stress signaling and its dynamic modulation.

Restricción del crecimiento intrauterino: una aproximación al diagnóstico, seguimiento y manejo / Intrauterine growth restriction: an approach to diagnosis, monitoring and management

ANTECEDENTES: La restricción del crecimiento intrauterino (RCIU) es una entidad de origen multifactorial que puede ser causada por una gran variedad de patologías a nivel materno, fetal o placentario, y que representa altas tasas de morbimortalidad materna y perinatal. Es importante realizar un diagnóstico certero de esta patología con el fin de llevar a cabo un enfoque de seguimiento y de manejo que pueda disminuir todas las complicaciones asociadas a la enfermedad. OBJETIVO: Realizar una revisión detallada y actualizada de la etiopatogenia, criterios diagnósticos, seguimiento con ecografía Doppler y manejo de la restricción del crecimiento intrauterino. MÉTODO: Se realizó una búsqueda de literatura publicada en inglés y español en bases de datos como PubMed/MEDLINE, Ovid, MDconsult utilizando las palabras clave: restricción del crecimiento intrauterino, feto pequeño para la edad gestacional, circulación fetal, ecografía Doppler, además de la búsqueda de estadísticas relacionadas con RCIU, de los resultados obtenidos se seleccionaron 61 artículos. La información obtenida fue clasificada y utilizada como soporte para la realización de esta revisión. RESULTADOS: Se presentan los estudios disponibles, las revisiones y artículos recomendados para la evaluación de pacientes con RCIU, adicionalmente aquellos que brindan nueva información con respecto al diagnóstico, evaluación de circulación fetal, seguimiento y manejo de esta entidad clínica. CONCLUSIÓN: La restricción del crecimiento intrauterino es una patología que presenta altas tasas de morbimortalidad perinatal. La identificación temprana de estos fetos y el seguimiento adecuado mediante la evalución Doppler permiten disminuir los resultados adversos y las secuelas a corto y largo plazo.

BACKGROUND: Intrauterine growth restriction has a multifactorial origin and can be caused by a variety of pathologies in the mother, fetus or placenta, representing high rates of maternal and perinatal morbidity and mortality. Therefore, it is important to accurately diagnose this condition in order to focus in the follow and management, which can reduce the complications associated with the disease. OBJECTIVE: To perform a detailed and up to date review of the etiology, diagnostic criteria, follow up with Doppler ultrasonography and management of intrauterine growth restriction. METHODS: A literature search was done in English and Spanish in databases such us PubMed/MEDLINE, Ovid, MDconsult using the following key words: intrauterine growth restriction, small for gestational age fetus, fetal circulation, Doppler ultrasonography. Also national statistics for intrauterine growth restriction were used, obtaining 61 documents. The information obtained was classified and used in the development of this review. RESULTS: The available studies are presented, and recommended revisions to the evaluation of patients with IUGR, additionally those that provide new information regarding the diagnosis, evaluation of fetal circulation, monitoring and management of this clinical entity. CONCLUSION: Intrauterine growth restriction is a pathology with a frequent diagnosis an represents high rates of perinatal morbidity and mortality. Early identification of these fetuses and adequate monitoring by Doppler evaluation allow reducing adverse outcomes and consequences in the short and long term.