Molecular classification and outcomes in pediatric aplastic anemia with myeloid neoplasm-associated gene variants

Background: The genetic variations in aplastic anemia (AA) patients are closely related to clonal hematopoiesis, but there is limited research on this topic in children with AA. The aim of this study is to investigate the molecular classification and outcomes of children with AA combined with myeloid neoplasm-associated gene variants.

Methods: The clinical features, types of gene variants, mechanisms of action of the mutated genes, and correlations between gene variants and the outcomes of AA patients with myeloid neoplasm-associated gene variants were retrospectively analyzed.

Results: Forty-six AA patients with myeloid neoplasm-associated gene variants were included, and a total of 20 gene variants were identified. The most frequent variant affected TET2 (9 patients, 19.6%), followed by ASXL1 (5 patients, 10.9%) and MPL (5 patients, 10.9%). Other variants, in descending order, affected TERT (4 patients); SH2B3, FLT3, ETV6, and JAK2 (3 patients each); BCOR, BCORL1, TP53, KIT, and SF3B1 (2 patients each); and CALR, GATA2, RUNX1, CBL, IDH1, IDH2, and WT1 (1 patient each). Six patients had 2 gene variants. The original mechanisms of action of the mutated genes mainly involved epigenetics and signal transduction pathways; both groups of genes were affected in 39.1% (18/46) of the patients. The difference in the efficacy of immunosuppressive therapy (IST) among the different gene groups was not significant. Disease severity (P = 0.046) and hematological response at 3 months (P = 0.002), 6 months (P = 0.001), 9 months (P = 0.001), and 1 year (P = 0.001) were important factors affecting survival time, but genotype was not. None of the patients experienced clonal evolution by the end of the follow-up cut-off time.

Conclusion: In patients with AA combined with myeloid tumor neoplasm-associated gene variants, TET2, ASXL1 and MPL variants were the most frequently observed and primarily involved epigenetics and signal transduction pathways. There was no significant difference in the efficacy of IST among patients with different gene variants. Survival time was associated with disease severity, and the development of a hematological response—particularly when achieved at 3 months—was an independent key factor, whereas genotype was not.

1 Introduction

Acquired aplastic anemia (AA) is a disease characterized by immune-mediated bone marrow failure caused by T-cell-mediated destruction of hematopoietic stem and progenitor cells (HSPCs). When immunosuppressive therapy (IST) is used as the first-line treatment for AA patients for whom a suitable donor cannot be identified, the overall survival (OS) rate can reach 80%–85% (1). After hematopoietic function recovery, owing to the relative growth or survival advantages of several somatic variants, AA patients may experience clonal expansion of cells, among whom 15%–20% will experience additional genetic changes and progress to secondary myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML). A prospective study of the late complications of AA revealed that the possibility that AA patients will progress to paroxysmal nocturnal hemoglobinuria (PNH)/MDS/AML increases with time (2).

With the application of next-generation sequencing technology, clonal hematopoiesis can be detected in the hematopoietic stem cells of more than 70% of AA patients, while somatic variants in myeloid neoplasm-associated genes can be detected in approximately 30% of AA patients at the time of diagnosis. These findings suggest that patients with myeloid neoplasm-associated gene variants may be at potential risk of developing MDS/AML, in whom clonal evolution is associated with adverse outcomes (3, 4). However, reports on the characterization of the somatic gene variants that lead to AA clonal hematopoiesis in children with AA are rare (5). This study retrospectively analyzed the clinical features, types, and mechanisms of these gene variants and the correlations between gene variants and outcomes in AA patients with myeloid neoplasm-associated gene variants to provide reference data for the clinical diagnosis and treatment of the disease.

2 Patients and methods

2.1 Patients

The clinical data of children with AA admitted to our hospital between January 2018 and September 2022 were retrospectively analyzed. The inclusion criteria were as follows: ① the diagnostic criteria of the Diagnosis and Treatment of AA in Children guidelines (2019 edition) (6) alongside the presence of myeloid neoplasm-associated gene mutations; and ② prior IST treatment. The exclusion criteria were as follows: ① no myeloid neoplasm-associated gene mutations; and ② congenital bone marrow failure.

2.2 Genetic evaluation

Blood or bone marrow samples were collected from the patients and analyzed with an H030 hematology-oncology panel (designed by Makino), which covers 30 myeloid neoplasm-associated genes—ASXL1, BCOR, BCOR1, CALR, CSF3R, EZH2, FLT3, PIGA, GATA2, ETV6, RUNX1, TP53, CBL, DNMT3A, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NRAS, SETBP1, SF3B1, SRSF2, SH2B3, TET2, TERT, U2AF1, WT1, and ZRSR2. The panel was run by Makino Company. Target gene regions are captured and enriched, followed by sequencing of the captured DNA fragments.The sequencing reads are then aligned to the human reference genome (GRCh37/hg19) for identification of gene mutation sites.

2.3 Detection of PNH clones

CD55 and CD59: The absence of CD55 and CD59 on granulocytes and erythrocytes was detected using flow cytometry. The proportions of CD55+ and CD59+ cells were classified as follows: <90%, abnormal; 90%–95%, approximately normal; and >95%, normal.

FLAER: Fluorescein-labeled proaerolysin (FLAER) is an Alexa-488-labeled inactive proaerolysin variant that specifically binds to all the glycated phosphatidylinositides on the granulocyte membrane (GPI) anchor protein. Patients were partially or completely negative for FLAER if the corresponding cell surface anchors were partially or completely absent, respectively, thus preventing or minimizing FLAER binding. If the percentage of FLAER-negative cells was more than 1%, they were considered to be abnormal clones. Testing was completed by Wuhan Kangshengda Medical Laboratory.

2.4 Treatment options for IST

All patients were treated orally with cyclosporine A (CsA) at 5 mg/(kg·d) once every 12 h, and the maintenance plasma concentration ranged from 100∼150 ng/mL (trough concentration).

2.5 Observation indicators

Efficacy evaluation: In accordance with 2019 guidelines for the Diagnosis and Treatment of AA in Children, efficacy was defined as follows: ① complete remission (CR): ANC > 1.5 × 10⁹/L, Hb > 110 g/L, PLT > 100 × 10⁹/L, independent from red blood cell and platelet transfusion. ② Partial remission (PR): ANC > 0.5 × 10⁹/L, Hb > 80 g/L, PLT >20 × 10⁹/L, independent from red blood cell and platelet transfusion. ③ No remission (NR): inability to achieve the criteria for PR or CR. The patients were followed up at 1, 3, 6, 9, and 12 months after IST, and the cut-off date was December 31, 2023. Early death was defined as death within 6 months after receiving IST, in which case the patient was classified as NR. Pediatric patients who underwent hematopoietic stem cell transplantation within 3–6 months after IST were also classified as NR. Acquisition of a hematological response (HR), including a PR and a CR, was defined as treatment effectiveness.

Clonal evolution was defined as the emergence of new abnormal cytogenetic clones or the transformation of myeloid patterns into MDS or AML.

2.6 Statistical methods

Data analysis was performed using SPSS 25.0 software. Measurement data that were not normally distributed are expressed as medians (ranges), and count data are expressed as percentages. Differences were analyzed using the chi-square test and Fisher's exact test. The patients were followed up until December 31, 2023, at which point OS was calculated. The log-rank test was used to assess the difference in survival between the groups. P < 0.05 was considered to indicate statistical significance.

3 Results

3.1 Basic characteristics

From January 2018 to September 2022, a total of 224 patients were diagnosed with AA, among whom 46 patients had myeloid neoplasm-associated gene mutations (46/224, 20.5%). There were 20 males and 26 females in this group; the male to female ratio was 1:1.3. The median age of onset was 5 years and 4 months (ranging from 10 months to 13 years and 11 months), and onset was most common between 3 and 5 years of age. The median duration of the disease was 30 (1–720) days. Twelve patients had nonsevere aplastic anemia (NSAA) (12/46, 26.1%), 16 had severe aplastic anemia (SAA) (16/46, 34.8%), and 18 had very severe aplastic anemia (VSAA) (18/46, 39.1%). Patients mainly sought medical attention due to pallor, fever, bleeding, and abnormal blood counts detected during physical examinations at the onset of the disease. Among them, 4 patients (4/46, 8.7%) had abnormal blood counts detected during physical examinations without typical symptoms. All patients completed bone marrow cytology and bone marrow biopsy examinations. Among them, 40 patients showed varying degrees of hypoplasia, while bone marrow cytology indicated active bone marrow hyperplasia in 6 patients (6/40, 13.0%).Of the 46 patients who underwent bone marrow biopsy, 2 patients showed active hyperplasia with a hematopoietic cell proportion of 40%–50%, and the rest showed hypoplasia. In 6 patients with active hyperplasia indicated by bone marrow cytology, bone marrow biopsy indicated hypoplasia; in 2 patients with active hyperplasia indicated by bone marrow biopsy, cytology indicated hypoplasia. All patients underwent complete chromosomal karyotype analysis, and no abnormal chromosomes were detected.

3.2 Genetic evaluation

Variants in twenty genes, namely, ASXL1, BCOR, BCORL1, CALR, FLT3, GATA2, ETV6, RUNX1, TP53, CBL, IDH1, IDH2, JAK2, KIT, MPL, SF3B1, SH2B3, TET2, TERT, and WT1, were detected in the 46 patients. The variants detected by gene sequencing examination in 46 patients, including the type of variant, variant allele frequency (VAF), clinical significance, and predicted effect on encoded protein (Table 1). The most frequently mutated gene was TET2 (9 patients, 19.6%), followed by ASXL1 (5 patients, 10.9%) and MPL (5 patients, 10.9%). In descending order of frequency, the mutated genes included TERT (4 patients); SH2B3, FLT3, ETV6, and JAK2 (3 patients each); and BCOR, BCORL1,TP53, KIT, and SF3B1 (2 patients each); and CALR, GATA2, RUNX1, CBL, IDH1, IDH2, and WT1 (1 patient each). Forty patients had only a single gene mutation; the other 6 patients had 2 gene variants: ASXL1 and IDH1, BCOR and TET2, MPL and TP53, TERT and TET2, TERT and MPL, and ETV6 and KIT (Figure 1).

Table 1

Table 1. Characteristics of gene variants in myeloid tumors among 46 patients.

Figure 1

Figure 1. Types of gene variants in myeloid tumors among 46 patients.

Based on the genes' mechanisms of action, the 20 mutated genes were divided into epigenetic genes, transcription regulator genes, signal transduction pathway genes, and spliceosome-related genes. The epigenetic genes included ASXL1, TET2, BCOR, BCORL1, IDH1, and IDH2. The transcription regulator genes were RUNX1, ETV6, TERT, TP53, GATA2, and WT1. The signal transduction pathway genes included JAK2, MPL, SH2B3, FLT3, CALR, CBL, and KIT. SF3B1 is the sole spliceosome-related gene. The genes associated with epigenetics and signal transduction pathways were the main genes. The KEGG database was used to perform pathway enrichment analysis on 20 genes, and the results showed that these genes are mainly involved in tumor related metabolic and transcriptional regulatory pathways. The main pathways involved are central carbon metabolism in cancer, transcriptional misregulation in cancer, AML, chronic myeloid leukemia, polycomb repressive complex, citrate cycle (TCA cycle), and 2-Oxocarboxylic acid metabolism (Figure 2).

Figure 2

Figure 2. KEGG enrichment analysis of 20 genes.

There was no statistical significance between gene mutations and baseline blood parameters (white blood cells, P = 0.086; absolute neutrophil count, P = 0.064; absolute lymphocyte count, P = 0.562; red blood cells, P = 0.276; hemoglobin, P = 0.091; platelets, P = 0.406; reticulocyte count, P = 0.189).There was no significant difference between the different types of gene variants and the sex, age, disease course, or severity of the disease (Table 2). The reduction in sample size in the figure is due to patients undergoing hematopoietic stem cell transplantation (HSCT), death, and loss to follow-up. These cases were directly excluded to conduct hematological assessments at each time point, aiming to determine whether there were statistical differences in response rates between the groups.

Table 2

Table 2. Clinical characteristics among different types of gene variants.

3.3 PNH detection

Forty-one patients underwent PNH clonal detection. In 4 patients, the CD55/CD59 ratio was between 90% and 95%, indicating approximately normal levels. The levels were normal in the remaining 37 patients. The percentage of FLAER-negative cells was >1% for two patients; thus, they were considered to harbor abnormal clones. One patient exhibited a monocyte clone percentage of 4.65% and a granulocyte clone percentage of 5.77%; this patient carried an FLT3 variant. Another patient exhibited a monocyte clone percentage of 0.67% and a granulocyte clone percentage of 0.91%; this patient carried an SH2B3 variant.

3.4 Evaluation of efficacy

Event-Free Survival (EFS) is defined as the time from diagnosis to the first event at the last follow-up. An event is defined as PR or CR. The overall effective rates for 1, 3, 6, 9, and 12 months of IST treatment were 7.1% (3/42), 33.3% (12/36), 48.1% (13/27), 66.7% (14/42), and 71.4% (15/21), respectively. There was no significant difference in the total effective rate of different degrees of AA at the time points noted above (Supplementary Table 1). Sixteen patients received allogeneic hematopoietic stem cell transplantation (HSCT). Of these patients, 11 underwent HSCT in our hospital, and 5 underwent HSCT in other hospitals. The median time from diagnosis to HSCT was 6 months (3.0–7.8 months). Two patients died after transplantation: 1 patient had severe intestinal GVHD, 1 patient had refractory septic shock and heart failure. The remaining patients survived and achieved CR.

Among the 46 patients, 40 had single gene variants (40/46, 86.9%). For 9 patients with TET2 variants, the effective rates at 3 and 6 months were 37.5% (3/8) and 42.9% (3/7), respectively. Of the 5 patients with ASXL1 variants, 1 also had an IDH1 variant. This patient died of septic shock 9 months after diagnosis. of the 5 patients with MPL variants, 2 patients had 2 gene variants. Significant differences in the platelet count (P = 0.053) or megakaryocyte count in the bone marrow (P = 0.567) were not observed between patients with MPL variants and those with other gene variants. There was no significant difference in the hematological reaction rate after IST in patients with different genotypes (Table 3). Differences in sample size occurred because patients underwent HSCT, died, or were lost to follow-up during treatment. Among the 40 patients with single-gene variants, 12 underwent HSCT, including 5 patients with single-gene variants in epigenetic regulators (3 with TET2 variants, 1 with IDH2 variant, 1 with ASXL1 variant), 2 with variants in transcriptional regulators (TP53, ETV6), and 5 with variants in signal transduction genes (SH2B3, CALR, CBL, MPL, KIT).

Table 3

Table 3. Hematologic response among different types of gene variants.

3.5 Survival and clonal evolution

The median follow-up time was 23 months (3 months to 37.25 months). Nine patients were lost to follow-up, 29 patients were alive, and 8 patients died. Among the 29 surviving patients, 15 patients received IST treatment, 7 patients achieved a PR, 7 patients achieved a CR, and 1 patient had NR and was dependent on blood component transfusion. Among the 8 patients who died, 6 underwent IST. In addition, 3 died from infectious diseases at 1 month (VSAA with WTI variant), 3 months (VSAA with FLT3 variant), and 9 months (VSAA with ASXL1 and IDH1 variant) after diagnosis. One SAA patient with an ETV6 variant died of cerebral hemorrhage 3 months after diagnosis, and one VSAA patient with a FLT3 variant died of airway bleeding 3 months after diagnosis. In addition, the cause and time of death of one SAA patient with a BCORL1 variant were unknown. At the end of follow-up, no patient had progressed to PNH or MDS/AML. Univariable survival analysis revealed that disease severity and the hematological response at 3, 6, 9, and 12 months of treatment were significant factors influencing survival time distribution (P < 0.05). In contrast, sex, age, disease course, gene variant status and prior HSCT were not significantly associated with survival outcomes. This study has a small sample size. Additionally, There is collinearity in efficacy data during the late-stage follow-up for patients who received treatment; coupled with a reduction in long-term efficacy data, these issues led to abnormal hazard ratios (HR) in the Cox proportional hazards model and meaningless P-values. Combining Kaplan–Meier (K–M) survival analysis with the Cox proportional hazards model, it can be concluded that achieving a hematological response at 3 months after treatment is a key factor that significantly influences survival. (Supplementary Table 2, Supplementary Figure 1).

4 Discussion

Currently, the remission rate of AA pediatric patients treated with IST is higher than that of adult patients. Pediatric patients can achieve a relatively high long-term survival rate, but they also experience long-term complications. The likelihood of long-term progression of AA patients to PNH/MDS/AML increases over time, but the cause of this progression remains unclear. Next-generation sequencing has shown that 70% of patients have somatic variants, and in some patients, these somatic variants affect myeloid neoplasm-associated genes (7, 8). This study revealed that patients with myeloid neoplasm-associated gene variants accounted for 20.5% of AA patients diagnosed during the same period. The most commonly mutated genes included TET2, ASXL1 and MPL, and the variant rate was higher in the SAA group compared with the NSAA group, which was similar to what has been reported in the literature (9).

On the basis of their mechanisms of action, myeloid neoplasm-associated gene that experienced variants in this group can be divided into epigenetic genes, transcription regulator genes, signal transduction pathway genes, and spliceosome-related genes. The epigenetic genome is closely related to the proliferation or targeted differentiation of hematopoietic cells (10). The epigenetic regulators ASXL1, TET2, IDH1, and IDH2 regulate chromatin structure (11). The most common somatic variants in AA patients are those of BCOR and its ligands BCORL1, DNMT3A, TET2, ASXL1, and PIGA (12). The risk of clonal transformation to MDS/AML in patients with ASXL1, TP53, RUNX1, and DNMT3 variants is 40% higher than that in patients with PIGA, BCOR, and BCORL1 variants (13). In this study, 43.4% of patients had epigenetic gene variants, most of which were TET2 and ASXL1 variants. Among them, 2 patients had 2 epigenetic genes (ASXL1 and IDH1; BCOR and TET2). The ASXL1 gene is associated with a poor prognosis in malignant hematological tumors. TET2, an epigenetic modifier gene, directly promotes osteogenesis and adipogenesis of bone marrow mesenchymal stem cells and promotes hematopoietic stem cell differentiation into the myeloid lineage (14). Among the transcription regulators, RUNX1 regulates the transcriptional programs of hematopoiesis. The tumor protein P53 (TP53) is a transcription factor that is frequently mutated in cancers and is critical for appropriate cell responses to stress and DNA damage (11). ETV6 regulates and promotes the differentiation of hematopoietic stem cells through bone marrow mesenchymal stem cells. TERT variant can lead to changes in telomerase activity. Leukocyte telomere length is an important predictor of the development of malignant clones and is associated with the recurrence and mortality of aplastic anemia. The shorter the telomeres are, the higher the likelihood of clonal evolution (15). The signal transduction genes JAK2, CALR and MPL are involved in JAK-STAT signaling pathway activation. Activated JAK-STAT signaling is at the core of the pathogenesis of BCR-ABL-negative myeloproliferative neoplasms (MPNs) (11). CBL mutations are rare in AA. In this study, one patient harbored a CBL variant, with the disease phenotype presenting as juvenile myelomonocytic leukemia (JMML). Protein function prediction indicated the variant was deleterious, and pathogenicity analysis suggested it was pathogenic. Both hematological tests and bone marrow examinations of this patient supported the diagnosis of VSAA. Previous studies have shown that CBL variants are associated with susceptibility to JMML (16); however, it remains unclear whether early-stage JMML caused the bone marrow hematopoietic dysfunction in this case. The patient developed the disease at the age of 13 years and 5 months, with a short disease duration and a disease severity of VSAA. HSCT was performed one month after diagnosis, and the patient's blood counts remained normal as of the last follow-up.

In this study, 34.7% (16/46) of the patients had gene variants in signaling pathways, including 5 with MPL mutations, 3 with JAK2, 3 with FLT3, and 3 with SH2B3. Mutation of the spliceosome-related gene SF3B1 is closely related to ineffective erythrocyte formation and is an early event in the development of hematopoietic stem cell (HSPC) dysfunction (17). SH2B3 is a negative regulator of the JAK-STAT pathway. Some people believe that SH2B3 variants are related to clonal hematopoiesis (18). In this study, 2 patients had SF3B1 variants. The KEGG enrichment analysis of genes clearly showed that 20 genes were significantly enriched in key biological pathways related to tumors. These pathways mainly focus on metabolic reprogramming of tumor cells and dysregulation of transcriptional regulatory networks, indicating that these genes play important driving roles in tumor occurrence, development, and maintenance of malignant phenotypes. Studies have found that the gene mutations and signaling pathways that drive the evolution of hematopoietic clones in AA patients significantly overlap with the tumor related metabolic and transcriptional regulatory pathways identified by the KEGG enrichment analysis mentioned above (19).

In this study, protein function prediction indicated 9 patients had pathogenic or likely pathogenic mutations, among whom 8 presented with SAA/VSAA. Pathogenicity analysis identified 5 patients with pathogenic variants, including 3 patients of SAA and 2 patients of AA. Due to the low incidence, the pathogenicity of most genes remains uncertain currently. Additionally, the pathogenicity of different variant sites within the same gene is unclear, requiring further long-term exploration and analysis. The differential diagnosis between AA and hypoplastic myelodysplastic syndrome (hypo-MDS) remains clinically and morphologically challenging. In this study, we identified somatic gene mutations in a subset of pediatric AA patients. Functional validation of the encoded proteins and in silico pathogenicity analyses suggested potential pathogenicity for some of these mutations. However, due to the extremely low incidence of such mutations in pediatric AA, there is currently no consensus or clinical guideline regarding whether they represent true driver mutations or merely passenger mutations. Therefore, the clinical significance of these mutations warrants further validation in larger, prospective studies.

Genetic sequencing can assist in the differential diagnosis of congenital bone marrow failure disorders. However, the comparison of IST efficacy between pediatric AA patients with myeloid neoplasm-associated gene expression and those without requires verification with additional data.One study of 279 AA patients with a median age of 39 years (14–85 years) revealed that somatic variants were not significantly associated with treatment response or long-term survival (20), and a significant correlation was noted between TET2 variant and a favorable response to IST treatment (21). However, in this study, compared with patients without TET2 variants, patients with TET2 variants exhibited no significant differences in the effectiveness of IST treatment for 1, 3, 6, 9, or 12 months. In addition, no deaths occurred among patients with TET2 variants. ASXL1 variants are common in AA and MDS/AML, and ASXL1 variants in AA are associated with clonal evolution and poor prognosis (22, 23). Patients with PIGA and BCOR/BCORL1 variants have better responses to IST treatment and longer OS and progression-free survival (PFS) (22, 24). In this study, 5 patients had ASXL1 variants, 2 of whom died. Four patients had BCOR/BCORL1 variants, and 2 patients achieved a PR and a CR at 3 months and 6 months of treatment, separately.

Adult AA patients with PNH clones respond well to IST (25), and one study revealed that 39% of children with AA had detectable PNH clones (26). SAA children who are newly diagnosed with a positive PNH clone have a poorer response to IST and are more likely to develop AA-PNH syndrome (27). In this study, 2 patients exhibited abnormal clones according to FLAER analysis, 1 patient was lost to follow-up, and 1 patient did not progress to PNH after transplantation. By the end of follow-up, no patient had progressed to AML/MDS. However, the follow-up time was short, and long-term follow-up is still needed. For patients with somatic variants, the course of the disease has a significant effect on the risk of MDS transformation (28). Studies have shown that 15%–20% of AA patients develop secondary MDS/AML after 10 years of follow-up (29). However, the probability of AA children developing MDS/AML is much lower than that of adult patients with AA. In a North American study of 314 AA children, 6 (1.9%) patients progressed to MDS/AML (30). In this study, none of the patients developed MDS/AML after 2 years of follow-up. This finding may be related to the short follow-up time; therefore, long-term follow-up is needed.

In conclusion, the majority of myeloid neoplasm-associated gene variants in AA patients affected TET2, ASXL1 and MPL, and epigenetic- and signal transduction pathway-related genes were the most commonly involved. No significant differences in IST efficacy were observed in patients with different gene variants. Survival was correlated to the severity of the disease and whether a hematological response was obtained after treatment. Although clonal evolution did not occur, follow-up monitoring still needs to be continued for these patients. This study has limitations including a single-center design, small sample size, and short follow-up duration. While the present study only presents data on gene variants in 46 patients in the figure, subsequent research can be advanced in multiple aspects to enhance its value, considering the limited sample size of pediatric AA and the low incidence of the disease itself. First, the sample size can be expanded through multicenter collaboration, and more in-depth investigations can be conducted in the field of molecular genetics to provide more sufficient evidence for clarifying the clinical significance of these mutations. Second, long-term follow-up of patients is necessary to track their treatment efficacy and outcomes; during follow-up, relevant genetic analyses and next-generation sequencing (NGS) can be supplemented to compare and evaluate dynamic changes in genes. Furthermore, basic experiments can be designed to verify the issues observed in clinical practice, making the research conclusions more persuasive and clinically translatable.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Ethics statement

The studies involving humans were approved by Institutional Review Board of Children's Hospital of Chongqing Medical University. The studies were conducted in accordance with the local legislation and institutional requirements. The ethics committee/institutional review board waived the requirement of written informed consent for participation from the participants or the participants' legal guardians/next of kin because This project is a retrospective study conducted using medical records obtained from previous clinical diagnosis and treatment, which did not pose unnecessary risks to the subjects. We promised not to disclose patient privacy information.

Author contributions

DL: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Writing – original draft, Writing – review & editing. ML: Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing. YL: Data curation, Formal analysis, Investigation, Software, Writing – review & editing. LZ: Data curation, Formal analysis, Methodology, Writing – review & editing. YH: Formal analysis, Investigation, Writing – review & editing. YG: Formal analysis, Investigation, Writing – review & editing. XG: Investigation, Supervision, Validation, Writing – review & editing. YD: Conceptualization, Investigation, Supervision, Validation, Writing – review & editing. XW: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Validation, Writing – original draft, Writing – review & editing.

Funding

The author(s) declare that financial support was received for the research and/or publication of this article. This work was supported by the Chongqing Municipal Science and Health Joint Medical Research Project, 2021MSXM065.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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The author(s) declare that no Generative AI was used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fped.2025.1700402/full#supplementary-material

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