Universidad del Desarrollo

Background FNIP1 (Folliculin Interacting Protein 1) deficiency is a rare inborn error of immunity characterized by heart defects and B-cell deficiency. Neutropenia is frequently reported in FNIP1-deficient patients, but its underlying mechanism has not been elucidated, with no evidence of neutropenia in the Fnip1 mouse model. Aims To test the hypothesis that FNIP1 deficiency could be directly associated with neutropenia we collected updated clinical and laboratory data of FNIP1-deficient patients. To unveil the role of FNIP1 in neutrophil development and function, we employed CRISPR/Cas9 genome editing to create a FNIP1-deficient model in HL-60 cells. Methods Ten (8 published and 2 unpublished) patients were enrolled. CRISPR/Cas9 was used to knock out (KO) FNIP1 in HL-60 cells. Whole RNA-sequencing, expression of pAKT, pS6, p4EBP1, mitochondrial abundance and membrane potential (MMP), oxygen consumption rate (OCR), ATP production and extracellular acidification rate (ECAR) of FNIP1-deficient and wild-type HL-60 cells were performed to assess effects of FNIP1 deficiency. Results All the patients (male:female = 5:5, age range &lt;1-40 years, median 5.5 years) showed hypogammaglobulinemia/agammaglobulinemia with B-cell deficiency, heart defects, severe/recurrent infections and were receiving immunoglobulin replacement treatment. Crohn disease and seronegative arthritis were diagnosed in 1 individual each. Eight out of 10 patients showed decreased absolute neutrophil count (ANC; four chronic and four intermittent neutropenia). All the patients with chronic neutropenia had ANC &lt;500/µl (severe neutropenia). Neutrophil oxidative burst was normal in 2 patients while chemotaxis resulted defective in 1 case. Bone marrow (BM) analysis showed left-shifted granulopoiesis with no promyelocyte/myelocyte arrest in 3/3 cases. Monocytosis and monocytopenia were present in 4 and 1 out of 10 patients, respectively. G-CSF was started in 3 patients, who responded to &lt;5 mcg/kg/day. To date, no individual has developed a clonal hematopoietic disorder. Six patients were alive at the last follow-up. Whole RNA-seq in FNIP1 KO HL-60 cells showed aberrant transcriptional control of differentiation from HSC to myeloid cells (increased C/EBPα and decreased MEIS1 and HOXA9). Enrichment in gene sets related to cell differentiation (Notch Transcriptional and post-Translation regulation), motility/cytoskeletal remodeling (Rap1, Rac1, Rho, RhoA and Cdc42 pathways), mitochondrial (oxidative stress induced senescence) and neutrophil (degranulation, chemotaxis and extracellular trap formation) function was observed in FNIP1 KO HL-60 cells. FNIP1 KO HL-60 cells did not show differences in total mitochondria but MMP was significantly reduced (p&lt;0.0001). Although transcripts involved in mitochondrial electron transport chain (COX16, COX19, COX6C, COX7A2 and COX7B) and OXPHOS (ATP5F1) were dysregulated, FNIP1 KO HL-60 cells showed no differences in ATP production and OCR, a measure of OXPHOS. Decrease of PPARγ and increase of key glycolytic enzymes (HK2 and LDHA) suggested altered glucose metabolism. ECAR, a measure of glycolysis, was increased in FNIP1 KO HL-60 cells (p&lt;0.0001). We examined the PI3K/AKT pathway and did not observe differences in pAKT473, p4EBP1 and pS6 between WT and FNIP1 KO HL-60 cells. Conclusions Neutropenia is a frequent finding and part of the phenotypic spectrum of FNIP1 deficiency. FNIP1-deficient patients do not lack mature neutrophils in the BM and respond to G-CSF treatment. FNIP1 deficiency profoundly impacts the mechanisms underlying critical neutrophil activities, ultimately impairing neutrophil differentiation and function. Similarly to FNIP1-deficienct B cells, FNIP1 acts in neutrophil's progenitors regulating glycolysis. Our work shows that FNIP1 deficiency alters previously unrecognized pathways (cytoskeletal dynamics/cell migration). We have previously described increased MMP and phosphorylation of pAKT473 and p4EBP in FNIP1-deficient B cells. As neutropenia is not associated with promyelocyte/myelocyte arrest, FNIP1 may have a prominent role in later stages of granulocyte's differentiation, thus explaining the difference between B cells and HL-60 observed in MMP and PI3K pathway. Future work will focus on targeted interventions to restore neutrophil function in FNIP1 deficiency.

Abstract Agammaglobulinemia is the most profound primary antibody deficiency that can occur due to an early termination of B-cell development. We here investigated 3 novel patients, including the first known adult, from unrelated families with agammaglobulinemia, recurrent infections, and hypertrophic cardiomyopathy (HCM). Two of them also presented with intermittent or severe chronic neutropenia. We identified homozygous or compound-heterozygous variants in the gene for folliculin interacting protein 1 (FNIP1), leading to loss of the FNIP1 protein. B-cell metabolism, including mitochondrial numbers and activity and phosphatidylinositol 3-kinase/AKT pathway, was impaired. These defects recapitulated the Fnip1−/− animal model. Moreover, we identified either uniparental disomy or copy-number variants (CNVs) in 2 patients, expanding the variant spectrum of this novel inborn error of immunity. The results indicate that FNIP1 deficiency can be caused by complex genetic mechanisms and support the clinical utility of exome sequencing and CNV analysis in patients with broad phenotypes, including agammaglobulinemia and HCM. FNIP1 deficiency is a novel inborn error of immunity characterized by early and severe B-cell development defect, agammaglobulinemia, variable neutropenia, and HCM. Our findings elucidate a functional and relevant role of FNIP1 in B-cell development and metabolism and potentially neutrophil activity.

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