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Early identification of resistant cancer cells is currently a major challenge, as their expansion leads to refractoriness. To capture the dynamics of these cells, we made a comprehensive analysis of disease progression and treatment response in a chronic lymphocytic leukemia (CLL) patient using a combination of single-cell and bulk genomic methods. At diagnosis, the patient presented with unfavorable genetic markers, including notch receptor 1 (NOTCH1) mutation and loss(11q). The initial and subsequent treatment lines did not lead to a durable response and the patient developed refractory disease. Refractory CLL cells featured substantial dysregulation in B-cell phenotypic markers such as human leukocyte antigen (HLA) genes, immunoglobulin (IG) genes, CD19 molecule (CD19), membrane spanning 4-domains A1 (MS4A1; previously known as CD20), CD79a molecule (CD79A) and paired box 5 (PAX5), indicating B-cell de-differentiation and disease transformation. We described the clonal evolution and characterized in detail two cell populations that emerged during the refractory disease phase, differing in the presence of high genomic complexity. In addition, we successfully tracked the cells with high genomic complexity back to the time before treatment, where they formed a rare subpopulation. We have confirmed that single-cell RNA sequencing enables the characterization of refractory cells and the monitoring of their development over time., (© 2024 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Germline mutations in ETV6 gene cause inherited thrombocytopenia with leukemia predisposition. Here, we report on functional validation of ETV6 W380R mutation segregating with thrombocytopenia in a family where two family members also suffered from acute lymphoblastic leukemia (ALL) or essential thrombocythemia (ET). In-silico analysis predicted impaired DNA binding due to W380R mutation. Functional analysis showed that this mutation prevents the ETV6 protein from localizing into the cell nucleus and impairs the transcriptional repression activity of ETV6. Based on the germline ETV6 mutation, ET probably started with somatic JAK2 V617F mutation, whereas ALL could be caused by diverse mechanisms: high-hyperdiploidity; somatic deletion of exon 1 IKZF1 gene; or somatic mutations of other genes found by exome sequencing of the ALL sample taken at the diagnosis.

The molecular genetics of well-characterized inherited diseases, such as phenylketonuria (PKU) and hyperphenylalaninemia (HPA) predominantly caused by mutations in the phenylalanine hydroxylase ( PAH ) gene, is often complicated by the identification of many novel variants, often with no obvious impact on the associated disorder. To date, more than 1100 PAH variants have been identified of which a substantial portion have unknown clinical significance. In this work, we study the functionality of seven yet uncharacterized PAH missense variants p.Asn167Tyr, p.Thr200Asn, p.Asp229Gly, p.Gly239Ala, p.Phe263Ser, p.Ala342Pro, and p.Ile406Met first identified in the Czech PKU/HPA patients. From all tested variants, three of them, namely p.Asn167Tyr, p.Thr200Asn, and p.Ile406Met, exerted residual enzymatic activity in vitro similar to wild type (WT) PAH, however, when expressed in HepG2 cells, their protein level reached a maximum of 72.1% ± 4.9%, 11.2% ± 4.2%, and 36.6% ± 7.3% compared to WT PAH, respectively. Remaining variants were null with no enzyme activity and decreased protein levels in HepG2 cells. The chaperone-like effect of applied BH4 precursor increased protein level significantly for p.Asn167Tyr, p.Asp229Gly, p.Ala342Pro, and p.Ile406Met. Taken together, our results of functional characterization in combination with in silico prediction suggest that while p.Asn167Tyr, p.Thr200Asn, and p.Ile406Met PAH variants have a mild impact on the protein, p.Asp229Gly, p.Gly239Ala, p.Phe263Ser, and p.Ala342Pro severely affect protein structure and function.

Activation-induced cytidine deaminase (AID) is a mutator enzyme essential for somatic hypermutation (SHM) and class switch recombination (CSR) during effective adaptive immune responses. Its aberrant expression and activity have been detected in lymphomas, leukemias, and solid tumors. In chronic lymphocytic leukemia (CLL) increased expression of alternatively spliced AID variants has been documented. We used real-time RT-PCR to quantify the expression of AID and its alternatively spliced transcripts (AIDΔE4a, AIDΔE4, AIDivs3, and AIDΔE3E4) in 149 CLL patients and correlated this expression to prognostic markers including recurrent chromosomal aberrations, the presence of complex karyotype, mutation status of the immunoglobulin heavy chain variable gene, and recurrent mutations. We report a previously unappreciated association between higher AID transcript levels and trisomy of chromosome 12. Functional analysis of AID splice variants revealed loss of their activity with respect to SHM, CSR, and induction of double-strand DNA breaks. In silico modeling provided insight into the molecular interactions and structural dynamics of wild-type AID and a shortened AID variant closely resembling AIDΔE4, confirming its loss-of-function phenotype.

Phenylketonuria (PKU) and hyperphenylalaninemia (HPA) are a group of genetic disorders predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene. To date, more than 950 variants have been identified, however the pathogenic mechanism of many variants remains unknown. In this study, in silico prediction and in vitro prokaryotic and eukaryotic expression systems were used to functionally characterize five PAH missense variants (p.F233I, p.R270I, p.F331S, p.S350Y, and p.L358F) previously identified in Slovak and Czech patients. p.F233I, p.R270I, and p.S350Y were classified as deleterious mutations since they showed no specific activity in functional assay and no response to chaperone co-expression. Protein levels of these PAH variants were very low when expressed in HepG2 cells, and only p.S350Y responded to BH4 precursor overload by significant increase in PAH monomer, probably due to reduced rate of protein degradation as the result of proper protein folding. Variants p.F331S and p.L358F exerted residual enzymatic activity in vitro. While the first can be classified as probably pathogenic due to its very low protein levels in HepG2 cells, the latter is considered to be mild mutation with protein levels of approximately 17.85% compared to wt PAH. Our findings contribute to better understanding of structure and function of PAH mutated enzymes and optimal treatment of PKU patients carrying these mutations using BH4 supplementation.