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.

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.

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.

Monitoring Calcium-Sensing Receptor (CaSR)-Induced Intracellular Calcium Flux Using an Indo-1 Flow Cytometry Assay.

The calcium-sensing receptor (CaSR) has a critical role in maintaining serum calcium concentrations within the normal physiological range, and mutations in the receptor, or components of its signaling and trafficking pathway, cause disorders of calcium homeostasis. Inactivating mutations cause neonatal severe hyperparathyroidism or familial hypocalciuric hypercalcemia (FHH), while gain-of-function mutations cause autosomal dominant hypocalcemia (ADH). Characterizing the functional impact of mutations of the CaSR, and components of the CaSR-signaling pathway, is clinically important to enable correct diagnoses of FHH and ADH, optimize management, and prevent inappropriate parathyroidectomy or vitamin D supplementation. CaSR signals predominantly by activating the G-alpha subunit-11 to mobilize calcium release from intracellular stores. Thus, measurement of CaSR-induced intracellular calcium (Ca2+i) signaling is the gold standard method to investigate the pathogenicity of CaSR genetic variants. This protocol describes a method to assess CaSR-induced Ca2+I signaling using the Indo-1 calcium indicator dye and flow cytometry. This method has been used to assess multiple genetic variants in CaSR and components of its signaling and trafficking pathway in HEK293 cells.

Ursolic acid interaction with transcription factors BRAF, V600E, and V600K: a computational approach towards new potential melanoma treatments.

CONTEXT: Melanoma is one of the cancers with the highest mortality rate for its ability to metastasize. Several targets have undergone investigation for the development of drugs against this pathology. One of the main targets is the kinase BRAF (RAF, rapidly accelerated fibrosarcoma). The most common mutation in melanoma is BRAFV600E and has been reported in 50-90% of patients with melanoma. Due to the relevance of the BRAFV600E mutation, inhibitors to this kinase have been developed, vemurafenib-OMe and dabrafenib. Ursolic acid (UA) is a pentacyclic triterpene with a privileged structure, the pentacycle scaffold, which allows to have a broad variety of biological activity; the most studied is its anticancer capacity. In this work, we reported the interaction profile of vemurafenib-OMe, dabrafenib, and UA, to define whether UA has binding capacity to BRAFWT, BRAFV600E, and BRAFV600K. Homology modeling of BRAFWT, V600E, and V600K; molecular docking; and molecular dynamics simulations were carried out and interactions and residues relevant to the binding of the inhibitors were obtained. We found that UA, like the inhibitors, presents hydrogen bond interactions, and hydrophobic interactions of van der Waals, and π-stacking with I463, Q530, C532, and F583. The ΔG of ursolic acid in complex with BRAFV600K (- 63.31 kcal/mol) is comparable to the ΔG of the selective inhibitor dabrafenib (- 63.32 kcal/mol) in complex to BRAFV600K and presents a ΔG like vemurafenib-OMe with BRAFWT and V600E. With this information, ursolic acid could be considered as a lead compound for design cycles and to optimize the binding profile and the selectivity towards mutations for the development of new selective inhibitors for BRAFV600E and V600K to new potential melanoma treatments. METHODS: The homology modeling calculations were executed on the public servers I-TASSER and ROBETTA, followed by molecular docking calculations using AutoGrid 4.2.6, AutoDockGPU 1.5.3, and AutoDockTools 1.5.6. Molecular dynamics and metadynamics simulations were performed in the Desmond module of the academic version of the Schrödinger-Maestro 2020-4 program, utilizing the OPLS-2005 force field. Ligand-protein interactions were evaluated using Schrödinger-Maestro program, LigPlot + , and PLIP (protein-ligand interaction profiler). Finally, all of the protein figures presented in this article were made in the PyMOL program.

Modulation of human-to-swine influenza a virus adaptation by the neuraminidase low-affinity calcium-binding pocket.

Frequent interspecies transmission of human influenza A viruses (FLUAV) to pigs contrasts with the limited subset that establishes in swine. While hemagglutinin mutations are recognized for their role in cross-species transmission, the contribution of neuraminidase remains understudied. Here, the NA's role in FLUAV adaptation was investigated using a swine-adapted H3N2 reassortant virus with human-derived HA and NA segments. Adaptation in pigs resulted in mutations in both HA (A138S) and NA (D113A). The D113A mutation abolished calcium (Ca2+) binding in the low-affinity Ca2+-binding pocket of NA, enhancing enzymatic activity and thermostability under Ca2+-depleted conditions, mirroring swine-origin FLUAV NA behavior. Structural analysis predicts that swine-adapted H3N2 viruses lack Ca2+ binding in this pocket. Further, residue 93 in NA (G93 in human, N93 in swine) also influences Ca2+ binding and impacts NA activity and thermostability, even when D113 is present. These findings demonstrate that mutations in influenza A virus surface proteins alter evolutionary trajectories following interspecies transmission and reveal distinct mechanisms modulating NA activity during FLUAV adaptation, highlighting the importance of Ca2+ binding in the low-affinity calcium-binding pocket.

Homologous recombination deficiency gene panel analysis results in synchronous endometrial and ovarian cancers.

OBJECTIVE: The objective of this study was to analyze the genetic alterations of tumors within the scope of the homologous recombination deficiency gene panel in patients diagnosed with synchronous endometrial ovarian cancer who have been followed for over 5 years using next-generation sequencing. METHODS: DNA was isolated from the patient's formalin-fixed, paraffin-embedded tissue blocks. Next-generation sequencing was performed using the Illumina capture-based sequencing method. Samples were sequenced using the Sophia HR Solution DNA Kit. RESULTS: Seven patients were included in this study. The ratios of likely pathogenic (LP)/pathogenic (P) somatic mutations in ATM (serine/threonine kinase or Ataxia-telangiectasia mutated gene), BRCA2 (breast cancer type 2 susceptibility gene), BARD1 (BRCA1 associated RING domain 1), TP53 (tumor protein p53), BIRP1 (BRCA1-interacting helicase 1 gene), PALB2 (partner and localizer of BRCA2), and CHECK2 were 21 (48.8%), 8 (18.6%), 5 (11.6%), 3 (6.9%), 2 (4.6%), 2 (4.6%), and 2 (4.6%), respectively, in endometrium, and the ratios of somatic mutations in ATM, BRCA2, TP53, BARD1, RAD54L (DNA repair/recombination protein like), BIRP1, and RAD51D (RAD51 recombinase paralog D) were 24 (60%), 6 (15%), 5 (12.5%), 2 (5%), 2 (5%), 1 (2.5%), and 1 (2.5%), respectively, in ovary. In endometrioid-synchronous endometrial ovarian cancer cases, P/LP mutations were observed in ATM and CHECK2 genes in endometrium and ATM, BRCA2, and TP53 genes in ovary. In two non-endometrioid-synchronous endometrial ovarian cancer cases, CHEK2 (checkpoint kinase 2) mutations were observed in endometrium and ATM and TP53 mutations in ovary, whereas in one case, P/LP mutations in ATM and TP53 genes were common in both tissues. CONCLUSION: Pathogenic variations confirming the diagnosis of synchronous endometrial ovarian cancer with genetic alterations were identified in all but one case. ATM gene mutation emerged as the most common alteration and has a potential association with a favorable prognosis.

Development and experimental validation of computational methods for human antibody affinity enhancement.

High affinity is crucial for the efficacy and specificity of antibody. Due to involving high-throughput screens, biological experiments for antibody affinity maturation are time-consuming and have a low success rate. Precise computational-assisted antibody design promises to accelerate this process, but there is still a lack of effective computational methods capable of pinpointing beneficial mutations within the complementarity-determining region (CDR) of antibodies. Moreover, random mutations often lead to challenges in antibody expression and immunogenicity. In this study, to enhance the affinity of a human antibody against avian influenza virus, a CDR library was constructed and evolutionary information was acquired through sequence alignment to restrict the mutation positions and types. Concurrently, a statistical potential methodology was developed based on amino acid interactions between antibodies and antigens to calculate potential affinity-enhanced antibodies, which were further subjected to molecular dynamics simulations. Subsequently, experimental validation confirmed that a point mutation enhancing 2.5-fold affinity was obtained from 10 designs, resulting in the antibody affinity of 2 nM. A predictive model for antibody-antigen interactions based on the binding interface was also developed, achieving an Area Under the Curve (AUC) of 0.83 and a precision of 0.89 on the test set. Lastly, a novel approach involving combinations of affinity-enhancing mutations and an iterative mutation optimization scheme similar to the Monte Carlo method were proposed. This study presents computational methods that rapidly and accurately enhance antibody affinity, addressing issues related to antibody expression and immunogenicity.

Mobilome impacts on physiology in the widely used non-toxic mutant Microcystis aeruginosa PCC 7806 ΔmcyB and toxic wildtype.

The Microcystis mobilome is a well-known but understudied component of this bloom-forming cyanobacterium. Through genomic and transcriptomic comparisons, we found five families of transposases that altered the expression of genes in the well-studied toxigenic type-strain, Microcystis aeruginosa PCC 7086, and a non-toxigenic genetic mutant, Microcystis aeruginosa PCC 7806 ΔmcyB. Since its creation in 1997, the ΔmcyB strain has been used in comparative physiology studies against the wildtype strain by research labs throughout the world. Some differences in gene expression between what were thought to be otherwise genetically identical strains have appeared due to insertion events in both intra- and intergenic regions. In our ΔmcyB isolate, a sulfate transporter gene cluster (sbp-cysTWA) showed differential expression from the wildtype, which may have been caused by the insertion of a miniature inverted repeat transposable element (MITE) in the sulfate-binding protein gene (sbp). Differences in growth in sulfate-limited media also were also observed between the two isolates. This paper highlights how Microcystis strains continue to "evolve" in lab conditions and illustrates the importance of insertion sequences / transposable elements in shaping genomic and physiological differences between Microcystis strains thought otherwise identical. This study forces the necessity of knowing the complete genetic background of isolates in comparative physiological experiments, to facilitate the correct conclusions (and caveats) from experiments.

Genomic signature for oligometastatic disease in non-small cell lung cancer patients with brain metastases.

Purpose/objectives: Biomarkers for extracranial oligometastatic disease remain elusive and few studies have attempted to correlate genomic data to the presence of true oligometastatic disease. Methods: Patients with non-small cell lung cancer (NSCLC) and brain metastases were identified in our departmental database. Electronic medical records were used to identify patients for whom liquid biopsy-based comprehensive genomic profiling (Guardant Health) was available. Extracranial oligometastatic disease was defined as patients having ≤5 non-brain metastases without diffuse involvement of a single organ. Widespread disease was any spread beyond oligometastatic. Fisher's exact tests were used to screen for mutations statistically associated (p<0.1) with either oligometastatic or widespread extracranial disease. A risk score for the likelihood of oligometastatic disease was generated and correlated to the likelihood of having oligometastatic disease vs widespread disease. For oligometastatic patients, a competing risk analysis was done to assess for cumulative incidence of oligometastatic progression. Cox regression was used to determine association between oligometastatic risk score and oligoprogression. Results: 130 patients met study criteria and were included in the analysis. 51 patients (39%) had extracranial oligometastatic disease. Genetic mutations included in the Guardant panel that were associated (p<0.1) with the presence of oligometastatic disease included ATM, JAK2, MAP2K2, and NTRK1, while ARID1A and CCNE1 were associated with widespread disease. Patients with a positive, neutral and negative risk score for oligometastatic disease had a 78%, 41% and 11.5% likelihood of having oligometastatic disease, respectively (p<0.0001). Overall survival for patients with positive, neutral and negative risk scores for oligometastatic disease was 86% vs 82% vs 64% at 6 months (p=0.2). Oligometastatic risk score was significantly associated with the likelihood of oligoprogression based on the Wald chi-square test. Patients with positive, neutral and negative risk scores for oligometastatic disease had a cumulative incidence of oligometastatic progression of 77% vs 35% vs 33% at 6 months (p=0.03). Conclusions: Elucidation of a genomic signature for extracranial oligometastatic disease derived from non-invasive liquid biopsy appears feasible for NSCLC patients. Patients with this signature exhibited higher rates of early oligoprogression. External validation could lead to a biomarker that has the potential to direct local therapies in oligometastatic patients.

CFTR Exon 10 deleterious mutations in patients with congenital bilateral absence of vas deferens in a cohort of Pakistani patients.

Congenital bilateral absence of vas deferens (CBAVD) is a urological syndrome of Wolffian ducts and is responsible for male infertility and obstructive azoospermia. This study is designed to explore the integrity of exon 10 of CFTR and its role in male infertility in a cohort of CBVAD patients in Pakistan. Genomic DNA was extracted from 17 male patients with CBAVD having clinical symptoms, and 10 healthy controls via phenol-chloroform method. Exon 10 of the CFTR gene was amplified, using PCR with specific primers and DNA screening was done by Sanger sequencing. Sequencing results were analyzed using freeware Serial Cloner, SnapGene, BioEdit and FinchTV. Furthermore, bioinformatics tools were used to analyze the mutations and their impact on the protein function and stability. We have identified 4 mutations on exon 10 of CFTR in 6 out of 17 patients. Two of the mutations were missense variants V456A, K464E, and the other two were silent mutations G437G, S431S. The identified variant V456A was present in 4 of the studied patients. Whereas, the presence of K464E in our patients further weighs on the crucial importance for its strategic location to influence the gene function at post-transcriptional and protein level. Furthermore, Polyphen-2 and SIFT analyze the mutations as harmful and deleterious. The recurrence of V456A and tactically conserved locality of K464E are evidence of their potential role in CBAVD patients and in male infertility. The data can contribute in developing genetic testing and treatment of CBAVD.

[Molecular pathogenesis of adult T-cell leukemia/lymphoma].

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive peripheral T-cell malignancy caused by human T-cell leukemia virus type-1 (HTLV-1) infection. Genetic alterations are thought to contribute to the pathogenesis of ATLL alongside HTLV-1 products such as Tax and HBZ. Several large-scale genetic analyses have delineated the entire landscape of somatic alterations in ATLL, which is characterized by frequent alterations in T-cell receptor/NF-κB pathways and immune-related molecules. Notably, up to one-fourth of ATLL patients harbor structural variations disrupting the 3'-UTR of the PD-L1 gene, which facilitate escape of tumor cells from anti-tumor immunity. Among these alterations, PRKCB and IRF4 mutations, PD-L1 amplification, and CDKN2A deletion are associated with poor prognosis in ATLL. More recently, several single-cell transcriptome and immune repertoire analyses have revealed phenotypic features of premalignant cells and tumor heterogeneity as well as virus- and tumor-related changes of the non-malignant hematopoietic pool in ATLL. Here we summarize the current understanding of the molecular pathogenesis of ATLL, focusing on recent progress made by genetic, epigenetic, and single-cell analyses. These findings not only provide a deeper understanding of the molecular pathobiology of ATLL, but also have significant implications for diagnostic and therapeutic strategies.

[Current state of genetic analysis in multiple myeloma and future perspectives].

Multiple myeloma (MM) is a hematologic malignancy characterized by clonal proliferation of plasma cells. Recent advances in next-generation sequencing technologies have facilitated in-depth genetic exploration of MM, unveiling a more comprehensive genomic landscape that extends beyond classical chromosomal alterations, such as IGH translocations and hyperdiploidy. These studies have elucidated recurrent mutations across various functional pathways including those involving MAPK, NF-κB, cell cycle regulation, and epigenetic modulation. With respect to clinical utility, studies have shown that the number of genetic alterations and biallelic events in TP53 are associated with worse prognosis, and CRBN mutations with resistance to immunomodulatory drugs. We recently analyzed the full landscape of genetic alterations in relapsed and refractory MM using circulating tumor DNA (ctDNA), revealing TP53 mutations as the most frequent driver mutation. Notably, more than half of TP53 mutations were present in only ctDNA, suggesting a subclonal origin. Mutations in six genes, including KRAS and TP53, were associated with poor progression-free survival. In addition, the number of ctDNA mutations was identified as a prognostic factor independent of IGH translocations and clinical factors. Here we summarize recent progress in genetic analysis of MM, focusing on clinical relevance.

[Pathophysiology of sideroblastic anemia].

Sideroblastic anemias (SAs) are a diverse group of congenital and acquired disorders, characterized by anemia and the presence of ring sideroblasts in bone marrow. Congenital SA is a rare disorder that results from genetic mutations that impair heme biosynthesis, iron-sulfur [Fe-S] cluster biosynthesis, and mitochondrial protein synthesis. The predominant type of congenital SA is X-linked sideroblastic anemia, caused by mutations in the erythroid-specific δ-aminolevulinate synthase (ALAS2) gene, a key enzyme in the heme biosynthesis pathway in erythroid cells. SAs can also arise due to exposure to certain drugs or alcohol or to copper deficiency (secondary SAs). They are also often associated with myelodysplastic syndrome (idiopathic SA), and idiopathic SAs are the most frequently encountered type. This review discusses the current understanding of the pathophysiology underlying SA.

[Novel therapies for MDS and future prospects].

Myelodysplastic syndromes (MDS) are clonal diseases resulting from the accumulation of genetic mutations. In general, MDS is categorized into two risk groups, with management and treatment varying significantly based on this classification. Over the past two decades, allogeneic transplantation and hypomethylating agents (HMAs) have become the standard of care and remain crucial for higher-risk MDS. Unfortunately, no new drugs have emerged to replace HMAs as the standard of care. However, the landscape of practice and research in MDS has evolved. In 2022, the focus of diagnostic classification of MDS shifted significantly from morphology to genetic alterations. As a result, treatment strategies centered on genetic mutations are now already used internationally. Revisions made to the International Working Group (IWG) criteria for assessing treatment response in 2023 are expected to further improve accuracy. Meanwhile, interest has increased in understanding the relationship between inflammation and the development and progression of lower-risk MDS. This year, luspatercept, an anti-anemic agent targeting the TGFß pathway, became available for clinical use in Japan. Various research initiatives are currently underway to develop new medicines targeting specific molecules within innate immune and inflammasome-signaling pathways, including IL-1ß, CD33, TLR, IRAK4, and p38MAPK.

Genotype-phenotype correlation and founder effect analysis in southeast Chinese patients with sialidosis type I.

BACKGROUND: Sialidosis type 1 (ST-1) is a rare autosomal recessive disorder caused by mutation in the NEU1 gene. However, limited reports on ST-1 patients in the Chinese mainland are available. METHODS: This study reported the genetic and clinical characteristics of 10 ST-1 patients from southeastern China. A haplotype analysis was performed using 21 single nucleotide polymorphism (SNP) markers of 500 kb flanking the recurrent c.544 A > G in 8 families harboring the mutation. Furthermore, this study summarized and compared previously reported ST-1 patients from Taiwan and mainland China. RESULTS: Five mutations within NEU1 were found, including two novel ones c.557 A > G and c.799 C > T. The c.544 A > G mutation was most frequent and identified in 9 patients, 6 patients were homozygous for c.544 A > G. Haplotype analysis revealed a shared haplotype surrounding c.544 A > G was identified, suggesting a founder effect presenting in southeast Chinese population. Through detailed assessment, 52 ST-1 patients from 45 families from Taiwan and mainland China were included. Homozygous c.544 A > G was the most common genotype and found in 42.2% of the families, followed by the c.544 A > G/c.239 C > T compound genotype, which was observed in 22.2% of the families. ST-1 patients with the homozygous c.544 A > G mutation developed the disease at a later age and had a lower incidence of cherry-red spots significantly. CONCLUSION: The results contribute to gaps in the clinical and genetic features of ST-1 patients in southeastern mainland China and provide a deeper understanding of this disease to reduce misdiagnosis.

Vitamin B12 responsive developmental and epileptic encephalopathy due to a novel mutation in the FUT2 gene: a case report.

BACKGROUND: Vitamin B12 deficiency is a recognised cause of neurological manifestations, including peripheral neuropathy, behavioural changes, and seizures. However, developmental and epileptic encephalopathy due to vitamin B12 deficiency is very rare. Here, we report an infant with vitamin B12-responsive developmental and epileptic encephalopathy due to a novel mutation in the fucosyltransferase 2 (FUT2) gene responsible for vitamin B12 absorption. CASE PRESENTATION: An 11-month-old girl of non-consanguineous parents presented with recurrent episodes of seizures since four months. Her seizures started as flexor epileptic spasms occurring in clusters resembling infantile epileptic spasms syndrome with hypsarrhythmia in the electroencephalogram. She was treated with multiple drugs, including high-dose prednisolone, vigabatrin, sodium valproate, levetiracetam and clobazam, without any response, and she continued to have seizures at 11 months. She had an early developmental delay with maximally achieving partial head control and responsive smile at four months. Her development regressed with the onset of seizure; at 11 months, her developmental age was below six weeks. On examination, she was pale and had generalised hypotonia with normal muscle power and reflexes. Her full blood count and blood picture revealed macrocytic anaemia with oval and round macrocytes. Bone marrow aspiration showed hypercellular marrow erythropoiesis with normoblastic and megaloblastic maturation. Due to the unusual association of refractory epilepsy and megaloblastic anaemia, a rare genetic disease of the vitamin B12 or folate pathways was suspected. The whole exome sequencing revealed a homozygous missense variant in exon 2 of the FUT2 gene associated with reduced vitamin B12 absorption and low plasma vitamin B12 levels, confirming the diagnosis of vitamin B12 deficiency related developmental and epileptic encephalopathy. She was started on intramuscular hydroxocobalamin, for which she showed a marked response with reduced seizure frequency. CONCLUSION: We report a novel variant in the FUT2 gene associated with vitamin B12-responsive developmental and epileptic encephalopathy and megaloblastic anaemia. This case report highlights the importance of timely genetic testing in children with refractory developmental and epileptic encephalopathy to identify treatable causes.

Identifying SETBP1 haploinsufficiency molecular pathways to improve patient diagnosis using induced pluripotent stem cells and neural disease modelling.

BACKGROUND: SETBP1 Haploinsufficiency Disorder (SETBP1-HD) is characterised by mild to moderate intellectual disability, speech and language impairment, mild motor developmental delay, behavioural issues, hypotonia, mild facial dysmorphisms, and vision impairment. Despite a clear link between SETBP1 mutations and neurodevelopmental disorders the precise role of SETBP1 in neural development remains elusive. We investigate the functional effects of three SETBP1 genetic variants including two pathogenic mutations p.Glu545Ter and SETBP1 p.Tyr1066Ter, resulting in removal of SKI and/or SET domains, and a point mutation p.Thr1387Met in the SET domain. METHODS: Genetic variants were introduced into induced pluripotent stem cells (iPSCs) and subsequently differentiated into neurons to model the disease. We measured changes in cellular differentiation, SETBP1 protein localisation, and gene expression changes. RESULTS: The data indicated a change in the WNT pathway, RNA polymerase II pathway and identified GATA2 as a central transcription factor in disease perturbation. In addition, the genetic variants altered the expression of gene sets related to neural forebrain development matching characteristics typical of the SETBP1-HD phenotype. LIMITATIONS: The study investigates changes in cellular function in differentiation of iPSC to neural progenitor cells as a human model of SETBP1 HD disorder. Future studies may provide additional information relevant to disease on further neural cell specification, to derive mature neurons, neural forebrain cells, or brain organoids. CONCLUSIONS: We developed a human SETBP1-HD model and identified perturbations to the WNT and POL2RA pathway, genes regulated by GATA2. Strikingly neural cells for both the SETBP1 truncation mutations and the single nucleotide variant displayed a SETBP1-HD-like phenotype.