Understanding complex disease-related mechanisms: Rational therapies for Diamond-Blackfan anaemia.

The rich history surrounding Diamond-Blackfan anaemia (DBA), originally described in 1938 as congenital hypoplastic anaemia2 reflects the evolution of paediatric haematology. In their paper, the authors1 present the results of a clinical trial using the thrombopoietin-mimetic agent eltrombopag to treat red cell failure in DBA. A low response rate belies the importance of this work. Commentary on: Duncan et al. Treatment of refractory/relapsed Diamond-Blackfan anaemia with eltrombopag. Br J Haematol 2024;204:2077-2085.

Treatment of refractory/relapsed Diamond-Blackfan anaemia with eltrombopag.

Diamond-Blackfan anaemia (DBA) is a rare, inherited bone marrow failure syndrome with a ribosomal defect causing slowed globin chain production with normal haem synthesis, causing an overabundance of reactive iron/haem and erythroid-specific cellular toxicity. Eltrombopag, a non-peptide thrombopoietin receptor agonist, is a potent intracellular iron chelator and induced a robust durable response in an RPS19-mutated DBA patient on another trial. We hypothesized eltrombopag would improve RBC production in DBA patients. We conducted a single-centre, single-arm pilot study (NCT04269889) assessing safety and erythroid response of 6 months of daily, fixed-dose eltrombopag for DBA patients. Fifteen transfusion-dependent (every 3-5 weeks) patients (median age 18 [range 2-56]) were treated. One responder had sustained haemoglobin improvement and >50% reduction in RBC transfusion frequency. Of note, 7/15 (41%) patients required dose reductions or sustained discontinuation of eltrombopag due to asymptomatic thrombocytosis. Despite the low response rate, eltrombopag has now improved erythropoiesis in several patients with DBA with a favourable safety profile. Dosing restrictions due to thrombocytosis may cause insufficient iron chelation to decrease haem production and improve anaemia in most patients. Future work will focus on erythropoiesis dynamics in patients and use of haem synthesis inhibitors without an impact on other haematopoietic lineages.

Effect of Glucocorticosteroids in Diamond-Blackfan Anaemia: Maybe Not as Elusive as It Seems.

Diamond-Blackfan anaemia (DBA) is a red blood cell aplasia that in the majority of cases is associated with ribosomal protein (RP) aberrations. However, the mechanism by which this disorder leads to such a specific phenotype remains unclear. Even more elusive is the reason why non-specific agents such as glucocorticosteroids (GCs), also known as glucocorticoids, are an effective therapy for DBA. In this review, we (1) explore why GCs are successful in DBA treatment, (2) discuss the effect of GCs on erythropoiesis, and (3) summarise the GC impact on crucial pathways deregulated in DBA. Furthermore, we show that GCs do not regulate DBA erythropoiesis via a single mechanism but more likely via several interdependent pathways.

Short Stature in Patients with Diamond-Blackfan Anemia: A Cross-Sectional Study.

OBJECTIVE: To systematically describe the short stature of patients with Diamond-Blackfan anemia and to explore factors affecting the height development of patients with Diamond-Blackfan anemia. STUDY DESIGN: This cross-sectional study was conducted at the Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, and the height, weight, and clinical data of 129 patients with Diamond-Blackfan anemia were collected from June 2020 to September 2020. RESULTS: The median height-age-z score (HAZ) of children affected by Diamond-Blackfan anemia was -1.54 (-6.36-1.96). Short stature was found in 37.98% of the patients. Specific Diamond-Blackfan anemia growth curves were developed for weight, height, and body mass index, separately for male and female patients. Multivariable logistic regression models showed that female sex (aOR 4.92; 95% CI 1.29-18.71; P = .0195), underweight (aOR 10.41, 95% CI 1.41-76.98, P = .0217), cardiovascular malformations (aOR 216.65; 95% CI 3.29-14279.79; P = .0118), and RPL11(aOR 29.14; 95% CI 1.18-719.10; P = .0392) or RPS26 (aOR 53.49; 95% CI 1.40-2044.30; P = .0323) mutations were independent risk factors for short stature. In the subgroup of patients who were steroid-dependent, patients with a duration of steroid therapy over 2 years (OR 2.95; 95% CI 1.00-8.66; P = .0494) or maintenance dose of prednisone >0.1 mg/kg per day (OR 3.30; 95% CI 1.02-10.72; P = .0470) had a higher incidence of short stature. CONCLUSIONS: Patients with Diamond-Blackfan anemia had a high prevalence of short stature. The risk of short stature increased with age and was associated with sex, underweight, congenital malformations, and RPL11 or RPS26 mutations. The duration of steroid therapy and maintenance dose of steroid was significantly associated with the incidence of short stature in steroid-dependent patients with Diamond-Blackfan anemia.

[Deferasirox and Complex Proximal Tubulopathy. Presentation of two clinical cases]. / Deferasirox y Tubulopatía Proximal Compleja. Presentación de dos casos clínicos.

INTRODUCTION: Treatment with iron chelators is essential for patients with iron overload secondary to repeated trans fusions. Deferasirox is the first once-daily oral active iron chelator. As a result, therapeutic adherence has improved, reducing the complications of iron overload, especially heart failure. However, it is not exempt from possible side effects, such as kidney involvement, which is more frequent in children. OBJECTIVE: To report 2 patients with Diamond-Blackfan anemia (DBA) who developed impaired renal function secondary to the administration of Deferasirox. CLINICAL CASES: Case 1. A 15-year-old adolescent diagnosed with DBA undergoing treatment with periodic transfusions and Deferasirox. During an acute gastroenteritis, she developed acute renal failure along with complex proximal tubu- lopathy. Case 2. A 5-year-old boy diagnosed with DBA receiving periodic transfusions and treatment with Deferasirox. He presented polyuria with laboratory abnormalities compatible with acute renal failure and proximal tubular dysfunction. In both cases, they were adequately hydrated and Deferasi rox was temporarily suspended, improving renal function. CONCLUSION: Based on these cases, close monitoring of renal and tubular function, as well as ferritin levels, is recommended in patients recei ving Deferasirox. In the presence of intercurrent processes, adequate hydration should be performed, and an early dose reduction or drug administration interruption should be considered in cases of kidney involvement.

Eltrombopag Improves Erythroid Differentiation in a Human Induced Pluripotent Stem Cell Model of Diamond Blackfan Anemia.

Diamond Blackfan Anemia (DBA) is a congenital macrocytic anemia associated with ribosomal protein haploinsufficiency. Ribosomal dysfunction delays globin synthesis, resulting in excess toxic free heme in erythroid progenitors, early differentiation arrest, and pure red cell aplasia. In this study, DBA induced pluripotent stem cell (iPSC) lines were generated from blood mononuclear cells of DBA patients with inactivating mutations in RPS19 and subjected to hematopoietic differentiation to model disease phenotypes. In vitro differentiated hematopoietic cells were used to investigate whether eltrombopag, an FDA-approved mimetic of thrombopoietin with robust intracellular iron chelating properties, could rescue erythropoiesis in DBA by restricting the labile iron pool (LIP) derived from excessive free heme. DBA iPSCs exhibited RPS19 haploinsufficiency, reduction in the 40S/60S ribosomal subunit ratio and early erythroid differentiation arrest in the absence of eltrombopag, compared to control isogenic iPSCs established by CRISPR/Cas9-mediated correction of the RPS19 point mutation. Notably, differentiation of DBA iPSCs in the presence of eltrombopag markedly improved erythroid maturation. Consistent with a molecular mechanism based on intracellular iron chelation, we observed that deferasirox, a clinically licensed iron chelator able to permeate into cells, also enhanced erythropoiesis in our DBA iPSC model. In contrast, erythroid maturation did not improve substantially in DBA iPSC differentiation cultures supplemented with deferoxamine, a clinically available iron chelator that poorly accesses LIP within cellular compartments. These findings identify eltrombopag as a promising new therapeutic to improve anemia in DBA.

Moxibustion with deferasirox results in safe, accelerated, and sustained cardiac iron chelation for a young Diamond Blackfan Anemia patient: An integrative case report.

Diamond Blackfan Anemia (DBA) is a rare blood disorder of bone marrow failure typically identified in the first year of life. Due to treatment limitations, nearly all DBA patients are reliant upon chronic red cell infusion for some period of their lives. Repeat blood transfusion invariably leads to iron overload, which creates significant and life-threatening side effects. In this case report, a female child of 2 years and 8 months with DBA was diagnosed via T2* MRI imaging with potentially emergent levels of cardiac iron overload as a result of chronic red cell infusion. According to Chinese, Japanese, and integrative medicine principles, moxibustion was applied externally in conjunction with the prescribed oral chelation agent, deferasirox (DFX), over a period of six months. Analysis of repeat imaging at six months showed complete removal of cardiac iron and a one-third decrease in hepatic iron. In addition, there was no evidence of known risks to high-dose chelation, including renal, aural, or ocular damage. Follow-up examination two years after initial imaging showed no evidence of cardiac iron deposition with hepatic iron levels below minimum levels recommended for chelation despite the medical necessity of continued red cell infusion. This case study suggests that an integrative medical approach combining DFX with moxibustion may safely accelerate and sustain iron chelation for transfusion-dependent DBA patients. Recommendations for a successful approach to integrative iron chelation are provided in the case discussion.

Calmodulin inhibitors improve erythropoiesis in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 (rps29) mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34+ hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34+ cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA.

Metformin-induced suppression of Nemo-like kinase improves erythropoiesis in preclinical models of Diamond-Blackfan anemia through induction of miR-26a.

Diamond-Blackfan anemia (DBA) results from haploinsufficiency of ribosomal protein subunits in hematopoietic progenitors in the earliest stages of committed erythropoiesis. Nemo-like kinase (NLK) is chronically hyperactivated in committed erythroid progenitors and precursors in multiple human and murine models of DBA. Inhibition of NLK activity and suppression of NLK expression both improve erythroid expansion in these models. Metformin is a well-tolerated drug for type 2 diabetes with multiple cellular targets. Here we demonstrate that metformin improves erythropoiesis in human and zebrafish models of DBA. Our data indicate that the effects of metformin on erythroid proliferation and differentiation are mediated by suppression of NLK expression through induction of miR-26a, which recognizes a binding site within the NLK 3' untranslated region (3'UTR) to facilitate transcript degradation. We propose that induction of miR-26a is a potentially novel approach to treatment of DBA and could improve anemia in DBA patients without the potentially adverse side effects of metformin in a DBA patient population.

The nuclear gene rpl18 regulates erythroid maturation via JAK2-STAT3 signaling in zebrafish model of Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a rare, inherited bone marrow failure syndrome, characterized by red blood cell aplasia, developmental abnormalities, and enhanced risk of malignancy. However, the underlying pathogenesis of DBA is yet to be understood. Recently, mutations in the gene encoding ribosomal protein (RP) L18 were identified in DBA patients. RPL18 is a crucial component of the ribosomal large subunit but its role in hematopoiesis remains unknown. To genetically model the ribosomal defect identified in DBA, we generated a rpl18 mutant line in zebrafish, using CRISPR/Cas9 system. Molecular characterization of this mutant line demonstrated that Rpl18 deficiency mirrored the erythroid defects of DBA, namely a lack of mature red blood cells. Rpl18 deficiency caused an increase in p53 activation and JAK2-STAT3 activity. Furthermore, we found inhibitors of JAK2 or STAT3 phosphorylation could rescue anemia in rpl18 mutants. Our research provides a new in vivo model of Rpl18 deficiency and suggests involvement of signal pathway of JAK2-STAT3 in the DBA pathogenesis.

Steroid resistance in Diamond Blackfan anemia associates with p57Kip2 dysregulation in erythroid progenitors.

Despite the effective clinical use of steroids for the treatment of Diamond Blackfan anemia (DBA), the mechanisms through which glucocorticoids regulate human erythropoiesis remain poorly understood. We report that the sensitivity of erythroid differentiation to dexamethasone is dependent on the developmental origin of human CD34+ progenitor cells, specifically increasing the expansion of CD34+ progenitors from peripheral blood (PB) but not cord blood (CB). Dexamethasone treatment of erythroid-differentiated PB, but not CB, CD34+ progenitors resulted in the expansion of a newly defined CD34+CD36+CD71hiCD105med immature colony-forming unit-erythroid (CFU-E) population. Furthermore, proteomics analyses revealed the induction of distinct proteins in dexamethasone-treated PB and CB erythroid progenitors. Dexamethasone treatment of PB progenitors resulted in the specific upregulation of p57Kip2, a Cip/Kip cyclin-dependent kinase inhibitor, and we identified this induction as critical; shRNA-mediated downregulation of p57Kip2, but not the related p27Kip1, significantly attenuated the impact of dexamethasone on erythroid differentiation and inhibited the expansion of the immature CFU-E subset. Notably, in the context of DBA, we found that steroid resistance was associated with dysregulated p57Kip2 expression. Altogether, these data identify a unique glucocorticoid-responsive human erythroid progenitor and provide new insights into glucocorticoid-based therapeutic strategies for the treatment of patients with DBA.

A Phenotypic Screening Assay Identifies Modulators of Diamond Blackfan Anemia.

Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome caused by mutations in ribosomal protein genes. Pathogenic mechanisms are poorly understood but involve severely reduced proliferation of erythroid precursors. Because current DBA therapies are ineffective and associated with severe side effects, disease-specific therapies are urgently needed. We hypothesized that druggable molecular pathways underlying the defect can be revealed through phenotypic small-molecule screens. Accordingly, a screening assay was developed using c-kit+ fetal liver erythroid progenitors from a doxycycline-inducible DBA mouse model. The addition of doxycycline to the culture medium induces the phenotype and reduces proliferation to <10% of normal, such that rescue of proliferation can be used as a simple readout for screening. Here, we describe the assay rationale and efforts toward validation of a microtiter plate-compatible assay and its application in a pilot screen of 3871 annotated compounds. Ten hits demonstrated concentration-dependent activity, and we report a brief follow-up of one of these compounds. In conclusion, we established a robust scalable assay for screening molecules that rescue erythropoiesis in DBA.

Identification of novel drug targets for diamond-blackfan anemia based on RPS19 gene mutation using protein-protein interaction network.

BACKGROUND: Diamond-Blackfan anemia (DBA) is a congenital erythroid aplasia that usually presents in infancy. In order to explore the molecular mechanisms of wild and mutated samples from DBA patients were exposed to bioinformatics investigation. Biological network of differentially expressed genes was constructed. This study aimed to identify novel therapeutic signatures in DBA and uncovered their mechanisms. The gene expression dataset of GSE14335 was used, which consists of 6 normal and 4 diseased cases. The gene ontology (GO), as well as Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed, and then protein-protein interaction (PPI) network of the identified differentially expressed genes (DEGs) was constructed by Cytoscape software. RESULTS: A total of 607 DEGs were identified in DBA, including 433 upregulated genes and 174 downregulated genes. GO analysis results showed that upregulated DEGs were significantly enriched in biological processes, negative regulation of transcription from RNA polymerase II promoter, chemotaxis, inflammatory response, immune response, positive regulation of cell proliferation, negative regulation of cell proliferation, response to mechanical stimulus, positive regulation of cell migration, response to lipopolysaccharide, and defence response. KEGG pathway analysis revealed the TNF signalling pathway, Osteoclast differentiation, Chemokine signalling pathway, Cytokine -cytokine receptor interaction, Rheumatoid arthritis, Biosynthesis of amino acids, Biosynthesis of antibiotics and Glycine, serine and threonine metabolism. The top 10 hub genes, AKT1, IL6, NFKB1, STAT3, STAT1, RAC1, EGR1, IL8, RELA, RAC3, mTOR and CCR2 were identified from the PPI network and sub-networks. CONCLUSION: The present study flagged that the identified DEGs and hub genes enrich our understanding of the molecular mechanisms underlying the development of DBA, and might shine some lights on identifying molecular targets and diagnostic biomarkers for DBA.

Successful long-term management with low-dose prednisolone in an adult patient with Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a rare congenital disease caused by mutations in ribosomal protein genes and is characterized by pure red cell aplasia. While the prognosis is relatively favorable, quality of life (QOL) among DBA patients is negatively impacted by the adverse effects of long-term prednisolone (PSL) therapy and blood transfusions. We describe a 43-year-old man who was diagnosed with DBA (Hb of 2.18 g/dl) at the age of two months. He was initially treated with PSL and blood transfusions, followed by cyclosporine and low-dose (6 mg/day) PSL, which resulted in a sustained hemoglobin level of 9 g/dl without severe adverse events or loss of QOL. High levels of eADA and GSH as well as a RPS19 gene mutation were confirmed. The only curative therapy is hematopoietic stem cell transplantation, which is associated with significant mortality. However, using low-dose PSL to maintain a stable hemoglobin level may improve QOL for patients who receive curative treatment.

Drug discovery for Diamond-Blackfan anemia using reprogrammed hematopoietic progenitors.

Diamond-Blackfan anemia (DBA) is a congenital disorder characterized by the failure of erythroid progenitor differentiation, severely curtailing red blood cell production. Because many DBA patients fail to respond to corticosteroid therapy, there is considerable need for therapeutics for this disorder. Identifying therapeutics for DBA requires circumventing the paucity of primary patient blood stem and progenitor cells. To this end, we adopted a reprogramming strategy to generate expandable hematopoietic progenitor cells from induced pluripotent stem cells (iPSCs) from DBA patients. Reprogrammed DBA progenitors recapitulate defects in erythroid differentiation, which were rescued by gene complementation. Unbiased chemical screens identified SMER28, a small-molecule inducer of autophagy, which enhanced erythropoiesis in a range of in vitro and in vivo models of DBA. SMER28 acted through autophagy factor ATG5 to stimulate erythropoiesis and up-regulate expression of globin genes. These findings present an unbiased drug screen for hematological disease using iPSCs and identify autophagy as a therapeutic pathway in DBA.

Pure red cell aplasia.

Pure red cell aplasia (PRCA) is a syndrome defined by a normocytic normochromic anemia with severe reticulocytopenia and marked reduction or absence of erythroid precursors from the bone marrow. Diamond-Blackfan anemia is a congenital form of PRCA. Acquired PRCA may be either a primary disorder or secondary to some other disorder or agent. Primary acquired PRCA is an autoimmune disorder that is frequently antibody-mediated. Myelodysplastic syndromes may also present with the morphologic appearance of PRCA. Secondary acquired PRCA may be associated with collagen vascular/autoimmune disorders such as systemic lupus erythematosus; lymphoproliferative disorders such as chronic lymphocytic leukemia or large granular lymphocyte leukemia; infections, particularly B19 parvovirus; thymoma and other solid tumors; or a variety of other disorders, drugs, or toxic agents. The therapeutic approach to PRCA typically involves immunosuppression, but specific pathogenic subtypes are associated with specific therapeutic approaches. Cyclosporine A, with or without concurrent corticosteroids, appears to be the single most effective immunosuppressive agent.

Fanconi Syndrome Secondary to Deferasirox in Diamond-Blackfan Anemia: Case Series and Recommendations for Early Diagnosis.

Deferasirox is an oral iron chelator used to treat patients with transfusion-related iron overload. We report, from two institutions, two children with Diamond-Blackfan anemia who developed Fanconi syndrome secondary to deferasirox administration, along with a review of the literature. The current recommendation for the laboratory monitoring of patients receiving deferasirox does not include serum electrolytes or urine analysis. Thus, despite routine clinic visits and bloodwork, these two patients presented with life-threatening electrolyte abnormalities requiring hospitalization. Hence, we propose the inclusion of serum electrolytes and urine analysis as part of routine monitoring to facilitate the early diagnosis of Fanconi syndrome in the context of high doses of deferasirox therapy.

Glucocorticoids improve erythroid progenitor maintenance and dampen Trp53 response in a mouse model of Diamond-Blackfan anaemia.

Diamond-Blackfan anaemia (DBA) is a rare congenital disease causing severe anaemia and progressive bone marrow failure. The majority of patients carry mutations in ribosomal proteins, which leads to depletion of erythroid progenitors in the bone marrow. As many as 40% of all DBA patients receive glucocorticoids to alleviate their anaemia. However, despite their use in DBA treatment for more than half a century, the therapeutic mechanisms of glucocorticoids remain largely unknown. Therefore we sought to study disease specific effects of glucocorticoid treatment using a ribosomal protein s19 (Rps19) deficient mouse model of DBA. This study determines for the first time that a mouse model of DBA can respond to glucocorticoid treatment, similar to DBA patients. Our results demonstrate that glucocorticoid treatment reduces apoptosis, rescues erythroid progenitor depletion and premature differentiation of erythroid cells. Furthermore, glucocorticoids prevent Trp53 activation in Rps19-deficient cells- in a disease-specific manner. Dissecting the therapeutic mechanisms behind glucocorticoid treatment of DBA provides indispensible insight into DBA pathogenesis. Identifying mechanisms important for DBA treatment also enables development of more disease-specific treatments of DBA.

RAP-011 improves erythropoiesis in zebrafish model of Diamond-Blackfan anemia through antagonizing lefty1.

Diamond-Blackfan Anemia (DBA) is a bone marrow failure disorder characterized by low red blood cell count. Mutations in ribosomal protein genes have been identified in approximately half of all DBA cases. Corticosteriod therapy and bone marrow transplantation are common treatment options for patients; however, significant risks and complications are associated with these treatment options. Therefore, novel therapeutic approaches are needed for treating DBA. Sotatercept (ACE-011, and its murine ortholog RAP-011) acts as an activin receptor type IIA ligand trap, increasing hemoglobin and hematocrit in pharmacologic models, in healthy volunteers, and in patients with ß-thalassemia, by expanding late-stage erythroblasts through a mechanism distinct from erythropoietin. Here, we evaluated the effects of RAP-011 in zebrafish models of RPL11 ribosome deficiency. Treatment with RAP-011 dramatically restored hemoglobin levels caused by ribosome stress. In zebrafish embryos, RAP-011 likely stimulates erythropoietic activity by sequestering lefty1 from erythroid cells. These findings identify lefty1 as a signaling component in the development of erythroid cells and rationalize the use of sotatercept in DBA patients.

Immune status of patients with inherited bone marrow failure syndromes.

Immune function abnormalities have been reported in patients with Fanconi anemia (FA), dyskeratosis congenita (DC) and, rarely, in Shwachman-Diamond syndrome (SDS), and Diamond-Blackfan anemia (DBA), but large systematic studies are lacking. We assessed immunological parameters in 118 patients with these syndromes and 202 unaffected relatives. We compared the results in patients with reference values, and with values in relatives after adjusting for age, sex, corticosteroid treatment, and severe bone marrow failure (BMF). Adult patients (≥18 years) with FA had significantly lower immunoglobulins (IgG, IgA and IgM), total lymphocytes, and CD4 T cells than reference values or adult relatives (P < 0.001); children with FA had normal values. Both children and adults with FA had lower B- and NK cells (P < 0.01) than relatives or reference values. Patients with DC had essentially normal immunoglobulins but lower total lymphocytes than reference values or relatives, and lower T-, B-, and NK-cells; these changes were more marked in children than adults (P < 0.01). Most patients with DBA and SDS had normal immunoglobulins and lymphocytes. Lymphoproliferative responses, serum cytokine levels, including tumor necrosis factor-α and interferon-γ, and cytokine levels in supernatants from phytohemagglutinin-stimulated cultures were similar across patient groups and relatives. Only patients with severe BMF, particularly those with FA and DC, had higher serum G-CSF and Flt3-ligand and lower RANTES levels compared with all other groups or relatives (P < 0.05). Overall, immune function abnormalities were seen mainly in adult patients with FA, which likely reflects their disease-related progression, and in children with DC, which may be a feature of early-onset severe disease phenotype.