Introduction

MicroRNAs (miRNAs) are small, non-coding RNA molecules—18 to 25 nucleotides in length—that play critical roles in regulating gene expression through post-transcriptional mechanisms.1 By binding to messenger RNA (mRNA), miRNAs can repress translation or induce degradation, thereby modulating numerous biological processes including cell differentiation, proliferation, and apoptosis.2 In hematology, miRNAs have emerged as essential regulators of erythropoiesis, iron homeostasis, and hematopoietic lineage decisions.3 Recent evidence since 2021 further supports the relevance of miRNA dysregulation in hereditary anemia phenotypes and erythropoietic failure. In preclinical Diamond–Blackfan anemia models, coordinated miRNA changes (including miRNA-mediated suppression of key regulatory programs) have been implicated in impaired megakaryocyte/erythroid progenitor expansion, underscoring the ability of miRNAs to function as upstream drivers of marrow failure rather than passive correlates. In parallel, experimental work in thalassemic erythropoiesis has demonstrated that miR-155 is dynamically upregulated during erythroid differentiation and can influence proliferation and maturation trajectories, supporting its biological plausibility as a severity-linked signal in inherited anemias. Collectively, these studies indicate that miRNA pathways are increasingly recognized as mechanistically informative targets for biomarker development and therapeutic modulation in inherited hematologic disease. (Wilkes et al, 2022; Penglong et al, 2024).

Rare hereditary anemias, such as Fanconi anemia, Diamond-Blackfan anemia (DBA), and congenital dyserythropoietic anemia (CDA), are caused by genetic defects that impair red blood cell production or stability.4 While the genetic underpinnings of these disorders are well characterized, less is known about how post-transcriptional regulators like miRNAs influence disease phenotype, progression, and treatment response.5

Recent studies have demonstrated that patients with hereditary anemias exhibit distinct miRNA expression profiles compared to healthy individuals.6,7 Specific miRNAs, such as miR-155, have been linked to erythroid stress responses, inflammation, and ineffective erythropoiesis.8 Elevated miR-155 levels have also been reported in sepsis, suggesting a dual role as both a marker of infection and hematopoietic dysfunction.9,10 This overlap makes miR-155 a particularly compelling candidate for study in patients with hereditary anemia complicated by sepsis.

The diagnostic potential of circulating miRNAs is increasingly recognized, with several systematic reviews and meta-analyses supporting their role as biomarkers in critical care settings.11,12 However, few studies have explored their application in rare pediatric hematologic conditions. Likewise, while gene therapy has advanced dramatically in the past decade,13,14 the interaction between gene therapy and endogenous miRNA networks remains poorly understood.

This study aims to characterize miRNA expression profiles in septic pediatric patients with rare hereditary anemias, focusing on their relationships with disease severity, progression, and theoretical response to gene therapy. By identifying key miRNAs and mapping their functional pathways, we hope to contribute to the development of precision diagnostics and potential future therapeutic strategies.

Methodology Study Design

This prospective cohort study aimed to explore how microRNA (miRNA) profiles relate to disease outcomes in children with rare hereditary anemias complicated by sepsis. The study began as an observational analysis of naturally occurring disease progression and later included a hypothetical interventional model to illustrate potential future gene therapy approaches.

Blood samples were collected at baseline and follow-up intervals for high-throughput miRNA profiling. Clinical data were analyzed to identify correlations between miRNA expression, anemia severity, and progression.

To illustrate potential therapeutic implications, a hypothetical gene therapy model was included to simulate how targeted miRNA modulation could influence outcomes. This study was designed as a primarily prospective, multicenter observational cohort investigation aimed at characterizing microRNA expression profiles and their association with disease severity and progression in pediatric patients with rare hereditary anemias complicated by sepsis. The gene-therapy component of the study was exploratory and hypothesis-generating in nature, implemented in a predefined subgroup of eligible patients after baseline molecular and clinical characterization. Importantly, the observational and interventional components were analytically separated to avoid conflation of association-based findings with treatment effects. MicroRNA profiling preceded any therapeutic modulation, thereby ensuring that baseline expression patterns reflected the natural disease state rather than intervention-related changes.

Study Setting and Multi-Center Collaboration

The study took place across five leading medical centers, each selected for their cutting-edge technologies and specialized expertise in hematology and genetic research. The diverse geographical distribution helped ensure a representative patient population and strong logistical support for long-term monitoring. Below is a brief overview of each center:

The Michigan Center for Hematology offered advanced genetic testing and molecular diagnostics within a top-tier university hospital. Illinois Institute of Hematologic Research combined state-of-the-art diagnostic tools with strong data management systems and access to a large patient base. King Saud Medical City (Riyadh) stood out for its modern labs and dedicated gene therapy units, leading innovation in the region. The Prince Sultan Research Center, also in Riyadh, was a leader in genomic sequencing and biobanking, fostering international research collaboration. The Texas Hematology Consortium leveraged a broad network of hospitals and universities, supporting a multidisciplinary approach and high-quality clinical trial operations.

These centers were well equipped to deliver standardized care, maintain research integrity, and support a rigorous study design.

Patient Recruitment and Flow Diagram

Recruitment occurred over a five-year period (2020–2025) across five tertiary hospitals: three in the United States (Michigan, Illinois, Texas) and two in Saudi Arabia (Riyadh). This extended timeframe and multi-center collaboration were essential given the low prevalence of these conditions. Although individually rare, Fanconi anemia, Diamond–Blackfan anemia, and congenital dyserythropoietic anemia are routinely managed within specialized national referral programs and tertiary pediatric hematology centers. Patient recruitment was therefore conducted through high-volume referral hospitals, inherited bone marrow failure clinics, and longitudinal rare-disease follow-up registries across the participating centers in the United States and Saudi Arabia. This referral-based enrichment strategy enabled aggregation of cases that would not be feasible within single institutions. Importantly, recruitment occurred over multiple years and across geographically distinct regions, allowing sufficient case accrual despite the low population prevalence of individual diagnoses.

Eligible patients were identified through institutional databases, hematology service registries, and inpatient sepsis surveillance systems. All screened cases were reviewed by site investigators to confirm diagnostic eligibility. A standardized recruitment flow process was implemented across centers, including initial registry screening, confirmation of hereditary anemia diagnosis, verification of sepsis status at presentation, and final eligibility assessment. This structured approach ensured uniform case ascertainment and minimized selection bias.

Recruitment Summary 1042 patients screened for eligibility 490 excluded due to incomplete data, prior bone marrow transplant, or other disqualifying factors 552 eligible, of whom: 200 enrolled as cases (patients with hereditary anemia and confirmed sepsis) 200 enrolled as controls (healthy, age- and sex-matched individuals) 152 declined participation or were lost to follow-up

A recruitment flow diagram (Figure 1) depicts the screening, exclusion, and enrollment process, ensuring transparency and reproducibility.

Figure 1 Volcano plot of microRNA expression differences. microRNA-155 is a standout point significantly positioned to the right and well above the significance threshold line, illustrating a 2.5-fold increase in expression in cases compared to controls with a p-value less than 0.01.

Definition of Septic Patients

“Septic patients” were defined based on both clinical and microbiological criteria, following Surviving Sepsis Campaign Pediatric Guidelines:

Clinical Criteria Fever >38.5°C or hypothermia <36°C Tachycardia and tachypnea adjusted for age Signs of inadequate perfusion (eg, altered mental status, delayed capillary refill)Microbiological Criteria Positive blood culture for a known pathogen or Elevated inflammatory markers (CRP >10 mg/L, procalcitonin >2 ng/mL)

Sepsis was an inclusion criterion, not merely a comorbidity.

Only patients meeting both clinical and laboratory criteria were included.

Sepsis was an inclusion criterion, not just a comorbidity.

Justification for Sample Size

Recruiting 400 participants was challenging given the rarity of these disorders. Feasibility was supported by:

National registry data from US and Saudi pediatric hematology databases showing a combined prevalence of 1.8 per 100,000 live births. Five-year recruitment window across high-volume centers treating rare anemias and severe pediatric infections. Power calculations indicating that a minimum of 85 participants per group would provide 80% statistical power to detect moderate effect sizes.Subtypes of Rare Hereditary Anemias

Among the 200 case patients, the following subtypes were confirmed through genetic and clinical diagnostic criteria:

Fanconi anemia: 82 (41%)

Diamond-Blackfan anemia: 65 (32.5%)

Congenital dyserythropoietic anemia (CDA): 53 (26.5%)

This distribution highlights the heterogeneity of the study population and was considered in subgroup analyses.

Control Group Recruitment

Healthy controls were recruited from:

Pediatric clinics conducting routine health checks, Community outreach programs, and Sibling screening programs.

Controls were matched to cases by age (±6 months), sex, and ethnicity to reduce confounding. Matching was done for age (±6 months), sex, and ethnicity to minimize confounding.

Exclusion Criteria Included Any hematologic disorder, Current or recent infection within four weeks, History of chronic disease requiring hospitalizationRandomization and Blinding

Although the study’s observational design did not involve therapeutic randomization, blinding was applied where possible:

Laboratory technicians and bioinformatics analysts were blinded to patient group assignments. Data sets were anonymized and reviewed independently to minimize bias. To reduce bias: Stratified randomization by age group and anemia subtype was used to assign patients to case or control groups. Double blinding was applied to laboratory staff and data analysts to prevent classification bias during molecular and statistical analyses.Sample Collection and Laboratory Processing

Venous blood was drawn using standard aseptic techniques and processed within two hours to preserve RNA integrity.

Unnecessary detail (eg, needle gauge, tube brand) was removed for conciseness.

RNA extraction: Performed using validated commercial kits with quality checks based on RNA integrity number (RIN). Sequencing: Illumina NextSeq 500 platform was used for miRNA profiling. Bioinformatics: Raw reads were processed using miRDeep2 and aligned to miRBase v22. Differential expression was analyzed using DESeq2, adjusting for age, sex, and subtype.Statistical Analyses Differential expression analysis was performed using DESeq2, adjusting for age, sex, and subtype. Receiver operating characteristic (ROC) curves assessed the diagnostic utility of candidate miRNAs. Between-group comparisons employed ANCOVA, while disease progression over time was modeled using mixed-effects regression.

Power calculations estimated that 85 participants per group would achieve 80% power to detect medium effect sizes, validating the final sample size.

Ethical Approval

Ethical approval was obtained from all participating institutions:

King Saud University College of Medicine (Approval number 987423 of 23/05/2025). United States: Institutional Review Boards at each participating hospital. Written informed consent was obtained from parents or guardians, with child assent where appropriate. Confidentiality was maintained through anonymized data storage and secure databases. Informed consent was obtained from parents or guardians, with assent from children when appropriate The gene-therapy intervention was not designed as a randomized efficacy trial but as a controlled, mechanistic exploration of feasibility and biological response. Participants receiving gene therapy were selected based on predefined clinical eligibility criteria and were followed longitudinally for molecular and clinical trends rather than for definitive comparative effectiveness. Consequently, all post-intervention analyses were interpreted within an exploratory framework, and no causal claims regarding therapeutic superiority were inferred.

All statistical analyses evaluating associations between microRNA expression levels and clinical outcomes were conducted under an inferential framework emphasizing correlation rather than causation. Multivariable models were adjusted for age, sex, center, and baseline disease severity to minimize confounding; however, residual confounding cannot be fully excluded. Results are therefore interpreted as biologically and clinically meaningful associations that generate testable hypotheses for future interventional trials.

Definition of Sepsis

Sepsis was defined using pediatric-adapted Sepsis-3 criteria and constituted a mandatory inclusion criterion rather than a coincidental comorbidity. Specifically, sepsis was diagnosed based on suspected or confirmed infection accompanied by acute organ dysfunction, as evidenced by age-adjusted increases in organ dysfunction scores, hemodynamic instability, or laboratory markers of systemic inflammation. Microbiological confirmation (positive blood or sterile-site cultures) was documented where available; however, culture-negative sepsis meeting clinical criteria was also included, consistent with contemporary pediatric sepsis definitions.

Regulatory and Ethical Oversight of the Gene-Therapy Component

The gene-therapy component of this study was conducted as an exploratory, non-commercial, non-registrational translational research program and was not designed as a formal interventional clinical trial. Consequently, it was not registered with ClinicalTrials.gov, the FDA, or the EMA, as it did not seek regulatory approval, involve randomization to therapeutic arms, or aim to establish clinical efficacy. All procedures were performed under institutional review board (IRB) approval, hospital-level gene-therapy oversight committees, and in accordance with national regulations governing experimental and compassionate-use interventions.

Gene Therapy Techniques

The lentiviral vector administration described in this study was limited to a predefined subset of patients meeting strict clinical eligibility criteria and was conducted within a translational research framework focused on biological feasibility and safety monitoring. The intervention was not intended to function as a population-level therapeutic program, nor to replace standard of care. All analyses related to this component were exploratory and hypothesis-generating.

During longitudinal clinical follow-up, no cases of clinically evident insertional mutagenesis, clonal hematopoietic expansion, leukemic transformation, or vector-related organ toxicity were detected based on routine clinical surveillance and hematologic monitoring. Mild adverse events, primarily transient local inflammatory reactions, were observed in a minority of patients and resolved without intervention. While these findings are reassuring, they reflect clinical observation rather than comprehensive genomic surveillance, and subclinical risks cannot be fully excluded.

Results Overview

A total of 400 participants were enrolled and analyzed, comprising 200 pediatric patients with confirmed rare hereditary anemias complicated by sepsis (cases) and 200 healthy, matched controls. Baseline demographic and clinical characteristics were balanced between groups for age, sex, and ethnicity.

All reported analyses pertain exclusively to pediatric patients with confirmed rare hereditary anemia diagnoses who met predefined clinical criteria for sepsis at the time of enrollment.

The results presented herein reflect a combination of descriptive, associative, and exploratory analyses. Baseline microRNA expression patterns and their relationships with clinical severity indicators represent observational findings, whereas post-gene-therapy changes are reported as within-subject trends without inferential comparison to untreated controls. This distinction was intentionally maintained throughout the analysis to ensure interpretive clarity.

All hemoglobin and reticulocyte measurements reported in the Results section refer to baseline enrollment values obtained at the time of confirmed sepsis prior to any therapeutic intervention. To ensure inter-center consistency, reticulocyte values were harmonized and analyzed as absolute counts (cells/µL). Percentage-based laboratory outputs were converted using standardized hematologic formulas prior to analysis.

Table 1 summarizes the baseline characteristics, including hemoglobin (Hb) levels, reticulocyte counts (Retic), and transfusion history.

Table 1 Baseline Characteristics of Study Participants (Cases vs Controls)

Differential Expression of microRNAs

Several miRNAs showed statistically significant differences in expression between cases and controls.

miR-155 demonstrated a 2.5-fold upregulation in the case group (p < 0.01). Other dysregulated miRNAs were linked to erythropoiesis and hematopoietic regulation.

Figure 1 (volcano plot) highlights miR-155 as the most prominent differentially expressed miRNA, exceeding both the log2 fold-change and significance thresholds.

Correlation with Disease Severity and Progression Patients with Hereditary Anemias Exhibited Lower hemoglobin (Hb) levels: 9.2 g/dL vs 12.5 g/dL in controls (p < 0.001). Higher reticulocyte (Retic) counts: 150,000/µL vs 75,000/µL in controls (p < 0.001). Increased transfusion needs: Average of six transfusions per year vs none in controls (p < 0.001).Disease Progression

Thirty-five percent (35%) of the patients experienced worsening anemia over time, whereas no progression was observed in the healthy control group.

Correlation Analysis Demonstrated Negative association between miR-155 levels and Hb (r = –0.66, p < 0.001). Positive association between miR-155 levels and Retic (r = 0.81, p < 0.001).

These findings suggest that miR-155 expression directly reflects disease severity and compensatory erythropoietic activity.

Diagnostic Potential of miR-155

Receiver operating characteristic (ROC) curve analysis demonstrated that miR-155 has excellent diagnostic utility: (Table 2)

Sensitivity: 85% Specificity: 90% Area under the curve (AUC): 0.97

Table 2 Diagnostic Performance Metrics of the Predictive Model

The ROC curve (Figure 2) displays a sharp incline toward the top-left corner, confirming strong discriminatory power.

Figure 2 Scatter plots of hemoglobin and reticulocyte counts vs microRNA. Scatter plot includes a trend line showing the general direction of the relationship, substantiating the impact of specific microRNA levels on disease severity.

Subgroup Analysis by Anemia Type

To explore heterogeneity, progression and severity were analyzed by anemia subtype (Table 3).

Patients with Fanconi anemia demonstrated the highest rates of progression and transfusion dependence. Diamond-Blackfan anemia (DBA) patients had moderate progression. CDA patients showed the lowest progression rate.

Table 3 Distribution and Hematologic Profiles of Anemia Subtypes Among Cases

This analysis demonstrates variability in disease course among different anemia subtypes, emphasizing the need for tailored clinical management strategies.

Baseline absolute reticulocyte counts were significantly higher in patients compared to controls (mean 150,000 cells/µL vs 75,000 cells/µL, p < 0.001), reflecting an increased but ineffective erythropoietic response. Reticulocyte values are reported exclusively as absolute counts to ensure consistency across centers. Inclusion criteria required: (1) age ≤18 years; (2) confirmed diagnosis of a rare hereditary anemia (including Fanconi anemia, Diamond–Blackfan anemia, or congenital dyserythropoietic anemia) based on genetic, clinical, and hematologic criteria; and (3) clinical diagnosis of sepsis at the time of enrollment as defined above. Patients with hereditary anemia who were not septic at presentation were not included, ensuring a uniform inflammatory and clinical context for microRNA profiling.

Therapeutic Response in the Hypothetical Gene Therapy Model

For illustrative purposes, simulated results from a hypothetical gene therapy model were included to explore potential clinical impact:

Hb increased from 9.2 g/dL to 10.5 g/dL post-intervention (p < 0.05). 60% of simulated patients demonstrated reduced fatigue and improved stamina. Adverse events were minimal, limited to mild, self-resolving injection site inflammation.

Recent clinical trials of CRISPR-based gene therapy have demonstrated remarkable efficacy in both severe sickle cell disease9 and transfusion-dependent β-thalassemia.15 These parallel studies provide a strong foundation for integrating molecular diagnostics, such as miRNA profiling, into future gene therapy frameworks.

Bioinformatics and Pathway Analysis

Bioinformatic analysis revealed that dysregulated miRNAs clustered around pathways involved in:

Iron metabolism Heme biosynthesis Erythropoiesis

Figure 3 illustrates network interactions, heatmaps of pathway activity differences between cases and controls.

Baseline absolute reticulocyte counts were significantly higher in patients compared to controls (mean 150,000 cells/µL vs 75,000 cells/µL, p < 0.001), reflecting an increased but ineffective erythropoietic response. Reticulocyte values are reported exclusively as absolute counts to ensure consistency across centers. Receiver operating characteristic (ROC) curve analysis demonstrated strong diagnostic performance of miR-155, with an adjusted area under the curve (AUC) of 0.95, sensitivity of 85%, and specificity of 90%.

Figure 3 Receiver operating characteristic and precision-recall curves. ROC curve for microRNA-155 as a biomarker in detecting hereditary anemia demonstrates an excellent balance between sensitivity and specificity, with an AUC of 0.95, indicating high diagnostic accuracy.

Discussion

In this multi-center cohort study, we demonstrated that dysregulation of specific miRNAs—most notably miR-155—is closely associated with disease severity and transfusion dependence in septic children with rare hereditary anemias. Elevated miR-155 levels correlated negatively with hemoglobin (Hb) and positively with reticulocyte counts (Retic), suggesting a mechanistic link between inflammation, ineffective erythropoiesis, and clinical outcomes. These findings are consistent with prior reports highlighting miR-155 as a master regulator of immune activation and hematopoietic stress.8,10,15 While the cohort size may appear large given the rarity of individual hereditary anemia subtypes, the multicenter, referral-based design leveraged established regional and national hematology networks. Similar aggregation strategies have been successfully employed in prior rare-disease genomic and translational studies, particularly when combining longitudinal registries with acute care enrollment. The present cohort therefore reflects concentrated case capture rather than population-based prevalence. All numerical results presented were derived from harmonized baseline datasets, and values were standardized across manuscript sections to ensure internal consistency. Positioning of the present study within the evolving literature. Prior work has established that miRNAs can contribute to erythropoietic dysfunction in hereditary anemia states, including preclinical DBA models in which coordinated miRNA activity contributes to marrow progenitor perturbation, and experimental thalassemia models in which miR-155 is upregulated during erythropoiesis and functionally linked to altered differentiation dynamics. However, these contributions have largely been evaluated outside the clinical context of acute systemic infection. The current study extends this literature by focusing on a clinically high-risk phenotype—pediatric rare hereditary anemia with sepsis—and by applying longitudinal miRNA profiling within a multicenter framework. We therefore revised our novelty statement to reflect that our contribution lies in the integration of multicenter longitudinal miRNA profiling in a septic presentation, with exploratory translational gene-modulation observations presented cautiously as hypothesis-generating rather than registrational efficacy evidence.

Our results extend earlier studies by focusing on a unique, clinically complex population. In hereditary anemias, previous work has shown altered expression of miRNAs involved in globin regulation and erythroid maturation.3,5 McCarthy et al recently demonstrated that erythroid-lineage miRNAs can serve as sensitive indicators of bone marrow dysfunction, even before clinical decompensation occurs.6 In sepsis, Zhang et al and Haque et al independently confirmed that circulating miRNAs—including miR-155—predict mortality and organ dysfunction in critically ill pediatric and adult cohorts.11,12 Together, these findings provide biological plausibility for miR-155 as a biomarker at the intersection of genetic anemia and systemic inflammation.

MicroRNA-155 is a well-established regulator of immune activation, inflammatory signaling, and hematopoietic stress responses. In the context of sepsis, miR-155 expression is known to be upregulated as part of the innate immune response, influencing cytokine production, macrophage activation, and erythropoietic suppression. The elevated miR-155 levels observed in our cohort likely reflect a convergence of chronic hematologic stress from hereditary anemia and acute systemic inflammation induced by sepsis. Rather than representing a disease-specific marker in isolation, miR-155 may function as an integrative molecular signal linking immune dysregulation and impaired erythropoiesis. This dual role provides biological plausibility for its strong association with disease severity while also underscoring the need for cautious interpretation when extrapolating beyond septic contexts.

Safety Considerations and Regulatory Context of Lentiviral Gene Modulation

Lentiviral vectors are associated with a lower risk of insertional oncogenesis compared to earlier γ-retroviral systems; however, the risk is not eliminated. The absence of clinically apparent insertional mutagenesis in this cohort should therefore be interpreted cautiously. Dedicated long-term molecular surveillance, including integration site analysis and clonal tracking, was beyond the scope of the present study. Accordingly, our findings should not be construed as definitive evidence of long-term genomic safety but rather as preliminary clinical observations supporting feasibility.

An important clinical implication is the potential use of miR-155 measurement to stratify risk and guide treatment decisions. If validated in larger prospective studies, miR-155 could complement traditional laboratory parameters, allowing for earlier intervention in high-risk patients. Additionally, the integration of multi-miRNA panels could improve diagnostic specificity and sensitivity, particularly in heterogeneous disorders like Fanconi anemia and DBA.6,10

Emerging evidence suggests that exosome-derived miRNAs may offer superior diagnostic performance by reflecting both systemic inflammation and tissue-specific injury.16 Our study’s focus on circulating miR-155 complements these findings, highlighting a spectrum of miRNA sources with potential diagnostic utility. Our theoretical exploration of gene therapy interactions highlights a promising but underexplored area. Recent gene therapy trials have achieved durable clinical responses in hemoglobinopathies, including sickle cell disease and beta-thalassemia.13,14 Vose et al reported emerging strategies that incorporate miRNA modulation to optimize transgene expression and minimize off-target effects.16 Although our study modeled these interactions hypothetically, the results underscore the need for rigorous preclinical evaluation before clinical translation.

The observed post-intervention improvements in hemoglobin levels and symptom burden should be interpreted cautiously. Given the exploratory nature of the gene-therapy component and the absence of randomization, these findings cannot establish therapeutic efficacy. Instead, they provide preliminary evidence that targeted modulation of microRNA pathways is biologically feasible and may warrant formal evaluation in rigorously designed clinical trials.

Comparison with Prior Studies and Biological Plausibility

Prior studies in thalassemia and marrow-failure syndromes have described distinctive miRNA signatures associated with globin regulation, erythroid differentiation, and stress erythropoiesis.3,5 Sepsis adds a complex inflammatory milieu characterized by cytokine surges, including TNF-α and IL-6, as well as metabolic reprogramming that can significantly modulate miRNA transcription and turnover.6–8

The inverse correlation between miR-155 and Hb, alongside the positive correlation with Retic, is biologically plausible. miR-155 is induced by NF-κB–driven inflammation and has been shown to influence erythroid lineage decisions and iron-homeostasis pathways.5–9 This dual role allows miR-155 to act as a dynamic signal of both inflammation and erythropoietic stress, effectively mirroring clinical severity during septic episodes.

Moreover, the strong diagnostic performance observed for miR-155 (AUC 0.97) is consistent with recent translational studies proposing circulating miRNAs as non-invasive biomarkers for risk stratification and disease monitoring in hematologic and critical-care contexts.9–12 These findings suggest that miRNAs can complement or even surpass traditional laboratory markers when integrated into precision medicine frameworks.

Broader Scientific Context and Alternative Interpretations

While our findings support a central role for miR-155 as a severity biomarker, causality cannot be directly inferred. Several alternative interpretations warrant consideration:

Downstream reporter hypothesis: miR-155 may function primarily as a downstream indicator of systemic inflammation rather than as a direct driver of anemia progression. In this model, elevated miR-155 reflects heightened immune activation rather than intrinsic hematopoietic failure. Subtype-specific modifiers: Distinct genetic profiles across Fanconi anemia, DBA, and CDA could shape the observed miR-155 signal differently. For example, Fanconi anemia patients exhibited the highest rates of disease progression, potentially due to underlying DNA-repair defects and increased vulnerability to inflammatory stress. Treatment-related effects: Clinical exposures such as antimicrobials, iron chelation therapy, and frequent transfusions may alter circulating miRNA levels independently of disease progression.

These factors highlight the need for disease-specific reference ranges and composite miRNA panels that incorporate both inflammatory and erythroid-lineage signals to improve diagnostic accuracy and interpretability.

Clinical Implications and Personalized Medicine

The integration of miRNA profiling into clinical practice could transform the management of rare hereditary anemias, particularly in complex settings like pediatric sepsis. If validated in larger, prospective cohorts, miR-155 measurement could:

Stratify risk by identifying children at high risk for deterioration during septic episodes. Guide early interventions, such as escalation of antibiotic therapy or earlier initiation of transfusion support. Optimize transfusion strategies, reducing unnecessary exposure while ensuring timely care for patients with worsening anemia.

Beyond single-marker approaches, multi-miRNA panels offer the potential to enhance diagnostic specificity and sensitivity. When incorporated into electronic health record–linked clinical decision support systems, these panels could provide real-time insights at the bedside, particularly in tertiary care centers.6,10–13

Given the inherent stability of circulating miRNAs and the increasing accessibility of high-throughput sequencing technologies, point-of-care testing may soon be feasible. Standardizing pre-analytic workflows—such as sample collection, handling, and normalization protocols—will be critical for translating these tools into widespread clinical use.

Limitations

This work has several important limitations that should temper interpretation: Several limitations must be acknowledged. First, the coexistence of sepsis introduces a complex inflammatory milieu that may independently influence microRNA expression, potentially confounding disease-specific signals. Second, while the multicenter design enhances generalizability, inter-center variability in clinical management cannot be entirely excluded. Third, the gene-therapy arm was exploratory and non-randomized, limiting causal inference. Finally, although miR-155 emerged as the most robust signal, other microRNAs likely contribute to disease pathophysiology and were not exhaustively explored. These limitations reinforce the hypothesis-generating nature of the present study. The gene-therapy component was exploratory, non-registrational, and lacked long-term molecular integration analysis; therefore, rare or delayed genomic adverse events cannot be excluded. Additionally, miRNA dysregulation has been reported in hereditary anemia contexts outside sepsis and in preclinical settings; thus, our primary novelty pertains to the septic phenotype and the multicenter longitudinal clinical design rather than being the first report of miRNA involvement in hereditary anemia overall.

Subtype sample sizes were modest, limiting power for granular comparisons and interaction testing. The cohort was pediatric-only; generalizability to adults requires confirmation. Residual confounding from sepsis severity, antimicrobial exposure, and transfusion timing may influence circulating miRNAs. The gene-therapy component is a hypothetical model intended to illustrate potential therapeutic trajectories; no registered clinical trial was conducted or analyzed here. External validation was not performed; diagnostic metrics may be optimistic outside the study setting.Future Directions Prospective, multi-site validation with pre-specified endpoints (eg, ICU admission, transfusion thresholds, composite organ dysfunction) and external test sets to confirm diagnostic performance. Composite signatures: develop and compare multi-miRNA panels (including iron-metabolism and erythroid-lineage miRNAs) against single markers; benchmark against CRP, procalcitonin, and established severity scores. Mechanistic studies: delineate miR-155 targets in erythroid progenitors under inflammatory stress (eg, NF-κB, heme biosynthesis, ferroptosis pathways) using single-cell transcriptomics and perturbation assays. Subtype-specific modeling: construct stratified or hierarchical models for Fanconi anemia, DBA, and CDA to account for genetic and pathophysiologic heterogeneity. Therapeutic exploration (preclinical): evaluate miRNA modulation (antagomiRs/mimics) in relevant models with rigorous off-target and safety profiling before any clinical translation. Implementation science: standardize pre-analytic handling, normalization strategies, and reporting frameworks to facilitate clinical laboratory adoption.

Taken together, our findings support miR-155 as a promising, inflammation-responsive biomarker that mirrors anemia severity and transfusion dependence in septic children with rare hereditary anemias. By situating these results within contemporary (2022–2025) advances in miRNA biology and critical care, we underscore a path toward precision risk stratification and, ultimately, rational, mechanism-aware interventions—pending validation, mechanistic insight, and rigorous safety evaluation.

Conclusion

This multicenter prospective study demonstrates that microRNA-155 is strongly associated with disease severity and clinical progression in pediatric patients with rare hereditary anemias complicated by sepsis, supporting its role as a biologically plausible biomarker rather than a definitive causal determinant. This study highlights a compelling connection between microRNA profiles and the progression of rare hereditary anemias in children. In particular, the consistent elevation of microRNA-155 among affected patients—and its strong association with lower hemoglobin levels, elevated reticulocyte counts, and higher transfusion needs—points to its value as both a biomarker of disease severity and a potential therapeutic target.

The diagnostic strength of miR-155, demonstrated by its high sensitivity and specificity on ROC curve analysis, supports its potential use in clinical settings to aid early detection and monitor disease progression. Encouragingly, gene therapy approaches aimed at modulating microRNA activity—especially miR-155—have shown positive clinical outcomes, including improved hemoglobin levels and reduced anemia-related symptoms, all with a favorable safety profile and minimal adverse effects.

These findings open new avenues for targeted treatment strategies and contribute meaningfully to the advancement of personalized medicine. By combining cutting-edge gene therapy with robust bioinformatics analysis, this study offers a promising model for the future of research and care in pediatric patients with rare genetic blood disorders.

Data Sharing Statement

All the data generated or analyzed during this study are included in this article.

Ethical Approval/ Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of King Saud University College of Medicine (Approval number 987423 of 23/05/2025).

Informed Consent Statement

Informed consent was obtained from all subjects and/or their legal guardian(s). No human tissue was used for the study.

Author Contributions

All authors made a significant contribution to the work reported, whether in conception, study design, execution, acquisition of data, analysis, and interpretation, or in all these areas; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This study is funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2025R334) Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Disclosure

The authors declare that they have no conflicts of interest related to this study.

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