Search

BackgroundAt present, the knowledge about disease-causing mutations in IRF2BP2 is very limited because only a few patients affected by this condition have been reported. As previous studies have described, the haploinsufficiency of this interferon transcriptional corepressors leads to the development of CVID. Very recently, a more accurate phenotype produced by truncating variants in this gene has been defined, manifesting CVID with gastrointestinal inflammatory symptoms and autoimmune manifestations.MethodsWe analyzed 5 index cases with suspected primary immunodeficiency by high throughput sequencing. They were submitted for a genetic test with a panel of genes associated with immune system diseases, including IRF2BP2. The screening of SNVs, indels and CNVs fulfilling the criteria with very low allelic frequency and high protein impact, revealed five novel variants in IRF2BP2. In addition, we isolated both wild-type and mutated allele of the cDNA from one of the families.ResultsIn this study, we report five novel loss-of-function (LoF) mutations in IRF2BP2 that likely cause primary immunodeficiency, with CVID as more frequent phenotype, variable expression of inflammatory gastrointestinal features, and one patient with predisposition of viral infection. All identified variants were frameshift changes, and one of them was a large deletion located on chromosome 1q42, which includes the whole sequence of IRF2BP2, among other genes. Both de novo and dominant modes of inheritance were observed in the families here presented, as well as incomplete penetrance.ConclusionsWe describe novel variants in a delimited low-complex region, which may be considered a hotspot in IRF2BP2. Moreover, this is the first time that a large CNV in IRF2BP2 has been reported to cause CVID. The distinct mechanisms than LoF in IRF2BP2 could cause different phenotype compared with the mainly described. Further investigations are necessary to comprehend the regulatory mechanisms of IRF2BP2, which could be under variable expression of the disease.

Syndromic 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.

BackgroundTwo years since the onset of the COVID-19 pandemic no predictive algorithm has been generally adopted for clinical management and in most algorithms the contribution of laboratory variables is limited.ObjectivesTo measure the predictive performance of currently used clinical laboratory tests alone or combined with clinical variables and explore the predictive power of immunological tests adequate for clinical laboratories. Methods: Data from 2,600 COVID-19 patients of the first wave of the pandemic in the Barcelona area (exploratory cohort of 1,579, validation cohorts of 598 and 423 patients) including clinical parameters and laboratory tests were retrospectively collected. 28-day survival and maximal severity were the main outcomes considered in the multiparametric classical and machine learning statistical analysis. A pilot study was conducted in two subgroups (n=74 and n=41) measuring 17 cytokines and 27 lymphocyte phenotypes respectively.Findings1) Despite a strong association of clinical and laboratory variables with the outcomes in classical pairwise analysis, the contribution of laboratory tests to the combined prediction power was limited by redundancy. Laboratory variables reflected only two types of processes: inflammation and organ damage but none reflected the immune response, one major determinant of prognosis. 2) Eight of the thirty variables: age, comorbidity index, oxygen saturation to fraction of inspired oxygen ratio, neutrophil-lymphocyte ratio, C-reactive protein, aspartate aminotransferase/alanine aminotransferase ratio, fibrinogen, and glomerular filtration rate captured most of the combined statistical predictive power. 3) The interpretation of clinical and laboratory variables was moderately improved by grouping them in two categories i.e., inflammation related biomarkers and organ damage related biomarkers; Age and organ damage-related biomarker tests were the best predictors of survival, and inflammatory-related ones were the best predictors of severity. 4) The pilot study identified immunological tests (CXCL10, IL-6, IL-1RA and CCL2), that performed better than most currently used laboratory tests.ConclusionsLaboratory tests for clinical management of COVID 19 patients are valuable but limited predictors due to redundancy; this limitation could be overcome by adding immunological tests with independent predictive power. Understanding the limitations of tests in use would improve their interpretation and simplify clinical management but a systematic search for better immunological biomarkers is urgent and feasible.

Multisystem Inflammatory Syndrome in Children (MIS-C) associated with COVID-19 is characterized by hypercytokinemia leading to overwhelming inflammation. We describe the use of a hemadsorption device as part of the supportive treatment for cytokine storm.

Activated phosphoinositide 3-kinase (PI3K) δ Syndrome (APDS), caused by autosomal dominant mutations in PIK3CD (APDS1) or PIK3R1 (APDS2), is a heterogeneous primary immunodeficiency. While initial cohort-descriptions summarized the spectrum of clinical and immunological manifestations, questions about long-term disease evolution and response to therapy remain. The prospective European Society for Immunodeficiencies (ESID)-APDS registry aims to characterize the disease course, identify outcome predictors, and evaluate treatment responses. So far, 77 patients have been recruited (51 APDS1, 26 APDS2). Analysis of disease evolution in the first 68 patients pinpoints the early occurrence of recurrent respiratory infections followed by chronic lymphoproliferation, gastrointestinal manifestations, and cytopenias. Although most manifestations occur by age 15, adult-onset and asymptomatic courses were documented. Bronchiectasis was observed in 24/40 APDS1 patients who received a CT-scan compared with 4/15 APDS2 patients. By age 20, half of the patients had received at least one immunosuppressant, but 2–3 lines of immunosuppressive therapy were not unusual before age 10. Response to rapamycin was rated by physician visual analog scale as good in 10, moderate in 9, and poor in 7. Lymphoproliferation showed the best response (8 complete, 11 partial, 6 no remission), while bowel inflammation (3 complete, 3 partial, 9 no remission) and cytopenia (3 complete, 2 partial, 9 no remission) responded less well. Hence, non-lymphoproliferative manifestations should be a key target for novel therapies. This report from the ESID-APDS registry provides comprehensive baseline documentation for a growing cohort that will be followed prospectively to establish prognostic factors and identify patients for treatment studies.

Autoimmune lymphoproliferative syndrome (ALPS) is a rare primary immune disorder characterized by impaired apoptotic homeostasis. The clinical characteristics include lymphoproliferation, autoimmunity (mainly cytopenia), and an increased risk of lymphoma. A distinctive biological feature is accumulation (>2.5%) of an abnormal cell subset composed of TCRαβ+ CD4-CD8- T cells (DNTs). The most common genetic causes of ALPS are monoallelic pathogenic variants in the FAS gene followed by somatic FAS variants, mainly restricted to DNTs. Identification of somatic FAS variants has been typically addressed by Sanger sequencing in isolated DNTs. However, this approach can be costly and technically challenging, and may not be successful in patients with normal DNT counts receiving immunosuppressive treatment. In this study, we identified a novel somatic mutation in FAS (c.718_719insGTCG) by Sanger sequencing on purified CD3+ cells. We then followed the evolutionary dynamics of the variant along time with an NGS-based approach involving deep amplicon sequencing (DAS) at high coverage (20,000-30,000x). Over five years of clinical follow-up, we obtained six blood samples for molecular study from the pre-treatment (DNTs>7%) and treatment (DNTs400 variants called. In summary, our study illustrates the evolutionary dynamics of a somatic FAS mutation before and during immunosuppressive treatment. The results show that pathogenic somatic FAS variants can be identified with the use of DAS in whole blood of ALPS patients regardless of their DNT counts.

X-linked agammaglobulinemia (XLA) is a clinically and genetically well-defined immunodeficiency and the most common form of agammaglobulinemia. It is characterized by susceptibility to recurrent bacterial infections, profound hypogammaglobulinemia, and few or no circulating B cells. XLA is caused by mutations in the BTK gene, which encodes Bruton's tyrosine kinase (BTK). Because of its X-linked recessive inheritance pattern, XLA virtually only affects males, and the mother is the carrier of the mutation in 80–85% of the males with this condition. In the remaining 15–20% of the cases, the affected male is considered to have a de novo mutation. Here, we present the case of a child with a diagnosis of XLA caused by a missense mutation in the BTK gene (c.494G>A/p.C165Y). Apparently, his mother was wild type for this gene, which implied that the mutation was de novo, but careful analysis of Sanger electropherograms and the use of high-coverage massive parallel sequencing revealed low-level maternal gonosomal mosaicism. The mutation was detected in various samples from the mother (blood, urine, buccal swab, and vaginal swab) at a low frequency of 2–5%, and the status of the patient's mutation changed from de novo to inherited. This study underscores the importance of accurately establishing the parents' status on detection of an apparently de novo mutation in a patient, as inadvertent low-level mosaicism may lead to misinterpretation of the risk of recurrence, vital for genetic counseling.

Primary 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.

Severe combined immunodeficiency (SCID), the most severe form of T-cell immunodeficiency, can be screened at birth by quantifying T-cell receptor excision circles (TRECs) in dried blood spot (DBS) samples. Early detection of this condition speeds up the establishment of appropriate treatment and increases the patient's life expectancy. Newborn screening for SCID started in January 2017 in Catalonia, the first Spanish and European region to universally include this testing. The results obtained in the first 2 years of experience are evaluated here. All babies born between January 2017 and December 2018 were screened. TREC quantification in DBS (1.5 mm diameter) was performed with the Enlite Neonatal TREC kit from PerkinElmer (Turku, Finland). In 2018, the retest cutoff in the detection algorithm was updated based on the experience gained in the first year, and changed from 34 to 24 copies/μL. This decreased the retest rate from 3.34 to 1.4% (global retest rate, 2.4%), with a requested second sample rate of 0.23% and a positive detection rate of 0.02%. Lymphocyte phenotype (T, B, NK populations), expression of CD45RA/RO isoforms, percentage and intensity of TCR αβ and TCR γδ, presence of HLA-DR+ T lymphocytes, and in vitro lymphocyte proliferation were studied in all patients by flow cytometry. Of 130,903 newborns screened, 30 tested positive, 15 of which were male. During the study period, one patient was diagnosed with SCID: incidence, 1 in 130,903 births in Catalonia. Thirteen patients had clinically significant T-cell lymphopenia (non-SCID) with an incidence of 1 in 10,069 newborns (43% of positive detections). Nine patients were considered false-positive cases because of an initially normal lymphocyte count with normalization of TRECs between 3 and 6 months of life, four infants had transient lymphopenia due to an initially low lymphocyte count with recovery in the following months, and three patients are still under study. The results obtained provide further evidence of the benefits of including this disease in newborn screening programs. Longer follow-up is needed to define the exact incidence of SCID in Catalonia.

Background: Immunodeficiencies (ID), in particular primary immunodeficiencies (PID), are often associated with haematological manifestations, such as peripheral cytopenias or lymphoproliferative syndromes. Early diagnosis and management have significant prognostic implications. Secondary immunodeficiencies (SID) may also be induced by oncohaematological diseases and their treatments. Haematologists and oncologists must therefore be aware of the association between blood disorders and cancer and ID, and be prepared to offer their patients appropriate treatment without delay. Our aim was to define the warning signs of primary and secondary IDs in paediatric and adult patients with oncohaematological manifestations.Methods: A multidisciplinary group of six experts (2 haematologists, 2 immunologists, and 2 paediatricians specializing in ID) conducted a literature review and prepared a document based on agreements reached an in-person meeting. An external group of 44 IDs specialists from all over Spain assessed the document and were consulted regarding their level of agreement.Results: This document identifies the haematological and extra-haematological diseases that should prompt a suspicion of PIDs in adults and children, in both primary care and haematology and oncology departments. Cytopenia and certain lymphoproliferative disorders are key diagnostic pointers. The diagnosis must be based on a detailed clinical history, physical exploration, complete blood count and standard laboratory tests. The immunological and haematological tests included in the diagnostic process will depend on the care level. Patients who are candidates for immunoglobulin replacement therapy must be carefully selected, and treatment should be offered as soon as possible to avoid the development of complications. Finally, this document recommends procedures for monitoring these patients.Conclusions: This document combines scientific evidence with the opinion of a broad panel of experts, and emphasizes the importance of an early diagnosis and treatment to avoid complications. The resulting document is a useful tool for primary care physicians and specialists who see both adult and paediatric patients with oncohaematological diseases.

LRBA deficiency was first described in 2012 as an autosomal recessive disorder caused by biallelic mutations in the LRBA gene (OMIM #614700). It was initially characterized as producing early-onset hypogammaglobulinemia, autoimmune manifestations, susceptibility to inflammatory bowel disease, and recurrent infection. However, further reports expanded this phenotype (including patients without hypogammaglobulinemia) and described LRBA deficiency as a clinically variable syndrome with a wide spectrum of clinical manifestations. We present the case of a female patient who presented with type 1 diabetes, psoriasis, oral thrush, and enlarged liver and spleen at the age of 8 months. She later experienced recurrent bacterial and viral infections, including pneumococcal meningitis and Epstein Barr viremia. She underwent two consecutive stem cell transplants at the age of 8 and 9 years, and ultimately died. Samples from the patient and her parents were subjected to whole exome sequencing, which revealed a homozygous 1-bp insertion in exon 23 of the patient's LRBA gene, resulting in frameshift and premature stop codon. The patient's healthy mother was heterozygous for the mutation and her father tested wild-type. This finding suggested that either one copy of the paternal chromosome 4 bore a deletion including the LRBA locus, or the patient inherited two copies of the mutant maternal LRBA allele. The patient's sequencing data showed a 1-Mb loss of heterozygosity region in chromosome 4, including the LRBA gene. Comparative genomic hybridization array of the patient's and father's genomic DNA yielded normal findings, ruling out genomic copy number abnormalities. Here, we present the first case of LRBA deficiency due to a uniparental disomy (UPD). In contrast to classical Mendelian inheritance, UPD involves inheritance of 2 copies of a chromosomal region from only 1 parent. Specifically, our patient carried a small segmental isodisomy of maternal origin affecting 1 Mb of chromosome 4.

Immunoglobulin replacement therapy (IGRT) has contributed critically to the management of primary antibody deficiencies (PAD) and the decrease in pneumonia rate. However, despite adequate IGRT and improved prognosis, patients with PAD continue to experience recurrent respiratory tract infections, leading to bronchiectasis and continuing decline in lung function with a severe impact on their quality of life. Moreover, non-infectious inflammatory and interstitial lung complications, such as granulomatous-lymphocytic interstitial lung disease, contribute substantially to the overall morbidity of PAD. These conditions develop much more often than appreciated and represent a major therapeutic challenge. Therefore, a regular assessment of the structural and functional condition of the lung and the upper airways with appropriate treatment is required to minimize the deterioration of lung function. This work summarizes the knowledge on lung complications in PAD and discusses the currently available diagnostic tools and treatment options.

Common variable immunodeficiency (CVID) is the most frequent symptomatic primary immunodeficiency characterized by recurrent infections, hypogammaglobulinemia and poor response to vaccines. Its diagnosis is made based on clinical and immunological criteria, after exclusion of other diseases that can cause similar phenotypes. Currently, less than 20% of cases of CVID have a known underlying genetic cause. We have analyzed whole-exome sequencing and copy number variants data of 36 children and adolescents diagnosed with CVID and healthy relatives to estimate the proportion of monogenic cases. We have replicated an association of CVID to p.C104R in TNFRSF13B and reported the second case of homozygous patient to date. Our results also identify five causative genetic variants in LRBA, CTLA4, NFKB1, and PIK3R1, as well as other very likely causative variants in PRKCD, MAPK8, or DOCK8 among others. We experimentally validate the effect of the LRBA stop-gain mutation which abolishes protein production and downregulates the expression of CTLA4, and of the frameshift indel in CTLA4 producing expression downregulation of the protein. Our results indicate a monogenic origin of at least 15–24% of the CVID cases included in the study. The proportion of monogenic patients seems to be lower in CVID than in other PID that have also been analyzed by whole exome or targeted gene panels sequencing. Regardless of the exact proportion of CVID monogenic cases, other genetic models have to be considered for CVID. We propose that because of its prevalence and other features as intermediate penetrancies and phenotypic variation within families, CVID could fit with other more complex genetic scenarios. In particular, in this work, we explore the possibility of CVID being originated by an oligogenic model with the presence of heterozygous mutations in interacting proteins or by the accumulation of detrimental variants in particular immunological pathways, as well as perform association tests to detect association with rare genetic functional variation in the CVID cohort compared to healthy controls.