Functional assessment of inherited myeloid neoplasm-associated SAMD9L germline variants via Monoallelic CRISPR modelling.

Comprehensive genomic analysis of five kindreds with multiple childhood leukemias: importance of individual functional analysis for rare ETV6 germline variants

Germline variants in leukemia predisposition genes are increasingly detected in pediatric patients. Nevertheless, the rare variants identified in diagnostic samples may be misinterpreted without variant-level functional evaluation and cautious clinical interpretation. Whole-exome sequencing was performed on 28 individuals from five kindreds in which at least two children developed acute leukemia. Results identified a rare ETV6 variant, c.604C > G (p.Arg202Gly), in monozygotic twins with ETV6::RUNX1-positive B-cell precursor acute lymphoblastic leukemia. To support variant interpretation, HeLa-cell populations stably expressing FLAG-tagged wild-type ETV6 and p.Arg202Gly and the known loss-of-function control p.Pro214Leu were established. Subcellular localization was assessed via immunofluorescence microscopy with quantitative scoring. Transcriptional repression was evaluated using luciferase reporter assays driven by ETV6 target promoters (MMP3 and PF4). p.Arg202Gly predominantly showed nuclear localization comparable to WT, whereas p.Pro214Leu was enriched in the cytoplasm. In the reporter assays, similar to WT, p.Arg202Gly retained repression activity. Meanwhile, p.Pro214Leu failed to repress both reporters. The in-silico prediction of nuclear export sequences suggested an additional export signal in p.Pro214Leu, but not in p.Arg202Gly. Collectively, these findings indicate that p.Arg202Gly behaves as WT-like in the assays performed and do not support a loss-of-function effect. Our study emphasizes the importance of variant-level functional assessment for rare ETV6 variants to inform clinical interpretation and avoid overestimation of pathogenicity.

Genomic Characterization of Epigenetic Regulator Gene Alterations in Juvenile Myelomonocytic Leukemia Through Whole-Exome Sequencing

Background/Objectives: Juvenile myelomonocytic leukemia (JMML) is a rare, highly aggressive form of pediatric myelodysplastic/myeloproliferative neoplasm characterized by molecular heterogeneity and constitutive activation of the RAS signaling pathway. This study aimed to characterize the mutational landscape, driver genes, mutational signatures, functional pathways, and therapeutic potential of mutated epigenetic regulator genes in JMML. Methods: Tumor and matched buccal swab samples were collected from 35 JMML patients, and whole-exome sequencing was performed. Somatic variants were called with GATK-Mutect2 and annotated with ANNOVAR. maftools and OncodriveCLUST were used for mutational profiling, co-occurrence analysis, driver gene identification, and protein domain mapping. Drug–gene interactions were explored using DGIdb, mutational signatures for genes were characterized by MutationalPatterns, and functional enrichment analysis was performed by the clusterProfiler package. Results: A total of 28 variants were detected in epigenetic regulator genes, with missense mutations being the most common class of variants and a C>T nucleotide substitution pattern being the most frequent. EP300, SETD2, and DNMT3B were the genes most frequently altered, with 8.57% of cases each, followed by ASXL1, BCORL1, ATRX, KMT2A, and TET2 (5.71% each). Driver gene analysis identified ASXL1 as the top candidate driver gene, followed by BCORL1, EP300, and SETD2. Functional enrichment analysis revealed a high number of genes involved in chromatin organization, histone modification, transcriptional regulation, and oncogenic signaling pathways. The mutational signatures identified were SBS5-like, which are dominated by C>T and T>C transitions, indicating endogenous mutational processes. Analysis of drug–gene interactions revealed KMT2A, EP300, and ATRX as the most interconnected and potentially actionable therapeutic targets. Conclusions: This study provides a comprehensive characterization of epigenetic regulator gene alterations in JMML and highlights the importance of epigenetic dysregulation in disease pathogenesis.

PTPRJ Drives Clonal Selection in CEBPA-mutated AML

Identification of novel UBE2E3::PDE1A and CTC1::MEF2C fusion transcripts in acute myeloid leukemia

Acute myeloid leukemia (AML) is a hematologic malignancy caused by the malignant proliferation and differentiation block of immature myeloid cells (blasts) in the bone marrow. The pathogenesis, diagnostic classification, treatment, and prognosis of AML are fundamentally linked to the accumulation of genetic variants, fusion gene formation, and chromosomal karyotype abnormalities. Using mRNA sequencing (next-generation sequencing, NGS), we identified and validated two novel fusion genes, UBE2E3::PDE1A and CTC1::MEF2C, in a case of AML-M2a. This patient also harbored six single nucleotide variants (SNV) (EP300, KRAS, NRAS, RAD21, TET2, and TP53) and complex karyotype (CK). The chromosomes exhibited extreme instability and high variability, with tumor cells containing multiple clones and showing continuous karyotype evolution as the disease progressed. The patient was treated with three chemotherapy regimens (VMA, VHAA, and Decitabine-Venetoclax), but none achieved remission. We propose that the combination of these novel fusion genes, multiple SNVs, and the complex karyotype collectively contributed to the patient’s multidrug resistance.

Characterisation of a novel p53 mutation (p53N263Tfs*7) associated with li-fraumeni syndrome

Li-Fraumeni Syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in the TP53 tumour suppressor gene, which encodes the multifunctional transcription factor p53. p53 is the most commonly mutated protein in human cancer, with the majority occurring within the DNA-binding domain, often disrupting transcriptional activity and resulting in a loss-of-function. Here, we characterise the novel p53 N263Tfs*7 truncated mutant, identified as a germline mutation from a patient with LFS who developed breast cancer. Functional assays revealed a partial loss-of-function across key cellular processes, including proliferation, cell death, cell motility, and transcriptional transactivation. This mutant lacked a dominant-negative effect, distinguishing it from common DNA-binding domain missense mutations. Our findings demonstrate that oncogenesis in LFS can be driven by partial impairment of functional p53, rather than dominant-negative or gain-of-function mutations alone. This underscores the clinical significance of recognising subtle TP53 variants for the refined molecular classification and clinical prediction of cancer risk in TP53 mutation carriers.

Shared inheritance reveals landscape of somatic and germline cancer risk in TP53

Pathogenic variants in TP53, the key tumour-suppressor gene underlying Li-Fraumeni syndrome (LFS), are among the best-established causes of inherited cancer predisposition. However, large-scale sequencing has revealed that many apparently pathogenic TP53 variants detected in blood are the result of somatic clonal expansions, complicating risk interpretation. Using blood-derived whole-exome data from 469,391 UK Biobank participants, we combined variant allele fraction (VAF) with haplotype-sharing analysis to distinguish germline and somatic TP53 variants. Germline variants were concentrated at sites linked to partial loss of p53 function and lower disease penetrance, whereas classic LFS alleles appeared almost entirely somatic. High-VAF carriers of classic LFS alleles conferred markedly increased risk of haematological malignancy but not solid tumours, consistent with large TP53-mutant clonal expansions. The prevalence of somatic clonal expansion also correlated with missense variant pathogenicity, suggesting that somatic activity provides an informative in vivo proxy for functional impact. These results provide new insights into TP53-associated cancer risk at the population level, demonstrate that somatic rather than germline risk predominates in middle-aged healthy adults and provide a scalable framework for variant classification in large-scale population genomics.