Maurizio Miano

The X-linked G-protein coupled receptor GPR174 is highly expressed in T and B lymphocytes and has immunoregulatory roles in mice, but its function in humans is unknown. We describe a cohort of six individuals who have function-disrupting variants in GPR174 and a clinical phenotype of lymphadenopathy and autoimmunity. Histological analysis of two patient lymph nodes revealed necrotizing lymphadenitis and lymphoproliferation resembling Kikuchi-Fujimoto disease. In-depth analysis of three patients and related carriers revealed overaccumulation of CD8 terminally differentiated effector memory cells re-expressing CD45RA (T _EMRA ). Patient cells and GPR174- deficient CD8 T cells generated from controls showed less repression of proliferation by the GPR174 ligand lysophosphatidylserine (lysoPS) and an effector-biased gene expression program. GPR174-deficient CD4 T cells were resistant to lysoPS-mediated suppression of IL2 production. In mice, chronic viral infection led to over-accumulation of GPR174-deficient effector CD8 T cells. We describe an inborn error of immunity associated with dysregulated lymphocyte responses that we propose predisposes to exaggerated lymphoproliferation and autoimmunity following viral infection. Summary: Six individuals with function-disrupting variants in the G-protein coupled receptor GPR174 provide insight into its immunoregulatory role. GPR174 loss emerges as a potential genetic risk factor for histiocytic necrotizing lymphadenitis (Kikuchi-Fujimoto disease) and autoimmunity via defects in T cell regulation.

WHIM syndrome is a rare primary immunodeficiency disorder caused by gain-of-function mutations of the chemokine receptor CXCR4, leading to abnormal and exacerbated leukocyte trafficking. It is associated with severe neutropenia and lymphopenia, and recurrent infections. Few pediatric cases have been reported, but comprehensive analyses of the composition and function of peripheral lymphocyte populations in pediatric WHIM syndrome are lacking. Here we report the case of a 6-year-old male patient carrying a CXCR4 c.1000C>T gain-of-function mutation, presenting with characteristic manifestations such as abnormal orientation of the cerebellar folia, myelokathexis and severe neutropenia as well as lymphopenia. In-depth analysis of peripheral lymphocyte subpopulations revealed an elevated CD4/CD8 T-cell ratio while T-cell activation, expansion and cytokine production were normal. B lymphopenia was accompanied with a shift in circulating B-cell populations towards transitional stages, and increased apoptosis. Interestingly, despite normal serum immunoglobulin levels, vaccination responses and B-cell receptor dependent activation, the patient’s B cells showed a reduced capacity for plasmablast differentiation in vitro . These findings warrant follow-up studies in larger patient cohorts, as longitudinal monitoring of B-cell function might reveal emerging humoral defects in WHIM pediatric patients reaching adolescence and adulthood.

We applied supervised and unsupervised machine learning (ML) analyses to a cohort of 140 patients referred to the Hematology Unit of the G. Gaslini Institute from 1989 to 2023 for persistent cytopenia and/or features suggestive of telomere biology disorders (TBDs). Patients were labeled as “TBD” ( n = 20, established molecular diagnosis of TBD), “other diagnosis” (OD, n = 27, established molecular diagnosis of congenital disease including marrow failures), and “undefined diagnosis” (UD, n = 93, no established molecular diagnosis). After training a random forest model on 47 patients with established molecular diagnosis (20 TBD and 27 OD), supervised analysis was applied to the UD group and predicted 16/93 patients as having potential TBD and 77/93 subjects with potential OD, accounting for 17.2% and 82.7% of possibly reallocated diagnoses, respectively. The unsupervised approach applied to the whole cohort ( n = 140) identified 4 distinct clusters to be significantly associated (P = 0.000001) with 47 molecular diagnoses, with TBD patients prevailing in Clusters 1 and 2 and OD patients in Clusters 3 and 4. Telomere length (TL) and mucocutaneous abnormalities were the most relevant drivers in discriminating between the TBD and OD groups in supervised and unsupervised analyses; they prevailed in Clusters 1 and 2. Interestingly, both analyses yielded similar results in the UD group, where all 16/93 patients without molecular diagnosis predicted to have TBD in the supervised approach were placed in “TBD clusters” 1–2 of the unsupervised analysis. This model might correctly reallocate a remarkable proportion of undefined or previously misclassified cases, thus potentially leading to substantially improved diagnostic work‐up of rare and challenging diseases like TBD.

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.

ANKRD26-related thrombocytopenia (ANKRD26-RT) is characterized by lifelong mild to moderate thrombocytopenia. Patients suffer from an increased susceptibility to acute or chronic myeloid leukemia, myelodysplastic syndrome, or chronic lymphocytic leukemia. We described here a patient with inherited thrombocytopenia initially misdiagnosed as immune thrombocytopenic purpura. A chromosomal deletion involving the ANKRD26 gene was identified. Gene and protein expression analyses suggest an alternative pathogenic mechanism of altered megakaryopoiesis: the synthesis of a chimeric protein with aberrant expression due to the unregulated action of a promoter from a gene located upstream of ANKRD26. This study highlights the importance of advanced genetic testing and functional analysis of patients’ primary cells in the case of the detection of previously unrecognized structural variants in order to understand pathogenic mechanisms. These investigations provided a definitive diagnosis for the patient and facilitated the development of a tailored clinical management strategy, especially concerning the potential for myeloid transformation.

We analyzed 193 Fanconi anemia patients from the Italian Registry, focusing on hematological outcome, cancer risk, and mortality, both in transplanted (n = 130, 67.4% of the cohort) and non‐transplanted (n = 63, 36.6% of the cohort) patients. After a median follow‐up of 7 years, almost all patients developed cytopenia that was more frequent in patients receiving hematopoietic stem cell transplantation (HSCT). The cumulative overall survival from birth was 91.0% at age 10 years, 71.6% at age 20, and 47.4% at age 30 years; the median survival age was 29.1 years. When stratifying patients by indication for transplantation (moderate vs. severe cytopenia vs. persistent poor prognosis cytogenetic alterations/acute myeloid leukemia), we found a 5‐year cumulative mortality higher, though not significantly (p = 0.281) in the last group. Cancers were the second most common cause of death in the whole cohort after infections. Head and neck squamous cell carcinoma was the most frequent cancer, followed by hematologic neoplasms. The cumulative incidence of solid/hematological malignancy remarkably increased after 20 years of age and was 51.7% at age 40 years. The risk of malignancies was greater in subjects who received HSCT (sub‐distribution azard ratio 2.9, 95% CI: 1.1–7.5, p = 0.024). We also identified a small group of patients with stable or even improved cytopenia over time without transplant, thus confirming that bone marrow failure is not automatic in all patients and heightening the importance of tight monitoring to surveil on the worsening of hematopoietic function and cancer occurrence. Overall, this study provides important findings that may help to make robust clinical decisions.

Immune dysregulation in Inborn Errors of Immunity (IEI) shows a broad phenotype, including autoimmune disorders, benign lymphoproliferation, and malignancies, driven by an increasing number of implicated genes. Recent findings suggest that childhood cancer survivors (CCSs) may exhibit immunological abnormalities potentially linked to an underlying IEI, along with a well-known increased risk of subsequent malignancies due to prior cancer treatments. We describe a patient with two composite heterozygous pathogenic variants in the interleukin-2-inducible T-cell kinase (ITK) gene and a history of multiple tumors, including recurrent Epstein–Barr virus (EBV)-related nodular sclerosis and Hodgkin’s lymphoma (NSHL), associated with unresponsive multiple hand warts, immune thrombocytopenia, and an impaired immunological profile (CD4+ lymphocytopenia, memory B-cell deficiency, reduction in regulatory T-cells, and B-cell- and T-cell-activated profiles). In our case, ITK-related immune dysregulation and prior exposure to oncological treatments seem to have simultaneously intervened in the same individual, leading to the development of a unique clinical profile. It is essential to raise awareness of the two-way association between immune dysregulation disorders and multiple tumors.

Introduction: Autoimmune cytopenias (AICs) are a group of disorders characterized by immune-mediated destruction of blood cells. In children, they are often secondary to immune dysregulation that may require long-lasting immunosuppression. Mycophenolate mofetil and sirolimus represent two well-tolerated options to treat these disorders, often as a steroid-sparing option. However, no data are available on the infection risk for patients undergoing long-lasting treatments.Patients and methods: The rate of severe infective events was calculated in episodes per 100 persons/months at risk (p/m/r) documented by the analysis of hospitalization charts between January 2015 and July 2023 of patients treated with mycophenolate mofetil or sirolimus given for isolated AIC or AICs associated with autoimmune lymphoproliferative syndrome (ALPS)/ALPS-like syndromes in two large Italian pediatric hematology units.Results: From January 2015 to July 2023, 13 out of 96 patients treated with mycophenolate mofetil or sirolimus developed 16 severe infectious events requiring hospitalization. No patients died. Overall infection rate was 0.24 person/*100 months/risk (95% CI 0.09–0.3). Serious infectious events incidence was higher in patients with ALPS-like compared to others (0.42 versus 0.09; p = 0.006) and lower in patients who underwent mycophenolate treatment alone compared to those who started sirolimus after mycophenolate failure (0.04 versus 0.29, p = 0.03). Considering only patients who started treatment at the beginning of study period, overall cumulative hazard was 18.6% at 60 months (95% CI 3.4–31.4) with higher risk of infectious events after 5 years in ALPS-like patients (26.1%; 95% CI 3.2–43.5) compared to other AICs (4%; 95% CI 0–11.4; p = 0.041).Discussion: To the best of our knowledge, this is the first study to describe the infectious risk related to mycophenolate and sirolimus chronic treatment in patients with AICs and immune dysregulation. Our data highlight that infection rate is very low and mainly related to the underlying hematological condition.Conclusions: Mycophenolate and sirolimus represent a safe immunosuppressive therapy in AICs and immune dysregulation syndromes.

Mutations in the small Rho-family guanosine triphosphate hydrolase RAC2, critical for actin cytoskeleton remodeling and intracellular signal transduction, are associated with neonatal severe combined immunodeficiency (SCID), infantile neutrophilic disorder resembling leukocyte adhesion deficiency (LAD), and later-onset combined immune deficiency (CID). We investigated 54 patients (23 previously reported) from 37 families yielding 15 novel RAC2 missense mutations, including one present only in homozygosity. Data were collected from referring physicians and literature reports with updated clinical information. Patients were grouped by presentation: neonatal SCID (n = 5), infantile LAD-like disease (n = 5), or CID (n = 44). Disease correlated to RAC2 activity: constitutively active RAS-like mutations caused neonatal SCID, dominant-negative mutations caused LAD-like disease, whereas dominant-activating mutations caused CID. Significant T- and B-lymphopenia with low immunoglobulins were seen in most patients; myeloid abnormalities included neutropenia, altered oxidative burst, impaired neutrophil migration, and visible neutrophil macropinosomes. Among 42 patients with CID with clinical data, upper and lower respiratory infections and viral infections were common. Twenty-three distinct RAC2 mutations, including 15 novel variants, were identified. Using heterologous expression systems, we assessed downstream effector functions including superoxide production, p21-activated kinase 1 binding, AKT activation, and protein stability. Confocal microscopy showed altered actin assembly evidenced by membrane ruffling and macropinosomes. Altered protein localization and aggregation were observed. All tested RAC2 mutant proteins exhibited aberrant function; no single assay was sufficient to determine functional consequence. Most mutants produced elevated superoxide; mutations unable to support superoxide formation were associated with bacterial infections. RAC2 mutations cause a spectrum of immune dysfunction, ranging from early onset SCID to later-onset combined immunodeficiencies depending on RAC2 activity. This trial was registered at www.clinicaltrials.gov as #NCT00001355 and #NCT00001467.