SCN1A intronic variants impact on Nav1.1 protein expression and sodium channel function, and associated with epilepsy phenotypic severity.

High-throughput sequencing has identified numerous intronic variants in the SCN1A gene in epilepsy patients. Abnormal mRNA splicing caused by these variants can lead to significant phenotypic differences, but the mechanisms of epileptogenicity and phenotypic differences remain unknown. Two variants, c.4853-1 G>C and c.4853-25 T>A, were identified in intron 25 of SCN1A, which were associated with severe Dravet syndrome (DS) and mild focal epilepsy with febrile seizures plus (FEFS+), respectively. The impact of these variants on protein expression, electrophysiological properties of sodium channels and their correlation with epilepsy severity was investigated through plasmid construction and transfection based on the aberrant spliced mRNA. We found that the expression of truncated mutant proteins was significantly reduced on the cell membrane, and retained in the cytoplasmic endoplasmic reticulum. The mutants caused a decrease in current density, voltage sensitivity, and an increased vulnerability of channel, leading to a partial impairment of sodium channel function. Notably, the expression of DS-related mutant protein on the cell membrane was higher compared to that of FEFS+-related mutant, whereas the sodium channel function impairment caused by DS-related mutant was comparatively milder than that caused by FEFS+-related mutant. Our study suggests that differences in protein expression levels and altered electrophysiological properties of sodium channels play important roles in the manifestation of diverse epileptic phenotypes. The presence of intronic splice site variants may result in severe phenotypes due to the dominant-negative effects, whereas non-canonical splice site variants leading to haploinsufficiency could potentially cause milder phenotypes.

Genomic Pipeline for Analysis of Mutational Events in Bacteria.

Unveiling the strategies of bacterial adaptation to stress constitute a challenging area of research. The understanding of mechanisms governing emergence of resistance to antimicrobials is of particular importance regarding the increasing threat of antibiotic resistance on public health worldwide. In the last decades, the fast democratization of sequencing technologies along with the development of dedicated bioinformatical tools to process data offered new opportunities to characterize genomic variations underlying bacterial adaptation. Thereby, research teams have now the possibility to dive deeper in the deciphering of bacterial adaptive mechanisms through the identification of specific genetic targets mediating survival to stress. In this chapter, we proposed a step-by-step bioinformatical pipeline enabling the identification of mutational events underlying biocidal stress adaptation associated with antimicrobial resistance development using Escherichia marmotae as an illustrative model.

A novel stoploss mutation CYB5R3 c.906A>G(p.*302Trpext*42) involved in the pathogenesis of hereditary methemoglobinemia.

Recessive congenital methemoglobinemia (RCM) is a hereditary autosomal disorder with an extremely low incidence rate. Here, we report a case of methemoglobinemia type I in a patient with congenital persistent cyanosis. The condition was attributed to a novel compound heterozygous mutation in CYB5R3, characterized by elevated methemoglobin levels (13.4 % of total hemoglobin) and undetectable NADH cytochrome b5 reductase (CYB5R3) activity. Whole-exome sequencing (WES) revealed two heterozygous mutations in CYB5R3: a previously reported pathogenic missense mutation c.611G>A(p.Cys204Tyr) inherited from the father, and a novel stop codon mutation c.906A>G(p.*302Trpext*42) from the mother, the latter mutation assessed as likely pathogenic according to ACMG guidelines. In cells overexpressing the CYB5R3 c.906A>G mutant construct, the CYB5R3 mRNA level was significantly lower than in cells overexpressing the wild-type (WT) CYB5R3 construct. However, there was no significant difference in protein expression levels between the mutant and WT constructs. Notably, an additional protein band of approximately 55 kDa was detected in the mutant cells. Immunofluorescence localization showed that, compared to wild-type CYB5R3, the subcellular localization of the CYB5R3 p.*302Trpext*42 mutant protein did not show significant changes and remained distributed in the endoplasmic reticulum and mitochondria. However, the c.906A>G(p.*302Trpext*42) mutation resulted in increased intracellular reactive oxygen species (ROS) levels and decreased NAD+/NADH ratio, suggesting impaired CYB5R3 function and implicating this novel mutation as likely pathogenic.

PBA2, a novel inhibitor of the ß-catenin/CBP pathway, eradicates chronic myeloid leukemia including BCR-ABL T315I mutation.

BACKGROUND: BCR-ABL is a constitutively active tyrosine kinase that stimulates multiple downstream signaling pathways to promote the survival and proliferation of chronic myeloid leukemia (CML) cells. The clinical application of specific BCR-ABL tyrosine kinase inhibitors (TKIs) has led to significantly improved prognosis and overall survival in CML patients compared to previous treatment regimens. However, direct targeting of BCR-ABL does not eradicate CML cells expressing T315I-mutated BCR-ABL. Our previous study revealed that inhibiting CREB binding protein (CBP) is efficacious in activating ß-catenin/p300 signaling, promoting cell differentiation and inducing p53/p21-dependent senescence regardless of BCR-ABL mutation status. We hypothesize that the specific inhibition of CBP may represent a novel strategy to promote ß-catenin/p300-mediated differentiation and suppress cancer cell proliferation for treating CML patients. METHODS: The anticancer efficacy of PBA2, a novel CBP inhibitor, in CML cells expressing wild-type or T315I-mutated BCR-ABL was investigated in vitro and in vivo. Cell differentiation was determined by the nitroblue tetrazolium (NBT) reduction assay. The extent of cellular senescence was assessed by senescence-associated ß-galactosidase (SA-ß-Gal) activity. Cytotoxicity was measured by MTS assay. RNA interference was performed to evaluate the cell proliferation effects of CBP knockdown. The interaction of ß-catenin and CBP/p300 was examined by co-immunoprecipitation assay. RESULTS: PBA2 exhibited significantly higher anticancer effects than imatinib in CML cells harboring either wild-type or T315I-mutated BCR-ABL both in vitro and in vivo. Mechanistically, PBA2 reduced CBP expression and promoted ß-catenin-p300 interaction to induce cell differentiation and senescence. CONCLUSION: Our data supported the rational treatment of CML by inhibiting the ß-catenin/CBP pathway regardless of BCR-ABL mutation status.

Identification and functional characteristics of CHD1L gene variants implicated in human Müllerian duct anomalies.

BACKGROUND: Müllerian duct anomalies (MDAs) are congenital developmental disorders that present as a series of abnormalities within the reproductive tracts of females. Genetic factors are linked to MDAs and recent advancements in whole-exome sequencing (WES) provide innovative perspectives in this field. However, relevant mechanism has only been investigated in a restricted manner without clear elucidation of respective observations. METHODS: Our previous study reported that 2 of 12 patients with MDAs harbored the CHD1L variant c.348-1G>C. Subsequently, an additional 85 MDAs patients were recruited. Variants in CHD1L were screened through the in-house database of WES performed in the cohort and two cases were identified. One presented with partial septate uterus with left renal agenesis and the other with complete septate uterus, duplicated cervices and longitudinal vaginal septum. The pathogenicity of the discovered variants was further assessed by molecular dynamics simulation and various functional assays. RESULTS: Ultimately, two novel heterozygous CHD1L variants, including a missense variant c.956G>A (p.R319Q) and a nonsense variant c.1831C>T (p.R611*) were observed. The variants were absent in 100 controls. Altogether, the contribution yield of CHD1L to MDAs was calculated as 4.12% (4/97). All three variants were assessed as pathogenic through various functional analysis. The splice-site variant c.348-1G>C resulted in a 11 bp sequence skipping in exon 4 of CHD1L and led to nonsense mediated decay of its transcripts. Unlike WT CHD1L, the truncated R611* protein mislocalized to the cytoplasm, abolish the ability of CHD1L to promote cell migration and failed to interact with PARP1 owing to the loss of macro domain. The R319Q variant exhibited conformational disparities and showed abnormal protein recruitment behavior through laser microirradiation comparing with the WT CHD1L. All these variants impaired the CHD1L function in DNA damage repair, thus participating in MDAs. CONCLUSIONS: The current study not only expands the mutational spectrum of CHD1L in MDAs but determines three variants as pathogenic according to ACMG guidelines with reliable functional evidence. Additionally, the impairment in DNA damage repair is an underlying mechanism involved in MDAs.

Deciphering the possible role of MmpL7 efflux pump in SQ109 resistance in Mycobacterium tuberculosis.

BACKGROUND: SQ109 is a promising candidate drug for the treatment of patients with drug-resistant tuberculosis (DR-TB). The purpose of this study was to investigate the activity of SQ109 against clinical isolates of Mycobacterium tuberculosis (MTB) from patients with multidrug-resistant TB (MDR-TB) and pre-extensively drug-resistant TB (pre-XDR-TB), and to explore new drug-resistant mechanisms of SQ109. METHODS: We evaluated the in vitro activity of SQ109 against clinical isolates from patients with MDR-TB and pre-XDR-TB using minimal inhibitory concentration (MIC) assay. The drug-resistant gene, mmpL3 of SQ109-resistant strains was sequenced, and a quantitative real-time PCR assay was used to analyze 28 efflux pump genes in SQ109-resistant strains without mmpL3 mutations. The role of candidate efflux pumps mmpL5 and mmpL7 on the MIC of SQ109 was evaluated using recombinantly cloned MmpL5 and MmpL7 expressed in Mycobacterium smegmatis. RESULTS: The MIC90, MIC95 and MIC99 values of SQ109 for 225 clinical isolates of MTB were 0.25 mg/L, 0.5 mg/L and 1.0 mg/L, respectively. Among the pre-XDR strains, six showed resistance to SQ109 despite the absence of gene mutations in mmpL3. In six resistant pre-XDR strains, the MIC of SQ109 decreased with the use of an efflux pump inhibitor, and there was significant upregulation of mmpL5 and mmpL7 in two strains after exposure to SQ109. The presence of MmpL7 in Mycobacterium smegmatis resulted in decreased susceptibility to SQ109, with the MIC increasing from 16 mg/L to 32 mg/L. CONCLUSIONS: Our data demonstrated that SQ109 exhibited excellent levels of in vitro activity against MTB. MmpL7 may be a potential gene for MTB resistance to SQ109, providing a useful target for detecting SQ109 resistance in MTB.

BRAF V600E mutation in thyroid carcinoma: a large-scale study in Han Chinese population.

BACKGROUND: The prevalence of genetic mutations in thyroid cancer varies significantly among different ethnic backgrounds. The present study aimed to investigate the clinical potential of BRAF V600E in a large group of homogenous Han Chinese patients. METHODS: From 2018 to 2021, 6232 thyroid disease patients who underwent thyroidectomy at our hospital were enrolled. We measured the diagnostic value of BRAF and plotted ROC curves. Patients with full clinical-pathological data were selected and divided into the BRAF mutation and wild type groups. We conducted univariate and multivariate analyses to quantify the differences in potential predictive factors of papillary thyroid carcinoma (PTC) patients between the groups. Kaplan-Meier survival analysis was used to estimate overall recurrence and recurrence rate. RESULTS: The prevalence of BRAF V600E mutation was 86.0% in PTCs. The sensitivity and specificity of BRAF mutation for diagnosing PTC from suspicious lesions were 85.5% and 100%, respectively. The sensitivity and specificity of BRAF analysis in the indeterminate cytology group were 72.5% and 100%, respectively. BRAF mutation showed an independent association with older age, negative HT, larger tumor size, extrathyroidal extension, and multifocality in PTCs. In micro-PTCs (tumor size ≤ 1), the mutation was also positively correlated with progressive phenotypes of extrathyroidal extension and multifocality. BRAF mutation was associated with poorer recurrence-free probability in Kaplan-Meier survival analysis. CONCLUSIONS: This large single-center study reveals that BRAF V600E is highly prevalent in the Han Chinese population and demonstrates BRAF V600E mutation testing has high diagnostic accuracy and its strong association with the progress of aggressiveness in PTCs and a higher probability of recurrence. BRAF mutation can serve as an accurate marker for diagnosis and decision-making with great value.

Whole-genome sequencing reveals cellular origin of concomitant chronic lymphocytic leukemia and multiple myeloma: a case report.

Chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) are hematological disorders affecting B cells. The clonal relationship between CLL and MM has not always been clarified, although this information is critical to understanding its pathogenesis. Here, we present a rare clinical case of synchronous CLL and MM. Whole-genome sequencing (WGS) was performed using malignant lymph node (LN) and bone marrow (BM) tissues. Based on the high consistency of single nucleotide variants (SNVs), significantly mutated genes (SMGs), copy number variations (CNVs), different B cell receptor (BCR) IGH rearrangement features in LN and BM, and the different light-chain expression patterns in CLL and MM cells, we concluded that CLL and MM cells from this patient originated from the same hematopoietic stem cell/progenitors, different pro-B cells and suffered oncogenic mutations at different B cell differentiation stages. Depth analysis of genome features using WGS provides a new method to explore the process of malignant B cell genesis.

AB019. Detailed analysis of DNA hydroxymethylation observed with the malignant progression of IDH-mutant gliomas.

BACKGROUND: Gliomas vary in prognosis with World Health Organization (WHO) grade. Low-grade gliomas can undergo malignant progression (MP), becoming aggressive high-grade tumors, worsening prognosis. This is prevalent in isocitrate dehydrogenase-mutant (IDH-mt) gliomas like astrocytoma and oligodendroglioma, but the mechanism of MP is still not fully understood. High-grade IDH-mt gliomas have been reported to exhibit TET-mediated DNA hydroxymethylation, which is suggested to potentially influence gene expression. We hypothesized that hydroxymethylation in specific regions could be implicated in triggering MP. METHODS: We collected glioma tumor samples over a decade, using WHO 2021 classification to study IDH-mt astrocytoma grade 2 progression to grades 3 or 4, indicating MP. Samples from five patients, demonstrating MP, were analyzed for DNA hydroxymethylation status across more than 850,000 genomic locations using the oxidative bisulfite process and Infinium EPIC methylation array. This was complemented by RNA sequencing for gene expression analysis and its correlation with hydroxymethylation, and motif-enrichment analysis to infer transcription factor involvement in hydroxymethylation-based gene regulation. Additionally, to delve into the fundamental causes of hydroxymethylation, we exposed an IDH-mt glioma cell line to hypoxic conditions and systematically explored the genomic locations where hydroxymethylation occurred. RESULTS: Our comprehensive analysis identified a significant overlap of hydroxymethylated genomic regions across samples during MP, with a notable enrichment in open sea and intergenic regions (P<0.001). These regions were significantly associated with cancer-related signalling pathways. Integration with RNA sequencing data revealed 91 genes with significant correlations between hydroxymethylation and gene expression, implying roles in cell cycle regulation and antineoplastic functions. Furthermore, motif-enrichment analysis suggested the potential regulatory role of KLF4 in these processes. The cell culture results revealed that a certain similarity exists between the hydroxymethylation patterns observed during MP and those in glioma cells cultured under hypoxic conditions. CONCLUSIONS: This study elucidates the importance of region-specific DNA hydroxymethylation in the MP of IDH-mt astrocytomas, suggesting its potential impact on gene expression relevant to cancer malignancy. Our findings propose a complex interplay between hydroxymethylation and gene regulation, which may offer new insights into the mechanisms driving glioma progression and highlight potential targets for therapeutic intervention.

AB066. From imaging to molecular diagnosis: VASARI magnetic resonance imaging features to predict isocitrate dehydrogenase mutation status in glioma.

BACKGROUND: Glioma, the most common brain tumor, poses significant challenges in patient care and economic burden. Clinicians often struggle with management strategies, especially under the 2021 World Health Organization (WHO) central nervous system (CNS) classification emphasizing molecular diagnosis. Isocitrate dehydrogenase (IDH) mutation status is crucial in glioma management. However, many facilities lack the capability for comprehensive molecular tests, and not all patients are candidates for invasive biopsies. MRI offers a non-invasive method to evaluate glioma characteristics. The Visually Accessible Rembrandt Images (VASARI) MRI feature set provides a systematic approach to analyzing brain glioma. This study examines the association of VASARI features with IDH mutation status and their predictive capability. METHODS: This study included 105 glioma patients treated between 2017 and 2022 who had not undergone surgery, chemotherapy, or radiotherapy. Brain MRIs were assessed using VASARI MRI features by two blinded radiologists. Pathological and molecular examinations were conducted per the 2021 WHO CNS tumor classification. IDH mutations were assessed using polymerase chain reaction (PCR) followed by DNA sequencing. Chi-squared analysis identified VASARI features significantly associated with IDH mutation status. A random forest model predicted IDH mutation status using these features. RESULTS: Brain MRI assessments using VASARI terminology showed good inter-observer agreement (kappa =0.714-0.831) and excellent intra-observer agreement (kappa =0.910). Thirteen VASARI features were significantly associated with IDH mutation status. The prediction model based on VASARI MRI features achieved an area under the curve (AUC) of 0.97, with 93.75% sensitivity, 75% specificity, and 84.38% accuracy on test data. CONCLUSIONS: The VASARI MRI feature set is a reliable method for evaluating glioma patients and is feasible for routine radiological practice. Several VASARI features significantly associate with IDH mutation status, aiding glioma patient management. The IDH mutation prediction model based on VASARI features performs excellently and warrants further validation before routine implementation.

AB003. Clinical analysis of central nervous system (CNS) World Health Organization (WHO) grade 4 gliomas with IDH-mutant versus IDH-wildtype focused on CDKN2A homozygous deletion.

BACKGROUND: We are primarily investigating the prognostic role of cell-cycle-dependent kinase inhibitor (CDKN)-2A homozygous deletion in central nervous system (CNS) World Health Organization (WHO) grade 4 gliomas. Additionally, traditional prognostic factors for grade 4 gliomas will be examined, and our results will be validated. METHODS: We conducted a retrospective analysis of glioma cohorts in our institute. Medical records were reviewed for 142 glioblastoma patients for 15 years, and pathological slides were examined again for the updated diagnosis according to the 2021 WHO classification of CNS tumors. The isocitrate dehydrase (IDH) mutation and CDKN2A deletion were examined by next generation sequencing (NGS) analysis using ONCO accuPanel®. Traditional prognostic factors including age, WHO performance status, extent of resection, and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation were examined. RESULTS: After the exclusion of 6 patients with poor status of pathologic samples, 136 glioblastoma that were diagnosed by previous WHO criteria were changed into 29 (21.3%) astrocytoma, IDH-mutant, CNS WHO grade 4 and 107 (78.7%) glioblastoma, IDH-wildtype, CNS WHO grade 4. Among them, 61 patients (56.0%) had CDKN2A deletion. Group A with IDH-wildtype and CDKN2A deletion had a mean overall survival (OS) of 15.70 months [95% confident interval (CI): 13.86-17.54], group B with IDH-mutant and CDKN2A deletion had a mean OS of 19.37 months (95% CI: 13.43-25.30), group C with IDH-wildtype and intact CDKN2A had a mean OS of 22.63 months (95% CI: 20.10-25.17), and group D with IDH-mutant and intact CDKN2A had a mean OS of 33.38 months (95% CI: 29.35-37.40). Multifactor analysis showed following factors were independently associated with OS: age [≥50 vs. <50 years; hazard ratio (HR) 4.642], extent of resection (gross total resection vs. others; HR 5.523), WHO performance (0, 1 vs. 2; HR 5.007), MGMT promoter methylation, (methylated vs. unmethylated; HR 5.075), IDH mutation (mutant vs. wildtype; HR 6.358), and CDKN2A deletion (absence vs. presence; HR 13.452). CONCLUSIONS: The presenting study suggests that CDKN2A deletion should play a powerful prognostic role in CNS WHO grade 4 gliomas as well as low-grade glioma. Even if CNS WHO grade 4 gliomas had mutant IDH, they can have poor clinical outcomes due to CDKN2A deletion.

SAAMBE-MEM: a sequence-based method for predicting binding free energy change upon mutation in membrane protein-protein complexes.

MOTIVATION: Mutations in protein-protein interactions can affect the corresponding complexes, impacting function and potentially leading to disease. Given the abundance of membrane proteins, it is crucial to assess the impact of mutations on the binding affinity of these proteins. Although several methods exist to predict the binding free energy change due to mutations in protein-protein complexes, most require structural information of the protein complex and are primarily trained on the SKEMPI database, which is composed mainly of soluble proteins. RESULTS: A novel sequence-based method (SAAMBE-MEM) for predicting binding free energy changes (ΔΔG) in membrane protein-protein complexes due to mutations has been developed. This method utilized the MPAD database, which contains binding affinities for wild-type and mutant membrane protein complexes. A machine learning model was developed to predict ΔΔG by leveraging features such as amino acid indices and position-specific scoring matrices (PSSM). Through extensive dataset curation and feature extraction, SAAMBE-MEM was trained and validated using the XGBoost regression algorithm. The optimal feature set, including PSSM-related features, achieved a Pearson correlation coefficient of 0.64, outperforming existing methods trained on the SKEMPI database. Furthermore, it was demonstrated that SAAMBE-MEM performs much better when utilizing evolution-based features in contrast to physicochemical features. AVAILABILITY AND IMPLEMENTATION: The method is accessible via a web server and standalone code at http://compbio.clemson.edu/SAAMBE-MEM/. The cleaned MPAD database is available at the website.

Relapse and resistance in acute myeloid leukemia post venetoclax: improving second lines therapy and combinations.

INTRODUCTION: The combined use of the BCL-2 inhibitor venetoclax with azacitidine now is the standard of care for patients with acute myeloid leukemia (AML) unfit for intensive chemotherapy with outcomes exceeding those achieved with hypomethylating agents alone. Venetoclax in combination with intensive chemotherapy is also increasingly used both as frontline as well as salvage therapy. However, resistance to and relapse after venetoclax-based therapies are of major concern and outcomes after treatment failure remain poor. AREAS COVERED: A comprehensive search was performed using PubMed database (up to April 2024). Studies evaluating venetoclax-based combination treatments in AML and studies assessing markers of response and resistance to venetoclax were investigated. We summarize the status of venetoclax-based therapies in the frontline and relapsed/refractory setting with focus on the main mechanisms of resistance to BCL-2 inhibition. Further, strategies to overcome resistance including combinatorial regimens of hypomethylating agent (HMA) + venetoclax + inhibitors targeting actionable mutations like IDH1/2 or FLT3-ITD and the introduction of novel agents like menin-inhibitors are addressed. EXPERT OPINION: Although venetoclax is reshaping the treatment of unfit and fit AML patients, prognosis of patients after HMA/VEN failure remains dismal, and strategies to abrogate primary and secondary resistance are an unmet clinical need.

Celastrol induces DNA damage and cell death in BCR-ABL T315I-mutant CML by targeting YY1 and HMCES.

BACKGROUND: Chronic myeloid leukemia (CML) is driven primarily by the constitutively active BCR-ABL fusion oncoprotein. Although the development of tyrosine kinase inhibitors has markedly improved the prognosis of CML patients, it remains a significant challenge to overcome drug-resistant mutations, such as the T315I mutation of BCR-ABL, and achieve treatment-free remission in the clinic. PURPOSE: The identification of new intervention targets beyond BCR-ABL could provide new perspectives for future research and therapeutic intervention. A network pharmacology analysis was conducted to identify the most promising natural product with anti-CML activity. Celastrol was selected for further analysis to gain insights into its mechanism of action (MoA), with the aim of identifying potential new intervention targets for BCR-ABL T315I-mutant CML. METHODS: Transcriptomic and proteomic analyses were conducted to systematically investigate the molecular MoA of celastrol in K562T315I cells. To identify the target proteins of celastrol, mass spectrometry-coupled cellular thermal shift assay (MS-CETSA) was carried out, followed by validations with genetic knockdown and overexpression, cell proliferation assay, comet assay, Western blotting, celastrol probe-based in situ labeling and pull-down assay, molecular docking, and biolayer interferometry. RESULTS: Our multi-omics analyses revealed that celastrol primarily induces DNA damage accumulation and the unfolded protein response in K562T315I cells. Among the twelve most potential celastrol targets, experimental evidence demonstrated that the direct interaction of celastrol with YY1 and HMCES increases the levels of DNA damage, leading to cell death. CONCLUSION: This study represents the first investigation utilizing a proteome-wide label-free target deconvolution approach, MS-CETSA, to identify the protein targets of celastrol. This study also develops a new systems pharmacology strategy. The findings provide new insights into the multifaceted mechanisms of celastrol and, more importantly, highlight the potential of targeting proteins in DNA damage and repair pathways, particularly YY1 and HMCES, to combat drug-resistant CML.

Recurrent SARS-CoV-2 spike mutations confer growth advantages to select JN.1 sublineages.

The recently dominant SARS-CoV-2 Omicron JN.1 has evolved into multiple sublineages, with recurrent spike mutations R346T, F456L, and T572I, some of which exhibit growth advantages, such as KP.2 and KP.3. We investigated these mutations in JN.1, examining their individual and combined effects on immune evasion, ACE2 receptor affinity, and in vitro infectivity. F456L increased resistance to neutralization by human sera, including those after JN.1 breakthrough infections, and by RBD class-1 monoclonal antibodies, significantly altering JN.1 antigenicity. R346T enhanced ACE2-binding affinity and modestly boosted the infectivity of JN.1 pseudovirus, without a discernible effect on serum neutralization, while T572I slightly bolstered evasion of SD1-directed mAbs against JN.1's ancestor, BA.2, possibly by altering SD1 conformation. Importantly, expanding sublineages such as KP.2 containing R346T, F456L, and V1104L, showed similar neutralization resistance as JN.1 with R346T and F456L, suggesting V1104L does not appreciably affect antibody evasion. Furthermore, the hallmark mutation Q493E in KP.3 significantly reduced ACE2-binding affinity and viral infectivity, without noticeably impacting serum neutralization. Our findings illustrate how certain JN.1 mutations confer growth advantages in the population and could inform the design of the next COVID-19 vaccine booster.

Novel ABCC8 mutation in the genetic diagnosis of familial hyperinsulinaemic hypoglycaemia.

Familial hyperinsulinaemic hypoglycaemia-1 arises from mutations within the genes of pancreatic beta cells, resulting in unregulated insulin secretion from pancreatic beta cells. A 4.06 kg female neonate, born to a second-degree consanguineously married couple, presented with repeated asymptomatic hypoglycaemia. There was a significant history of a previous sibling's death from nesidioblastosis. Despite treatment with intravenous glucose, diazoxide, hydrochlorothiazide and octreotide, she continued to experience hypoglycaemic episodes. Despite efforts to manage sepsis, including antibiotics, antifungals and intravenous immunoglobulin/granulocyte-macrophage colony-stimulated factor, her condition worsened. She succumbed on day 34. This case underscores the complexities of managing congenital hyperinsulinaemic hypoglycaemia, especially in the context of concurrent infections and the need for multidisciplinary care. Early genetic diagnosis proved invaluable in facilitating timely and effective treatment. Furthermore, the genetic results enabled us to counsel the parents regarding the recurrence risk in subsequent pregnancies and the necessity for antenatal diagnosis.

Severe lupus nephritis in a young adult with PTEN hamartoma tumour syndrome.

On chromosome 10q23 is found the PTEN gene, which encodes a phosphate and tension homologue. The protein dephosphorylates phosphatidylinositol-(3,4,5)-trisphosphate at the plasma membrane to produce inorganic phosphatidylinositol-(4,5)-bisphosphate. This enzymatic activity inhibits the phosphatidylinositol-3-kinase, protein kinase B and mammalian target of the rapamycin signalling cascade. Consequently, essential cellular functions, including metabolic regulation, cellular growth, proliferation and viability, are affected. A mutation in this gene gives rise to hamartoma tumour syndrome, which exhibits a range of phenotypes, including Bannayan-Riley-Ruvalcaba syndrome, Cowden syndrome and proteus-like disease. A man in his late 20s with a PTEN tumour-like arteriovenous malformation in the right thigh was recently diagnosed with lupus nephritis. The patient's nephritic symptoms, pleural effusion, dyslipidaemia and splenomegaly demonstrate systemic lupus erythematosus (SLE) multisystem involvement. The case report identifies an association between a PTEN mutation and a new diagnosis of SLE that might have been triggered by PTEN-associated immune dysregulation.

Case of B-acute lymphoblastic leukaemia with t(1;19)(q23;p13.3) TCF3::PBX1 and co-occurring CBL mutation in an elderly patient.

The t(1;19) (q23;p13) TCF3::PBX1 is a well-described, recurring chromosomal abnormality in B-acute lymphoblastic leukaemia (B-ALL) that has historically been associated with a worse prognosis in paediatric patients. Gene expression profiling has demonstrated that TCF3::PBX1 results in a distinct subtype of B-ALL, leading to its recognition in the most recent WHO and ICC classifications. Though initially believed to be a poor prognostic sign in the adult population, emerging evidence suggests its presence may instead be intermediate or even favourable in B-ALL. However, adults with TCF3::PBX1 are typically younger and often qualify for treatment with paediatric-inspired regimens. Thus, the prognostic significance in this population remains unclear. This translocation appears to be very rare in older adults with B-ALL and its predictive and prognostic nature in this population is unknown. Herein, we explore a case of this translocation occurring in a patient in her 70s. She initially presented to the emergency department with abdominal pain and thrombocytopenia and was subsequently diagnosed with B-ALL. In addition to t(1;19) (q23;p13), a pathologic mutation in the CBL gene was identified. CBL mutations have been implicated in cancer progression and are mostly described in paediatric B-ALL. She was treated with modified Ph-negative EWALL induction (Vincristine, Idarubicin, dexamethasone) and achieved a complete remission. However, she subsequently experienced an early relapse and was refractory to targeted therapy with blinatumomab. After treatment with inotuzumab ozogamicin, she achieved a second complete remission. Unfortunately, she then suffered a central nervous system (CNS) relapse and passed away from complications of her disease. This case serves as an example of the heterogeneous nature of B-ALL. It demonstrates that patients with ostensibly favourable prognostic factors may experience poor response rates to traditional chemotherapy as well as targeted salvage agents. It also illustrates the challenges of treating B-ALL in the elderly population.

Circulating tumor DNA (ctDNA) as a biomarker of response to therapy in advanced Hepatocellular carcinoma treated with Nivolumab.

BACKGROUND: Circulating tumor DNA (ctDNA) is a promising non-invasive marker for detection, diagnosis, treatment selection, and prognosis of hepatocellular carcinoma (HCC). OBJECTIVE: This study aimed to examine the utility of ctDNA as a prognostic and predictive tool in HCC patients treated with nivolumab. METHODS: We analyzed pre-treatment ctDNA from 44 HCC patients using comprehensive genomic testing on a commercially available platform. We utilized log rank test and univariate Cox models to correlate overall survival (OS) and progression-free survival (PFS) with ctDNA expressions. RESULTS: Of 44 patients, 77.3% were men with median age of 67 years. All but 3 patients had at least one alteration identified, and TP53 was the most commonly altered gene (52.3%). Median OS was 17.5 months (95% CI: 12.7, NA). Mutations involving PIK3CA, BRCA1, and CCND1 amplification were associated with shorter OS (P 0.0001, 0.0001 and 0.01, respectively). Median PFS time was 4.01 months (95% CI: 3.06, 9.33). Mutations involving KIT and PIK3CA were associated with shorter PFS (P 0.0001 and 0.0004, respectively), while mutation involving CTNNB1 were associated with longer PFS (p= 0.04). CONCLUSIONS: ctDNA profiling may provide a benefit for prediction of survival and progression of HCC patients treated with nivolumab. Future studies are needed for confirmation.