IntroductionSTING-associated vasculopathy with onset in infancy (SAVI) is a rare, severe type I interferonopathy caused by gain-of-function mutations in STING1, leading to early-onset systemic inflammation, cutaneous vasculopathy, and life-threatening interstitial lung disease. Current treatments attenuate downstream inflammation without fully addressing the pathogenic driver and carry significant adverse effects. Recently, gene editing has emerged as a paradigm-shifting approach for IEI, with substantial potential for gain-of-function disorders that require allele-specific correction. We present a highly efficient and specific base- and prime-editing strategy that supports both ex vivo and in vivo therapeutic applications.ResultsK562 were transduced to generate stable STING1-WT and STING1-V155M lines (K562wt, K562mut). 8 sgRNAs were screened for base editing across PAM-flexible nucleases (SpCas9-NG, SpRY) in K562mut, and sgRNA2 and sgRNA5 were selected based on on-target editing. Given the high bystander activity observed with sgRNA5, we leveraged a previously developed library-derived in silico TadA design tool to identify >15 variants for testing. The TadA variant prioritized after experimental screening (var_17) reduced bystander editing (p<0.05). To overcome bystander constraints, PEGsm was selected for prime editing after comprehensive screening. Top-performing guides and selected editor variants were delivered by electroporation as in vitro–transcribed mRNA, achieving high on-target editing (90.10% ± 2.73%) for sgRNA5 and PEGsm (PE2 and PE3) and 53.48% ± 2.02% for sgRNA2. To functionally assess editing, interferon-stimulated gene transcripts (IFIT1, ISG15, and IFI44L) were quantified by droplet digital PCR (ddPCR) before and after 2′3′-cGAMP stimulation. ISG15 and IFIT1 were significantly reduced in unstimulated K562mut-edited cells (sgRNA2, PEGsm) versus K562mut, while after stimulation, K562wt and K562mut-edited reached the K562mut plateau as expected. In healthy donor hematopoietic stem and progenitor cells (HSPCs), a surrogate sgRNA was used to benchmark editing efficiency within the target window.Surrogate sgRNA achieved a mean bystander editing rate of 75.0% ± 4.4%. Finally, peripheral blood mononuclear cells (PBMCs) were reprogrammed into patient-derived induced pluripotent stem cells (iPSCs) using non-integrating Sendai vectors. iPSCs were prime edited to generate fully corrected isogenic single-cell–derived clones.ConclusionWe developed a comprehensive, bespoke base- and prime-editing platform for SAVI, enabling efficient and specific STING1 variant correction with functional normalization of interferon-stimulated genes (ISGs) expression, paving the way for future therapeutic applications. We also generated multiple fully corrected isogenic iPSC clones to support mechanistic studies of SAVI pathogenesis.Figure 1.Engineering of a STING1-V155M K562 model and functional assessment of bespoke base and prime editing. (A) Bidirectional third-generation lentiviral (LV) vector used to generate stable K562wt and K562mut lines expressing STING1-WT or STING1-V155M together with an EGFRt surface marker. (B) In vitro transcription (IVT) mRNA electroporation of top guide–editor pairs yields high A-to-G editing in K562mut, including base editing with sgRNA2/sgRNA5, a bystander-reducing TadA variant (var17) with sgRNA5, and prime editing with PEGsm in PE2 and PE3 configurations; editing was quantified by Sanger sequencing (EditR). (C) Functional readout by ddPCR showing IFIT1 and ISG15 expression (normalized to HPRT1) in K562mut across editing conditions and after STING inhibition (H-151). Statistics were computed by one-way ANOVA with multiple comparisons correction; significance is indicated, and where not otherwise specified, **** denotes p < 0.0001.

OBJECTIVES:H syndrome is a rare autosomal recessive disorder caused by mutations in SLC29A3, encoding the nucleoside transporter hENT3. Its heterogeneous clinical presentation often includes skin hyperpigmentation, systemic inflammation, endocrinopathies and sensorineural hearing loss. However, typical cutaneous signs may be absent, leading to diagnostic challenges. The objective was to describe a patient with H syndrome misdiagnosed until adulthood as having cryopyrin-associated periodic syndrome (CAPS), due to overlapping clinical and functional features. METHODS:We conducted clinical, immunological, genetic and functional assessments in a 24-year-old male with a complex history of early-onset urticarial rash, fever, hearing loss, oral ulcers, colitis and episodic inflammation. Genetic analyses included whole-exome sequencing (WES) and segregation study by quantitative PCR (qPCR). Functional assays evaluated IL-1β secretion, ASC speck formation, reactive oxygen species (ROS) production and type I interferon signature. RESULTS:The patient showed enhanced and accelerated IL-1β secretion and increased ASC speck formation in CD14+ cells after lipopolysaccharide (LPS) stimulation, indicating NLRP3 inflammasome hyperactivation, hallmark features of CAPS. ROS production was significantly elevated in both granulocytes and monocytes, even at baseline. A type I interferon signature was intermittently positive. Genetic testing ultimately revealed a homozygous deletion of exon 2 in the SLC29A3 gene, confirming H syndrome. CONCLUSION:This case highlights the phenotypic overlap between H syndrome and CAPS, including shared inflammasome dysregulation. Absence of typical skin hyperpigmentation delayed diagnosis despite early-onset systemic inflammation and partial response to IL-1 blockade. Functional assays may support diagnostic refinement in autoinflammatory syndromes with atypical features or inconclusive genetics.

BACKGROUND:Newborn screening (NBS) for inborn errors of immunity increasingly uses T-cell receptor excision circles (TREC) and, in some programs, Kappa-deleting recombination excision circles (KREC) to detect early T- and B-cell lymphopenia. While TREC-based screening is well established, the significance and management of isolated low KREC remain unclear. OBJECTIVE:To evaluate the implications of two different regional post-screening algorithms for isolated low KREC and to characterize the clinical course, immunological profile, and follow-up of term newborns with transient B-cell lymphopenia. METHODS:We performed a retrospective multicenter study of term newborns with isolated low KREC identified through NBS, confirmed B-cell lymphopenia, and subsequent normalization during follow-up. KREC levels, B-cell counts, and serum immunoglobulins were assessed longitudinally by RT-PCR and flow cytometry. RESULTS:Eighteen newborns were enrolled. At the first evaluation (V1; mean age 13.5 days), all had marked peripheral B-cell lymphopenia (CD19+ ≤ 2%; mean 54 cells/μL), although repeat dried blood spot (DBS) testing already showed KREC values above the diagnostic cutoff in 78%. By the second visit (V2; mean age 50 days), B-cell percentages and absolute counts normalized in all infants, with emerging IgA and IgM production, and normal KREC on whole blood. No infectious or immunological complications were recorded over 39.5 person-years of follow-up (mean 2.3 ± 1.7 years). CONCLUSION:Isolated low KREC at birth may identify newborns with transient B-cell lymphopenia that resolves during early infancy. Repeat KREC testing on a second DBS before referral may represent a pragmatic triage step to reduce unnecessary immunological evaluations, while preserving early assessment for newborns with persistent abnormalities. Prospective studies are needed to refine post-screening strategies.

Background: Deficiency of adenosine deaminase 2 (DADA2) is complex monogenic disease caused by recessive mutations in the ADA2 gene. DADA2 exhibits a broad clinical spectrum encompassing vasculitis, immunodeficiency, and hematologic abnormalities. Yet, the impact of DADA2 on the bone marrow (BM) microenvironment is largely unexplored. Objective: This study comprehensively examined the BM and peripheral blood of pediatric and adult patients with DADA2 presenting with rheumatologic/immunologic symptoms or severe hematologic manifestations. Methods: Immunophenotyping of hematopoietic stem cells (HSCs), progenitor cells, and mature cell populations was performed for 18 patients with DADA2. We also conducted a characterization of mesenchymal stromal cells. Results: Our study revealed a significant decrease in primitive HSCs and progenitor cells, alongside their reduced clonogenic capacity and multilineage differentiation potential. These BM defects were evident in patients with both severe and nonsevere hematologic manifestations, including pediatric patients, demonstrating that BM disruption can emerge silently and early on, even in patients who do not show obvious hematologic symptoms. Beyond stem cells, there was a reduction in mature cell populations in the BM and peripheral blood, affecting myeloid, erythroid, and lymphoid populations. Furthermore, BM mesenchymal stromal cells in patients with DADA2 exhibited reduced clonogenic and proliferation capabilities and were more prone to undergo cellular senescence marked by elevated DNA damage. Conclusions: Our exploration into the BM landscape of patients with DADA2 sheds light on the critical hematologic dimension of the disease and emphasizes the importance of vigilant monitoring, even in the case of subclinical presentation. (J Allergy Clin Immunol 2025;155:616-27.)

Adenosine deaminase 2 Deficiency (DADA2) is an autoinflammatory disease characterized by systemic vasculopathy, strokes and mild immunodeficiency. Recently NETosis has been implicated in the pathogenesis of Deficiency of Adenosine Deaminase 2. To deep investigate the possible effects of NETs on the immune system we characterized proteomic profile of NETs from DADA2 as compared to HD and Polyarteritis Nodosa (PAN) patients. To determine if NETs contain possibly immunogenic antigens we study functional aspects on Dendritic Cells after in vitro stimulation with NETs. Twenty-three DADA2 patients were enrolled. We analyzed NETosis by Imaging Flow Citometry. We evaluated NETs remnants and DNAse in the plasma samples by ELISA assay whereas DNAse activity by DNA digestion. We used quantitative proteomics approach and network analysis to identify NET proteins and pathways in 6 DADA2, 7 PAN and 7 HD. We analyzed circulating and monocyte-derived dendritic cells by flow cytometry. Neutrophils from DADA2 patients show a significant increased suicidal NETosis. DNAse enzymes were not normal in the level or activity. By proteomic analysis we identified 1356 proteins among which a hundred of proteins were significantly up or down-modulated in DADA2 NETs as compared to normal and disease control NETs in resting condition and after stimulation with PMA, Adenosine and TNFα. DADA2 NETs are significantly more efficient than normal NETs in stimulating patients’ monocyte-derived dendritic cells. We identified different pathways significantly modulated in DADA2 NETs versus PAN/HD NETs. This peculiar protein profile could contribute in activating inflammatory pathways in Dendritic cells in DADA2.

Suppressor of cytokine signaling 1 (SOCS1) haploinsufficiency is a recently described inborn error of immunity characterized by autoimmunity, inflammation, lymphoproliferation, and increased infection susceptibility. SOCS1, a negative regulator of cytokine signaling via the JAK/STAT pathway, explains the condition’s broad phenotypic variability. Single nucleotide polymorphisms in SOCS1 have been linked to multiple sclerosis (MS), and SOCS1 mimetics have shown efficacy in MS animal models. However, neurological involvement has not been previously reported in patients with SOCS1 insufficiency. We describe a family with a heterozygous SOCS1 variant, highlighting neurological manifestations such as MS, autoimmune encephalitis, and recurrent complex regional pain syndrome as novel features. Next-Generation Sequencing and segregation analysis were performed on PBMCs from patients and healthy donors. Functional studies included luciferase reporter assays in HeLa cells expressing the SOCS1 mutant, flow cytometry for phenotypic analysis, and gene expression profiling of the type-I interferon (IFN) signature. Intraepidermal nerve fiber density was evaluated via immunohistochemistry on skin biopsy. Genetic analysis confirmed the variant’s inheritance. Transfected cells carrying the SOCS1 variant showed increased STAT1 transcriptional activity after IFN-γ stimulation. Elevated STAT5 phosphorylation and T-cell proliferation were observed in response to IL-2. Peripheral blood revealed an elevated IFN signature during relapse. Skin biopsy showed reduced intraepidermal nerve fiber density. This report expands the clinical spectrum of SOCS1-related disorders to include neurological symptoms, emphasizing SOCS1’s critical role in regulating inflammation in the central and peripheral nervous systems.

Deficiency of adenosine deaminase 2 (DADA2) is a monogenic autoinflammatory disorder characterized by systemic inflammation, vasculopathy, immunodeficiency, and bone marrow failure. Current therapies—including anti-TNF agents and allogeneic hematopoietic stem cell transplantation (HSCT)—have limitations, especially for patients with hematologic involvement or no matched donor. We developed a lentiviral vector (LV.ADA2) to restore ADA2 expression in patient hematopoietic stem and progenitor cells (HSPCs) and evaluated its safety and efficacy in preclinical models. LV.ADA2 transduction of mobilized peripheral blood HSPCs from healthy donors resulted in stable ADA2 expression and secretion without impairing clonogenicity, multilineage differentiation, or long-term engraftment in immunodeficient mice. In HSPCs from 12 DADA2 patients, LV.ADA2 restored ADA2 protein and enzymatic activity preserving colony-forming ability and multilineage differentiation in vitro. In vivo, gene-corrected patient-derived HSPCs sustained long-term engraftment and multilineage reconstitution comparable to healthy controls. Integration site analysis confirmed a polyclonal pattern of hematopoietic reconstitution across all experimental settings, with no evidence of clonal dominance. Together, these findings demonstrate that ADA2 gene therapy with a LV is safe, maintains HSPC functionality, and enables durable hematopoietic reconstitution in a mouse model, providing a strong foundation for clinical translation and as a curative alternative to allogeneic HSCT in DADA2.

Introduction Deficiency of adenosine deaminase 2 (DADA2) is a monogenic autoinflammatory syndrome caused by biallelic mutations in the ADA2 gene characterized by early-onset inflammatory vasculopathy, strokes and immunodeficiency. Haematologic manifestations include lymphopenia, and bone marrow failure (BMF) mainly pure red cell aplasia. The diagnosis of DADA2 is confirmed by decreased enzymatic activity of ADA2 and genetic testing. TNFα inhibitors control inflammatory symptoms whereas hematopoietic stem cell transplant may be needed to treat refractory cytopenia. Objectives The aim of this study is to characterize the mechanisms of bone marrow damage in DADA2 patients. Methods Bone marrow(BM) samples from DADA2 patients and healthy donors (HD) were analyzed for different in-vitro assays to test in vitro potential therapeutic agents. In-vitro colony forming unit assay were performed from fresh bone marrow mononuclear cells (BMMNCs) in presence of anti-TNFα, human recombinant ADA2 or Eltrombopag. After 14 days CFU colonies were scored. Pro-inflammatory cytokines in the BM plasma were also measured by flow cytometry bead array. Results Fourteen patients (median age 17yo) were studied. Eight /14 (57%) showed reduced erythroid (CFU-E 0.5, normal range 27-81/2 × 104) and myeloid (CFU-GM 3.5, normal range 33-100/2 ×104) progenitor cell growth; The addition of anti-TNFα and eltrombopag 1 ug/ml, had a statistically significant stimulatory effect on the growth of myeloid progenitors (p=0.01 and p= 0.05, respectively). The addition of ADA2 (1 ug/ml and 10 ug/ml) had a stimulatory effect on myeloid progenitors, although not statistically significant. TNFα marrow plasma levels were higher in 4/9 patients (44 %) compared to 1/6 (17%) of healthy controls (p=0,002). No differences were noted in marrow plasma IFNγ levels. Conclusion Our study shows that the bone marrow of DADA2 patients is characterized by an inflammatory milieu and by a reduced growth of marrow progenitor cells, partially rescued in vitro by anti-TNFα and Eltrombopag. Further studies are needed to better understand the mechanisms of BM damage and to develop novel potential therapeutic approaches.

Deficiency of Adenosine deaminase 2 (DADA2) is a monogenic autoinflammatory disorder presenting with a broad spectrum of clinical manifestations, including immunodeficiency, vasculopathy and hematologic disease. Biallelic mutations in ADA2 gene have been associated with a decreased ADA2 activity, leading to reduction in deamination of adenosine and deoxyadenosine into inosine and deoxyinosine and subsequent accumulation of extracellular adenosine. In the early reports, the pivotal role of innate immunity in DADA2 pathogenic mechanism has been underlined, showing a skewed polarization from the M2 macrophage subtype to the proinflammatory M1 subtype, with an increased production of inflammatory cytokines such as TNF-α. Subsequently, a dysregulation of NETosis, triggered by the excess of extracellular Adenosine, has been implicated in the pathogenesis of DADA2. In the last few years, evidence is piling up that adaptive immunity is profoundly altered in DADA2 patients, encompassing both T and B branches, with a disrupted homeostasis in T-cell subsets and a B-cell skewing defect. Type I/type II IFN pathway upregulation has been proposed as a possible core signature in DADA2 T cells and monocytes but also an increased IFN-β secretion directly from endothelial cells has been described. So far, a unifying clear pathophysiological explanation for the coexistence of systemic inflammation, immunedysregulation and hematological defects is lacking. In this review, we will explore thoroughly the latest understanding regarding DADA2 pathophysiological process, with a particular focus on dysregulation of both innate and adaptive immunity and their interacting role in the development of the disease.

BACKGROUND:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may result in a severe pneumonia associated with elevation of blood inflammatory parameters, reminiscent of cytokine storm syndrome. Steroidal anti-inflammatory therapies have shown efficacy in reducing mortality in critically ill patients; however, the mechanisms by which SARS-CoV-2 triggers such an extensive inflammation remain unexplained. OBJECTIVES:To dissect the mechanisms underlying SARS-CoV-2-associated inflammation in patients with severe coronavirus disease 2019 (COVID-19), we studied the role of IL-1β, a pivotal cytokine driving inflammatory phenotypes, whose maturation and secretion are regulated by inflammasomes. METHODS:We analyzed nod-like receptor protein 3 pathway activation by means of confocal microscopy, plasma cytokine measurement, cytokine secretion following in vitro stimulation of blood circulating monocytes, and whole-blood RNA sequencing. The role of open reading frame 3a SARS-CoV-2 protein was assessed by confocal microscopy analysis following nucleofection of a monocytic cell line. RESULTS:We found that circulating monocytes from patients with COVID-19 display ASC (adaptor molecule apoptotic speck like protein-containing a CARD) specks that colocalize with nod-like receptor protein 3 inflammasome and spontaneously secrete IL-1β in vitro. This spontaneous activation reverts following patient's treatment with the IL-1 receptor antagonist anakinra. Transfection of a monocytic cell line with cDNA coding for the ORF3a SARS-CoV-2 protein resulted in ASC speck formation. CONCLUSIONS:These results provide further evidence that IL-1β targeting could represent an effective strategy in this disease and suggest a mechanistic explanation for the strong inflammatory manifestations associated with COVID-19.

Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive disease associated with a highly variable clinical presentation, including systemic vasculitis, immunodeficiency, and cytopenia. We report a case of a 16-year-old girl affected by recurrent viral infections [including cytomegalovirus (CMV)-related hepatitis and measles vaccine virus-associated manifestations] and persistent inflammation, which occurred after Parvovirus infection and complicated by secondary hemophagocytic lymphohistiocytosis (HLH). HLH’s first episode presented at 6 years of age and was preceded by persistent fever and arthralgia with evidence of Parvovirus B19 infection. The episode responded to intravenous steroids but relapsed during steroids tapering. High-dose intravenous immunoglobulin (IVIG) helped manage her clinical symptoms and systemic inflammation. The frequency of IVIG administration and the dosage were progressively reduced. At the age of 9, she experienced varicella zoster virus (VZV) reactivation followed by the recurrence of the inflammatory phenotype complicated by HLH with neurological involvement. Again, high-dose steroids and monthly IVIG resulted in a quick response. Targeted next-generation sequencing (NGS) for autoinflammatory diseases and immunodeficiencies revealed the homozygous Leu183Pro ADA2 mutation, which was confirmed by Sanger analysis. ADA2 enzymatic test showed a complete loss of ADA2 activity. For about 3 years, IVIG alone was completely effective in preventing flares of inflammation and neurological manifestations. Anti-TNF treatment was started at the age of 13 for the appearance of recurrent genital ulcers, with a complete response. This case further expands the clinical spectrum of DADA2 and emphasizes the importance of extensive genetic testing in clinical phenotypes characterized by persistent unspecific inflammatory syndromes. The use of high doses of IVIG might represent a possible effective immune modulator, especially in combination with anti-TNF treatment.

Neutrophil extracellular traps (NETs) are macromolecular structures programmed to trap circulating bacteria and viruses. The accumulation of NETs in the circulation correlates with the formation of anti-double-stranded (ds) DNA antibodies and is considered a causative factor for systemic lupus erythematosus (SLE). The digestion of DNA by DNase1 and DNases1L3 is the rate- limiting factor for NET accumulation. Mutations occurring in one of these two DNase genes determine anti-DNA formation and are associated with severe Lupus-like syndromes and lupus nephritis (LN). A second mechanism that may lead to DNase functional impairment is the presence of circulating DNase inhibitors in patients with low DNase activity, or the generation of anti-DNase antibodies. This phenomenon has been described in a relevant number of patients with SLE and may represent an important mechanism determining autoimmunity flares. On the basis of the reviewed studies, it is tempting to suppose that the blockade or selective depletion of anti-DNase autoantibodies could represent a potential novel therapeutic approach to prevent or halt SLE and LN. In general, strategies aimed at reducing NET formation might have a similar impact on the progression of SLE and LN.

BACKGROUND-AIMAdenosine Deaminase 2 Deficiency (DADA2) (OMIM: 607575) is a monogenic autoinflammatory disease caused by loss of function homozygous or heterozygous mutations in ADA 2 gene (previously CECR1, Cat Eye Syndrome Chromosome Region 1.A timely diagnosis is crucial to start Anti-TNF therapies that are efficacious in controlling the disease.The confirmation of DADA2 is based on DNA sequencing and enzymatic assay.It is thus very important to have robust and reliable assays that can be rapidly utilized in specialized laboratories that can centralize samples from other centers. METHODSIn this paper we show a novel enzymatic assay based on liquid chromatography-tandem mass spectrometry that allows the accurate determination of the ADA2 enzyme activity starting from very small amounts of plasma spotted on filter paper (dried plasma spot).ADA2 activity was determined in dried plasma spots (DPS) from 44 healthy donors, 18 DADA2 patients and 4 carriers. RESULTSADA2 activity, expressed as mean ± SD, was 2.63 ± 1.7 mU/mL in healthy controls, 0.02 ± 0.03 mU/mL in DADA2 patients and 0.025 ± 0.18 ± mU/mL in carriers. CONCLUSIONSThe method allows to significantly distinguish healthy controls from affected patients and carriers and could be of help in implementing the diagnostic workflow of DADA2.

Adenosine deaminase 2 deficiency (DADA2) is a rare inherited disorder that is caused by autosomal recessive mutations in the ADA2 gene. Clinical manifestations include early-onset lacunar strokes, vasculitis/vasculopathy, systemic inflammation, immunodeficiency, and hematologic defects. Anti-tumor necrosis factor therapy reduces strokes and systemic inflammation. Allogeneic hematopoietic stem/progenitor cell (HSPC) transplantation can ameliorate most disease manifestations, but patients are at risk for complications. Autologous HSPC gene therapy may be an alternative curative option for patients with DADA2. We designed a lentiviral vector encoding ADA2 (LV-ADA2) to genetically correct HSPCs. Lentiviral transduction allowed efficient delivery of the functional ADA2 enzyme into HSPCs from healthy donors. Supranormal ADA2 expression in human and mouse HSPCs did not affect their multipotency and engraftment potential in vivo. The LV-ADA2 induced stable ADA2 expression and corrected the enzymatic defect in HSPCs derived from DADA2 patients. Patients' HSPCs re-expressing ADA2 retained their potential to differentiate into erythroid and myeloid cells. Delivery of ADA2 enzymatic activity in patients' macrophages led to a complete rescue of the exaggerated inflammatory cytokine production. Our data indicate that HSPCs ectopically expressing ADA2 retain their multipotent differentiation ability, leading to functional correction of macrophage defects. Altogether, these findings support the implementation of HSPC gene therapy for DADA2.

Adenosine Deaminase 2 Deficiency (DADA2) (OMIM: 607575) is a monogenic, autoinflammatory disease caused by the loss of functional homozygous or heterozygous mutations in the ADA 2 gene (previously CECR1, Cat Eye Syndrome Chromosome Region 1). A timely diagnosis is crucial to start Anti-TNF therapies that are efficacious in controlling the disease. The confirmation of DADA2 is based on DNA sequencing and enzymatic assay. It is, thus, very important to have robust and reliable assays that can be rapidly utilized in specialized laboratories that can centralize samples from other centers. In this paper, we show a novel enzymatic assay based on liquid chromatography-tandem mass spectrometry that allows the accurate determination of the ADA2 enzyme activity starting from very small amounts of plasma spotted on filter paper (dried plasma spot). The method allows significantly distinguishing healthy controls from affected patients and carriers and could be of help in implementing the diagnostic workflow of DADA2.

PURPOSE:Italian expenditure for vitamin D greatly increased in the last few years, reaching €314 million ($376.8 million) in 2019. In Italy, the main cause of the increase in public spending for vitamin D is the marketing of high-cost medicines. At national and regional levels, some interventions have been performed to reduce expenditure, but spending has continued to increase. The aim of this work is to propose a new saving strategy determined by an analysis of a significant sample of the market.METHODS:Data on the use of vitamin D formulations, including data for the different active substances that represent its pharmaceutical analogue and composition of groups of equivalence, were extrapolated from the Italian Medicines Agency transparency lists and from the Farmadati database. Data on pharmaceutical expenditure were obtained from the Data Warehouse of Liguria Region; the composition of this expenditure was analyzed in detail, focusing on the characteristics of the pharmaceutical preparations and their cost (price per defined daily dose).FINDINGS:Vitamin D expenditure paralleled that of cholecalciferol, the most used active ingredient, which in Liguria increased from €643,352 ($772,022.4) in 2010 to €8,006,574 ($9,607,888.8) in 2019 (increase of 1144%). Spending focused on high-cost formulations, exceeding 90% of total cholecalciferol cost in 2019. We simulated a possible optimization of the expense for cholecalciferol by applying a revised price to all the cholecalciferol consumptions in high-cost products because these formulations do not have an added therapeutic value, finding that the saving would be at least 60%. National data on the detailed expenditure composition for vitamin D are not available, but we found a strong resemblance between total cholecalciferol expenditure time series in Italy and the Liguria Region.IMPLICATIONS:The expense of cholecalciferol and consequently the expense of vitamin D could be optimized by modifying the reimbursement of high-cost formulations. At a national level, savings should be proportional to that estimated for the Liguria Region. On the basis of the 2019 data, Italian savings with respect to total cholecalciferol expenditure should be €170.65 million ($204.78 million); per capita cholecalciferol expenditure would shift from €4.66 ($5.59) to €1.84 ($2.21).