BACKGROUND:Vaccination helps prevent SARS-CoV-2 infection and severe COVID-19. However, vaccine-induced humoral immune responses vary among individuals and wane over time. We aimed to describe the SARS-CoV-2 anti-spike IgG antibody response to vaccination and identify health and demographic factors associated with this response among children and adults. METHODS:We studied a subset of double-vaccinated children (n = 151; mean age: 12 ± 1.5 years, 46% female) and adults (n = 995; 44 ± 6.0 years, 60% female) from the cross-provincial Canadian CHILD Cohort. Dried blood spots were collected over two time periods (March 2021 to September 2021; October 2021 to January 2022). Antibody levels were quantified using automated chemiluminescent ELISAs. Demographic, vaccination, and health data (e.g. location, age, ethnicity, prior SARS-CoV-2 infection, vaccine type, comorbidities) were collected via online questionnaires. Associations were tested using multivariable linear mixed-effect analysis accounting for household membership. RESULTS:Our cohort had SARS-CoV-2 anti-spike seropositivity rate of 97% following two COVID-19 vaccine doses. In both children and adults, the highest antibody levels were observed around three months post-vaccination and did not differ by biological sex. Higher antibody levels were associated with: prior SARS-CoV-2 infection (β = 0.16 scaled luminescence units; 95%CI 0.07,0.25), age < 18 years (13-17 years old, β = 0.14; 95%CI 0.03,0.25 | <13 years old: β = 0.17; 95%CI 0.03,0.32) and receiving mRNA vaccines (two Moderna: β = 0.21; 95%CI 0.10,0.33 | two Pfizer-BioNTech (β = 0.10; 95%CI 0.02,0.18 | one Modern and one Pfizer-BioNTech β = 0.14; 95%CI 0.01,0.27) vs. receiving Oxford-AstraZeneca viral vector vaccines. There were no antibody level differences among study sites, ethnicities, household essential worker status, education levels, immunosuppressive comorbidity presence, or when comparing a 3-8 vs. 9-16-week interval between vaccine doses. INTERPRETATION:We identified key factors (i.e. prior SARS-CoV-2 infection, age, vaccine type) associated with post-vaccination antibody responses in children and adults across Canada. This may help improve future vaccine development and deployment among different population subgroups.

Identification of early interventions to reduce/eliminate asthma - the most common chronic disease among children - could significantly reduce burden on the healthcare system. Large-scale asthma Exposome-Wide Association Studies (ExWAS) could identify potential interventions, however integration of diverse data is required to address association confounders. The CHILD Cohort Study has followed 3,454 healthy Canadian children and their families from early pregnancy, collecting exceptionally diverse data including 27,006 variables from participant questionnaires, clinical data, household and neighbourhood-level exposures, and sample-derived chemical analytic/omic datasets. Here, we report integration of these datasets into the CHILDdb database platform, and use these data to perform ExWAS and machine learning analyses, identifying and further characterizing associations between childhood asthma and 2,954 diverse early exposures (pregnancy-age 5). Significant asthma associations include antibiotic use, human milk components, DEHP-phthalate, and mother's prenatal cleaning product/disinfectant exposure. Subsequent analysis revealed epigenetic changes in the cord blood at birth, after prenatal cleaner exposure, and different microbiome and/or inflammatory cytokine changes associated with different asthma-associated exposures in the child. Collective results support asthma as a heterogeneous condition involving multiple etiologies, with associated endotypes, including significant prenatal exposures with potential transgenerational effects, and suggest targets for early interventions. ### Competing Interest Statement TE reports to have acted or acts as local PI for company-sponsored trials by DBV Therapeutics, Greer Stallergens, Novartis, and sub-investigator ALK-Abelló. He is Co-Investigator or scientific lead in three investigator-initiated oral immunotherapy trials supported by the SickKids Food Allergy and Anaphylaxis Program and serves as an associate editor for Allergy. He/his lab received unconditional/in-kind contributions from Macro Array Diagnostics and an unrestricted grant from ALK-Abelló. He holds scientific advisory board roles for ALK-Abelló, VAMED, Nutricia/Danone, Hipp, Sanofi, Greer-Stallergens, Allergy Therapeutics and Aimmune. TE reports lecture fees from Novartis, ThermoFisher, Nutricia/Danone, Aimmune, Sanofi, Schwalbe, MADX, ALK-Abelló. ### Funding Statement Key support for this work was provided by the Schroeder Allergy and Immunology Research Institute, Genome Canada, Genome BC, and CIHR, with additional support by Simon Fraser University and the Digital Research Alliance of Canada. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethical approval for the CHILD Cohort Study, including the oversight of the CHILD biological samples and the CHILD database (CHILDdb), was obtained from the local Research Ethics Board of each study site: the University of British Columbia, the University of Alberta, the University of Manitoba, the Hospital for Sick Children, and McMaster University. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data described in the manuscript are available by registration to the CHILD database https://childstudy.ca/childdb/ and the submission of a formal request. More information about data access for the CHILD Cohort Study can be found at https://childstudy.ca/for-researchers/dataaccess/. Researchers interested in accessing CHILD Cohort Study data for their research should contact child{at}mcmaster.ca.

IntroductionInborn errors of immunity are genetic disorders that result in the absence or dysfunction of critical components of the immune system. Spondyloenchondrodysplasia (SPENCD) is one of these diseases, causing immune dysfunction, abnormal bone development, and neurological changes. SPENCD is caused by damaging variants in ACP5, which encodes tartaric acid-resistant acid phosphatase (TRAP). TRAP has dual roles, acting as a phosphatase and generating reactive oxygen species (ROS). While previous studies have shown that some SPENCD variants impair phosphatase activity, the impact of these variants on ROS production has not, to our knowledge, been investigated.MethodsClinical assessments and panel sequencing were performed. ACP5 variant-expressing constructs were generated using site-directed mutagenesis and expressed in HEK293 cells. Immunoblotting and p-nitrophenyl phosphate (PNPP) assay were performed on cells transfected with wild-type, patient, or known SPENCD variants. Flow cytometry was performed on PBMCs and THP-1 cells treated with the TRAP inhibitor AubipyOMe and stained for ROS with H2DCF.ResultsWe report a novel case, elucidating the functional impact of two ACP5 variants: ACP5 (NM_001611.5) c. 550C>T, p.Q184* and c. 740T>G, p.L247R. This patient presented with hallmark SPENCD characteristics, including autoimmune hemolytic anemia, autism, intracranial calcification, radiolucent lesions, and short stature. In silico variant assessment confirmed a high likelihood of each variant being damaging with deleterious scores in multiple models. TRAP protein expression was comparable to wild type for patient and known SPENCD variants, except for truncating variants, which resulted in higher levels of truncated protein. Phosphatase activity was abolished for both the patient and known SPENCD variants. ROS production was impaired in cells treated with the TRAP inhibitor.ConclusionThis work describes a newly identified SPENCD case, demonstrating the damaging effects of two ACP5 variants (p.Q184* and p.L247R) on TRAP production and function, reclassifying p.L247R as pathogenic. We also show impairment of ROS production with TRAP inhibition and are assessing how the patient’s ACP5 variants influence ROS production. These findings elucidate the dual roles of TRAP in dephosphorylation and ROS production, showing TRAP’s physiological role and SPENCD’s biological mechanism.

BACKGROUND & AIMS:Puberty is a critical period of development during which nutritional exposures are known to shape long-term health and the risk of chronic diseases. Current dietary assessment methods have limitations for use in large cohorts of adolescent populations. We aimed to evaluate the relative validity of Keenoa (not an acronym), an artificial intelligence-enhanced image-assisted mobile application, against the validated Automated Self-Administered 24 h recall (ASA24)-Canada web-based platform, among adolescents in the CHILD Cohort Study. METHODS:Using a randomized crossover design, participants aged 11-15 years old completed three days (two weekdays and one weekend day) of both Keenoa food tracking and ASA24 food recalls. Differences in reported intakes were analyzed using paired t-tests or Wilcoxon signed-rank test and deattenuated correlations by Spearman's coefficient. Agreement and bias were determined using Bland-Altman's test, and inter-quartile cross-classification agreement was assessed using weighted Cohen kappa. RESULTS:This study included 141 participants with a mean age of 12.2 ± 0.8 years; of them 74 (52.5 %) males; and 88 (62.4 %) identified as Caucasian/White. Mean ± SD reported energy intakes (kcal/d) were 1976 ± 451 and 1978 ± 425, with ASA24 and Keenoa, respectively (P = 0.95). Mean reported macronutrient, iron, and potassium intakes did not significantly differ between tools. Reported fiber intake was higher, while sodium, calcium and vitamin D intakes were lower with Keenoa compared to ASA24 (P values < 0.001-0.025). Deattenuated correlations between tools ranged from r = 0.77 to 1.00 (all p< 0.01) and weighted Cohen κ scores ranged from 0.22 to 0.42 (all p < 0.001). Among all participants, 121 (85.8 %) and 78 (55.3 %) completed all 3 requested days with Keenoa and ASA24, respectively (P< 0.01). CONCLUSION:The artificial intelligence-enhanced image-assisted Keenoa mobile application showed strong to moderate relative validity against ASA24 for energy, macronutrient, potassium and iron intakes. Vitamin D, calcium, fiber and sodium showed limited relative agreement based on mean differences. This novel tool may facilitate dietary assessment and reduce attrition bias in cohort studies. Future validation using objective biomarker measures will help establish true validity.

Importance:Infant milk feeding type (eg, human milk vs formula) and infant gut microbes have each been associated with differences in microbial metabolites and childhood blood pressure; however, evidence remains limited regarding how specific infant gut microbes, at a species or strain level, in combination with milk feeding type, shape microbial metabolites and blood pressure. Objective:To investigate whether human milk feeding and infant gut microbes, including Bifidobacterium longum subsp infantis (B infantis) and other milk-degrading microbes, are associated with infant fecal metabolites and childhood systolic blood pressure (SBP). Design, Setting, and Participants:This cohort study was part of the Canadian Healthy Infant Longitudinal Development (CHILD) cohort study, a prospective multicenter, contemporary, population-based cohort of pregnant mothers and their offspring recruited between 2009 and 2012. Data were collected from 2009 to 2018 and analyzed from January to December 2024. Participants included a subset of children born at 35 weeks of gestation or later without congenital abnormalities or respiratory distress syndrome and with available data on gut microbiome, fecal metabolome, SBP, and covariates. Exposures:Gut microbiome, fecal metabolome, and human milk feeding status at ages 3 months and 1 year. Main Outcomes and Measures:Age-, sex-, and height-specific SBP percentile, measured at ages 3 and 5 years. Results:A total of children (610 [46.1%] girls; 982 children [74.2%] delivered vaginally; mean [SD] maternal age at delivery, 33.3 [4.5] years) were included. At age 3 months, but not at age 1 year, human milk feeding and presence of B infantis showed interactive associations with infant fecal metabolites at ages 3 months and 1 year and SBP at ages 3 and 5 years. Among infants harboring B infantis at age 3 months, mixed feeding (difference, -14.81 [95% CI, -27.05 to -2.56] percentile) and exclusive human milk feeding (difference, -17.16 [95% CI, -29.48 to -4.83] percentile) were associated with a lower childhood SBP, whereas no association was observed among infants without B infantis. Several additional infant gut microbes (eg, Eggerthella lenta, Veillonella dispar) and fecal metabolites (eg, creatinine, succinic acid) also demonstrated feeding- or B infantis-dependent associations with childhood SBP. Conclusions and Relevance:In this cohort study, early-life interactions between human milk feeding and B infantis, among other bacteria, were associated with the infant fecal metabolome and childhood SBP, underscoring the potential importance of early-life nutrition-microbe interplay in cardiometabolic health.

NCKAP1L encodes hematopoietic protein-1 (HEM1), a component of the WASp family verproline-homologous protein (WAVE) actin regulatory complex, activated downstream of multiple immune receptors. HEM1 deficiency disrupts WAVE-mediated regulation of F-actin polymerization and actin capping in neutrophils and lymphocytes, resulting in impaired immune synapse formation, cell migration, and phagocytosis, and excessive cytokine release due to dysregulated granule exocytosis (1, 2). HEM1 deficiency also impacts mTORC2 signaling, T cell proliferation, and other T cell effector functions (3, 4). Thus, the totality of HEM1 deficiency is impaired specific immunity, in addition to nonspecific immune system hyperactivation.Biallelic loss-of-function NCKAP1L variants, including the homozygous c.1076C>T (p.P359L) variant, were first described in 2020 and cause an inborn error of immunity marked by early-onset infection susceptibility, autoimmunity, and excessive inflammation (4). Only nine patients from seven kindreds with HEM1 deficiency have been reported (1, 3–5).We describe two previously unreported patients and provide updated clinical information on two previously published patients, all homozygous for NCKAP1L c.1076C>T (p.P359L) (4, 6), comparing their features with prior cases. Three patients presented in early life with severe bacterial and viral infections accompanied by excessive inflammatory responses resembling hemophagocytic lymphohistiocytosis (HLH), but with uncharacteristic leukocytosis, and improving spontaneously without targeted immunosuppression. One fetus was identified at 25 weeks’ gestation with hydrops, including cardiomegaly, pleural effusion, ascites, echogenic bowel, and Doppler findings consistent with severe anemia; subsequent imaging revealed hepatosplenomegaly, and fetal demise occurred at 31 weeks.While hydrops fetalis has not been previously reported, it is noteworthy that the older sister of the two patients reported by Salzer et al. (3) died shortly after birth of unknown cause.Over time, the three surviving patients experienced fewer infections, even without prophylaxis, but experienced lymphoproliferative, atopic, and immune-mediated organ disease, without classical autoimmunity. Three of the four patients are alive at ages 4–17 years and receive supportive care or immunomodulation; none have undergone hematopoietic stem cell transplantation.This report expands the clinical and immunologic spectrum of HEM1-associated immune actinopathy and increases the total number of reported cases to 12. The shared homozygous c.1076C>T variant among unrelated Ojibway Indigenous Canadian families suggests a possible founder effect.Tabular data are included as downloadable supplement files.

X-linked inhibitor of apoptosis (XIAP) is essential for innate and adaptive immune responses, including programmed cell death inhibition, NFkB and MAPK activation, NLRP3 inflammasome activity regulation, and TNFR signaling (1, 2). XIAP deficiency is an immune dysregulation syndrome caused by hemizygous loss-of-function variants in XIAP, with prominent clinical features, including hemophagocytic lymphohistiocytosis, splenomegaly, and inflammatory bowel disease (IBD) (2, 3).A 6-week-old previously healthy term male infant born to nonconsanguineous parents of West African heritage presented to the hospital with bloody diarrhea, hypoalbuminemia, anemia, elevated C-reactive protein (CRP), fever, and growth faltering. The family history was notable for a male sibling’s death at 10 weeks from sepsis following a diarrheal illness.A full septic workup was negative, and the child was empirically treated for culture-negative sepsis with minimal improvement. The working diagnosis was cow’s milk protein-induced allergic proctocolitis, but his severe gastrointestinal symptoms persisted despite dietary modifications, including complete gut rest. Upper and lower endoscopy revealed flat ileal mucosa without villi, and edematous and friable colonic mucosa, while biopsies showed active duodenitis with villous blunting, and chronic active colitis.Screening immune investigations, including neutrophil oxidative burst, were unremarkable. Whole-exome sequencing (WES) was reported as negative; however, chromosomal microarray (CMA) revealed a 5.7 Kb deletion in Xq25, encompassing the noncoding exon 1 of XIAP. Flow cytometry at Cincinnati Children’s Hospital Diagnostic Immunology Lab showed absent XIAP expression in peripheral blood mononuclear cells. A functional defect in XIAP was demonstrated by absent production of TNF-alpha and IL-8 production by monocytes following stimulation with L18-MDP, with intact cytokine response to LPS stimulation.We describe a case of XIAP deficiency resulting from a copy number loss at Xq25 that was functionally supported by absent XIAP expression and NOD2-mediated cytokine production. This case highlights the phenotypic spectrum of XIAP deficiency, whereby IBD can present very early in life, and the importance of a prompt and thorough genetic evaluation for those with suspected monogenic inborn errors of immunity. Any patient suspected of having XIAP deficiency should have a CMA performed, as pathogenic deletions in the noncoding exon 1 of XIAP have been reported (4, 5), and may be missed on gene panels or WES.Figure 1.A. Whole blood from the index case and a healthy control were stained for intracellular XIAP protein and gated for monocytes, lymphocytes, and granulocytes. Histograms show the lack of XIAP protein staining in all leukocyte subsets. B. Isolated peripheral blood mononuclear cells were stimulated with either LPS as a positive control, L18-MDP as the experimental condition, or PBS as an unstimulated control. Gated monocytes from the control show upregulation of both TNF-a or IL-8 expression when stimulated with either LPS or muramyl dipeptide (MDP), but the patient did not show any upregulation with MDP stimulation. Normal responses noted with LPS indicating the patient’s monocytes are viable and responsive.

Abstract Although genome-wide association studies (GWASs) have been widely applied to investigate the genetic basis of common traits and diseases in human populations, the associated loci do not fully account for the estimated heritability. The missing heritability may be explained, in part, by epistasis or gene-gene interactions. Existing methods for detecting epistasis, however, are limited to pair-wise interactions and/or targeted genomic regions. Here, we present a novel model, termed the Epistatic SNP Network Analysis ( ESNA ), which detects higher-order epistatic interactions using genome-wide SNP data. ESNA employs a scale-free network algorithm within a parallel computing framework that identifies modules of correlated SNPs, potentially interacting variants that converge on common biological pathways, while enhancing computational efficiency. We applied ESNA to investigate epistatic interactions contributing to respiratory outcomes such as recurrent wheeze and asthma among preschool-aged children in the CHILD Cohort Study. Using genome-wide data comprising 775,569 SNPs from 1,899 children, ESNA identified 914 SNP network modules, 9 of which were significantly associated with recurrent wheeze between ages 2 and 5 years (P<5.47×10 -5 ). Furthermore, 7 of these wheeze-associated modules were also associated with asthma by age 5 years (P<5.47×10 -5 ). Pathway enrichment analysis revealed that the associated modules consist of SNPs located in genes previously implicated in asthma and related biological processes, such as cellular response to stimuli and nervous system development. Compared to existing network-based methods for epistasis, ESNA demonstrated substantial improvements in computational efficiency, reducing memory usage by 50% and processing genome-wide SNP data 48 times faster. The code implementation and documentation are available at https://github.com/ComputationalGenomicsLaboratory/ESNA .

The COVID-19 pandemic, caused by SARS-CoV-2, highlighted the need for rapid, large-scale research initiatives to better understand the genomic, clinical, and epidemiological factors underlying disease susceptibility and diverse health outcomes. Our study leveraged federally-funded COVID-19 research programs which sequenced SARS-CoV-2 (VirusSeq) and human (HostSeq) genomes to generate an integrated dataset linking viral and host genomic data with clinical and epidemiological information. This study focused on overcoming challenges in participant recruitment and sample collection during the pandemic, particularly within the context of stringent public health measures. We aimed to ensure the inclusion of individuals from rural and remote regions of BC, who are often underrepresented in national research programs. To facilitate this, we utilized the British Columbia Centre for Disease Control’s (BC CDC) Consent to Contact Registry (CCRD) to recruit individuals who had tested positive for SARS-CoV-2 and had agreed to be contacted for research purposes. Recruitment was conducted through phone calls and emails, and participants provided either blood or saliva samples for genomic analysis. Participant comments shared during recruitment were reviewed descriptively to identify common motivations and concerns. Participants could mail back kits allowing those from remote communities to participate. Despite challenges, such as skepticism about remote data collection, we successfully enrolled 793 participants. We used a descriptive mixed methods approach that combined quantitative recruitment, participation and geographic metrics with a narrative summary of participant comments, integrating these data to explain participation patterns, motivations, concerns and barriers in the context of remote, registry-based recruitment. Our experiences underscore the importance of agile and inclusive research approaches during public health emergencies. Key strategies, such as leveraging existing public health registries, virtual consent processes and remote sample collection, can facilitate the recruitment of geographically diverse populations and should be considered in future large-scale genomic research initiatives.

BackgroundSevere combined immunodeficiency (SCID) is a life-threatening condition that requires urgent medical attention. Newborn screening (NBS) using the T cell receptor excision circle (TREC) assay facilitates early diagnosis of SCID, which is crucial for improving outcomes.ObjectivesDescribe the diagnoses and outcomes of the first 2.5 years of NBS for SCID in British Columbia and the Yukon, Canada.MethodsAn interdisciplinary working group of health professionals met at regular intervals prior to and after implementing NBS for SCID. We developed an algorithm for the evaluation and initial management of infants with an abnormal NBS for SCID based on a literature review and local expert consensus. The diagnoses and outcomes of infants with a positive NBS were monitored prospectively. TRECs were measured from dried blood spots of newborns, with the threshold for an abnormal NBS determined using multiples of the median to achieve a positivity rate of 0.1%. Absolute T cell counts less than 1,500 cells/mcL or naïve CD4 or CD8 T cell percentages less than 50% were considered abnormal and prompted immunology consultation.Results and DiscussionA total of 110,156 infants were screened between October 2022 and April 2025. There were 2 infants diagnosed with SCID, resulting in a population-based incidence of 1 in 55,078. Both underwent stem cell transplantation within 4 months of life and had no infections at the time of transplant. There was one case of congenital athymia secondary to 22q11 microdeletion syndrome identified by NBS for SCID, who underwent a successful thymic transplantation at 5 months of age. There were no cases of SCID diagnosed outside of the NBS program within this time frame.ConclusionOur experience supports the value of NBS using the TREC assay in facilitating early diagnosis and treatment of infants with SCID.

ObjectiveLower respiratory tract infections (LRTIs) in early life are one of the strongest risk factors for childhood asthma and are often treated with systemic antibiotics (IV or oral). We aimed to explore the association between early-life LRTIs and systemic antibiotics on asthma development and the potential mediating role of antibiotics in this relationship.MethodsData were collected as part of the longitudinal, general Canadian population CHILD Study. LRTIs during the first 18 months of life were identified through parental symptom report at regular study visits. Systemic antibiotic use was defined as at least one dose of oral/intravenous antibiotics between birth and the 18-month visit and were further categorized by indication as either given for a respiratory indication (upper or lower respiratory symptoms) or non-respiratory indication. Asthma was diagnosed by in-study pediatricians at the 5-year study visit. Adjusted logistic regression models and mediation analyses via systemic antibiotics use were performed.ResultsAmong 2,073 participants included in our analysis, 72 (4.9%) had asthma age 5, and 609 (29.3%) used systemic antibiotics before the 18-month visit. Among children who had taken antibiotics, 61.6% also had an LRTI in that period compared to 49.7% among children without exposure to systemic antibiotics (p < .001). Moderate-severe LRTIs before age 18 months were associated with higher odds of 5-year asthma [aOR 4.12 (95%CI 2.04–7.95) p < .001]. Antibiotics taken for respiratory indications were associated with higher odds of asthma at age 5 [aOR 2.36 (95%CI 1.59–3.48) p < .001]. Children who received systemic antibiotics for only non-respiratory indications during the first 18 months of life were not associated with increased odds of asthma [aOR 1.08 (95%CI 0.44–2.30) p = .851]. Using mediation analysis, 21.7% of the association between LRTI and asthma is estimated to be mediated through use of early-life systemic antibiotics. However, a significant direct effect of moderate-to-severe LRTIs on asthma risk remained in adjusted mediation models (p = .014).ConclusionThrough mediation modeling we estimate that the increased risk of asthma at age 5 that is associated with moderate-severe LRTIs in infancy may be partially mediated by systemic antibiotics taken during the first 18 months of life. This underscores the importance of public health strategies focused on antibiotic stewardship and reducing early life LRTIs to mitigate asthma risk.

P47phox deficient phagocytic oxidase Chronic Granulomatous Disease (p47phox CGD) is an inherited immunodeficiency caused by mutations in the NCF1 gene which encodes the p47phox protein, a subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which is a critical first line of innate immune system defense against bacterial and fungal pathogens. The mutation in NCF1 prevents NADPH oxidase production of oxidative bursts that destroy pathogens and control infection. Individuals with p47phox CGD experience recurrent severe infections, and inflammation of multiple organs, most notably the bowel, lung, liver and genitourinary system. Allogeneic hematopoietic stem cell transplantation (HSCT) is the only potential cure, but is associated with significant limitations due to donor availability, and the risks of transplant related morbidity and mortality including graft failure, graft-versus-host disease and post-transplant immunosuppression. The prevalent causative mutation giving rise to p47phox CGD is the ‘delGT’ dinucleotide deletion in exon 2 of the NCF1 gene. The NCF1 locus is complex as the gene encoding p47phox is flanked by two nearly identical nonfunctional pseudogenes which bear the inactivating delGT mutation. Prime Editing is uniquely well-suited to correct the delGT NCF1 variant, both because of its versatile ability to precisely replace targeted and specific DNA sequences, and because it does not induce double-strand breaks, which carry the risk of chromosomal instability at this complex locus. PM359 is an autologous CD34+ hematopoietic stem cell suspension drug product that is Prime Edited at the NCF1 locus resulting in correction of the delGT mutation. In preclinical studies, >80% of p47phox CGD CD34⁺ cells were Prime Edited to precisely correct the delGT mutation. The Prime Edited corrected p47phox CGD CD34+ cells reconstituted human hematopoiesis in NBSGW immunodeficient mice and the frequency of engrafted precisely corrected Prime Edited p47phox CGD CD34+ cells remained stable for 16 weeks in vivo, resulting in quantitative restoration of NADPH oxidase activity, with no perturbation of multilineage human blood production, no detectable off-target edits, and no detectable chromosomal alterations (a result that contrasts to CRISPR nuclease editing at this locus). Prime-0101 is a first-in-human study of PM359 in adult and pediatric participants with p47phox CGD. Two study participants, Participant 1 (18yo male) and Participant 2 (57yo male) underwent HSC mobilization with G-CSF and plerixafor, and the apheresis product was transferred to a central manufacturing facility to generate PM359. Study participants received myeloablative conditioning with targeted busulfan prior to infusion of PM359. Both Participants achieved rapid neutrophil engraftment at 13 and 19 days and platelet engraftment at 14 and 12 days respectively following infusion of PM359. By one month after treatment, 69% of Participant 1's and 80% of Participant 2's peripheral neutrophils expressed normal levels of NADPH oxidase activity as measured by the dihydrorhodamine (DHR) assay. These results correlated to the frequency of Prime Edited CD34+ cells measured in the drug products (Participant 1: 68% and Participant 2: 91% Prime Edited colony forming cells). DHR results for Participant 1 have remained stable through month 3. Importantly, frequency of both DHR+ neutrophils and Prime Edited CD34+ cells exceed the 20% threshold expected to be sufficient for restoration of NADPH oxidase anti-pathogen activity and amelioration of disease pathology. Safety was consistent with busulfan conditioning; neither participant required platelet or red blood cell transfusion support. The initial results from the Prime-0101 study provide the first-in-human demonstration of the safety and efficacy of Prime Editing and offer an autologous cell therapy for individuals with p47phox CGD.

ThPOK is a transcription factor that acts as a master regulator of CD4+ T cell lineage commitment. We report the first human disease caused by a genetic alteration in ThPOK, specifically, a damaging heterozygous de novo variant in ThPOK (NM_001256455.2:c.1080A>C, p.K360N). This patient exhibited the unusual constellation of persistent CD4+ T cell deficiency, allergy, interstitial lung disease, corneal vascularization and scarring, developmental delay, and growth failure. The ThPOKK360N variant displayed abnormal multimorphic activity, interfering with ThPOKWT (antimorph), failing to bind wild-type ThPOK consensus sequences (amorph), and showing novel DNA-binding specificity (neomorph). Single-cell RNA sequencing revealed defects in CD4+ and CD8+ T cell maturation and activation (hypomorph). Recapitulated in lentivirally transduced healthy control T cells and fibroblasts, the transcriptomic analysis showed ThPOKK360N-transduced T cells had impaired TCR activation and ThPOKK360N-transduced fibroblasts with increased profibrotic gene expression. This novel human disease confirms ThPOK's role in CD4+ T cell development but also uncovers novel roles in TCR activation and regulation of fibrotic pathways in fibroblasts.

Early child development sets the stage for lifelong health. Identifying early life factors related to child development can help guide programs and policies to bolster child health and wellbeing. The objective of this research was to examine how a broad range of predictors, measured prenatally to the third year of life, are related to child development at kindergarten. We linked survey data from the Manitoba site of the CHILD Cohort Study with data from the Early Development Instrument (EDI) assessment, completed in kindergarten by the Manitoba public school system (n = 442 children). The EDI measures five domains of development (ex. language, physical), scored to indicate the bottom 10% (i.e., 'vulnerable') of the population on one or more domains. Using structural equation modelling, we grouped 23 predictors of child development into six latent factors including prenatal exposures, child health and lifestyle, family stress, and socioeconomic status (SES). We examined the associations between each latent factor and EDI vulnerability. Overall, 20.1% of children were vulnerable on one or more EDI domains. Higher family stress at 1 year and 3 years was related to a 0.20 (p-value ≤0.001) and 0.33 (p-value ≤0.001) standardized increase of EDI vulnerability. Higher SES was related to a-0.26 (p-value =0.01) standardized decrease of EDI vulnerability, and this link was partially mediated through family stress at 3 years (10.6% mediated). Prenatal exposures (e.g., maternal diet quality), as well as child health and lifestyle factors (e.g., weekday sleep) were not related to EDI vulnerability. Supporting parental mental health and programs to reduce early life parenting stress, as well as targeting supports to those living with low SES, appear to be priority areas that could help to improve early child development.

Exposure to plastic additives, such as phthalates and bisphenols, has been associated with a higher risk of allergic conditions, but the evidence is inconsistent for children younger than five. To examine the association between pre- and postnatal urinary phthalates and bisphenols, and allergic conditions, and potential effect modification by sex, in pre-school children, through a pooled analysis. We pooled data from the Barwon Infant Study (Australia), the Canadian Healthy Infant Longitudinal Development Study (Canada), the Health Outcomes and Measures of the Environment (United States) and the Environmental Influences on Child Health Outcomes–wide cohorts (United States). Urinary phthalates and bisphenols were measured during pregnancy and early childhood. We estimated daily intakes from urinary concentrations, except for mono-(3-carboxypropyl) phthalate (MCPP). Outcomes, including asthma, wheeze, eczema, and rhinitis, were assessed up to five years of age through questionnaires and clinical assessments. We used generalised estimating equations for single compounds and quantile G-computation for the chemical mixtures. 5306 children were included. A two-fold increase in prenatal dibutyl phthalates (DBP; risk ratio [RR] = 1.08; 95

AIM:To determine relationships between paediatric use of acid-suppressive medications (ASMs) (proton pump inhibitors (PPI) and histamine-2 receptor antagonists (H2RA)) and preschool body mass index (BMI) and mediation by C. difficile colonisation. METHODS:ASMs were parent-reported at age 3, 6 and 12 months in 1025 infants from the CHILD Cohort Study. Targeted qPCR for C. difficile was conducted in stool collected at 3 months, and age/sex adjusted BMI-z scores were calculated at age 5 years. ASM use and BMI-z score associations were assessed by regression, and mediation analyses were conducted. RESULTS:Approximately 4% of infants received H2RAs, most before age 3 months. C. difficile colonisation was twice as likely in 3-month-old infants receiving H2RAs (OR: 2.27, 95% CI: 1.13-4.51) and was associated with higher BMI-z scores at age 5 years (β: 0.31, 95% CI: 0.16, 0.46). Mediation models showed an indirect positive effect of H2RA use on 5-year BMI-z scores, mediated through C. difficile colonisation (β: 0.065, 95% CI: 0.01, 0.13). CONCLUSIONS:We found evidence of increased preschool BMI following H2RA use in infants that was mediated through C. difficile colonisation. Further study on ASM use in infancy, the gut microbiome, and pathways to body weight health outcomes is warranted.

Monogenic defects that impair the control of inflammation and tolerance lead to profound immune dysregulation, including autoimmunity and atopy. Studying these disorders reveals important molecular and cellular factors that regulate human immune homeostasis and identifies potential precision medicine targets. Here, we provide a detailed immunological assessment of a pediatric patient with a recently discovered syndrome causing Immunodysregulation, Craniofacial anomalies, Hearing impairment, Athelia, and Developmental delay (or ICHAD syndrome). The immunodysregulation resulted in autoimmune hemolytic anemia (AIHA) and atopic dermatitis. The patient carried a de novo germline heterozygous c.406+540_574+13477dup;p.Gly136_Ser191dup variant in IKAROS family zinc finger 2 (IKZF2), which encodes HELIOS. This variant led to reduced HELIOS protein expression and dominant interference of wild-type HELIOS-mediated repression of the IL2 promoter. Multi-parameter flow cytometry analyses of patient peripheral blood mononuclear cells revealed strongly impaired natural killer cell differentiation and function, and increased CD8+ T cell activation and cytokine secretion. Strikingly, patient CD4+ T cells were hyperactive, produced elevated levels of nearly all T helper (TH) cytokines, and readily proliferated in response to stimulation. Patient regulatory T cells (Tregs) developed normally but aberrantly produced high levels of many TH cytokines. Single-cell RNA sequencing revealed largely normal Tregs (albeit mostly memory), but naïve CD4+ T cells that were more enriched in genes related to activation, proliferation, metabolism, and TH differentiation. This work describes the immunological phenotype of one of the first reported cases of germline dominant negative HELIOS deficiency, expands our understanding of the pathogenesis of AIHA on a single cell level, and provides valuable insights into HELIOS function in a variety of lymphocyte subsets.

The Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling cascade is an evolutionarily conserved signal transduction pathway that regulates many vital cellular processes, including immune function and hematopoiesis. Human genetic variants that disrupt JAK-STAT signaling are being found to cause a rapidly increasing number of diseases, including both germline-encoded inborn errors of immunity (IEI) and acquired somatic variants, causing a so-called phenocopy of the IEI. Multiple genetic mechanisms are responsible for this growing group of JAK-STAT diseases including loss-of-function, gain-of-function, and dominant negative effects. In this review, we discuss the clinical presentation and pathogenesis of all currently described JAK-STAT defects, as well as provide an overview of the guiding principles to consider in diagnosing and treating these conditions. (J Allergy Clin Immunol 2025;155:357-67.)

Age- and sex-related differences in immune cell compositions and immune outcomes have been identified across the life course, but a comprehensive and nuanced characterization of these changes during the rapid developmental window of early life is lacking. We explore immune associated DNA methylation (DNAm) changes in the context of age and sex leveraging whole blood samples collected at ages one and five from CHILD, a Canadian longitudinal pediatric cohort (n = 760: 356 female and 404 male). DNAm-based computational cell type deconvolution reveals significant changes in all estimated immune cell types across time, with notable sex differences. In addition, we identify distinct DNAm signatures reflecting age- and sex-associated immune profiles in early life. While age-related DNAm changes are relatively limited, sex-associated differences are consistent across this developmental window and partially validate in independent pediatric cohorts. Together, these findings provide insights into early immune system maturation, underscoring the presence of sex differences prior to puberty.