Subclinical atherosclerosis and accelerated epigenetic age mediated by inflammation: a multi-omics study.

AIMS: Epigenetic age is emerging as a personalized and accurate predictor of biological age. The aim of this article is to assess the association of subclinical atherosclerosis with accelerated epigenetic age and to investigate the underlying mechanisms mediating this association. METHODS AND RESULTS: Whole blood methylomics, transcriptomics, and plasma proteomics were obtained for 391 participants of the Progression of Early Subclinical Atherosclerosis study. Epigenetic age was calculated from methylomics data for each participant. Its divergence from chronological age is termed epigenetic age acceleration. Subclinical atherosclerosis burden was estimated by multi-territory 2D/3D vascular ultrasound and by coronary artery calcification. In healthy individuals, the presence, extension, and progression of subclinical atherosclerosis were associated with a significant acceleration of the Grim epigenetic age, a predictor of health and lifespan, regardless of traditional cardiovascular risk factors. Individuals with an accelerated Grim epigenetic age were characterized by an increased systemic inflammation and associated with a score of low-grade, chronic inflammation. Mediation analysis using transcriptomics and proteomics data revealed key pro-inflammatory pathways (IL6, Inflammasome, and IL10) and genes (IL1B, OSM, TLR5, and CD14) mediating the association between subclinical atherosclerosis and epigenetic age acceleration. CONCLUSION: The presence, extension, and progression of subclinical atherosclerosis in middle-aged asymptomatic individuals are associated with an acceleration in the Grim epigenetic age. Mediation analysis using transcriptomics and proteomics data suggests a key role of systemic inflammation in this association, reinforcing the relevance of interventions on inflammation to prevent cardiovascular disease.

Early insulin resistance in normoglycemic low-risk individuals is associated with subclinical atherosclerosis.

BACKGROUND: Elevated glycated hemoglobin (HbA1c) is associated with a higher burden of subclinical atherosclerosis (SA). However, the association with SA of earlier insulin resistance markers is poorly understood. The study assessed the association between the homeostatic model assessment of insulin resistance index (HOMA-IR) and SA in addition to the effect of cardiovascular risk factors (CVRFs) in individuals with normal HbA1c. METHODS: A cohort of 3,741 middle-aged individuals from the Progression of Early Subclinical Atherosclerosis (PESA) study with basal HbA1c < 6.0% (< 42 mmol/mol) and no known CV disease underwent extensive imaging (multiterritorial vascular ultrasound and coronary artery calcium score, CACS) to assess the presence, burden, and extent of SA. RESULTS: Individuals with higher HOMA-IR values had higher rates of CVRFs. HOMA-IR showed a direct association with the multiterritorial extent of SA and CACS (p < 0.001) and with global plaque volume measured by 3-dimensional vascular ultrasound (p < 0.001). After adjusting for key CVRFs and HbA1c, HOMA-IR values ≥ 3 were associated with both the multiterritorial extent of SA (odds ratio 1.41; 95%CI: 1.01 to 1.95, p = 0.041) and CACS > 0 (odds ratio 1.74; 95%CI: 1.20 to 2.54, p = 0.004), as compared with the HOMA-IR < 2 (the reference HOMA-IR category). In a stratified analysis, this association remained significant in individuals with a low-to-moderate SCORE2 risk estimate (75.6% of the cohort) but not in high-risk individuals. CONCLUSIONS: The use of HOMA-IR identified low-risk individuals with a higher burden of SA, after adjusting for the effects of key traditional CVRFs and HbA1c. HOMA-IR is a simple measure that could facilitate earlier implementation of primary CV prevention strategies in clinical practice.

Emerging Role of Acquired Mutations and Clonal Hematopoiesis in Atherosclerosis　- Beyond Conventional Cardiovascular Risk Factors.

Accumulating evidence suggests that conventional cardiovascular risk factors are incompletely predictive of cardiovascular disease, as a substantial risk remains even when these factors are apparently managed well. In this context, clonal hematopoiesis has emerged as a new and potent risk factor for atherosclerotic cardiovascular disease and other cardiometabolic conditions. Clonal hematopoiesis typically arises from somatic mutations that confer a competitive advantage to a mutant hematopoietic stem cell, leading to its clonal expansion in the stem cell population and its progeny of blood leukocytes. Human sequencing studies and experiments in mice suggest that clonal hematopoiesis, at least when driven by certain mutations, contributes to accelerated atherosclerosis development. However, the epidemiology, biology and clinical implications of this phenomenon remain incompletely understood. Here, we review the current understanding of the connection between clonal hematopoiesis and atherosclerosis, and highlight knowledge gaps in this area of research.

A novel and efficient tandem CD19- and CD22-directed CAR for B cell ALL.

CD19-directed chimeric antigen receptor (CAR) T cells have yielded impressive response rates in refractory/relapse B cell acute lymphoblastic leukemia (B-ALL); however, most patients ultimately relapse due to poor CAR T cell persistence or resistance of either CD19+ or CD19- B-ALL clones. CD22 is a pan-B marker whose expression is maintained in both CD19+ and CD19- relapses. CD22-CAR T cells have been clinically used in B-ALL patients, although relapse also occurs. T cells engineered with a tandem CAR (Tan-CAR) containing in a single construct both CD19 and CD22 scFvs may be advantageous in achieving higher remission rates and/or preventing antigen loss. We have generated and functionally validated using cutting-edge assays a 4-1BB-based CD22/CD19 Tan-CAR using in-house-developed novel CD19 and CD22 scFvs. Tan-CAR-expressing T cells showed similar in vitro expansion to CD19-CAR T cells with no increase in tonic signaling. CRISPR-Cas9-edited B-ALL cells confirmed the bispecificity of the Tan-CAR. Tan-CAR was as efficient as CD19-CAR in vitro and in vivo using B-ALL cell lines, patient samples, and patient-derived xenografts (PDXs). Strikingly, the robust antileukemic activity of the Tan-CAR was slightly more effective in controlling the disease in long-term follow-up PDX models. This Tan-CAR construct warrants a clinical appraisal to test whether simultaneous targeting of CD19 and CD22 enhances leukemia eradication and reduces/delays relapse rates and antigen loss.

Bone marrow activation in response to metabolic syndrome and early atherosclerosis.

AIMS: Experimental studies suggest that increased bone marrow (BM) activity is involved in the association between cardiovascular risk factors and inflammation in atherosclerosis. However, human data to support this association are sparse. The purpose was to study the association between cardiovascular risk factors, BM activation, and subclinical atherosclerosis. METHODS AND RESULTS: Whole body vascular 18F-fluorodeoxyglucose positron emission tomography/magnetic resonance imaging (18F-FDG PET/MRI) was performed in 745 apparently healthy individuals [median age 50.5 (46.8-53.6) years, 83.8% men] from the Progression of Early Subclinical Atherosclerosis (PESA) study. Bone marrow activation (defined as BM 18F-FDG uptake above the median maximal standardized uptake value) was assessed in the lumbar vertebrae (L3-L4). Systemic inflammation was indexed from circulating biomarkers. Early atherosclerosis was evaluated by arterial metabolic activity by 18F-FDG uptake in five vascular territories. Late atherosclerosis was evaluated by fully formed plaques on MRI. Subjects with BM activation were more frequently men (87.6 vs. 80.0%, P = 0.005) and more frequently had metabolic syndrome (MetS) (22.2 vs. 6.7%, P < 0.001). Bone marrow activation was significantly associated with all MetS components. Bone marrow activation was also associated with increased haematopoiesis-characterized by significantly elevated leucocyte (mainly neutrophil and monocytes) and erythrocyte counts-and with markers of systemic inflammation including high-sensitivity C-reactive protein, ferritin, fibrinogen, P-selectin, and vascular cell adhesion molecule-1. The associations between BM activation and MetS (and its components) and increased erythropoiesis were maintained in the subgroup of participants with no systemic inflammation. Bone marrow activation was significantly associated with high arterial metabolic activity (18F-FDG uptake). The co-occurrence of BM activation and arterial 18F-FDG uptake was associated with more advanced atherosclerosis (i.e. plaque presence and burden). CONCLUSION: In apparently healthy individuals, BM 18F-FDG uptake is associated with MetS and its components, even in the absence of systemic inflammation, and with elevated counts of circulating leucocytes. Bone marrow activation is associated with early atherosclerosis, characterized by high arterial metabolic activity. Bone marrow activation appears to be an early phenomenon in atherosclerosis development.[Progression of Early Subclinical Atherosclerosis (PESA); NCT01410318].

Analyzing the role of reoperation in recurrent glioblastoma: a 15-year retrospective study in a single institution.

BACKGROUND: Multiple treatment options at glioblastoma progression exist, including reintervention, reirradiation, additional systemic therapy, and novel strategies. No alternative has been proven to be superior in terms of postprogression survival (PPS). A second surgery has shown conflicting evidence in the literature regarding its prognostic impact, possibly affected by selection bias, and might benefit a sparse subset of patients with recurrent glioblastoma. The present study aims to determine the prognostic influence of salvage procedures in a cohort of patients treated in the same institution over 15 years. METHODS: Three hundred and fifty patients with confirmed primary glioblastoma diagnosed and treated between 2005 and 2019 were selected. To examine the role of reoperation, we intended to create comparable groups, previously excluding all diagnostic biopsies and patients who were not actively treated after the first surgery or at disease progression. Uni- and multivariate Cox proportional hazards regression models were employed, considering reintervention as a time-fixed or time-dependent covariate. The endpoints of the study were overall survival (OS) and PPS. RESULTS: At progression, 33 patients received a second surgery and 84 were treated with chemotherapy only. Clinical variables were similar among groups. OS, but not PPS, was superior in the reintervention group. Treatment modality had no impact in our multivariate Cox regression models considering OS or PPS as the endpoint. CONCLUSIONS: The association of reoperation with improved prognosis in recurrent glioblastoma is unclear and may be influenced by selection bias. Regardless of our selective indications and high gross total resection rates in second procedures, we could not observe a survival advantage.

New Cases of Hypochromic Microcytic Anemia Due to Mutations in the SLC11A2 Gene and Functional Characterization of the G75R Mutation.

Divalent metal-iron transporter 1 (DMT1) is a mammalian iron transporter encoded by the SLC11A2 gene. DMT1 has a vital role in iron homeostasis by mediating iron uptake in the intestine and kidneys and by recovering iron from recycling endosomes after transferrin endocytosis. Mutations in SLC11A2 cause an ultra-rare hypochromic microcytic anemia with iron overload (AHMIO1), which has been described in eight patients so far. Here, we report two novel cases of this disease. The first proband is homozygous for a new SLC11A2 splicing variant (c.762 + 35A > G), becoming the first ever patient reported with a SLC11A2 splicing mutation in homozygosity. Splicing studies performed in this work confirm its pathogenicity. The second proband harbors the previously reported DMT1 G75R mutation in homozygosis. Functional studies with the G75R mutation in HuTu 80 cells demonstrate that this mutation results in improper DMT1 accumulation in lysosomes, which correlates with a significant decrease in DMT1 levels in patient-derived lymphoblast cell lines (LCLs). We also suggest that recombinant erythropoietin would be an adequate therapeutic approach for AHMIO1 patients as it improves their anemic state and may possibly contribute to mobilizing excessive hepatic iron.

Fratricide-resistant CD1a-specific CAR T cells for the treatment of cortical T-cell acute lymphoblastic leukemia.

Relapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL) has a dismal outcome, and no effective targeted immunotherapies for T-ALL exist. The extension of chimeric antigen receptor (CAR) T cells (CARTs) to T-ALL remains challenging because the shared expression of target antigens between CARTs and T-ALL blasts leads to CART fratricide. CD1a is exclusively expressed in cortical T-ALL (coT-ALL), a major subset of T-ALL, and retained at relapse. This article reports that the expression of CD1a is mainly restricted to developing cortical thymocytes, and neither CD34+ progenitors nor T cells express CD1a during ontogeny, confining the risk of on-target/off-tumor toxicity. We thus developed and preclinically validated a CD1a-specific CAR with robust and specific cytotoxicity in vitro and antileukemic activity in vivo in xenograft models of coT-ALL, using both cell lines and coT-ALL patient-derived primary blasts. CD1a-CARTs are fratricide resistant, persist long term in vivo (retaining antileukemic activity in re-challenge experiments), and respond to viral antigens. Our data support the therapeutic and safe use of fratricide-resistant CD1a-CARTs for relapsed/refractory coT-ALL.

Matched sibling donor stem cell transplantation for sickle cell disease: Results from the Spanish group for bone marrow transplantation in children.

OBJECTIVES: The prevalence of sickle cell disease (SCD) in Spain is markedly inferior compared with other European and Mediterranean countries. However, the diagnosis of new patients with SCD is expected to increase. In this multicenter retrospective study, we analyze the hematopoietic stem cell transplantation (HSCT) results obtained in Spain. METHODS: Forty-five patients who underwent a matched sibling donor (MSD) HSCT between 1999 and 2018 were included. Primary endpoint was event-free survival (EFS), and secondary endpoints included acute and chronic graft-versus-host disease (GvHD) and overall survival (OS). RESULTS: Bone marrow was the most frequent stem cell source (93.3%). Most patients received a conditioning regimen based on busulfan and cyclophosphamide (69%). Cumulative incidence of grade III-IV acute GvHD and chronic GvHD was 6.8% (95% CI: 2.3%-20.1%) and 5.4% (95% CI: 1.38%-19.9%), respectively. EFS and overall survival (OS) at 3 years post-HSCT were 89.4% (95% CI: 73.9%-95.9%) and 92.1% (95% CI: 77.2%-97.4%), respectively. All patients aged ≤ 5 presented 100% EFS and OS. CONCLUSIONS: An early referral to HSCT centers should be proposed early in life, before severe complications occur. MSD HSCT should be considered a curative option for all patients aged ≤ 5 years and for older pediatric patients who present complications derived from the disease.

Haploidentical transplantation in pediatric non-malignant diseases: A retrospective analysis on behalf of the Spanish Group for Hematopoietic Transplantation (GETH).

OBJECTIVE: Describe the GETH haploidentical stem cell transplantation (haplo-HSCT) activity in non-malignant disease (NMDs). METHODS: We retrospectively analyzed data from children with NMDs who underwent haplo-HSCT. RESULTS: From January 2001 to December 2016, 26 pediatric patients underwent 31 haplo-HSCT through ex vivo T cell-depleted (TCD) graft platforms or post-transplantation cyclophosphamide (PT-Cy) at 7 Spanish centers. Five cases employed unmanipulated PT-Cy haplo-HSCT, 16 employed highly purified CD34+ cells, and 10 employed ex vivo TCD grafts manipulated either with CD3+ CD19+ depletion, TCRαß+ CD19+ selection or naive CD45RA+ T-cell depletion. Peripheral blood stem cells were the sole source for patients following TCD haplo-HSCT, and bone marrow was the source for one PT-Cy haplo-HSCT. The most common indications for transplantation were primary immunodeficiency disorders (PIDs), severe aplastic anemia, osteopetrosis, and thalassemia. The 1-year cumulative incidence of graft failure was 27.4%. The 1-year III-IV acute graft-versus-host disease (GvHD) and 1-year chronic GvHD rates were 34.6% and 16.7%, respectively. The 2-year overall survival was 44.9% for PIDs, and the 2-year graft-versus-host disease-free and relapse-free survival rate was 37.6% for the other NMDs. The transplantation-related mortality at day 100 was 30.8%. CONCLUSION: Although these results are discouraging, improvements will come if procedures are centralized in centers of expertise.

Design of Acoustic Bifocal Lenses Using a Fourier-Based Algorithm.

In this work, we develop a new design method based on fast Fourier transform (FFT) for implementing zone plates (ZPs) with bifocal focusing profiles. We show that the FFT of the governing binary sequence provides a discrete sequence of the same length, which indicates the location of the main foci at the ZP focusing profile. Then, using reverse engineering and establishing a target focusing profile, we are capable of generating a binary sequence that provides a ZP with the desired focusing profile. We show that this design method, based on the inverse fast Fourier transform (IFFT), is very flexible and powerful and allows to tailor the design of bifocal ZPs to achieve focusing profiles with the desired foci locations and resolutions. The key advantage of our design algorithm, compared to other alternatives presented in previous works, is that our method provides bifocal focusing profiles with an absolute control of the foci locations. Moreover, although we analyze the performance of this novel design algorithm for underwater ultrasonics, it can also be successfully extended to different fields of physics, such as optics or microwaves, where ZPs are widely employed.

On the Design of Soret Zone Plates Based on Binary Sequences Using Directional Transducers.

In this work, we analyze the effect of the distribution of transparent Fresnel regions over the focusing profile of Soret Zone Plates (SZP) based on binary sequences. It is shown that this effect becomes very significant in those fields where directional transducers are employed, such as microwaves or acoustics. A thorough analysis of both the SZP transmission efficiency and the focusing enhancement factor is presented. Moreover, experimental measurements are also carried out for a particular type of binary sequence, the Cantor ternary set, validating the theoretical model and demonstrating that the distribution of transparent Fresnel regions becomes a critical parameter in applications requiring directional emitters.

Analysis of Predistortion Techniques on Fresnel Zone Plates in Ultrasound Applications.

In this work, we analyze the effect of predistortion techniques on the focusing profile of Fresnel Zone Plates (FZPs) in ultrasound applications. This novel predistortion method is based on either increasing or decreasing the width of some of the FZP Fresnel rings by a certain amount. We investigate how the magnitude of the predistortion, as well as the number and location of the predistorted rings, influences the lens focusing profile. This focusing profile can be affected in different ways depending on the area of the lens where the predistortion is applied. It is shown that when the inner area of the lens, closer to its center, is predistorted, this technique allows the control of the focal depth at the main focus. However, when the predistortion is applied to an area farther from the center of the lens, the acoustic intensity distribution among the main focus and the closest adjacent secondary foci can be tailored at a certain degree. This predistortion technique shows great potential and can be used to control, modify and shape the FZP focusing profile in both industrial and therapeutic applications.

Hereditary Hyperferritinemia Cataract Syndrome: Ferritin L Gene and Physiopathology behind the Disease-Report of New Cases.

Hereditary hyperferritinemia-cataract syndrome (HHCS) is a rare disease characterized by high serum ferritin levels, congenital bilateral cataracts, and the absence of tissue iron overload. This disorder is produced by mutations in the iron responsive element (IRE) located in the 5' untranslated regions (UTR) of the light ferritin (FTL) gene. A canonical IRE is a mRNA structure that interacts with the iron regulatory proteins (IRP1 and IRP2) to post-transcriptionally regulate the expression of proteins related to iron metabolism. Ferritin L and H are the proteins responsible for iron storage and intracellular distribution. Mutations in the FTL IRE abrogate the interaction of FTL mRNA with the IRPs, and de-repress the expression of FTL protein. Subsequently, there is an overproduction of ferritin that accumulates in serum (hyperferritinemia) and excess ferritin precipitates in the lens, producing cataracts. To illustrate this disease, we report two new families affected with hereditary hyperferritinemia-cataract syndrome with previous known mutations. In the diagnosis of congenital bilateral cataracts, HHCS should be taken into consideration and, therefore, it is important to test serum ferritin levels in patients with cataracts.

Blinatumomab and inotuzumab for B cell precursor acute lymphoblastic leukaemia in children: a retrospective study from the Leukemia Working Group of the Spanish Society of Pediatric Hematology and Oncology (SEHOP).

Blinatumomab and inotuzumab ozogamycin represent promising alternatives to conventional chemotherapy in acute lymphoblastic leukaemia (ALL). We analysed data from 29 children with ALL treated under compassionate use with blinatumomab, inotuzumab or both. The complete remission (CR) rate in a heavily pretreated population with overt relapse was 47·6%. At earlier stages (first/second CR), both antibodies represented a useful tool to reduce minimal residual disease, and/or avoid further toxic chemotherapy until stem cell transplantation. Six patients developed grade 3 reversible non-haematological toxicity. The 12-month overall survival and event-free survival rates were 50·8 ± 26·4% and 38·9 ± 25·3% with blinatumomab, 45·8 ± 26% and 27·5 ± 25% with inotuzumab.

Somatic Mutations and Clonal Hematopoiesis: Unexpected Potential New Drivers of Age-Related Cardiovascular Disease.

Increasing evidence shows that conventional cardiovascular risk factors are incompletely predictive of cardiovascular disease, particularly in elderly individuals, suggesting that there may still be unidentified causal risk factors. Although the accumulation of somatic DNA mutations is a hallmark of aging, its relevance in cardiovascular disease or other age-related conditions has been, with the exception of cancer, largely unexplored. Here, we review recent clinical and preclinical studies that have identified acquired mutations in hematopoietic stem cells and subsequent clonal hematopoiesis as a new cardiovascular risk factor and a potential major driver of atherosclerosis. Understanding the mechanisms underlying the connection between somatic mutation-driven clonal hematopoiesis and cardiovascular disease will be highly relevant in the context of personalized medicine, as it may provide key information for the design of diagnostic, preventive, or therapeutic strategies tailored to the effects of specific somatic mutations.

Haploidentical transplantation in high-risk pediatric leukemia: A retrospective comparative analysis on behalf of the Spanish working Group for bone marrow transplantation in children (GETMON) and the Spanish Grupo for hematopoietic transplantation (GETH).

A total of 192 pediatric patients, median age 8.6 years, with high-risk hematological malignancies, underwent haploidentical stem cell transplantation (haplo-HSCT) using post-transplantation cyclophosphamide (PT-Cy), or ex vivo T cell-depleted (TCD) graft platforms, from January 1999 to December 2016 in 10 centers in Spain. Some 41 patients received an unmanipulated graft followed by PT-Cy for graft-vs-host disease (GvHD) prophylaxis. A total of 151 patients were transplanted with CD3-depleted peripheral blood stem cells (PBSCs) by either CD34+ selection, CD3+ CD19+ depletion, TCRαß+ CD19+ depletion or CD45RA+ depletion, added to CD34+ selection for GvHD prophylaxis. The PBSCs were the only source in patients following ex vivo TCD haplo-HSCT; bone marrow was the source in 9 of 41 patients following PT-CY haplo-HSCT. Engraftment was achieved in 91.3% of cases. A donor younger than 30 years, and the development of chronic GvHD were positive factors influencing survival, whereas positive minimal residual disease (MRD) before transplant and lymphoid disease were negative factors. The probability of relapse increased with lymphoid malignancies, a donor killer-cell immunoglobulin-like receptor (KIR) haplotype A and positive MRD pretransplant. No difference was found in overall survival, disease-free survival or relapse incidence between the two platforms. Relapse is still of concern in both platforms, and it should be the focus of future efforts. In conclusion, both platforms for haplo-HSCT were effective and could be utilized depending on the comfort level of the center.

Bifocal Ultrasound Focusing Using Bi-Fresnel Zone Plate Lenses.

In this work, we present a bifocal Fresnel zone plate (BiFZP) capable of generating focusing profiles with two different foci. The performance of the BiFZP is demonstrated in the ultrasound domain, with a very good agreement between the experimental measurements and the finite element method (FEM) simulations. This lens becomes an appealing alternative to other dual-focusing lenses, in which the foci location can only be set at a limited range of positions, such as M-bonacci zone plates. Moreover, the variation of the operating frequency has also been analyzed, providing an additional dynamic control parameter in this type of lenses.

Unexpected relevant role of gene mosaicism in patients with primary immunodeficiency diseases.

BACKGROUND: Postzygotic de novo mutations lead to the phenomenon of gene mosaicism. The 3 main types are called somatic, gonadal, and gonosomal mosaicism, which differ in terms of the body distribution of postzygotic mutations. Mosaicism has been reported occasionally in patients with primary immunodeficiency diseases (PIDs) since the early 1990s, but its real involvement has not been systematically addressed. OBJECTIVE: We sought to investigate the incidence of gene mosaicism in patients with PIDs. METHODS: The amplicon-based deep sequencing method was used in the 3 parts of the study that establish (1) the allele frequency of germline variants (n = 100), (2) the incidence of parental gonosomal mosaicism in families with PIDs with de novo mutations (n = 92), and (3) the incidence of mosaicism in families with PIDs with moderate-to-high suspicion of gene mosaicism (n = 36). Additional investigations evaluated body distribution of postzygotic mutations, their stability over time, and their characteristics. RESULTS: The range of allele frequency (44.1% to 55.6%) was established for germline variants. Those with minor allele frequencies of less than 44.1% were assumed to be postzygotic. Mosaicism was detected in 30 (23.4%) of 128 families with PIDs, with a variable minor allele frequency (0.8% to 40.5%). Parental gonosomal mosaicism was detected in 6 (6.5%) of 92 families with de novo mutations, and a high incidence of mosaicism (63.9%) was detected among families with moderate-to-high suspicion of gene mosaicism. In most analyzed cases mosaicism was found to be both uniformly distributed and stable over time. CONCLUSION: This study represents the largest performed to date to investigate mosaicism in patients with PIDs, revealing that it affects approximately 25% of enrolled families. Our results might have serious consequences regarding treatment and genetic counseling and reinforce the use of next-generation sequencing-based methods in the routine analyses of PIDs.