Maternal smoking during pregnancy increases the risk of gut microbiome-associated childhood overweight and obesity.

Childhood obesity is linked to maternal smoking during pregnancy. Gut microbiota may partially mediate this association and could be potential targets for intervention; however, its role is understudied. We included 1,592 infants from the Canadian Healthy Infants Longitudinal Development Cohort. Data on environmental exposure and lifestyle factors were collected prenatally and throughout the first three years. Weight outcomes were measured at one and three years of age. Stool samples collected at 3 and 12 months were analyzed by sequencing the V4 region of 16S rRNA to profile microbial compositions and magnetic resonance spectroscopy to quantify the metabolites. We showed that quitting smoking during pregnancy did not lower the risk of offspring being overweight. However, exclusive breastfeeding until the third month of age may alleviate these risks. We also reported that maternal smoking during pregnancy significantly increased Firmicutes abundance and diversity. We further revealed that Firmicutes diversity mediates the elevated risk of childhood overweight and obesity linked to maternal prenatal smoking. This effect possibly occurs through excessive microbial butyrate production. These findings add to the evidence that women should quit smoking before their pregnancies to prevent microbiome-mediated childhood overweight and obesity risk, and indicate the potential obesogenic role of excessive butyrate production in early life.

Pre-labor and post-labor cesarean delivery and early childhood adiposity in the Canadian Healthy Infant Longitudinal Development (CHILD) Cohort Study.

BACKGROUND/OBJECTIVES: Delivery by cesarean section (CS) compared to vaginal delivery has been associated with increased risk of overweight in childhood. Our study examined if the presence or absence of labor events in CS delivery altered risk of overweight in early childhood (1-5 years) compared to vaginal delivery and if this association differed according to infant sex. SUBJECTS/METHODS: The study included 3073 mother-infant pairs from the CHILD Cohort Study in Canada. Data from birth records were used to categorize infants as having been vaginally delivered, or delivered by CS, with or without labor events. Age and sex adjusted weight-for-length (WFL) and body mass index (BMI) z scores were calculated from height and weight data from clinic visits at 1, 3 and 5 years and used to classify children as overweight. Associations between delivery mode and child overweight at each timepoint were assessed using regression models, adjusting for relevant confounding factors including maternal pre-pregnancy BMI. Effect modification by infant sex was tested. RESULTS: One in four infants (24.6%) were born by CS delivery; 13.0% involved labor events and 11.6% did not. Infants born by CS without labor had an increased odds of being overweight at age 1 year compared to vaginally delivered infants after adjustment for maternal pre-pregnancy BMI, maternal diabetes, smoking, infant sex and birthweight-for-gestational age (aOR 1.68 [95% CI 1.05-2.67]). These effects did not persist to 3 or 5 years of age and, after stratification by sex, were only seen in boys (aOR at 1 year 2.21 [95% CI 1.26-3.88]). CONCLUSION AND RELEVANCE: Our findings add to the body of evidence that CS, in particular CS without labor events, may be a risk factor for overweight in early life, and that this association may be sex-specific. These findings could help to identify children at higher risk for developing obesity.

Transcription factor defects in inborn errors of immunity with atopy.

Transcription factors (TFs) are critical components involved in regulating immune system development, maintenance, and function. Monogenic defects in certain TFs can therefore give rise to inborn errors of immunity (IEIs) with profound clinical implications ranging from infections, malignancy, and in some cases severe allergic inflammation. This review examines TF defects underlying IEIs with severe atopy as a defining clinical phenotype, including STAT3 loss-of-function, STAT6 gain-of-function, FOXP3 deficiency, and T-bet deficiency. These disorders offer valuable insights into the pathophysiology of allergic inflammation, expanding our understanding of both rare monogenic and common polygenic allergic diseases. Advances in genetic testing will likely uncover new IEIs associated with atopy, enriching our understanding of molecular pathways involved in allergic inflammation. Identification of monogenic disorders profoundly influences patient prognosis, treatment planning, and genetic counseling. Hence, the consideration of IEIs is essential for patients with severe, early-onset atopy. This review highlights the need for continued investigation into TF defects to enhance our understanding and management of allergic diseases.

Integrating knowledge of protein sequence with protein function for the prediction and validation of new MALT1 substrates.

We developed a bioinformatics-led substrate discovery workflow to expand the known substrate repertoire of MALT1. Our approach, termed GO-2-Substrates, integrates protein function information, including GO terms from known substrates, with protein sequences to rank substrate candidates by similarity. We applied GO-2-Substrates to MALT1, a paracaspase and master regulator of NF-κB signalling in adaptive immune responses. With only 12 known substrates, the evolutionarily conserved paracaspase functions and phenotypes of Malt1 -/- mice strongly implicate the existence of undiscovered substrates. We tested the ranked predictions from GO-2-Substrates of new MALT1 human substrates by co-expression of candidates transfected with the oncogenic constitutively active cIAP2-MALT1 fusion protein or CARD11/BCL10/MALT1 active signalosome. We identified seven new MALT1 substrates by the co-transfection screen: TANK, TAB3, CASP10, ZC3H12D, ZC3H12B, CILK1 and ILDR2. Using catalytically inactive cIAP2-MALT1 (Cys464Ala), a MALT1 inhibitor, MLT-748, and noncleavable P1-Arg to Ala mutant versions of each substrate in dual transfections, we validated the seven new substrates in vitro. We confirmed the cleavage of endogenous TANK and the RNase ZC3H12D in B cells by Western blotting and mining TAILS N-terminomics datasets, where we also uncovered evidence for these and 12 other candidate substrates by endogenous MALT1. Thus, protein function information improves substrate predictions. The new substrates and other high-ranked MALT1 candidate substrates should open new biological frontiers for further validation and exploration of the function of MALT1 within and beyond NF-κB regulation.

Can tandem alternative splicing and evasion of premature termination codon surveillance contribute to attenuated Peutz-Jeghers syndrome?

Alternative use of short distance tandem sites such as NAGNn AG are a common mechanism of alternative splicing; however, single nucleotide variants are rarely reported as likely to generate or to disrupt tandem splice sites. We identify a pathogenic intron 5 STK11 variant (NM_000455.4:c.[735-6A>G];[=]) segregating with the mucocutaneous features but not the hamartomatous polyps of Peutz-Jeghers syndrome in two individuals. By RNAseq analysis of peripheral blood mRNA, this variant was shown to generate a novel and preferentially used tandem proximal splice acceptor (AAGTGAAG). The variant transcript (NM_000455.4:c.734_734 + 1insTGAAG), which encodes a frameshift (p.[Tyr246Glufs*43]) constituted 36%-43% of STK11 transcripts suggesting partial escape from nonsense mediated mRNA decay and translation of a truncated protein. A review of the ClinVar database identified other similar variants. We suggest that nucleotide changes creating or disrupting tandem alternative splice sites are a pertinent disease mechanism and require contextualization for clinical reporting. Additionally, we hypothesize that some pathogenic STK11 variants cause an attenuated phenotype.

Human complete NFAT1 deficiency causes a triad of joint contractures, osteochondromas, and B-cell malignancy.

The discovery of humans with monogenic disorders has a rich history of generating new insights into biology. Here we report the first human identified with complete deficiency of nuclear factor of activated T cells 1 (NFAT1). NFAT1, encoded by NFATC2, mediates calcium-calcineurin signals that drive cell activation, proliferation, and survival. The patient is homozygous for a damaging germline NFATC2 variant (c.2023_2026delTACC; p.Tyr675Thrfs∗18) and presented with joint contractures, osteochondromas, and recurrent B-cell lymphoma. Absence of NFAT1 protein in chondrocytes caused enrichment in prosurvival and inflammatory genes. Systematic single-cell-omic analyses in PBMCs revealed an environment that promotes lymphomagenesis with accumulation of naïve B cells (enriched for oncogenic signatures MYC and JAK1), exhausted CD4+ T cells, impaired T follicular helper cells, and aberrant CD8+ T cells. This work highlights the pleiotropic role of human NFAT1, will empower the diagnosis of additional patients with NFAT1 deficiency, and further defines the detrimental effects associated with long-term use of calcineurin inhibitors.

DNA methylation changes in cord blood and the developmental origins of health and disease - a systematic review and replication study.

BACKGROUND: Environmental exposures in utero which modify DNA methylation may have a long-lasting impact on health and disease in offspring. We aimed to identify and replicate previously published genomic loci where DNA methylation changes are attributable to in utero exposures in the NutriGen birth cohort studies Alliance. METHODS: We reviewed the literature to identify differentially methylated sites of newborn DNA which are associated with the following five traits of interest maternal diabetes, pre-pregnancy body mass index (BMI), diet during pregnancy, smoking, and gestational age. We then attempted to replicate these published associations in the Canadian Healthy Infant Longitudinal Development (CHILD) and the South Asian birth cohort (START) cord blood epigenome-wide data. RESULTS: We screened 68 full-text articles and identified a total of 17 cord blood epigenome-wide association studies (EWAS) of the traits of interest. Out of the 290 CpG sites reported, 19 were identified in more than one study; all of them associated with maternal smoking. In CHILD and START EWAS, thousands of sites associated with gestational age were identified and maintained significance after correction for multiple testing. In CHILD, there was differential methylation observed for 8 of the published maternal smoking sites. No other traits tested (i.e., folate levels, gestational diabetes, birthweight) replicated in the CHILD or START cohorts. CONCLUSIONS: Maternal smoking during pregnancy and gestational age are strongly associated with differential methylation in offspring cord blood, as assessed in the EWAS literature and our birth cohorts. There are a limited number of reported methylation sites associated in more than two independent studies related to pregnancy. Additional large studies of diverse populations with fine phenotyping are needed to produce robust epigenome-wide data in order to further elucidate the effect of intrauterine exposures on the infants' methylome.

Assessing secondhand and thirdhand tobacco smoke exposure in Canadian infants using questionnaires, biomarkers, and machine learning.

BACKGROUND: As smoking prevalence has decreased in Canada, particularly during pregnancy and around children, and technological improvements have lowered detection limits, the use of traditional tobacco smoke biomarkers in infant populations requires re-evaluation. OBJECTIVE: We evaluated concentrations of urinary nicotine biomarkers, cotinine and trans-3'-hydroxycotinine (3HC), and questionnaire responses. We used machine learning and prediction modeling to understand sources of tobacco smoke exposure for infants from the CHILD Cohort Study. METHODS: Multivariable linear regression models, chosen through a combination of conceptual and data-driven strategies including random forest regression, assessed the ability of questionnaires to predict variation in urinary cotinine and 3HC concentrations of 2017 3-month-old infants. RESULTS: Although only 2% of mothers reported smoking prior to and throughout their pregnancy, cotinine and 3HC were detected in 76 and 89% of the infants' urine (n = 2017). Questionnaire-based models explained 31 and 41% of the variance in cotinine and 3HC levels, respectively. Observed concentrations suggest 0.25 and 0.50 ng/mL as cut-points in cotinine and 3HC to characterize SHS exposure. This cut-point suggests that 23.5% of infants had moderate or regular smoke exposure. SIGNIFICANCE: Though most people make efforts to reduce exposure to their infants, parents do not appear to consider the pervasiveness and persistence of secondhand and thirdhand smoke. More than half of the variation in urinary cotinine and 3HC in infants could not be predicted with modeling. The pervasiveness of thirdhand smoke, the potential for dermal and oral routes of nicotine exposure, along with changes in public perceptions of smoking exposure and risk warrant further exploration.

A Novel Germline Heterozygous BCL11B Variant Causing Severe Atopic Disease and Immune Dysregulation.

B-cell lymphoma/leukemia 11B (BCL11B) is a C2H2 zinc finger transcription factor that is critically important for regulating the development and function of a variety of systems including the central nervous system, the skin, and the immune system. Germline heterozygous variants are associated with a spectrum of clinical disorders, including severe combined immunodeficiency as well as neurological, craniofacial, and dermal defects. Of these individuals, ~50% present with severe allergic disease. Here, we report the detailed clinical and laboratory workup of one of the most severe BCL11B-dependent atopic cases to date. Leveraging a zebrafish model, we were able to confirm a strong T-cell defect in the patient. Based on these data, we classify germline BCL11B-dependent atopic disease as a novel primary atopic disorder.

Factors associated with breast-feeding initiation and continuation in Canadian-born and non-Canadian-born women: a multi-centre study.

OBJECTIVE: To identify factors associated with breast-feeding initiation and continuation in Canadian-born and non-Canadian-born women. DESIGN: Prospective cohort of mothers and infants born from 2008 to 2012: the Canadian Healthy Infant Longitudinal Development (CHILD) Cohort Study. SETTING: General community setting in four Canadian provinces. PARTICIPANTS: In total, 3455 pregnant women from Vancouver, Edmonton, Winnipeg and Toronto between 2008 and 2012. RESULTS: Of 3010 participants included in the current study, the majority were Canadian-born (75·5 %). Breast-feeding initiation rates were high in both non-Canadian-born (95·5 %) and Canadian-born participants (92·7 %). The median breast-feeding duration was 10 months in Canadian-born participants and 11 months in non-Canadian-born participants. Among Canadian-born participants, factors associated with breast-feeding initiation and continuation were older maternal age, higher maternal education, living with their partner and recruitment site. Rooming-in during the hospital stay was also associated with higher rates of breast-feeding initiation, but not continuation at 6-month postpartum. Factors associated with non-initiation of breast-feeding and cessation at 6-month postpartum were maternal smoking, living with a current smoker, caesarean birth and early-term birth. Among non-Canadian-born participants, maternal smoking during pregnancy was associated with lower odds of breast-feeding initiation and lower odds of breast-feeding continuation at 6 months, and older maternal age and recruitment site were associated with breast-feeding continuation at 6 months. CONCLUSIONS: Although Canadian-born and non-Canadian-born women in the CHILD cohort have similar breast-feeding initiation rates, breast-feeding initiation and continuation are more strongly associated with socio-demographic characteristics in Canadian-born participants. Recruitment site was strongly associated with breast-feeding continuation in both groups and may indicate geographic disparities in breast-feeding rates nationally.

MALT1-Dependent Cleavage of HOIL1 Modulates Canonical NF-κB Signaling and Inflammatory Responsiveness.

Nuclear factor kappa B (NF-κB) is a critical transcription factor involved in regulating cell activation, inflammation, and survival. The linear ubiquitin chain assembly complex (LUBAC) which consists of HOIL1, HOIP, and SHARPIN, catalyzes the linear ubiquitination of target proteins-a post-translational modification that is essential for NF-κB activation. Human germline pathogenic variants that dysregulate linear ubiquitination and NF-κB signaling are associated with immunodeficiency and/or autoinflammation including dermatitis, recurrent fevers, systemic inflammation and enteropathy. We previously identified MALT1 paracaspase as a novel negative regulator of LUBAC by proteolytic cleavage of HOIL1. To directly investigate the impact of HOIL1 cleavage activity on the inflammatory response, we employed a stable transduction system to express and directly compare non-cleavable HOIL1 with wild-type HOIL1 in primary HOIL1-deficient patient skin fibroblasts. We discovered that non-cleavable HOIL1 resulted in enhanced NF-κB signaling in response to innate stimuli. Transcriptomics revealed enrichment of inflammation and proinflammatory cytokine-related pathways after stimulation. Multiplexed cytokine assays confirmed a 'hyperinflammatory' phenotype in these cells. This work highlights the physiological importance of MALT1-dependent cleavage and modulation of HOIL1 on NF-κB signaling and inflammation, provides a mechanism for the autoinflammation observed in MALT1-deficient patients, and will inform the development of therapeutics that target MALT1 paracaspase and LUBAC function in treating autoinflammatory skin diseases.

Mechanistic understanding of the combined immunodeficiency in complete human CARD11 deficiency.

BACKGROUND: Germline pathogenic variants impairing the caspase recruitment domain family member 11 (CARD11)-B cell chronic lymphocytic leukemia/lymphoma 10 (BCL10)-MALT1 paracaspase (MALT1) (CBM) complex are associated with diverse human diseases including combined immunodeficiency (CID), atopy, and lymphoproliferation. However, the impact of CARD11 deficiency on human B-cell development, signaling, and function is incompletely understood. OBJECTIVES: This study sought to determine the cellular, immunological, and biochemical basis of disease for 2 unrelated patients who presented with profound CID associated with viral and fungal respiratory infections, interstitial lung disease, and severe colitis. METHODS: Patients underwent next-generation sequencing, immunophenotyping by flow cytometry, signaling assays by immunoblot, and transcriptome profiling by RNA-sequencing. RESULTS: Both patients carried identical novel pathogenic biallelic loss-of-function variants in CARD11 (c.2509C>T; p.Arg837∗) leading to undetectable protein expression. This variant prevented CBM complex formation, severely impairing the activation of nuclear factor-κB, c-Jun N-terminal kinase, and MALT1 paracaspase activity in B and T cells. This functional defect resulted in a developmental block in B cells at the naive and type 1 transitional B-cell stage and impaired circulating T follicular helper cell (cTFH) development, which was associated with impaired antibody responses and absent germinal center structures on lymph node histology. Transcriptomics indicated that CARD11-dependent signaling is essential for immune signaling pathways involved in the development of these cells. Both patients underwent hematopoietic stem cell transplantations, which led to functional normalization. CONCLUSIONS: Complete human CARD11 deficiency causes profound CID by impairing naive/type 1 B-cell and cTFH cell development and abolishing activation of MALT1 paracaspase, NF-κB, and JNK activity. Hematopoietic stem cell transplantation functionally restores impaired signaling pathways.

Human MALT1 deficiency and predisposition to infections.

Human germline MALT1 deficiency is an inborn error of immunity characterized by recurrent bacterial, viral, and fungal infections, periodontal disease, enteropathy, dermatitis, and failure to thrive. The number of identified MALT1-deficient patients have greatly increased in the past two years, which has significantly improved our understanding of the clinical features of this disorder. Patients frequently experience infections affecting the respiratory, skin, gastrointestinal, and blood systems. The most frequently detected pathogens are Staphylococcus aureus, Candida albicans, and cytomegalovirus. Enhanced susceptibility to S. aureus and C. albicans is likely due to impaired Th17 immunity, similar to STAT3 and IL-17 pathway deficiencies.

Exome sequencing enables diagnosis of X-linked hypohidrotic ectodermal dysplasia in patient with eosinophilic esophagitis and severe atopy.

X-linked hypohidrotic ectodermal dysplasia (XLHED) is the most common form of ectodermal dysplasia. Clinical and genetic heterogeneity between different ectodermal dysplasia types and evidence of incomplete penetrance and variable expressivity increase the potential for misdiagnosis. We describe a family with X-linked hypohidrotic ectodermal dysplasia (XLHED) presenting with variable expressivity of symptoms between affected siblings. In addition to the classical signs of hypohidrosis, hypotrichosis and hypodontia, the index patient-a 5 year old boy, also presented with a severe atopy phenotype that was not observed in the other two affected brothers. Exome sequencing in the index and the mother identified a pathogenic nonsense variant in EDA (NM_001399.4: c.766 C>T; p. Gln256Ter). This study highlights how exome sequencing was crucial in establishing a precise molecular diagnosis of XLHED by enabling us to rule out other differential diagnoses including NEMO deficiency syndrome, that was initially presented as a clinical diagnosis to the family.

Idiopathic splenomegaly in childhood and the spectrum of RAS-associated lymphoproliferative disease: a case report.

BACKGROUND: KRAS (KRAS proto-oncogene, GTPase; OMIM: 190,070) encodes one of three small guanosine triphosphatase proteins belonging to the RAS family. This group of proteins is responsible for cell proliferation, differentiation and inhibition of apoptosis. Gain-of-function variants in KRAS are commonly found in human cancers. Non-malignant somatic KRAS variants underlie a subset of RAS-associated autoimmune leukoproliferative disorders (RALD). RALD is characterized by splenomegaly, persistent monocytosis, hypergammaglobulinemia and cytopenia, but can also include autoimmune features and lymphadenopathy. In this report, we describe a non-malignant somatic variant in KRAS with prominent clinical features of massive splenomegaly, thrombocytopenia and lymphopenia. CASE PRESENTATION: A now-11-year-old girl presented in early childhood with easy bruising and bleeding, but had an otherwise unremarkable medical history. After consulting for the first time at 5 years of age, she was discovered to have massive splenomegaly. Clinical follow-up revealed thrombocytopenia, lymphopenia and increased polyclonal immunoglobulins and C-reactive protein. The patient had an unremarkable bone marrow biopsy, flow cytometry showed no indication of expanded double negative T-cells, while malignancy and storage disorders were also excluded. When the patient was 8 years old, whole exome sequencing performed on DNA derived from whole blood revealed a heterozygous gain-of-function variant in KRAS (NM_004985.5:c.37G > T; (p.G13C)). The variant was absent from DNA derived from a buccal swab and was thus determined to be somatic. CONCLUSIONS: This case of idiopathic splenomegaly in childhood due to a somatic variant in KRAS expands our understanding of the clinical spectrum of RAS-associated autoimmune leukoproliferative disorder and emphasizes the value of securing a molecular diagnosis in children with unusual early-onset presentations with a suspected monogenic origin.

Rhinovirus Infection Drives Complex Host Airway Molecular Responses in Children With Cystic Fibrosis.

Early-life viral infections are responsible for pulmonary exacerbations that can contribute to disease progression in young children with cystic fibrosis (CF). The most common respiratory viruses detected in the CF airway are human rhinoviruses (RV), and augmented airway inflammation in CF has been attributed to dysregulated airway epithelial responses although evidence has been conflicting. Here, we exposed airway epithelial cells from children with and without CF to RV in vitro. Using RNA-Seq, we profiled the transcriptomic differences of CF and non-CF airway epithelial cells at baseline and in response to RV. There were only modest differences between CF and non-CF cells at baseline. In response to RV, there were 1,442 and 896 differentially expressed genes in CF and non-CF airway epithelial cells, respectively. The core antiviral responses in CF and non-CF airway epithelial cells were mediated through interferon signaling although type 1 and 3 interferon signaling, when measured, were reduced in CF airway epithelial cells following viral challenge consistent with previous reports. The transcriptional responses in CF airway epithelial cells were more complex than in non-CF airway epithelial cells with diverse over-represented biological pathways, such as cytokine signaling and metabolic and biosynthetic pathways. Network analysis highlighted that the differentially expressed genes of CF airway epithelial cells' transcriptional responses were highly interconnected and formed a more complex network than observed in non-CF airway epithelial cells. We corroborate observations in fully differentiated air-liquid interface (ALI) cultures, identifying genes involved in IL-1 signaling and mucin glycosylation that are only dysregulated in the CF airway epithelial response to RV infection. These data provide novel insights into the CF airway epithelial cells' responses to RV infection and highlight potential pathways that could be targeted to improve antiviral and anti-inflammatory responses in CF.

STIM1-mediated calcium influx controls antifungal immunity and the metabolic function of non-pathogenic Th17 cells.

Immunity to fungal infections is mediated by cells of the innate and adaptive immune system including Th17 cells. Ca2+ influx in immune cells is regulated by stromal interaction molecule 1 (STIM1) and its activation of the Ca2+ channel ORAI1. We here identify patients with a novel mutation in STIM1 (p.L374P) that abolished Ca2+ influx and resulted in increased susceptibility to fungal and other infections. In mice, deletion of STIM1 in all immune cells enhanced susceptibility to mucosal C. albicans infection, whereas T cell-specific deletion of STIM1 impaired immunity to systemic C. albicans infection. STIM1 deletion impaired the production of Th17 cytokines essential for antifungal immunity and compromised the expression of genes in several metabolic pathways including Foxo and HIF1α signaling that regulate glycolysis and oxidative phosphorylation (OXPHOS). Our study further revealed distinct roles of STIM1 in regulating transcription and metabolic programs in non-pathogenic Th17 cells compared to pathogenic, proinflammatory Th17 cells, a finding that may potentially be exploited for the treatment of Th17 cell-mediated inflammatory diseases.

Immunomodulatory function of the cystic fibrosis modifier gene BPIFA1.

BACKGROUND: Cystic fibrosis (CF) is characterized by a progressive decline in lung function due to airway obstruction, infection, and inflammation. CF patients are particularly susceptible to respiratory infection by a variety of pathogens, and the inflammatory response in CF is dysregulated and prolonged. BPI fold containing family A, member 1 (BPIFA1) and BPIFB1 are proteins expressed in the upper airways that may have innate immune activity. We previously identified polymorphisms in the BPIFA1/BPIFB1 region associated with CF lung disease severity. METHODS: We evaluated whether the BPIFA1/BPIFB1 associations with lung disease severity replicated in individuals with CF participating in the International CF Gene Modifier Consortium (n = 6,365). Furthermore, we investigated mechanisms by which the BPIFA1 and BPIFB1 proteins may modify lung disease in CF. RESULTS: The association of the G allele of rs1078761 with reduced lung function was replicated in an independent cohort of CF patients (p = 0.001, n = 2,921) and in a meta-analysis of the full consortium (p = 2.39x10-5, n = 6,365). Furthermore, we found that rs1078761G which is associated with reduced lung function was also associated with reduced BPIFA1, but not BPIFB1, protein levels in saliva from CF patients. Functional assays indicated that BPIFA1 and BPIFB1 do not have an anti-bacterial role against P. aeruginosa but may have an immunomodulatory function in CF airway epithelial cells. Gene expression profiling using RNAseq identified Rho GTPase signaling pathways to be altered in CF airway epithelial cells in response to treatment with recombinant BPIFA1 and BPIFB1 proteins. CONCLUSIONS: BPIFA1 and BPIFB1 have immunomodulatory activity and genetic variation associated with low levels of these proteins may increase CF lung disease severity.