FOXO1 Single-Nucleotide Polymorphisms Are Associated with Bleeding Severity and Sensitivity of Glucocorticoid Treatment of Pediatric Immune Thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune-mediated hemorrhagic disease. Emerging evidence indicates that FOXO1 SNPs are related to the immune dysregulation of several autoimmune diseases suggesting that FOXO1 may be involved in inflammation and pathologic activities in patients with ITP. This study aimed to evaluate whether FOXO1 gene single-nucleotide polymorphisms (SNPs) are associated with susceptibility to ITP and clinical priorities of concern include bleeding severity and sensitivity of glucocorticoid treatment. This study recruited 327 newly diagnosed ITP and 220 healthy controls. Four SNPs (rs17446593, rs17446614, rs2721068, and rs2721068) of the FOXO1 gene were detected using the Sequenom MassArray system. Bleeding severity were classified into the mild and severe groups based on the bleeding scores. ITP patients were classified as sensitive and insensitive to glucocorticoid treatment according to the practice guideline for ITP (2019 version). The frequencies of the four SNPs did not show any significant differences between the ITP and healthy control groups. Patients with AA genotype at rs17446593 (p = 0.009) and GG genotype at rs17446614 (p = 0.009) suffered more severe bleeding than patients without them. Carriers of haplotype Grs17446593Ars17446614Crs2721068Trs2755213 were protective to severe bleeding (p = 0.002). The AA genotype at rs17446593 was significantly higher in ITP patients sensitive to glucocorticoid treatment than in those insensitive to glucocorticoid treatment (p = 0.03). Haplotype Grs17446593Grs17446614Trs2721068Trs2755213 increases the risk of glucocorticoid resistance (p = 0.007). Although FOXO1 gene polymorphisms were not associated with susceptibility to ITP, the AA genotype at rs17446593 and GG genotype at rs17446614 were associated with bleeding severity. Haplotype GACT have a protective effect against severe bleeding. Patients with AA genotype at rs17446593 may tend to have good responds to glucocorticoid treatment. However, the FOXO1 gene haplotype GGTT increases the risk of glucocorticoid-resistant. Trial registration: ChiCTR1900022419.

HIF-1α induced by hypoxic condition regulates Treg/Th17 axis polarization in chronic immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an acquired immune disorder characterized by increased platelet destruction and reduced platelet (Plt) production. Hypoxia-inducible factor-1α (HIF-1α) have regulatory effects on Treg/Th17 axis balance and may represent relevant factors in the pathogenesis of ITP. Treg/Th17 ratio, serum levels and gene expression were investigated in new diagnosed ITP (NITP) and chronic ITP (CITP). The Treg/Th17 ratio obviously decreased in CITP (P = 0.001). The ratio of Treg/Th17 was correlated with the level of HIF-1α level both in mRNA (r = 0.49, P < 0.0001) and serum level (r = 0.50, P < 0.0001). However, none statistical upregulation of HIF-1α was observed in CITP. In vitro, There was significant polarization difference of Treg/Th17 axis (P = 0.042) and Foxp3-MFI/IL17-MFI (P = 0.0003) in hypoxic condition between NITP and CITP. These findings suggest that HIF-1α induced by hypoxia plays a crucial role in the chronicity of ITP by mediating the imbalance of the Treg/Th17 axis.

Machine learning models developed and internally validated for predicting chronicity in pediatric immune thrombocytopenia.

BACKGROUND: Primary immune thrombocytopenia (ITP) in children is typically self-limiting; however, 20% to 30% of patients may experience prolonged thrombocytopenia lasting over a year. The challenge is predicting chronicity to ensure personalized treatment approaches. OBJECTIVES: To address this issue, we developed and internally validated 4 machine learning (ML) models using demographic and immunologic characteristics to predict the likelihood of chronicity. METHODS: The present study was conducted at Beijing Children's Hospital from June 2018 to December 2021, aiming to develop predictive models for determining the chronicity of pediatric ITP. Four ML models, based on a logistic regression classifier, random forest classifier, eXtreme Gradient Boosting (XGBoost), and support vector machine, were employed. These models used a set of 16 variables, including 14 immunologic and 2 demographic predictors. The performance evaluation criteria included prediction accuracy, precision, recall, F1 score, and area under the receiver operating characteristic curve (AUROC). RESULTS: Data were collected from 662 patients who were randomly assigned to either a training dataset or a testing dataset using a random number generator. Among them, 26.5% had chronic disease. All models performed well, with AUROC values ranging from 0.81 to 0.84, and XGBoost was selected for its highest AUROC score and interpretability in constructing the predictive model. Age, T helper 17, T helper 17-to-regulatory T cell ratio, T helper 1, and double-negative T cells were identified as significant predictors by the XGBoost algorithm. CONCLUSION: We developed a precise predictive model for chronicity in pediatric ITP using ML during the initial phase. The XGBoost model achieved high predictive accuracy by using individual patient clinical parameters and demonstrated commendable interpretability.

Elevated TCR-αß+ double-negative T cells in pediatric patients with acquired aplastic anemia.

BACKGROUND AND AIMS: The pathophysiology of acquired aplastic anemia (aAA) is most associated with T cell mediated immune dysfunction, but the role of CD4- CD8- double negative T cells (DNTs) in pediatric patients with aAA is unclear. In this study, we aimed to investigate the proportion of TCR-αß+ DNTs in pediatric patients with aAA and correlation with the response to immunosuppressive therapy (IST). MATERIALS AND METHODS: Assessment of DNTs from peripheral blood was done by sensitive multi-color flow cytometry. The potential clinical value of TCR-αß+ DNTs was then assessed by the receiver operating characteristic (ROC) curves. RESULTS: The retrospective study evaluated 164 pediatric patients with aAA and 105 healthy donors (HD). Our data showed higher proportion of TCR-αß+ DNTs in total lymphocytes [1.04% (0.79%-1.40%) vs 0.69% (0.47%-0.87%), p < 0.001] and CD3+ T cells [1.52% (1.10%-1.96%) vs 1.10% (0.70%-1.40%), p < 0.001] in aAA compared to HD. Patients with SAA/VSAA achieving complete response (CR) after IST had a higher proportion of TCR-αß+ DNTs at initial diagnosis, than those not achieving CR for total (1.21%±0.39 vs 0.78%±0.38, p < 0.05) and CD3+ T cells (1.74%±0.53 vs 1.15%±0.59, p < 0.05). The ROC analysis showed areas under the curves (AUCs) for TCR-αß+ DNT proportion in lymphocytes and CD3+ T cells were 0.756 (cutoff value 1.33, p < 0.05) and 0.758 (cutoff value 1.38, p < 0.05), respectively. And the complete response rate was higher in TCR-αß+ DNT proportion high group than in TCR-αß+ DNT proportion low group at baseline (p < 0.001). CONCLUSION: Our observations suggest that elevated TCR-αß+ DNTs seems to play a role in the pathogenesis of aAA, and it was involve in immune response to IST.

Anti-glycoprotein autoantibodies are related to bleeding severity in children with newly diagnosed ITP and very low platelet counts.

BACKGROUND AND OBJECTIVE: Immune thrombocytopenia (ITP) is an autoimmune-mediated hemorrhagic disease. Anti-glycoprotein autoantibodies play a key role in the pathophysiology of ITP, but the relationship between platelet-specific antibodies and bleeding severity is unclear. This study aimed to analyze the relationship between anti-glycoprotein autoantibodies and bleeding severity in children with newly diagnosed ITP and platelet count less than 10 × 109 /L. METHOD: This was a single-center prospective observational study that analyzed children with newly diagnosed ITP and platelet count less than 10 × 109 /L between June 2018 and September 2021 at our hospital. The children were classified into the mild and severe groups based on the bleeding scores. The type and titer of anti-glycoprotein autoantibodies were detected using an enzyme-linked immunosorbent assay (ELISA) kit (PAKAUTO). We analyzed the relationship between bleeding severity and anti-glycoprotein autoantibodies. RESULTS: A total of 86 cases were enrolled, including 42 in the mild group and 44 in the severe group. Patients with anti-GPIIb/IIIa or anti-GPIb/IX antibodies suffered more severe bleeding than patients without them (χ2 = 7.303, p = .007; χ2 = 3.875, p = .049), but there was no significant difference between patients with or without anti-GPIa/IIa antibodies (χ2 = 0.745, p = .388). When antibodies were analyzed together, patients with three antibodies suffered more severe bleeding than those without three antibodies (χ2 = 5.053, p = .025). Patients with higher antibody titer in the eluent, but not in the plasma, suffered more severe bleeding in all three antibodies (Z = -2.389, p = .017; Z = -2.108, p = .035; Z = -2.557, p = .011). CONCLUSION: Anti-glycoprotein autoantibodies led to more severe bleeding in children under 18 years of age without drug treatment with newly diagnosed ITP and platelet count less than 10 × 109 /L.

Single Nucleotide Polymorphisms of the HIF1A Gene are Associated With Sensitivity of Glucocorticoid Treatment in Pediatric ITP Patients.

BACKGROUND: Hypoxia-inducible factor-1α (HIF-1α) plays a crucial role in both innate and adaptive immunity. Emerging evidence indicates that HIF-1α is associated with the inflammation and pathologic activities of autoimmune diseases, suggesting that HIF1α may be involved in immune dysregulation in patients with immune thrombocytopenia (ITP). The purpose of this study was to evaluate whether single nucleotide polymorphisms (SNPs) of the HIF1A gene are associated with susceptibility to ITP and its clinical prognosis including incidence of chronic ITP and glucocorticoid sensitivity. MATERIALS AND METHODS: This study involved 197 Chinese ITP pediatric patients (discovery cohort) and 220 healthy controls. The Sequenom MassArray system (Sequenom, San Diego, CA) was used to detect 3 SNPs genotypes in the HIF1A gene: rs11549465, rs1957757, and rs2057482. We also used another ITP cohort (N=127) to validate the significant results of SNPs found in the discovery cohort. RESULTS: The frequencies of the three SNPs did not show any significant differences between the ITP and healthy control groups. The CT genotype at rs11549465 was significantly higher in ITP patients sensitive to glucocorticoid treatment than in those insensitive to glucocorticoid treatment ( P =0.025). These results were validated using another ITP cohort (N=127, P =0.033). Moreover, the CC genotype was a risk factor for insensitive to GT the odds ratio (95% confidence interval) was 5.96 (5.23-6.69) in standard prednisone ( P =0.0069) and 6.35 (5.33-7.37) in high-dose dexamethasone ( P =0.04). CONCLUSIONS: Although HIF1A gene polymorphisms were not associated with susceptibility to ITP, the CT genotype at rs11549465 was associated with the sensitivity to glucocorticoid treatment of ITP patients, suggesting that the rs11549465 SNP may contribute to the sensitivity of glucocorticoid treatment in pediatric ITP patients.

Low-dose immune tolerance induction in children with severe hemophilia A with high-titer inhibitors: Type of factor 8 mutation and outcomes.

Background: No studies evaluated the role of F8 mutations in outcomes for low-dose immune tolerance induction (ITI) in people with severe hemophilia A (SHA) with high-titer inhibitors. Objectives: To explore the association between F8 mutation types and low-dose ITI outcomes in children with SHA with high-titer inhibitors. Methods: Children SHA with high-titer inhibitors who received low-dose ITI therapy at least for 1 year were included in this study. Based on the risk of inhibitor development, F8 mutations were classified into a high-risk group and a non-high-risk group. Rapid tolerance and the final ITI outcomes were assessed at the 12th and 24th month of treatment, respectively, and the predictor of outcomes was analyzed. Results: Of 104 children included, 101 had F8 mutations identified. The children with non-high-risk mutations presented a higher rate of rapid tolerance than those with high-risk mutations (61.0% vs. 29.2%; p = 0.006). Among 72 children beyond 24 months of ITI, 55 children (76.4%) achieved success, 3 (4.2%) achieved partial success, and 14 (19.4%) failed. The children in the non-high-risk group showed a higher success rate (86.8% vs. 43.8%; p = 0.001) and a shorter time to success (mean time, 9.3 months vs. 13.2 months; p = 0.04) compared to those in the high-risk group. In multivariable logistic regression, F8 mutations were an independent predictor of ITI success (non-high-risk group vs. high-risk group, adjusted odds ratio [OR], 20.3; 95% confidence interval [CI], 3.5-117.8), as was the interval from inhibitor diagnosis to ITI start (adjusted OR, 0.95; 95% CI, 0.90-0.99). They remained the significant predictors when success time was taken into account in a Cox model. Conclusions: Types of F8 mutation were a key predictor of outcomes for low-dose ITI in children with SHA with high-titer inhibitors. It can help to stratify the prognosis and guide clinical decisions.

F8 gene mutation spectrum in severe hemophilia A with inhibitors: A large cohort data analysis from a single center in China.

Introduction: Type of F8 gene mutation is the most important risk factor for inhibitor development in people with severe hemophilia A. However, there are few large cohort studies on the F8 mutation spectrum of people with severe hemophilia A with inhibitors. Objective: This was the first large cohort study in children with severe hemophilia A with inhibitors from China that aimed to analyze the association between F8 variant types and inhibitor status. Methods: The single-center retrospective cohort study was conducted on children with severe hemophilia A with inhibitors admitted from January 2015 to December 2021. The clinical data were collected, and F8 genetic tests were performed. Results: Among the 203 patients investigated, a mutation in F8 was identified in 196 cases. Most patients had deleterious mutations (153; 75.4%), including 82 cases of intron 22 inversions (40.4%); 40 cases of nonsense mutations (19.7%), with 15 cases in the light chain and 25 cases in the heavy chain; and 31 cases of large deletions or insertions (15.3%), with 29 cases involving more than one exon and 2 cases involving one exon. The large deletions or insertions encompassing multiple exons and nonsense mutations residing in the light chain were associated with not only the progression to a high-titer inhibitor (P < .05) but also higher peak inhibitor titer (P < .05). Conclusion: The F8 gene deleterious mutations, including intron 22 inversions, nonsense mutations, and large deletions or insertions, constitute the main mutation types in people with severe hemophilia A with inhibitors in China, with the latter mutation types (large deletions or insertions in multiple exons, and nonsense mutations in the light chain) signifying for a higher peak titer of inhibitor.

Case report: Effectiveness of sirolimus in treating partial DiGeorge Syndrome with Autoimmune Lymphoproliferative Syndrome (ALPS)-like features.

Background: DiGeorge Syndrome (DGS) is a rare disease associated with 22q11.2 chromosomal microdeletion, also known as a velocardiofacial syndrome, based on the frequent involvements of the palate, facial, and heart problems. Hematologic autoimmunity is rare in DGS but presents with a refractory course and poor prognosis. Herein, we report a case of partial DGS in a patient with refractory immune cytopenia and autoimmune lymphoproliferative syndrome (ALPS)-like manifestations. Case description: A 10-year-old boy with growth retardation presented initially with a ventricular septal defect at 7 months old, which had been repaired soon after. The patient suffered from thrombocytopenia and progressed into chronic refractory immune thrombocytopenia (ITP) at 30 months old. One year later, the patient developed multilineage cytopenias including thrombocytopenia, neutropenia, and anemia. First-line treatment of ITP, like high-dose dexamethasone and intravenous immunoglobulin, had little or short-term effect on controlling symptoms. Whole-exome sequencing revealed the presence of a de novo heterozygous 2.520 Mb deletion on chromosome 22q11.21. Moreover, decreased proportion of naive T cells and elevated double-negative T cells were found. The patient was given sirolimus therapy (1.5 mg/m2, actual blood concentration range: 4.0-5.2 ng/ml) without adding other immunosuppressive agents. The whole blood cell count was gradually restored after a month, and the disease severity was soothed with less frequency of infections and bleeding events. Decreased spleen size and restrained lymph node expansion were achieved after 3-month sirolimus monotherapy. Conclusions: This case is the first description on the efficacy of sirolimus monotherapy to treat refractory multilineage cytopenias of DGS presented with ALPS-like features.