Outcomes of hematopoietic stem cell gene therapy for Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, autoimmunity, and lymphoid malignancies. Gene therapy (GT) to modify autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplantation for patients who lack well-matched donors, avoiding graft-versus-host-disease. We report the outcomes of a phase 1/2 clinical trial in which 5 patients with severe WAS underwent GT using a self-inactivating lentiviral vector expressing the human WAS complementary DNA under the control of a 1.6-kB fragment of the autologous promoter after busulfan and fludarabine conditioning. All patients were alive and well with sustained multilineage vector gene marking (median follow-up: 7.6 years). Clinical improvement of eczema, infections, and bleeding diathesis was universal. Immune function was consistently improved despite subphysiologic levels of transgenic WAS protein expression. Improvements in platelet count and cytoskeletal function in myeloid cells were most prominent in patients with high vector copy number in the transduced product. Two patients with a history of autoimmunity had flares of autoimmunity after GT, despite similar percentages of WAS protein-expressing cells and gene marking to those without autoimmunity. Patients with flares of autoimmunity demonstrated poor numerical recovery of T cells and regulatory T cells (Tregs), interleukin-10-producing regulatory B cells (Bregs), and transitional B cells. Thus, recovery of the Breg compartment, along with Tregs appears to be protective against development of autoimmunity after GT. These results indicate that clinical and laboratory manifestations of WAS are improved with GT with an acceptable safety profile. This trial is registered at clinicaltrials.gov as #NCT01410825.

Mammalian Hbs1L deficiency causes congenital anomalies and developmental delay associated with Pelota depletion and 80S monosome accumulation.

Hbs1 has been established as a central component of the cell's translational quality control pathways in both yeast and prokaryotic models; however, the functional characteristics of its human ortholog (Hbs1L) have not been well-defined. We recently reported a novel human phenotype resulting from a mutation in the critical coding region of the HBS1L gene characterized by facial dysmorphism, severe growth restriction, axial hypotonia, global developmental delay and retinal pigmentary deposits. Here we further characterize downstream effects of the human HBS1L mutation. HBS1L has three transcripts in humans, and RT-PCR demonstrated reduced mRNA levels corresponding with transcripts V1 and V2 whereas V3 expression was unchanged. Western blot analyses revealed Hbs1L protein was absent in the patient cells. Additionally, polysome profiling revealed an abnormal aggregation of 80S monosomes in patient cells under baseline conditions. RNA and ribosomal sequencing demonstrated an increased translation efficiency of ribosomal RNA in Hbs1L-deficient fibroblasts, suggesting that there may be a compensatory increase in ribosome translation to accommodate the increased 80S monosome levels. This enhanced translation was accompanied by upregulation of mTOR and 4-EBP protein expression, suggesting an mTOR-dependent phenomenon. Furthermore, lack of Hbs1L caused depletion of Pelota protein in both patient cells and mouse tissues, while PELO mRNA levels were unaffected. Inhibition of proteasomal function partially restored Pelota expression in human Hbs1L-deficient cells. We also describe a mouse model harboring a knockdown mutation in the murine Hbs1l gene that shared several of the phenotypic elements observed in the Hbs1L-deficient human including facial dysmorphism, growth restriction and retinal deposits. The Hbs1lKO mice similarly demonstrate diminished Pelota levels that were rescued by proteasome inhibition.

Neonatal-Onset Chronic Diarrhea Caused by Homozygous Nonsense WNT2B Mutations.

Homozygous nonsense mutations in WNT2B were identified in three individuals from two unrelated families with severe, neonatal-onset osmotic diarrhea after whole-exome sequencing was performed on trios from the two families. Intestinal biopsy samples from affected individuals were used for histology and immunofluorescence and to generate enteroids ex vivo. Histopathologic evaluation demonstrated chronic inflammatory changes in the stomach, duodenum, and colon. Immunofluorescence demonstrated diminished staining for OLFM4, a marker for intestinal stem cells (ISCs). The enteroids generated from WNT2B-deficient intestinal epithelium could not be expanded and did not survive passage. Addition of CHIR-99021 (a GSK3A and GSK3B inhibitor and activator of canonical WNT/ß-CATENIN signaling) could not rescue WNT2B-deficient enteroids. Addition of supplemental recombinant murine WNT2B was able to perpetuate small enteroids for multiple passages but failed to expand their number. Enteroids showed a 10-fold increase in the expression of LEF1 mRNA and a 100-fold reduction in TLR4 expression, compared with controls by quantitative RT-PCR, indicating alterations in canonical WNT and microbial pattern-recognition signaling. In summary, individuals with homozygous nonsense mutations in WNT2B demonstrate severe intestinal dysregulation associated with decreased ISC number and function, likely explaining their diarrheal phenotype. WNT2B deficiency should be considered for individuals with neonatal-onset diarrhea.

Primary immunodeficiency testing in a Massachusetts tertiary care NICU: persistent challenges in the extremely premature population.

BACKGROUND: Prematurity presents a diagnostic challenge in interpreting primary immunodeficiency (PID) testing. METHODS: We retrospectively reviewed the charts of all infants in our level IV referral neonatal intensive care unit (NICU) in Massachusetts, with immunologic testing performed from 2006 to 2018. RESULTS: The overall rate of PID testing was enriched in our population, with 1% of admitted patients having extended immunologic testing. The addition of TREC (T cell receptor excision circle) newborn screening in Massachusetts in 2009 increased the proportion of infants tested for PID in our NICU by 3-fold (1.21% post-newborn screening (NBS) vs. 0.46% pre-NBS). A majority of the term and late preterm (≥34 weeks) infants (31 of 41, 76%), as well as very premature (29-33 weeks) infants (12 of 17, 71%), who had immune testing, had a genetic diagnosis associated with secondary immunodeficiency or a PID. Most infants who were born extremely premature (EP, <29 weeks) (25 of 29, 86%) had no identifiable cause of immunodeficiency besides prematurity, despite a mean postmenstrual age of 40.1 weeks at the time of testing. CONCLUSIONS: Persistent immune derangements were present within a subgroup of the EP population through term postmenstrual age. EP infants with significant infectious history and abnormal immune testing at term-corrected age should be considered for genetic testing. IMPACT: The role of immunologic testing in the premature population is unclear, we therefore reviewed the records of all infants in our NICU who had immunologic testing, to rule out immunodeficiency, done from 2006 to 2018. The addition of newborn screening for SCID in 2009 doubled the number of infants who had immune investigations. The extremely premature cohort included many infants with persistent immune derangements through term-corrected gestational age, suggesting a persistent effect of prematurity on immune development and potential function. We propose that former premature infants with clinical evidence of immunodeficiency and sustained immune abnormalities by term-corrected age undergo genetic testing for immunodeficiency.

Hypersensitivity to tetracyclines: Skin testing, graded challenge, and desensitization regimens.

BACKGROUND: Hypersensitivity reactions (HSRs) to tetracyclines and the related compound, tigecycline, can limit the use of these medications and compromise optimal patient care. Despite this, there is little discussion in the literature describing the presentation of these reactions or guiding clinicians on the management of these reactions in adult and pediatric patients. OBJECTIVE: To describe the clinical features, optimal diagnostic approach, and management of HSRs to tetracyclines. METHODS: Patients with reactions to tetracyclines at our institution from 2011 to 2019 were identified by retrospective chart review. Skin testing protocols were designed for each antibiotic. Graded challenge and desensitization procedures were devised based on medical history, skin testing results when available, and need for readministration. RESULTS: The HSRs to tetracyclines, their workup, and management are described for 10 patients, aged 7 to 68 years. Our skin testing protocols for doxycycline, minocycline, and tigecycline described herein had good negative predictive value. When skin testing was negative and the initial reaction was not severe, graded challenge to the culprit drug was performed. Using the included procedures, 3 patients were desensitized to oral doxycycline, 3 to oral minocycline, and 2 to intravenous tigecycline. All the desensitizations were successful. CONCLUSION: Once identified, HSRs to tetracyclines can be further evaluated with skin testing and graded challenge and managed in appropriate cases with desensitization. These procedures can facilitate first-line therapy for patients who require tetracyclines but developed hypersensitivity reactions.

Risk-factors Associated With Poor Outcomes in VEO-IBD Secondary to XIAP Deficiency: A Case Report and Literature Review.

Very early onset inflammatory bowel disease (VEO-IBD) represents a diagnostic and treatment challenge. Here we present a case of VEO-IBD secondary to a mutation in BIRC4 gene, which encodes X-linked inhibitor of apoptosis protein (XIAP), in a 17-month-old boy with severe failure to thrive, intractable diarrhea, and hepatosplenomegaly. Endoscopy and histology identified only mild duodenitis and ileitis, but severe pancolitis with crypt abscesses and epithelium apoptosis. Minimal improvement in symptoms was achieved with total parenteral nutrition (TPN), intravenous (IV) corticosteroids, and tacrolimus, whereas induction and maintenance therapy with adalimumab led to complete remission. After 6 months, the patient developed hemophagocytic lymphohistiocytosis and eventually died due to multisystem organ failure. A review of the literature revealed that some patients with VEO-IBD secondary to XIAP deficiency develop symptoms that are refractory to medical and surgical management, while initial reports suggest that allogeneic hematopoietic stem cell transplantation (HSCT), with reduced intensity conditioning, can successfully induce long-lasting remission and may even be curative. We propose that in patients with XIAP deficiency a constellation of symptoms including colitis at an early age, severe failure to thrive, and splenomegaly/hepatosplenomegaly can identify a subgroup of patients at high risk of experiencing medically refractory IBD phenotype and increased mortality. Hematopoietic stem cell transplant should be considered early in these high-risk patients, as it may resolve both their intestinal inflammation and a risk of developing life threatening hemophagocytic lymphohistiocytosis .

Applying the bronchopulmonary dysplasia framework to necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating disease of the neonatal intestine, causing widespread intestinal necrosis as well systemic illness that frequently results in death. Because the clinical onset of NEC is sudden and difficult to predict, NEC is considered an acute event. However, NEC does not occur in utero, meaning that postnatal exposures are required, and it does not typically occur right after birth, suggesting that longitudinal changes may be occurring before NEC can develop. In this perspective, the author considers whether NEC should be re-considered as a problem of disordered intestinal epithelial development, with required maladaptation over time prior to the onset of the necrotic event. This framework is similar to how bronchopulmonary dysplasia is currently conceptualized. They also advocate that NEC researchers incorporate this possibility into future studies on NEC susceptibility and pathogenesis.

WNT2B Deficiency Causes Enhanced Susceptibility to Colitis Due to Increased Inflammatory Cytokine Production.

BACKGROUND & AIMS: Humans with WNT2B deficiency have severe intestinal disease, including significant inflammatory injury, highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis. METHODS: We investigated the intestinal health of Wnt2b knock out (KO) mice. We assessed the baseline histology and health of the small intestine and colon, and the impact of inflammatory challenge using dextran sodium sulfate (DSS). We also evaluated human intestinal tissue. RESULTS: Mice with WNT2B deficiency had normal baseline histology but enhanced susceptibility to DSS colitis because of an increased early injury response. Although intestinal stem cells markers were decreased, epithelial proliferation was similar to control subjects. Wnt2b KO mice showed an enhanced inflammatory signature after DSS treatment. Wnt2b KO colon and human WNT2B-deficient organoids had increased levels of CXCR4 and IL6, and biopsy tissue from humans showed increased neutrophils. CONCLUSIONS: WNT2B is important for regulation of inflammation in the intestine. Absence of WNT2B leads to increased expression of inflammatory cytokines and increased susceptibility to gastrointestinal inflammation, particularly in the colon.

Human and mouse macrophages collaborate with neutrophils to kill larval Strongyloides stercoralis.

Macrophages are multifunctional cells that are active in TH1- and TH2-mediated responses. In this study, we demonstrate that human and mouse macrophages collaborate with neutrophils and complement to kill the parasite Strongyloides stercoralis in vitro. Infection of mice with worms resulted in the induction of alternatively activated macrophages (AAM) within the peritoneal cavity. These cells killed the worms in vivo and collaborated with neutrophils and complement during the in vitro killing process. AAM generated in vitro killed larvae more rapidly than naive macrophages, which killed larvae after a longer time period. In contrast, classically activated macrophages were unable to kill larvae either in vitro or in vivo. This study adds macrophages to the armamentarium of immune components that function in elimination of parasitic helminths and demonstrate a novel function by which AAM control large extracellular parasites.

M1 and M2 Macrophages Differentially Regulate Colonic Crypt Renewal.

BACKGROUND: The colonic epithelium is the most rapidly renewing tissue in the body and is organized into a single cell layer of invaginations called crypts. Crypt renewal occurs through Lgr5 + gut stem cells situated at the crypt base, which divide, produce daughter cells that proliferate, migrate, differentiate into all the cells required for normal gut function, and are finally shed into the crypt lumen. In health, this rapid renewal helps maintain barrier function next to the hostile gut microbial luminal environment. Inflammation results in an influx of immune cells including inflammatory M1 macrophages into the gut mucosa next to the crypt epithelium, but the direct effect of macrophages on crypt regeneration and renewal are poorly understood. METHODS: Using an in vitro macrophage-crypt coculture model, we show that homeostatic M2 macrophages and inflammatory M1 macrophages confer different effects on the crypt epithelium. RESULTS: Both M1 and M2 increase crypt cell proliferation, with M2 macrophages requiring physical contact with the crypt epithelium, whereas M1 macrophages exert their effect through a secreted factor. Only M1 macrophages reduce goblet and Tuft cell numbers and increase Lgr5 + crypt stem cell numbers, all dependent on physical contact with the crypt epithelium. Further studies showed that M1 macrophages increase the Wnt signaling pathways cyclin D1 and LEF1 through physical contact rather than a secreted factor. CONCLUSIONS: These findings highlight the importance of understanding distinct cellular interactions and direct dialogue between cells and increase our understanding of the contribution of different immune cell subtypes on crypt cell biology during inflammation.

Inflammatory macrophages but not homeostatic macrophages modulate crypt epithelial cell differentiation. Direct physical contact between an inflammatory macrophage and the crypt epithelium is required for regulation of differentiation, but crypt proliferation is via a secreted factor.

A sepsis trigger tool reduces time to antibiotic administration in the NICU.

OBJECTIVE: Prolonged time to antibiotic administration is associated with increased morbidity and mortality. Interventions to decrease the time to antibiotic administration may improve mortality and morbidity. STUDY DESIGN: We identified possible change concepts for reducing time to antibiotic usage in the NICU. For the initial intervention, we developed a sepsis screening tool based on NICU-specific parameters. The main goal of the project was to reduce time to antibiotic administration by 10%. RESULTS: The project was conducted from April 2017 until April 2019. There were no missed cases of sepsis in the project period. Time to antibiotic administration for patients who were started on antibiotics decreased during the project, with the mean shifting from 126 to 102 min, a reduction of 19%. CONCLUSIONS: We successfully reduced time to antibiotic delivery in our NICU using a trigger tool to identifying potential cases of sepsis in the NICU environment. The trigger tool requires broader validation.

Ectopic Rod Photoreceptor Development in Mice with Genetic Deficiency of WNT2B.

Wnt/ß-catenin signaling is essential for embryonic eye development in both the anterior eye and retina. WNT2B, a ligand and activator of the Wnt/ß-catenin pathway, assists in the development of the lens and peripheral regions of the eye. In humans WNT2B mutations are associated with coloboma and WNT2B may also assist in retinal progenitor cell differentiation in chicken, yet the potential role of WNT2B in retinal neuronal development is understudied. This study explored the effects of WNT2B on retinal neuronal and vascular formation using systemic Wnt2b knockout (KO) mice generated by crossing Wnt2bflox/flox (fl/fl) mice with CMV-cre mice. Wnt2b KO eyes exhibited relatively normal anterior segments and retinal vasculature. Ectopic formation of rod photoreceptor cells in the subretinal space was observed in Wnt2b KO mice as early as one week postnatally and persisted through nine-month-old mice. Other retinal neuronal layers showed normal organization in both thickness and lamination, without detectable signs of retinal thinning. The presence of abnormal photoreceptor genesis was also observed in heterozygous Wnt2b mice, and occasionally in wild type mice with decreased Wnt2b expression levels. Expression of Wnt2b was found to be enriched in the retinal pigment epithelium compared with whole retina. Together these findings suggest that WNT2B is potentially involved in rod photoreceptor genesis during eye development; however, potential influence by a yet unknown genetic factor is also possible.

Antimicrobial peptides modulate lung injury by altering the intestinal microbiota.

Mammalian mucosal barriers secrete antimicrobial peptides (AMPs) as critical host-derived regulators of the microbiota. However, mechanisms that support homeostasis of the microbiota in response to inflammatory stimuli such as supraphysiologic oxygen remain unclear. Here, we show that neonatal mice breathing supraphysiologic oxygen or direct exposure of intestinal organoids to supraphysiologic oxygen suppress the intestinal expression of AMPs and alters the composition of the intestinal microbiota. Oral supplementation of the prototypical AMP lysozyme to hyperoxia exposed neonatal mice reduced hyperoxia-induced alterations in their microbiota and was associated with decreased lung injury. Our results identify a gut-lung axis driven by intestinal AMP expression and mediated by the intestinal microbiota that is linked to lung injury. Together, these data support that intestinal AMPs modulate lung injury and repair. In Brief: Using a combination of murine models and organoids, Abdelgawad and Nicola et al. find that suppression of antimicrobial peptide release by the neonatal intestine in response to supra-physiological oxygen influences the progression of lung injury likely via modulation of the ileal microbiota. Highlights: Supraphysiologic oxygen exposure alters intestinal antimicrobial peptides (AMPs).Intestinal AMP expression has an inverse relationship with the severity of lung injury.AMP-driven alterations in the intestinal microbiota form a gut-lung axis that modulates lung injury.AMPs may mediate a gut-lung axis that modulates lung injury.

Antimicrobial peptides modulate lung injury by altering the intestinal microbiota.

BACKGROUND: Mammalian mucosal barriers secrete antimicrobial peptides (AMPs) as critical, host-derived regulators of the microbiota. However, mechanisms that support microbiota homeostasis in response to inflammatory stimuli, such as supraphysiologic oxygen, remain unclear. RESULTS: We show that supraphysiologic oxygen exposure to neonatal mice, or direct exposure of intestinal organoids to supraphysiologic oxygen, suppresses the intestinal expression of AMPs and alters intestinal microbiota composition. Oral supplementation of the prototypical AMP lysozyme to hyperoxia-exposed neonatal mice reduced hyperoxia-induced alterations in their microbiota and was associated with decreased lung injury. CONCLUSIONS: Our results identify a gut-lung axis driven by intestinal AMP expression and mediated by the intestinal microbiota that is linked to lung injury in newborns. Together, these data support that intestinal AMPs modulate lung injury and repair. Video Abstract.

WNT2B Deficiency Causes Increased Susceptibility to Colitis in Mice and Impairs Intestinal Epithelial Development in Humans.

Background and aims: WNT2B is a canonical Wnt ligand previously thought to be fully redundant with other Wnts in the intestinal epithelium. However, humans with WNT2B deficiency have severe intestinal disease, highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis. Methods: We investigated the intestinal health of Wnt2b knock out (KO) mice. We assessed the impact of inflammatory challenge to the small intestine, using anti-CD3χ antibody, and to the colon, using dextran sodium sulfate (DSS). In addition, we generated human intestinal organoids (HIOs) from WNT2B-deficient human iPSCs for transcriptional and histological analyses. Results: Mice with WNT2B deficiency had significantly decreased Lgr5 expression in the small intestine and profoundly decreased expression in the colon, but normal baseline histology. The small intestinal response to anti-CD3χ antibody was similar in Wnt2b KO and wild type (WT) mice. In contrast, the colonic response to DSS in Wnt2b KO mice showed an accelerated rate of injury, featuring earlier immune cell infiltration and loss of differentiated epithelium compared to WT. WNT2B-deficient HIOs showed abnormal epithelial organization and an increased mesenchymal gene signature. Conclusion: WNT2B contributes to maintenance of the intestinal stem cell pool in mice and humans. WNT2B deficient mice, which do not have a developmental phenotype, show increased susceptibility to colonic injury but not small intestinal injury, potentially due to a higher reliance on WNT2B in the colon compared to the small intestine.WNT2B deficiency causes a developmental phenotype in human intestine with HIOs showing a decrease in their mesenchymal component and WNT2B-deficient patients showing epithelial disorganization. Data Transparency Statement: All RNA-Seq data will be available through online repository as indicated in Transcript profiling. Any other data will be made available upon request by emailing the study authors.

Premature Infants Have Normal Maturation of the T Cell Receptor Repertoire at Term.

Premature infants are known to have immature immune systems compared to term infants; however, the impacts of ex utero immune development are not well characterized. Our previous retrospective clinical review showed prolonged T cell lymphopenia in a subset of extremely premature infants, suggesting that they may have lasting abnormalities in their T cell compartments. We used T cell receptor (TCR) repertoire sequencing to analyze the composition of the T cell compartment in premature and term infants in our NICU. We collected twenty-eight samples from individual subjects and analyzed the number of clonotypes, repertoire diversity, CDR3 length, and V gene usage between groups based on gestational age at birth and postmenstrual age at the time of sample collection. Further, we examined the TCR repertoire in infants with severe bronchopulmonary dysplasia (BPD) and those with abnormal T cell receptor excision circle (TREC) assays. Former extremely premature infants who were corrected to term postmenstrual age had TCR repertoire diversity that was more similar to term born infants than extremely premature infants, supporting normal maturation of the repertoire. Infants with severe BPD did not appear to have increased abnormalities in repertoire diversity. Decreased TCR repertoire diversity was associated with repeatedly abnormal TREC screening, although the diversity was within the normal range for subjects without low TRECs. This study suggests that extremely premature infants demonstrate normal maturation of the T cell repertoire ex utero. Further work is needed to better characterize postnatal T cell development and function in this population.

Insights into the Role of Commensal-Specific T Cells in Intestinal Inflammation.

Trillions of microorganisms exist in the human intestine as commensals and contribute to homeostasis through their interactions with the immune system. In this review, we use previous evidence from published papers to elucidate the involvement of commensal-specific T cells (CSTCs) in regulating intestinal inflammatory responses. CSTCs are generated centrally in the thymus or peripherally at mucosal interfaces and present as CD4+ or CD8+ T cells. Bacteria, fungi, and even viruses act commensally with humans, warranting consideration of CSTCs in this critical relationship. Dysregulation of this immunological balance can result in both intestinal inflammation or damaging autoimmune responses elsewhere in the body. Given the relative novelty of CSTCs in the literature, we aim to introduce the importance of their role in maintaining immune homeostasis at barrier sites such as the intestine.

Major basic protein from eosinophils and myeloperoxidase from neutrophils are required for protective immunity to Strongyloides stercoralis in mice.

Eosinophils and neutrophils contribute to larval killing during the primary immune response, and neutrophils are effector cells in the secondary response to Strongyloides stercoralis in mice. The objective of this study was to determine the molecular mechanisms used by eosinophils and neutrophils to control infections with S. stercoralis. Using mice deficient in the eosinophil granule products major basic protein (MBP) and eosinophil peroxidase (EPO), it was determined that eosinophils kill the larvae through an MBP-dependent mechanism in the primary immune response if other effector cells are absent. Infecting PHIL mice, which are eosinophil deficient, with S. stercoralis resulted in development of primary and secondary immune responses that were similar to those of wild-type mice, suggesting that eosinophils are not an absolute requirement for larval killing or development of secondary immunity. Treating PHIL mice with a neutrophil-depleting antibody resulted in a significant impairment in larval killing. Naïve and immunized mice with neutrophils deficient in myeloperoxidase (MPO) infected with S. stercoralis had significantly decreased larval killing. It was concluded that there is redundancy in the primary immune response, with eosinophils killing the larvae through an MBP-dependent mechanism and neutrophils killing the worms through an MPO-dependent mechanism. Eosinophils are not required for the development or function of secondary immunity, but MPO from neutrophils is required for protective secondary immunity.

Inborn Errors of Immunity in the Premature Infant: Challenges in Recognition and Diagnosis.

Due to heightened awareness and advanced genetic tools, inborn errors of immunity (IEI) are increasingly recognized in children. However, diagnosing of IEI in premature infants is challenging and, subsequently, reports of IEI in premature infants remain rare. This review focuses on how common disorders of prematurity, such as sepsis, necrotizing enterocolitis, and bronchopulmonary dysplasia, can clinically overlap with presenting signs of IEI. We present four recent cases from a single neonatal intensive care unit that highlight diagnostic dilemmas facing neonatologists and clinical immunologists when considering IEI in preterm infants. Finally, we present a conceptual framework for when to consider IEI in premature infants and a guide to initial workup of premature infants suspected of having IEI.

Novel variants in the stem cell niche factor WNT2B define the disease phenotype as a congenital enteropathy with ocular dysgenesis.

WNT2B is a member of the Wnt family, a group of signal transduction proteins involved in embryologic development and stem cell renewal and maintenance. We recently reported homozygous nonsense variants in WNT2B in three individuals with severe, neonatal-onset diarrhea, and intestinal failure. Here we present a fourth case, from a separate family, with neonatal diarrhea associated with novel compound heterozygous WNT2B variants. One of the two variants was a frameshift variant (c.423del [p.Phe141fs]), while the other was a missense change (c.722 G > A [p.G241D]) that we predict through homology modeling to be deleterious, disrupting post-translational acylation. This patient presented as a neonate with severe diet-induced (osmotic) diarrhea and growth failure resulting in dependence on parenteral nutrition. Her gastrointestinal histology revealed abnormal cellular architecture particularly in the stomach and colon, including oxyntic atrophy, abnormal distribution of enteroendocrine cells, and a paucity of colonic crypt glands. In addition to her gastrointestinal findings, she had bilateral corneal clouding and atypical genital development later identified as a testicular 46,XX difference/disorder of sexual development. Upon review of the previously reported cases, two others also had anterior segment ocular anomalies though none had atypical genital development. This growing case series suggests that variants in WNT2B are associated with an oculo-intestinal (and possibly gonadal) syndrome, due to the protein's putative involvement in multiple developmental and stem cell maintenance pathways.