Genetic testing guides therapy in children with refractory cytopenias

Immune cytopenias of childhood encompass a heterogeneous group of disorders characterized by immune-mediated destruction of red blood cells (autoimmune hemolytic anemia [AIHA]), platelets (immune thrombocytopenia [IPT]), neutrophils (autoimmune neutropenia [AIN]) or multiple lineages (Evans syndrome). Most primary or “idiopathic” cases present acutely but respond to first- or second-line therapy (steroids, intravenous immunoglobulin replacement therapy, ± rituximab) without long-term sequelae.¹ In contrast, cytopenias that fail to respond to repeated courses of multi-agent immunosuppressive therapy constitute a severe and often debilitating condition with substantial long-term morbidity.² Such non-responsiveness should prompt evaluation for an underlying comorbid disorder, malignancy, infection, lymphoproliferation, rheumatoid disease, or an inborn error of immunity (IEI). Therapy-refractory cytopenias in children frequently conceal unrecognized IEI or other hematopoietic defects that are amenable to targeted treatment. This study was approved by Stanford Institutional Review Board in accordance with the Declaration of Helsinki, and written informed consent was obtained.

We analyzed 24 pediatric and young adult patients with presumed immune cytopenias referred to our multidisciplinary clinic after failing multiple lines of immunosuppressive therapy (Figure 1). Comprehensive immune evaluation and genetic testing were performed in all cases. A contributory diagnosis was identified in 19 of 24 patients, directly guiding therapeutic decisions. Four patients initially thought to have autoimmune cytopenias were subsequently diagnosed with bone marrow failure syndromes: two carried pathogenic variants in DNAJC21 or SBDS consistent with Shwachman-Diamond–like syndrome,⁴ and one harbored a FANCA mutation consistent with Fanconi anemia. All four underwent hematopoietic stem cell transplantation (HSCT) and were excluded from subsequent analyses.

Among the remaining 20 patients, 14 had multilineage cytopenias, two had isolated AIN (both with monoallelic pathogenic NFKB1 variants), one had isolated AIHA (CTLA4 haploinsufficiency), and three had isolated ITP (1 with common variable immunodeficiency [CVID], 1 with systemic lupus erythematosus [SLE], and 1 without an identifiable cause). Clinical and genetic features of these 20 patients are summarized in Table 1.

An underlying genetic variant was identified in 75% (15/20) of patients, conferring a likely predisposition to autoimmunity or autoinflammation in 73% (11/15). Isolated cytopenias were observed in 25% (5/20) of genetically predisposed patients. Among the six patients without any identifiable genetic abnormality, two presented with isolated thrombocytopenia and four with combined anemia and thrombocytopenia (Tables 1 and 2). Genetic diagnoses clustered into four mechanistic categories: (i) disorders driven by excessive type I interferon signaling, (ii) inflammasome activation syndromes, (iii) diseases characterized by imbalance between effector and regulatory T-cell (Treg) activity, and (iv) other rare monogenic disorders. This framework provided a biologic rationale for targeted therapeutic approaches (Figure 1).

(i) Interferonopathy: three patients carried pathogenic variants in ACP5, confirming the diagnosis of spondyloenchondrodysplasia with immune dysregulation (SPENCDI), a type I interferonopathy. Ruxolitinib and baricitinib, which inhibit JAK1 and JAK2 downstream of the type I interferon receptor, were used successfully in two patients. The third patient experienced an acute SLE flare with pericarditis and responded to treatment with anifrolumab-fnia, a type I interferon receptor inhibitor approved for SLE.⁵

(ii) Inflammasomopathy: an infant presented with life-threatening AIHA, severe jaundice, recurrent fevers, urticarial-like rash, severe pain, and markedly elevated IL-18 levels. Genetic testing identified a C-terminal CDC42 variant associated with aberrant inflammasome activation. Treatment with the interleukin-1 receptor antagonist anakinra led to complete and durable resolution of symptoms.⁶ Notably, the cytopenia in this patient was secondary to hypersplenism rather than immune-mediated destruction. (ii) Imbalance between effector and Treg activation: seven patients had IEI associated with dysregulated effector and Treg balance. Three carried a 22q11.2 deletion, a defect in thymic development and function known to predispose to autoimmunity.⁷ All were treated with rapamycin, which enhances Treg expansion and promotes immune tolerance.^8-10 Three sisters with variable immune cytopenias were found to have autosomal dominant NFKB1 loss-of-function variants, a common monogenic cause of CVID.¹¹ Two of the three responded to rapamycin, achieving normalization of neutrophil and platelet counts. The seventh patient harbored a FOXP3 variant consistent with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome and presented with aplastic anemia and nephrotic syndrome. After initial therapy with steroids and tacrolimus, he was successfully transitioned to rapamycin following molecular diagnosis; both HSCT and FOXP3 gene therapy were subsequently offered.

(iv) Conditions with specific or directly targeted therapy: a 28-year-old man with short stature, severe neutropenia, oral ulcers, and recurrent bacterial infections, previously treated with steroids and granulocyte colony-stimulating factor (G-CSF) for presumed autoimmune neutropenia and thrombocytopenia, was found to harbor a pathogenic variant in G6PC3, confirming G6PC3 deficiency, a congenital neutropenia unresponsive to immunosuppression.¹² G6PC3 deficiency causes neutrophil dysfunction due to accumulation of 1,5-anhydroglucitol-6-phosphate (1,5-AG6P). Reducing 1,5-AG6P via inhibition of the sodium-glucose co-transporter 2 (SGLT2) restores neutrophil counts and function. Accordingly, treatment with the SGLT2 inhibitor empagliflozin led to complete normalization of neutrophil counts and resolution of symptoms.

Table 1.Mechanism-based classification of genetic immune dysregulation underlying therapy-refractory cytopenias.

A 2-year-old boy presenting with ITP and anemia carried a MAGT1 variant causing a magnesium transporter defect that disrupts intracellular magnesium homeostasis and immune function. Although magnesium supplementation alone was insufficient, his cytopenias stabilized on thrombopoietin mimetics. Another patient with CTLA4 haploinsufficiency achieved sustained remission of autoimmune hemolytic anemia on CTLA-4-Ig (abatacept).

No pathogenic variants predisposing to immune dysregulation were identified in six of the 20 patients. Two of these fulfilled clinical criteria for SLE and responded well to rapamycin, which suppresses the expansion of proinflammatory double-negative T cells in SLE. A third patient with an SLE-like phenotype harbored a heterozygous variant of uncertain significance (VUS) in TNFRSF6B, potentially predisposing to autoimmunity.¹³ Another patient with severe ITP met clinical criteria for CVID. The remaining two patients with ITP, one of whom was status post Fontan procedure, achieved sustained remission on rapamycin. Distinguishing germline from somatic variants in patients (Pat) with (i) a confirmed de novo pathogenic mutation (Pat#4) or (ii) a pathogenic variant without available parental testing (Pat#3 and Pat#13) remains challenging without multi-tissue analysis. Age at presentation, clinical severity, and an allele frequency near 50% are insufficient to determine variant origin. Given the variable expressivity and incomplete penetrance often seen in IEI, phenotypic overlap between germline and somatic cases is common.¹⁴ In the patient with a de novo CDC42 mutation (Pat#3), low-level parental mosaicism cannot be excluded. In the patient carrying a MAGT1 variant (Pat#13), an allele frequency of 50% in blood is consistent with a hemizygous germline change but does not exclude somatic mosaicism.

Table 2.Clinical, genetic, and therapeutic characteristics of the patient cohort before precision-guided treatment.

Notably, somatic MAGT1 variants have not been reported. Genetic causes of immune dysregulation should be systematically considered in children with therapy-refractory cytopenias, as molecular diagnoses carry prognostic significance and directly inform treatment. Patients with identified monogenic disorders experienced substantial benefit from targeted therapies, which were generally well tolerated. Those with primary immune regulatory diseases (PIRD) involving effector-Treg imbalance or thymic dysfunction improved with mTOR inhibition; patients with interferonopathies responded to JAK inhibitors; and those with inflammasomopathies achieved remission with IL-1 blockade. In select ultra-rare conditions, such as G6PC3 deficiency, repurposed therapies like SGLT2 inhibitors produced transformative outcomes.

Hadjadj et al. demonstrated that pediatric Evans syndrome is associated with a high frequency of potentially damaging variants in immune-related genes.^16-20 In a national cohort of 203 children with early-onset Evans syndrome, systematic genetic testing of 80 consecutive cases revealed that 65% carried pathogenic or likely pathogenic variants. These were identified in genes associated with autoimmune lymphoproliferative syndrome (e.g., FAS), primary immunodeficiency (CTLA4, LRBA, STAT3), or genes not previously linked to autoimmunity (e.g., KRAS).¹⁸ Consistent with our findings, patients with such variants exhibited more severe disease, required more intensive therapy, and frequently presented with additional immunopathologic features compared to those without identified variants. Long-term immunomodulation is often required to maintain remission, underscoring the need for coordinated multidisciplinary care among immunologists, hematologists, geneticists, and transplant specialists. As genomic testing becomes routine, the number of patients with genetic immune disorders identified in hematology clinics will continue to grow. Broad implementation of these tools will expand the hematologist’s therapeutic repertoire. Currently available testing is aimed at identifying germline variants in IEI-defining genes,¹⁵ however, detecting IEI variants on a clonal level could become the next diagnostic frontier, rendering “idiopathic” immune cytopenias a diagnosis of the past.

Figure 1.Genetic immune diseases in the hematologist’s waiting room. Breakdown of genetic variants identified in our patient cohort according to disease mechanism and corresponding targeted therapies. Comprehensive overview of the diagnostic evaluation of 20 children with therapy-refractory cytopenias. For each case, age, clinical presentation, laboratory findings, bone-marrow morphology, and identified genetic variants are shown, together with relevant comorbidities and prior therapies. Variants were identified by primary immunodeficiency panel (PID) or whole-exome sequencing (WES) and classified according to American College of Medical Genetics and Genomics (ACMG) guidelines. The table highlights the diagnostic heterogeneity of cytopenias and underscores how genetic testing clarified disease mechanism and therapeutic direction. All variants were classified according to ACMG guidelines. ACP5: acid phosphatase 5, tartrate resistant; CAPS: cryopyrin-associated periodic syndrome; CDC42: cell division cycle 42; CTLA4: cytotoxic T lymphocyte-associated protein 4; G6PC3: glucose-6-phosphatase catalytic subunit 3; F: female; IPEX: immune dysregulation, polyendocrinopathy, enteropathy, X-linked; MAGT1: magnesium transporter 1; NLRP12-AID: NLRP12 associated autoinflammatory disease; NFKB1: nuclear factor NF-κB subunit 1; SAVI: STING-associated vasculopathy with onset in infancy; SPENCDI: spondyloenchondrodysplasia with immune dysregulation; SGLT2: sodium-glucose cotransporter 2; SLE: systemic lupus erythematosus; TBX1: T-box transcription factor 1.

Footnotes

• Received August 8, 2025
• Accepted December 24, 2025

Correspondence

Y. GERNEZ - yaelg@stanford.edu

K. G. WEINACHT - kgw1@stanford.edu

Disclosures

No conflicts of interest to disclose.

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