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Background: Deficient Anterior pituitary with common Variable Immune Deficiency (DAVID) syndrome is a rare condition characterized by the association of adrenocorticotropic hormone deficiency (ACTHD) and primary hypogammaglobulinemia, caused byNFKB2heterozygous mutations. Nuclear factor kappa B (NFKB) signaling is a key regulator of the immune system; however, the underlying mechanism of its association with endocrine symptoms remains unknown. Two main hypotheses explain the effects of mutant NFKB2 on the pituitary gland: an autoimmune hypophysitis, preferentially affecting corticotroph function, or a primary developmental defect. The role of NFKB2 in the development of the human pituitary was called into question byNfkb2-deficientLym1mice, which have normal pituitary functions.Purpose: The aim of this study was to create a human disease model to define the role of NFKB2 in human pituitary development.Methods: We established pituitary organoids in three dimensions (3D) culture after directed differentiation from CRISPR/Cas9-edited human induced pluripotent stem cells (hiPSC). First, we conducted a proof-of-concept study, introducing a homozygousTBX19K146R/K146Rmissense pathogenic variant in hiPSC, an allele found in patients with congenital isolated ACTHD. Then, we used the same method to produceNFKB2D865G/D865Gmutant organoids, harboring the pathogenic missense variant previously identified in DAVID patients. This mutation causes a failure of NFKB2 p100 phosphorylation that blocks processing to form active NFKB2 p52. We then characterized pituitary organoid development by transcriptomics using bulk RNA sequencing and quantitative RT-PCR, and by immunofluorescence in section and whole-mount.Results: Analysis of wild-type (WT) organoids demonstrated that this in vitro model recapitulates corticotroph cell differentiation.TBX19K146R/K146Rorganoids conserved early expression ofHESX1, but had significantly decreasedPITX1, TBX19, LHX3, andPOMCtranscription.NFKB2D865G/D865Gorganoids also had dramatically reduced corticotrophs. Furthermore,NFKB2D865G/D865Gperturbs the normal expression of 66 genes known to contribute to pituitary development, among which 21 transcription factors.Conclusions: We used a combination of CRISPR/Cas9 editing and refinement of a 3D organoid culture protocol to model human ACTHD due toTBX19orNFKB2mutations. TheNFKB2variant studied induced a significant decrease in corticotroph differentiation, demonstrating for the first time a direct functional role of NFKB2 in human pituitary development. Signaling through NFKB2 is thus a valid new candidate pathway in the pathogenesis of isolated or syndromic ACTHD.

Deficient anterior pituitary with common variable immune deficiency (DAVID) syndrome is a rare condition characterized by adrenocorticotropic hormone (ACTH) deficiency and primary hypogammaglobulinemia. It is due to heterozygous mutations of the nuclear factor kappa‐B subunit 2 (NFKB2) gene. Only a few isolated cases have been reported since its first description by our team. Through the international multicenter GENHYPOPIT network, we identified a new case of DAVID syndrome. We then conducted an extensive review of the DAVID syndrome cases published from 2012 to 2022. A 7‐year‐old boy was diagnosed with symptomatic hypoglycemia revealing ACTH deficiency. Laboratory tests showed asymptomatic hypogammaglobulinemia. He harbored a heterozygous point mutation in NFKB2 gene (c.2600C > T, p.Ala867Val). His management included hydrocortisone replacement treatment, and he also received subcutaneous immunoglobulins during the Covid‐19 pandemic. We analyzed 28 cases of DAVID syndrome with ACTH deficiency. ACTH deficiency was the only hormone deficiency in 79% of patients, but some patients harbored growth hormone (GH) and thyroid stimulating hormone (TSH) deficiencies. The first presenting symptoms were sinus/pulmonary infections (82%, mean age of 3 years) and alopecia (mean age of 4.7 years). ACTH deficiency was the third presenting condition (mean age at diagnosis of 8.6 years). All patients had hypogammaglobulinemia (decreased IgA and IgM levels), and 57% of patients had at least one autoimmune manifestation. Heterozygous mutations at the 3′end of the NFKB2 gene, coding for the C‐terminal domain of the protein, were identified in all cases. Better knowledge of DAVID syndrome will help clinicians make an early diagnosis to avoid life‐threatening complications.