Mutations In Transcription Factors Research Articles

Chromatin endogenous cleavage provides a global view of RNA polymerase II transcription kinetics.

Chromatin immunoprecipitation (ChIP-seq) is the most common approach to observe global binding of proteins to DNA in vivo . The occupancy of transcription factors (TFs) from ChIP-seq agrees well with an alternative method, chromatin endogenous cleavage (ChEC-seq2). However, ChIP-seq and ChEC-seq2 reveal strikingly diUerent patterns of enrichment of yeast RNA polymerase II. We hypothesized that this reflects distinct populations of RNAPII, some of which are captured by ChIP-seq and some of which are captured by ChEC-seq2. RNAPII association with enhancers and promoters - predicted from biochemical studies - is detected well by ChEC-seq2 but not by ChIP-seq. Enhancer/promoter bound RNAPII correlates with transcription levels and matches predicted occupancy based on published rates of enhancer recruitment, preinitiation assembly, initiation, elongation and termination. The occupancy from ChEC-seq2 allowed us to develop a stochastic model for global kinetics of RNAPII transcription which captured both the ChEC-seq2 data and changes upon chemical-genetic perturbations to transcription. Finally, RNAPII ChEC-seq2 and kinetic modeling suggests that a mutation in the Gcn4 transcription factor that blocks interaction with the NPC destabilizes promoter-associated RNAPII without altering its recruitment to the enhancer.

A fungal metabolic regulator underlies infectious synergism during Candida albicans-Staphylococcusaureus intra-abdominal co-infection

Candida albicans and Staphylococcus aureus are two commonly associated pathogens that cause nosocomial infections with high morbidity and mortality. Our prior and current work using a murine model of polymicrobial intra-abdominal infection (IAI) demonstrates that synergistic lethality is driven by Candida-induced upregulation of functional S. aureus α-toxin leading to polymicrobial sepsis and organ damage. In order to determine the candidal effector(s) mediating enhanced virulence, an unbiased screen of C. albicans transcription factor mutants was undertaken revealing that zcf13Δ/Δ fails to drive augmented α-toxin or lethal synergism during co-infection. A combination of transcriptional and phenotypic profiling approaches shows that ZCF13 regulates genes involved in pentose metabolism, including RBK1 and HGT7 that contribute to fungal ribose catabolism and uptake, respectively. Subsequent experiments reveal that ribose inhibits the staphylococcal agr quorum sensing system and concomitantly represses toxicity. Unlike wild-type C. albicans, zcf13Δ/Δ did not effectively utilize ribose during co-culture or co-infection leading to exogenous ribose accumulation and agr repression. Forced expression of RBK1 and HGT7 in the zcf13Δ/Δ mutant fully restores pathogenicity during co-infection. Collectively, our results detail the interwoven complexities of cross-kingdom interactions and highlight how intermicrobial metabolism impacts polymicrobial disease pathogenesis with devastating consequences for the host.

TORC2 is required for the accumulation of γH2A in response to DNA damage

TOR protein kinases serve as the catalytic subunit of the TORC1 and TORC2 complexes, which regulate cellular growth, proliferation and survival. In the fission yeast, Schizosaccharomyces pombe, cells lacking TORC2 or its downstream kinase Gad8 (AKT or SGK1 in human cells) exhibit sensitivity to a wide range of stress conditions, including DNA damage stress. One of the first responses to DNA damage is the phosphorylation of C-terminal serine residues within histone H2AX in human cells (γH2AX), or histone H2A in yeast cells (γH2A). The kinases responsible for γH2A in S. pombe are the two DNA damage checkpoint kinases Rad3 and Tel1 (ATR and ATM, respectively, in human cells). Here we report that TORC2-Gad8 signaling is required for accumulation of γH2A in response to DNA damage and during quiescence. Using the TOR specific inhibitor, Torin1, we demonstrate that the effect of TORC2 on γH2A in response to DNA damage is immediate, rather than adaptive. The lack of γH2A is restored by deletion mutations of transcription and chromatin modification factors, including loss of components of Paf1C, SAGA, Mediator and the bromo-domain proteins Bdf1/Bdf2. Thus, we suggest that TORC2-Gad8 may affect the accumulation of γH2A by regulating chromatin structure and function.

Effect of bromodomain PHD-finger transcription factor (BPTF) on trophoblast epithelial-to-mesenchymal transition

The trophoblast epithelial-to-mesenchymal transition (EMT) is a procedure related to embryo implantation, spiral artery establishment and fetal-maternal communication, which is a key event for successful pregnancy. Inadequate EMT is one of the pathological mechanisms of recurrent miscarriage (RM). Whole-exome sequencing revealed that the mutation of bromodomain PHD-finger transcription factor (BPTF) was strongly associated with RM. In the present study, the effects of BPTF on EMT and the underlying mechanism were investigated. We found that the expression of BPTF in the villi of RM patients was significantly downregulated. Gene Ontology (GO) analysis revealed that BPTF participated in cell adhesion. The knockdown of BPTF prevented EMT and attenuated trophoblast invasion in vitro. BPTF activated Slug transcription by binding directly to the promoter region of the Slug gene. Interestingly, the protein levels of both Slug and BPTF were decreased in the villous cytotrophoblasts (VCTs) of RM villi. In conclusion, BPTF participates in the regulation of trophoblast EMT by activating Slug expression, suggesting that BPTF defects are an important factor in RM pathogenesis.

Mutations in myeloid transcription factors and activated signaling genes predict chronic myeloid leukemia outcomes

AbstractAdvancements in genomics are transforming the clinical management of chronic myeloid leukemia (CML) toward precision medicine. The impact of somatic mutations on treatment outcomes is still under debate. We studied the association of somatic mutations in epigenetic modifier genes and activated signaling/myeloid transcription factors (AS/MTFs) with disease progression and treatment failure in patients with CML after tyrosine kinase inhibitor (TKI) therapy. A total of 394 CML samples were sequenced, including 254 samples collected at initial diagnosis and 140 samples taken during follow-up. Single-molecule molecular inversion probe (smMIP)–based next-generation sequencing (NGS) was conducted targeting recurrently mutated loci in 40 genes, with a limit of detection of 0.2%. Seventy mutations were detected in 57 diagnostic samples (22.4%), whereas 64 mutations were detected in 39 of the follow-up samples (27.9%). Carrying any mutation at initial diagnosis was associated with worse outcomes after TKI therapy, particularly in AS/MTF genes. Patients having these mutations at initial diagnosis and treated with imatinib showed higher risks of treatment failure (hazard ratio, 2.53; 95% confidence interval, 1.13-5.66; P = .0239). The adverse prognostic impact of the mutations was not clear for patients treated with second-generation TKIs. The multivariate analysis affirmed that mutations in AS/MTF genes independently serve as adverse prognostic factors for molecular response, failure-free survival, and progression risk. Additionally, there was an observable nonsignificant trend indicating a heightened risk of progression to advanced disease and worse overall survival. In conclusion, mutations in the AS/MTF genes using smMIP-based NGS can help identify patients with a potential risk of both treatment failure and progression and may help upfront TKI selection.

Characterization of barley (Horduem vulgare) lys3 mutants identifies genes under the regulation of the prolamin-box binding transcription factor and elucidates its role in endosperm promoter methylation during grain development.

Barley ranks fourth in global cereal production and is primarily grown for animal feed and malt. Hordeins, the principal barley seed storage proteins, are homologous to wheat gluten and when ingested elicit an immune response in people with Coeliac disease. Risø 1508 is a chemically induced barley mutant with low hordein levels imparted by the lys3.a locus that is reported to be caused by an SNP in the barley prolamin-box binding factor gene (BPBF). Reports suggest the lys3.a locus prevents CG DNA demethylation at the Hor2 (B-hordein) promoter during grain development subsequently causing hypermethylation and inhibiting gene expression. In lys3.a mutants, endosperm-specific β-amylase (Bmy1) and Hor2 are similarly downregulated during grain development and thus we hypothesize that the inability to demethylate the Bmy1 promoter CG islands is also causing Bmy1 downregulation. We use whole-genome bisulfite sequencing and mRNA-seq on developing endosperms from two lys3.a mutants and a lys3.b mutant to determine all downstream genes affected by lys3 mutations. RNAseq analysis identified 306 differentially expressed genes (DEGs) shared between all mutants and their parents and 185 DEGs shared between both lys3.a mutants and their parents. Global DNA methylation levels and promoter CG DNA methylation levels were not significantly different between the mutants and their parents and thus refute the hypothesis that the lys3.a mutant's phenotype is caused by dysregulation of demethylation during grain development. The majority of DEGs were downregulated (e.g., B- and C-hordeins and Bmy1), but some DEGs were upregulated (e.g., β-glucosidase, D-hordein) suggesting compensatory effects and potentially explaining the low β-glucan phenotype observed in lys3.a germplasm. These findings have implications on human health and provide novel insight to barley breeders regarding the use of BPBF transcription factor mutants to create gluten-free barley varieties.

Absence of meningeal mast cells in the Mitf mutant mouse.

Mast cells (MCs) are located in the meninges of the central nervous system (CNS), where they play key roles in the immune response. MC-deficient mice are advantageous in delineating the role of MCs in the immune response in vivo. In this study, we illustrate that a mutation in microphthalmia-associated transcription factor (Mitf) affects meningeal MC number in a dosage-dependent manner. C57BL/6J Mitf null mice lack meningeal MCs completely, whereas heterozygous mice have on average 25% fewer MCs. Mitf heterozygous mice might be a valuable tool to study the role of MCs in the meninges.

Lignin reduction in sugarcane by performing CRISPR/Cas9 site-direct mutation of SoLIM transcription factor

Genetic engineering of plant cell walls is limited for reducing lignocellulose recalcitrance, so mild and/or green-like pretreatment is still required for sequential enzymatic saccharification. Here, we report a method to reduce lignin content in sugarcane stalks using the CRISPR/Cas 9 technique. Three target sequences of SoLIM were designed and fused to pRGEB32. The cassette constructs were introduced into sugarcane calli cv. KK3 through Agrobacterium-mediated transformation. We produced one base substitution and one insertion line for the 1st target site; two insertions, one deletion, and one base substitution for the 2nd target site; and one base substitution and insertion for the 3rd target site. qRT-PCR analysis of SoLIM, SoPAL, SoC4H, and SoCAD showeded that downregulation of SoLIM by single nucleotide insertions or deletions reduced the expression of SoPAL, SoC4H, and SoCAD. Consequently, the edited lines contained 9.74 to 51.46% less lignin content compared to that in the wild-type plants. The syringyl/guaiacyl (S/G) ratio of the edited lines ranged between 0.23 and 0.49, while the wild-type was 0.22. The histochemical evaluation and scanning electron microscopy of the cell walls supported this observation. A low lignin content sugarcane will provide a better feedstock for second-generation bioethanol production.

Evo-devo applied to sleep research: an approach whose time has come.

Sleep occurs in all animals but its amount, form, and timing vary considerably between species and between individuals. Currently, little is known about the basis for these differences, in part, because we lack a complete understanding of the brain circuitry controlling sleep-wake states and markers for the cell types which can identify similar circuits across phylogeny. Here, I explain the utility of an "Evo-devo" approach for comparative studies of sleep regulation and function as well as for sleep medicine. This approach focuses on the regulation of evolutionary ancient transcription factors which act as master controllers of cell-type specification. Studying these developmental transcription factor cascades can identify novel cell clusters which control sleep and wakefulness, reveal the mechanisms which control differences in sleep timing, amount, and expression, and identify the timepoint in evolution when different sleep-wake control neurons appeared. Spatial transcriptomic studies, which identify cell clusters based on transcription factor expression, will greatly aid this approach. Conserved developmental pathways regulate sleep in mice, Drosophila, and C. elegans. Members of the LIM Homeobox (Lhx) gene family control the specification of sleep and circadian neurons in the forebrain and hypothalamus. Increased Lhx9 activity may account for increased orexin/hypocretin neurons and reduced sleep in Mexican cavefish. Other transcription factor families specify sleep-wake circuits in the brainstem, hypothalamus, and basal forebrain. The expression of transcription factors allows the generation of specific cell types for transplantation approaches. Furthermore, mutations in developmental transcription factors are linked to variation in sleep duration in humans, risk for restless legs syndrome, and sleep-disordered breathing. This paper is part of the "Genetic and other molecular underpinnings of sleep, sleep disorders, and circadian rhythms including translational approaches" collection.

Case report and functional verification of a novel mutation in the interferon regulatory transcription factor 6 gene in a family with orofacial clefts

Case report and functional verification of a novel mutation in the interferon regulatory transcription factor 6 gene in a family with orofacial clefts

Systematic identification and characterization of five transcription factors mediating the oxidative stress response in Candida albicans

Candida albicans is an opportunistic human fungal pathogen that causes superficial and systemic infections, particularly in immunocompromised individuals. In response to C. albicans infection, innate immune cells of the host produce and accumulate reactive oxygen species (ROS), which can lead to irreversible damage and apoptosis of fungal cells. Several transcription factors involved in this oxidative stress response have been identified; however, a systematic study to identify the transcription factors that mediate the oxidative stress response has not yet been conducted. Here, we screened a comprehensive transcription factor mutant library consisting of 211 transcription factor deletion mutant strains in the presence and absence of hydrogen peroxide (H2O2), a potent ROS inducer, and identified five transcription factors (Skn7, Dpb4, Cap1, Dal81, and Stp2) that are sensitive to H2O2. Genome-wide transcriptional profiling revealed that H2O2 induces a discrete set of differentially regulated genes among the five identified transcription factor mutant strains. Functional enrichment analysis identified KEGG pathways pertaining to glycolysis/gluconeogenesis, amino sugar and nucleotide sugar metabolism, and ribosome synthesis as the most enriched pathways. GO term analysis of the top common differentially expressed genes among the transcription factor mutant strains identified hexose catabolism and iron transport as the most enriched GO terms upon exposure to H2O2.This study is the first to systematically identify and characterise the transcription factors involved in the response to H2O2. Based on our transcriptional profiling results, we found that exposure to H2O2 modulates several downstream genes involved in fungal virulence. Overall, this study sheds new light on the metabolism, physiological functions, and cellular processes involved in the H2O2-induced oxidative stress response in C. albicans.

A KDPG sensor RccR governs Pseudomonas aeruginosa carbon metabolism and aminoglycoside antibiotic tolerance.

Pseudomonas aeruginosa harbors sophisticated transcription factor (TF) networks to coordinately regulate cellular metabolic states for rapidly adapting to changing environments. The extraordinary capacity in fine-tuning the metabolic states enables its success in tolerance to antibiotics and evading host immune defenses. However, the linkage among transcriptional regulation, metabolic states and antibiotic tolerance in P. aeruginosa remains largely unclear. By screening the P. aeruginosa TF mutant library constructed by CRISPR/Cas12k-guided transposase, we identify that rccR (PA5438) is a major genetic determinant in aminoglycoside antibiotic tolerance, the deletion of which substantially enhances bacterial tolerance. We further reveal the inhibitory roles of RccR in pyruvate metabolism (aceE/F) and glyoxylate shunt pathway (aceA and glcB), and overexpression of aceA or glcB enhances bacterial tolerance. Moreover, we identify that 2-keto-3-deoxy-6-phosphogluconate (KDPG) is a signal molecule that directly binds to RccR. Structural analysis of the RccR/KDPG complex reveals the detailed interactions. Substitution of the key residueR152, K270 or R277 with alanine abolishes KDPG sensing by RccR and impairs bacterial growth with glycerol or glucose as the sole carbon source. Collectively, our study unveils the connection between aminoglycoside antibiotic tolerance and RccR-mediated central carbon metabolism regulation in P. aeruginosa, and elucidates the KDPG-sensing mechanism by RccR.

Clonal Evolution of Der(1;7)(q10;p10) Myeloid Neoplasms

Background der(1;7)(q10;p10) (der(1;7)) is a recurrent unbalanced translocation found in 1.5~6% of MDS and AML patients. It is caused by rearrangement of centromere alpha satellites in chromosomes 1 and 7 resulting in an amplification of 1q and a deletion of 7q. Previous studies comparing der(1;7)(+) cases to those with other lesions of 7q loss (-7/del(7q)) demonstrated a higher frequency of RUNX1 mutations, no TP53 mutations and a better prognosis for der(1;7) cases suggesting that der(1;7)(+) may define a unique entity of myeloid neoplasms (MN). However, with the lack of comprehensive molecular characterization using unbiased platforms such as whole genome/exome sequencing, the entire picture of der(1;7)(+) MN still remains unclear. Methods We enrolled a total of 148 MN cases harboring der(1;7) and an additional 3,238 der(1;7)(−) MN cases: -7/del(7q), +1q and ‘OTHER’. We first analyzed paired tumor/normal DNA from 26 der(1;7) cases using whole exome sequencing (WES), followed by targeted-capture sequencing of 329 genes, including those commonly mutated in MN and others newly identified in der(1;7) cases in WES. To investigate the origin of der(1;7)(+) MN, we also enrolled 11,234 healthy individuals to evaluate der(1;7) in clonal hematopoiesis, in which der(1;7) was detected on based on SNP array-based karyotyping. Results In accordance with previous reports, der(1;7) MDS cases showed an improved overall survival (OS) compared to -7/del(7q) MDS cases (Hazard Ratio (HR) =0.71, p=0.098), which however, was shorter than OS in +1q (HR=1.36, p=0.11) and ‘OTHER’ (HR=1.8, p<0.001) MDS cases. der(1;7)(+) cases were characterized by very high mortality from infection (45.5%), while mortality from disease progression was less common (36.4%), showing a sharp contrast to -7/del(7q) and ‘OTHER’ MDS cases which had 13.9% and 10.8% infection-related deaths, and 72.3% and 76.9% disease progression, respectively (Figure 1A.). WES in 26 der(1;7)(+) cases revealed a novel transcription factor, MYB in 2 cases, which was found in 18 (12%) of 148 der(1;7) cases using targeted-capture sequencing. der(1;7)(+) cases were characterized by high frequency of mutations in transcription factors, including RUNX1 (37.8%), GATA2 (12%), ETV6 (13%), and BCOR (14%) (Figure 1B.), which were significantly more frequent in der(1;7) cases than other sub-types (Odds Ratio (OR)<6)(Figure 1B.). Very high frequency of ETNK1 mutations is highly unique to der(1;7)(+) cases (18.9%) compared to that in other cases (<10%). High frequency of mutations in transcription factors seems to be a unique feature of der(1;7)(+) cases. Trisomy 8 and del(20q) CNAs were also common in der(1;7) cases (16% and 21%, respectively). Together with DNMT3A and del(20q), der(1;7) showed the highest variant allele frequency (VAF), suggesting the early origin of these lesions. Additionally, we identified der(1;7) in 6 out of 11,234 healthy individuals using SNP array-based highly sensitive copy number detection, of which 4 died from MDS, suggesting that der(1;7)(+) clonal hematopoiesis might be the origin of MDS in these cases. Moreover, we also analyzed longitudinal samples from 9 der(1;7)(+) cases using duplex-sequencing, der(1;7) showed consistently high VAF, further supporting that der(1;7) tended to represent the major clone. Conclusion Showing a unique set of co-mutation pattern and clinical features, der(1;7) MN defines a distinct subset of myeloid neoplasms, which may originate from der(1;7)(+) clonal hematopoiesis. der(1;7)(+) patients were at higher risk of life-threatening infection, which needs to be carefully prevented and monitored during their clinical course.

Pre-Transplant Somatic Co-Occurring Mutations (by next generation sequencing) in Acute Myeloid Leukemia: Frequency and Impact on Clinical Outcomes after Allogeneic Hematopoietic Cell Transplantation - a Large Study on Behalf of the EBMT Acute Leukemia Working Party

Background: Acute myeloid leukemia (AML) is a very heterogeneous hematological malignancy, which includes numerous genetically defined subsets. The genomic classification of AML with the identification of mutations in transcription factors, epigenetic modifiers, spliceosome, cohesin complex, and signaling pathways has led to a more accurate risk stratification model. The main genetic aberrations included in the European LeukemiaNet (ELN) 2022 classification are NPM1 (risk group according to karyotype and FLT3-ITD status), FLT3-ITD (intermediate risk), bZIP in-frame mutated CEBPA (favorable risk), and RUNX1, ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2 and TP53 mutations (all belonging to the adverse risk group). In the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT), the frequency and prognostic value of different gene-gene interactions has not been studied and may differ from that of patients treated with chemotherapy alone. We aimed at evaluating, through the European Society for Blood and Marrow Transplantation (EBMT) database, the frequency and impact of different recurrent somatic mutations, either alone or in association, on prediction of relapse and survival in patients receiving allo-HSCT. Methods: This is a retrospective, registry-based, multicenter analysis from the EBMT with the approval of the EBMT Acute Leukemia Working Party. Adult patients aged more than 18 years with a diagnosis ofAML who received an allo-HSCT between 2013-2022, with an available genetic profile determined at diagnosis by next generation sequencing (NGS) were included. Results: We identified 836 allografted AML patients who had NGS performed at diagnosis. Most of these patients had de novo AML (88%), with a median age of 53 years (range: 18-78 years). Karyotype was favorable in 7%, intermediate in 69% and adverse in 24% of patients. At transplant, 74% of patients were in first remission (CR1) and 13% in CR2. The most frequent detectable mutations by frequency were DNMT3A (33%), FLT3 (29%), NPM1 (29%), TET2 (28%), NRAS (23%), RUNX1 (22%), WT1 (22%), BCOR (19%), ASXL1 (17%), IDH2 (17%), IDH1 (15%), SRSF2 (13%), STAG2 (12%), CEBPA (11%), TP53 (10%), KRAS (10%), and PTPN11 (10%) . By multiple correspondence analysis, two independent groups of co-occurring mutations were identified, the first group included DNMT3A, NPM1 and FLT3, the second group included ASXL1, SRSF2 and RUNX1. Outcome analysis was performed on the subset of 298 patients allografted in CR1 with available data for the aforementioned six genes ( DNMT3A, NPM1, FLT3, ASXL1, SRSF2 and RUNX1). Most of these patients had de novo AML (90%), with a median age of 53 years (range: 19-75 years). Patients received primarily reduced intensity conditioning (58%) and peripheral blood stem cells (93%) from matched sibling donors (35%), matched unrelated (28%), and haploidentical donors (21%). Seventy percent of these patients had intermediate-risk cytogenetics, while 27% were classified as adverse-risk. Median follow up calculated by the reverse Kaplan-Meier method was 2.5 years. Overall, the 2-year relapse incidence (RI), leukemia-free survival (LFS) and overall survival (OS) were 24%, 62% and 69%, respectively. When outcome analysis was performed according to the presence or absence of single mutations, none of the six mutations significantly affected RI or LFS. The 2-year OS was positively affected by the presence of NPM1 mutation (78% vs 66%; p=0.02) and FLT3 (79% vs 65%, p=0.01) but not significantly affected by the other 4 mutations. When mutations were investigated in groups, the 2-year RI, LFS and OS were 24%, 70% and 78%, respectively, for patients with NPM1 mutation regardless of other co-mutations, 35%, 56% and 69% for patients with FLT3-ITDand/or DNMT3A mutation, wild type NPM1, regardless of other co-mutations, 17%, 70% and 74% for patients with RUNX1 and/or ASXL1 and/or SRSF2 mutation without FLT3-ITD and with wild type NPM1 and wild type DNMT3A and 20%, 56% and 61% for patients with all six genes unmutated. Conclusion: NGS at diagnosis can be extremely useful in risk stratification of AML patients undergoing allo-HSCT, potentially allowing adequate post-transplant interventions. Notably, the 2-year LFS of 70% for patients harboring RUNX1 and/or ASXL1 and/or SRSF2 mutation indicates that allo-HSCT can overcome the adverse risk associated with these somatic mutations at diagnosis.

Juvenile Alexander Disease and Cleidocranial Dysplasia: A Rare Combination of Genetic Abnormalities in a Pediatric Patient

Cleidocranial dysplasia (CCD) and Alexander disease (AxD) are rare, autosomal dominant disorders that are characterized by a mutation in the runt-related transcription factor 2 ( RUNX2) and the glial fibrillary acidic protein ( GFAP) genes, respectively. There is no known relationship between RUNX2 and GFAP which would cause co-morbidity. We report a rare case of a 13-year-old with CCD who came to the clinic complaining of a 2-year history of progressively worsening episodic exacerbations of bulbar, ataxia, nystagmus, kyphoscoliosis, and nausea, but was intact cognitively. Initial diagnosis was a difficult process because preliminary symptoms for Juvenile AxD overlapped with previously diagnosed CCD and the initial genetic test identified our patient’s GFAP gene as a variant of uncertain significance. Our experience emphasizes the importance of continuing to report pathogenic variants of GFAP for AxD to build on our existing compendium of variants for GFAP for quicker and more efficient diagnosis of AxD.

Genetic Evolution from Chronic Myeloproliferative Neoplasms to Acute Myeloid Leukemia: An Analysis of Forty-Six Paired Samples

Background: Secondary acute myeloid leukemia (sAML) may arise from chronic myeloproliferative neoplasm (MPN). The genetic evolution pattern may be understood by paired sample analysis. Knowledge of genetic change in MPN/sAML is still limited and more extensive studies should be done. Patients and Methods: Forty-six paired samples (27 males) at diagnosis of MPN (12 polycythemia vera, 6 essential thrombocythemia, 25 primary myelofibrosis and 3 MPN-unclassified) and sAML transformation were available. Mutational analyses of 27 genes ( JAK2V617F, CALR, MPL, ASXL1, IDH1, IDH2, TET2, DNMT3A, EZH2, SRSF2, U2AF1, SF3B1, ZRSR2, SMC1, STAG2, SMC3, RUNX1, SETBP1, IKZF1, WT1, K-RAS, N-RAS, C-CBL, TP53, RAD21, BCOR and BCORL1) were performed. Mutations and their variant allele frequencies (VAF) were measured by next generation sequencing. Results: At baseline (MPN), the mutation number was 2 (maximal 5). Forty-five samples harbored driver gene mutations (31 JAK2V617F, 13 CALR and 1 MPL). Twenty-eight samples harbored at least one epigenetic regulator mutations (11 TET2, 8 DNMT3A,7 ASXL1, 5 EZH2, 2 IDH2, 1 IDH1). Twelve samples harbored spliceosome mutations which were mutually exclusive (6 SRSF2, 3 SF3B1, 2 U2AF1 1 ZRSR2). Other mutations were RUNX1, BCORL1 (n=2 each), CBL and BCOR (n=1 each). In sAML, the median mutation number was 3 (maximal 7). Loss of mutation was found for JAK2V617F (9/31), TET2 (1/11) and SRSF2 (1/6). Acquisition was most common for RUNX1 (n=9), followed by TP53, ASXL1 (n=5 each), K-RAS (n=4) , ZRSR2, STAG2 (n=3 each). The median number of acquired mutation was 1 (maximal 5). The frequencies of gene mutations are summarized in Figure 1A. The baseline VAF of JAK2V617F was high (>35%) in 80.6% (25/31). In progression to sAML, VAF was constant in 29.0% (9/31, VAF changes <10%), increased in 20.0% (9/31, increased VAF> 10%), decreased in 41.9% (13/31, decreased VAF>10%) (Figure 1B). The baseline VAF in CALR mutation was high (>35%) in 91.3% (12/13). VAF remained stable in 76.9% (10/13), and loss of CALR mutation was not observed (Figure 1B). TET2 mutations tended to be stable (7/16), but clonal expansion (5/16 of all mutation events at MPN), or acquisition (n=2) could be found during transformation to sAML. DNMT3A mutations remained stable (5/8), exhibited clonal expansion (2/8), or acquisition (n=1) in sAML progression. ASXL1 mutations remained stable (6/7), with clonal expansion (1/7), or acquisition (n=5) during MPN/sAML progression. All EZH2 mutants had clonal expansion (5/5) and one acquired in sAML (Figure 1B). We found that CALR mutationwas often the founding clone with high VAF at MPN phase, stable clone during sAML, and might acquire mutations in signaling pathways (1 CBL, 1 KRAS), epigenetic regulators (2 ASXL1, 1 IDH2), transcription factors (2 RUNX1, 1 BCOR), spliceosome (2 ZRSR2, 1 SF3B1), and cohesin complex (1 STAG2). JAK2 mutationwas often preceded by other ancestral mutations such as TET2, ASXL1, DNMT3A, EZH2, IDH2, SRSF2 or ZRSR2. During evolution, leukemia arose from non- JAK2 mutated clones with mutations in EZH2, DNMT3A, RUNX1, EZH2, SETBP1, or acquisition of TP53, SF3B1, BCOR and STAG2 mutations) in sAML. In outcome, the median time from MPN to sAML was 82 (range 3.7 to 338.5) months. Female gender, loss of JAK2V617F, presence of U2AF1 and absence of IDH1 were associated with shorter time to sAML transformation. No other genetic mutation in our gene panel or the number of mutations harbored by patients in our cohort had significant impact on time to sAML. Marked increase of VAF was observed for JAK2 (7/22) , DNMT3A (1/7) , IDH1 (2/3), IDH2 (1/3), TET2 (3/10) , EZH2 (4/5), C-CBL (1/1) and SRSF2 (1/4). Marked decrease of VAF was observed for JAK2 (4/22), MPL (1/1), DNMT3A (1/7) , ASXL1 (1/8), SF3B1 (2/3), SRSF2 (1/4) and U2AF1 (1/2). Multiple mutational clones were detected for TET2 (two clones in 4 samples and 3 clones in 1). Clonal expansion and reduction were observed in two pairs of samples upon sAML transformation. Conclusions: At the MPN stage, most patients had driver mutations with frequent co-mutations of epigenetic modifier or spliceosome genes. CALR mutant clones were stable while for JAK2, sAML often arose from clones without JAK2V617F which acquired RUNX1, TP53, ASXL1 and K-RAS. Research funding:Ministryof Science and Technology Council,Taiwan (NSTC 112-2314-B-182-055) and Ministry of Health and Welfare, Taiwan (112-TDU-B-222-124001)

Impact of Mechanical Strain and Nicotinamide on RUNX2-Deficient Osteoblast Mimicking Cleidocranial Dysplasia

Cleidocranial dysplasia (CCD) is a rare genetic defect caused by a heterozygous mutation of runt-related transcription factor 2 (RUNX2), which is important for osteoblast and skeletal development. RUNX2-deficiency causes extra- and intra-oral malformations that often require orthodontic treatment. Nicotinamide (NAM) affects bone remodelling processes. As these are crucial for orthodontic therapy, NAM could improve orthodontic treatment in CCD patients. This study investigates the effect of NAM in control and RUNX2-deficient osteoblasts under mechanical strain mimicking orthodontic treatment. First, the optimal NAM concentration and the differences in the expression profile of control and RUNX2-deficient osteoblasts were determined. Subsequently, osteoblasts were exposed to tensile and compressive strain with and without NAM, and the expression of genes critically involved in bone remodelling was investigated. NAM increased the expression of bone remodelling genes. RUNX2-deficient osteoblasts expressed more receptor activator of NFkB ligand (RANKL) and interleukin-6 (IL6), but less colony-stimulating factor-1 (CSF1). Most of the positive effects of NAM on bone remodelling genes were impaired by mechanical loading. In conclusion, NAM stimulated osteoblast differentiation by increasing the expression of RUNX2 and regulated the expression of osteoclastogenic factors. However, the positive effects of NAM on bone metabolism were impaired by mechanical loading and RUNX2 deficiency.

Hypoglycaemia in adrenal insufficiency.

Adrenal insufficiency encompasses a group of congenital and acquired disorders that lead to inadequate steroid production by the adrenal glands, mainly glucocorticoids, mineralocorticoids and androgens. These may be associated with other hormone deficiencies. Adrenal insufficiency may be primary, affecting the adrenal gland's ability to produce cortisol directly; secondary, affecting the pituitary gland's ability to produce adrenocorticotrophic hormone (ACTH); or tertiary, affecting corticotrophin-releasing hormone (CRH) production at the level of the hypothalamus. Congenital causes of adrenal insufficiency include the subtypes of Congenital Adrenal Hyperplasia, Adrenal Hypoplasia, genetic causes of Isolated ACTH deficiency or Combined Pituitary Hormone Deficiencies, usually caused by mutations in essential transcription factors. The most commonly inherited primary cause of adrenal insufficiency is Congenital Adrenal Hyperplasia due to 21-hydroxylase deficiency; with the classical form affecting 1 in 10,000 to 15,000 cases per year. Acquired causes of adrenal insufficiency can be subtyped into autoimmune (Addison's Disease), traumatic (including haemorrhage or infarction), infective (e.g. Tuberculosis), infiltrative (e.g. neuroblastoma) and iatrogenic. Iatrogenic acquired causes include the use of prolonged exogenous steroids and post-surgical causes, such as the excision of a hypothalamic-pituitary tumour or adrenalectomy. Clinical features of adrenal insufficiency vary with age and with aetiology. They are often non-specific and may sometimes become apparent only in times of illness. Features range from those related to hypoglycaemia such as drowsiness, collapse, jitteriness, hypothermia and seizures. Features may also include signs of hypotension such as significant electrolyte imbalances and shock. Recognition of hypoglycaemia as a symptom of adrenal insufficiency is important to prevent treatable causes of sudden deaths. Cortisol has a key role in glucose homeostasis, particularly in the counter-regulatory mechanisms to prevent hypoglycaemia in times of biological stress. Affected neonates particularly appear susceptible to the compromise of these counter-regulatory mechanisms but it is recognised that affected older children and adults remain at risk of hypoglycaemia. In this review, we summarise the pathogenesis of hypoglycaemia in the context of adrenal insufficiency. We further explore the clinical features of hypoglycaemia based on different age groups and the burden of the disease, focusing on hypoglycaemic-related events in the various aetiologies of adrenal insufficiency. Finally, we sum up strategies from published literature for improved recognition and early prevention of hypoglycaemia in adrenal insufficiency, such as the use of continuous glucose monitoring or modifying glucocorticoid replacement.

Transcriptional reprogramming by mutated IRF4 in lymphoma

Disease-causing mutations in genes encoding transcription factors (TFs) can affect TF interactions with their cognate DNA-binding motifs. Whether and how TF mutations impact upon the binding to TF composite elements (CE) and the interaction with other TFs is unclear. Here, we report a distinct mechanism of TF alteration in human lymphomas with perturbed B cell identity, in particular classic Hodgkin lymphoma. It is caused by a recurrent somatic missense mutation c.295 T > C (p.Cys99Arg; p.C99R) targeting the center of the DNA-binding domain of Interferon Regulatory Factor 4 (IRF4), a key TF in immune cells. IRF4-C99R fundamentally alters IRF4 DNA-binding, with loss-of-binding to canonical IRF motifs and neomorphic gain-of-binding to canonical and non-canonical IRF CEs. IRF4-C99R thoroughly modifies IRF4 function by blocking IRF4-dependent plasma cell induction, and up-regulates disease-specific genes in a non-canonical Activator Protein-1 (AP-1)-IRF-CE (AICE)-dependent manner. Our data explain how a single mutation causes a complex switch of TF specificity and gene regulation and open the perspective to specifically block the neomorphic DNA-binding activities of a mutant TF.

The Presence of Somatic Mutations on Specific Pathways Such As Chromatin Modifier, Spliceosome, and Myeloid Transcription Factor at Diagnosis Was Found to Have Benefit from Allogeneic Hematopoietic Stem Cell Transplantation (HCT) in 1,228 Patients with Acute Myeloid Leukemia in First Complete Remission (CR1)

Introduction Allogeneic stem cell transplant (HCT) brings survival benefit to certain subgroups of acute myeloid leukemia (AML) patients, although it may not be suitable for all cases. Decision for HCT referral is based on the ELN risk grouping, which classifies pts into favorable, intermediate, and adverse risk categories. While HCT is recommended for pts in the intermediate and adverse risk, those in the favorable risk are deferred to receive HCT. However, currently, there is a lack of data directly demonstrating benefits of HCT in specific mutation groups. Patients and Method In this retrospective study, out of 1477 newly diagnosed AML pts 1,228 pts achieved CR1 and had undergone baseline next-generation sequencing (NGS) at diagnosis from January 1997 to January 2020. The data were collected from five different centers located in Toronto, Vancouver, and Montreal, Canada, as well as Hwasun and Seoul, South Korea. NGS was performed in each institution with each institution's standard targeted sequencing panel. To assess the effectiveness of HCT, particular attention will be given to the time from achieving complete remission 1 (CR1) to HCT. To handle immortal time bias, the Mantel-Byar method was used to depict outcomes during the follow-up period. Kaplan-Meier curves were avoided as they can introduce immortal bias, considering the period of time before treatment as part of the treatment arm. Instead, the MBT and landmark methods were employed to address this issue. Allogeneic HCT was treated as a time-dependent variable to prevent immortal bias. Gene mutations were grouped into 6 biologic pathways, including DNA methylation, chromatin modifiers, cohesion complex, activated signaling, tumor suppressor, spliceosome, and myeloid transcription factor (TF) mutations. In the multivariate (MVA) analysis, any mutations with a frequency of >5% were included as well as TP53 which has very grave prognosis. The primary objective of this study is to identify specific groups of AML pts who can derive significant benefits from receiving HCT in CR1, treating HCT as a time-dependent (td) covariate in the analysis. Statistical analyses were conducted using EZR version 1.41. Results The median duration of follow-up among survivors after achieving CR1 was 22.6 months (range: 1-217.9 months). Among the 593 pts, who underwent allogeneic HCT in CR1, the HCT group was composed of younger pts, with a median age of 50 years compared to 56 years in the non-HCT group (p < 0.001). Also, there were fewer pts with NPM1 mutation in the HCT group, accounting for 20.7% compared to 32.4% in the non-HCT group (p < 0.001). Apart from these characteristics, the two groups were similar in other parameters. The 2-year overall survival (OS) was higher in the HCT group, at 66.4%, compared to 54.6% in the non-HCT group (p < 0.0001). Similarly, the 2-year RFS was higher in the HCT group, with a rate of 64.8%, compared to 52.1% in the non-HCT group (p =1.82e-15). Furthermore, HCT as a time-dependent covariate was found to be favorable (Hazard ratio [HR] 0.76, 95% C.I. [0.63-0.91]; p=0.003), as demonstrated in the Simon-Makuch plot (p = 0.003). In the MVA, HCT as a time-dependent covariate retained its favorable impact on RFS (HR 0.67, [0.56-0.82]; p<0.0001). The MVA showed several prognostic factors besides HCT such as NPM1 and CEBPA mutations as favorable factors for RFS, being classified under the European Leukemia Network (ELN) favorable risk group. Conversely, FLT3-ITD, DNMT3A, IDH1, KIT, and TP53 mutations, monosomal karyotype, age > 60 years, and white blood count (WBC) at diagnosis > 55,600/mm^3 were associated with worse RFS. In terms of RFS, the greatest benefit of HCT was observed in the pts having mutations in ASXL1 (HR 0.34 [0.15-0.78]), BCOR (HR 0.29 [0.14-0.61]), IDH1/2 (HR 0.62 [0.42-0.92]), and RUNX1 (HR 0.17 [0.08-0.40]) but no significant benefit in those with KRAS (HR 1.59), NRAS (HR 1.21), PTPN11 (HR 1.23), and WT1 (HR 1.30). According to the biological pathway of somatic mutations, the greatest benefit of HCT in terms of RFS was observed in pts with mutations in the chromatin modifier (HR 0.37 [0.22-0.62]), spliceosome (HR 0.32 [0.16-0.65]), and myeloid TF pathway (HR 0.52 [0.34-0.79]). Conclusion Allogeneic HCT exerts a greater benefit on RFS in AML pts with mutations in the chromatin modifier, spliceosome, and myeloid TF pathways. Further study is warranted to replicate this finding.