Improved constructs for bait RNA display in a bacterial three-hybrid assay.

We have previously developed a transcription-based bacterial three-hybrid (B3H) assay as a genetic approach to probe RNA-protein interactions inside of E. coli cells. This system offers a straightforward path to identify and assess the consequences of mutations in RBPs with molecular phenotypes of interest. One limiting factor in detecting RNA-protein interactions in the B3H assay is RNA misfolding arising from incorrect base-pair interactions with neighboring RNA sequences in a hybrid RNA. To support correct folding of hybrid bait RNAs, we have explored the use of a highly stable stem ("GC clamp") to isolate regions of a hybrid RNA as discrete folding units. In this work, we introduce new bait RNA constructs to 1) insulate the folding of individual components of the hybrid RNA with GC clamps and 2) express bait RNAs that do not encode their own intrinsic terminator. We find that short GC clamps (5 or 7 bp long) are more effective than a longer 13bp GC clamp in the B3H assay. These new constructs increase the number of Hfq-sRNA and -5'UTR interactions that are detectable in the B3H system and improve the signal-to-noise ratio of many of these interactions. We therefore recommend the use of constructs containing short GC clamps for the expression of future B3H bait RNAs. With these new constructs, a broader range of RNA-protein interactions are detectable in the B3H assay, expanding the utility and impact of this genetic tool as a platform to search for and interrogate mechanisms of additional RNA-protein interactions.

The impact and future of artificial intelligence in medical genetics and molecular medicine: an ongoing revolution.

Artificial intelligence (AI) platforms have emerged as pivotal tools in genetics and molecular medicine, as in many other fields. The growth in patient data, identification of new diseases and phenotypes, discovery of new intracellular pathways, availability of greater sets of omics data, and the need to continuously analyse them have led to the development of new AI platforms. AI continues to weave its way into the fabric of genetics with the potential to unlock new discoveries and enhance patient care. This technology is setting the stage for breakthroughs across various domains, including dysmorphology, rare hereditary diseases, cancers, clinical microbiomics, the investigation of zoonotic diseases, omics studies in all medical disciplines. AI's role in facilitating a deeper understanding of these areas heralds a new era of personalised medicine, where treatments and diagnoses are tailored to the individual's molecular features, offering a more precise approach to combating genetic or acquired disorders. The significance of these AI platforms is growing as they assist healthcare professionals in the diagnostic and treatment processes, marking a pivotal shift towards more informed, efficient, and effective medical practice. In this review, we will explore the range of AI tools available and show how they have become vital in various sectors of genomic research supporting clinical decisions.

Influence of genetic co-mutation on chemotherapeutic outcome in NPM1-mutated and FLT3-ITD wild-type AML patients.

BACKGROUND: Nucleophosmin 1 (NPM1) gene-mutated acute myeloid leukemia (NPM1mut AML) is classified as a subtype with a favorable prognosis. However, some patients fail to achieve a complete remission or relapse after intensified chemotherapy. Genetic abnormalities in concomitant mutations contribute to heterogeneous prognosis of NPM1mut AML patients. METHODS: In this study, 91 NPM1-mutated and FLT3-ITD wild-type (NPM1mut/FLT3-ITDwt) AML patients with intermediate-risk karyotype were enrolled to analyze the impact of common genetic co-mutations on chemotherapeutic outcome. RESULTS: Our data revealed that TET1/2 (52/91, 57.1%) was the most prevalent co-mutation in NPM1mut AML patients, followed by IDH1/2 (36/91, 39.6%), DNMT3A (35/91, 38.5%), myelodysplastic syndrome related genes (MDS-related genes) (ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1 and ZRSR2 genes) (35/91, 38.5%), FLT3-TKD (27/91, 29.7%) and GATA2 (13/91, 14.3%) mutations. Patients with TET1/2mut exhibited significantly worse relapse-free survival (RFS) (median, 28.7 vs. not reached (NR) months; p = 0.0382) compared to patients with TET1/2wt, while no significant difference was observed in overall survival (OS) (median, NR vs. NR; p = 0.3035). GATA2mut subtype was associated with inferior OS (median, 28 vs. NR months; p < 0.0010) and RFS (median, 24 vs. NR months; p = 0.0224) compared to GATA2wt. By multivariate analysis, GATA2mut and MDS-related genesmut were independently associated with worse survival. CONCLUSION: Mutations in TET1/2, GATA2 and MDS-related genes were found to significantly influence the chemotherapeutic outcome of patients with NPM1mut AML. The findings of our study have significant clinical implications for identifying patients who have an adverse response to frontline chemotherapy and provide a novel reference for further prognostic stratification of NPM1mut/FLT3-ITDwt AML patients.

The plasmid-encoded members of paralogous gene family 52 are dispensable to the enzootic cycle of Borrelia burgdorferi.

Lyme disease, the leading vector-borne disease in the United States and Europe, develops after infection with Borrelia burgdorferi sensu lato bacteria. Transmission of the spirochete from the tick vector to a vertebrate host requires global changes in gene expression that are controlled, in part, by the Rrp2/RpoN/RpoS alternative sigma factor cascade. Transcriptional studies defining the B. burgdorferi RpoS regulon have suggested that RpoS activates the transcription of paralogous family 52 (PFam52) genes. In strain B31, PFam52 genes (bbi42, bbk53, and bbq03) encode a set of conserved hypothetical proteins with >89% amino acid identity that are predicted to be surface-localized. Extensive homology among members of paralogous families complicates studies of protein contributions to pathogenicity as the potential for functional redundancy will obfuscate findings. Using a sequential mutagenesis approach, we generated clones expressing a single PFam52 paralog, as well as a strain deficient in all three. The single paralog expressing strains were used to confirm BBI42, BBK53, and BBQ03 surface localization and RpoS regulation. Surprisingly, the PFam52-deficient strain was able to infect mice and complete the enzootic cycle similar to the wild-type parental strain. Indeed, the presence of numerous pseudogenes that contain frameshifts or internal stop codons among the PFam52 genes suggests that they may be subjected to gene loss in B. burgdorferi's reduced genome. Alternatively, the lack of phenotype might reflect the limitations of the experimental mouse infection model.

Advancing the molecular understanding of renal tumorigenesis: Acquired cystic disease-associated renal cell carcinoma (ACD-RCC) as a model system.

In summary, the study's investigation of KMT2C and TSC2 variants in ACD-RCC marks a significant advancement in comprehending this distinct kidney tumor. By illuminating the molecular landscape of ACD-RCC, the research sets the stage for future studies aimed at revealing the complex mechanisms driving tumor development and progression. This understanding could eventually lead to more effective management and treatment strategies for renal cancer patients.

Atypical chronic myeloid leukemia found in a patient with eosinophilia for six years: a case report.

BACKGROUND: Atypical chronic myeloid leukemia (aCML) is a highly aggressive type of blood cancer that falls under the category of myelodysplastic/myeloproliferative neoplasms (MDS/MPN). In the fifth edition of the WHO classification of tumors, this category has been renamed MDS/MPN with neutrophilia. Although eosinophilia is commonly observed in blood cancers, it is rarely seen in aCML. CASE PRESENTATION: This study presents a case of aCML that was diagnosed six years after the patient developed eosinophilia. The patient had undergone tests to rule out other primary and secondary diseases, but the eosinophilia remained unexplained. Treatment with corticosteroids and hydroxyurea had proven ineffective. Six years later, the patient experienced an increase in white blood cells, primarily neutrophils. After ruling out other possible diagnoses, a combination of morphologic and molecular genetic findings led to the diagnosis of aCML. The patient responded well to treatment with azacitidine. CONCLUSIONS: This study summarizes the current state of aCML diagnosis and management and discusses the possible connection between eosinophilia and aCML.

Vhl safeguards thymic epithelial cell identity and thymopoietic capacity by constraining Hif1a activity during development.

The thymus is a physiologically hypoxic organ and fulfills its role of generating T cells under low-oxygen conditions. We have therefore investigated how thymic epithelial cells (TECs) cope with physiological hypoxia by focusing on the role of the Hif1a-Vhl axis. In most cell types, the oxygen-labile transcriptional regulator Hif1a is a central player in co-ordinating responses to low oxygen: under normoxic conditions Hif1a is rapidly degraded in a Vhl-guided manner; however, under hypoxic conditions Hif1a is stabilized and can execute its transcriptional functions. Unexpectedly, we find that, although TECs reside in a hypoxic microenvironment, they express little Hif1a protein and do not require Hif1a for their development or function. Instead, we find that Vhl function in TECs is vital to constrain Hif1a activity, as loss of Vhl results in dramatic defects in TEC differentiation and thymopoiesis, which can be rescued by Hif1a co-depletion.

Near-tetraploidy/tetraploidy acute myeloid leukemia with clinical, pathologic and molecular characteristics.

Near-tetraploidy/tetraploidy (NT/T) is a cytogenetic alteration in acute myeloid leukemia (AML). In AML, specific chromosomal alterations are associated with clinical, morphological, and immunophenotypic features. The impact of cytogenetics on the prognosis of AML is well established. However, the prognostic implication of NT/T on AML remains unclear. Our aim is to further characterize the clinical, morphologic, immunophenotypic, molecular mutational, and prognostic features of NT/T AML. This retrospective chart review of NT/T AML cases showed NT/T AML was more common in older adult males, with predominately large blasts and myelodysplasia-related features. The most common lineage of dysplasia was dysgranulopoiesis in 77.8% of cases. Cases displayed multiple cytogenetic abnormalities, with only four showing NT/T as the sole abnormality. TP53 was the most common molecular mutation associated with NT/T AML (44.5%). Of the patients receiving treatment for NT/T AML, 80% achieved a CR. The median overall survival for the entire cohort was 4.5 months.

Blockade of mesenteric and omental adipose tissue sensory neurons improves cardiac remodeling through sympathetic pathway.

Mesenteric and omental adipose tissue (MOAT) communicates directly with the heart through the secretion of bioactive molecules and indirectly through afferent signaling to the central nervous system. Myocardial infarction (MI) may induce pathological alterations in MOAT, which further affects cardiac function. Our study revealed that MI induced significant MOAT transcriptional changes in genes related with signal transduction, including adiponectin (APN), neuropeptide Y (NPY), and complement C3 (C3), potentially influencing afferent activity. We further found that MOAT sensory nerve denervation with capsaicin (CAP) prevented cardiac remodeling, improved cardiac function, and reversed cardiac sympathetic nerve hyperactivation in the MI group, accompanied by reduced serum norepinephrine. In addition, CAP reversed the elevated MOAT afferent input and brain-heart sympathetic outflow post-MI, increasing APN and NPY and decreasing C3 and serum proinflammatory factors. These results demonstrated that blockade of the MOAT afferent sensory nerve exerts a cardioprotective effect by inhibiting the brain-heart sympathetic axis.

Proteasome-mediated degradation of long-range nucleases negatively regulates resection of DNA double-strand breaks.

Homologous recombination is initiated by the nucleolytic degradation (resection) of DNA double-strand breaks (DSBs). DSB resection is a two-step process. In the short-range step, the MRX (Mre11-Rad50-Xrs2) complex, together with Sae2, incises the 5'-terminated strand at the DSB end and resects back toward the DNA end. Then, the long-range resection nucleases Exo1 and Dna2 further elongate the resected DNA tracts. We found that mutations lowering proteasome functionality bypass the need for Sae2 in DSB resection. In particular, the dysfunction of the proteasome subunit Rpn11 leads to hyper-resection and increases the levels of both Exo1 and Dna2 to such an extent that it allows the bypass of the requirement for either Exo1 or Dna2, but not for both. These observations, along with the finding that Exo1 and Dna2 are ubiquitylated, indicate a role of the proteasome in restraining DSB resection by negatively controlling the abundance of the long-range resection nucleases.

Congenital Myasthenic Syndromes in Belgium: Genetic and Clinical Characterization of Pediatric and Adult Patients.

BACKGROUND: Congenital myasthenic syndromes (CMS) are a group of genetic disorders characterized by impaired neuromuscular transmission. CMS typically present at a young age with fatigable muscle weakness, often with an abnormal response after repetitive nerve stimulation (RNS). Pharmacologic treatment can improve symptoms, depending on the underlying defect. Prevalence is likely underestimated. This study reports on patients with CMS followed in Belgium in 2022. METHODS: Data were gathered retrospectively from the medical charts. Only likely pathogenic and pathogenic variants were included in the analysis. RESULTS: We identified 37 patients, resulting in an estimated prevalence of 3.19 per 1,000,000. The patients harbored pathogenic variants in CHRNE, RAPSN, DOK7, PREPL, CHRNB1, CHRNG, COLQ, MUSK, CHRND, GFPT1, and GMPPB. CHRNE was the most commonly affected gene. Most patients showed disease onset at birth, during infancy, or during childhood. Symptom onset was at adult age in seven patients, caused by variants in CHRNE, DOK7, MUSK, CHRND, and GMPPB. Severity and distribution of weakness varied, as did the presence of respiratory involvement, feeding problems, and extraneuromuscular manifestations. RNS was performed in 23 patients of whom 18 demonstrated a pathologic decrement. Most treatment responses were predictable based on the genotype. CONCLUSIONS: This is the first pooled characterization of patients with CMS in Belgium. We broaden the phenotypical spectrum of pathogenic variants in CHRNE with adult-onset CMS. Systematically documenting larger cohorts of patients with CMS can aid in better clinical characterization and earlier recognition of this rare disease. We emphasize the importance of establishing a molecular genetic diagnosis to tailor treatment choices.

Regulation of root growth and elongation in wheat.

Currently, the control of rhizosphere selection on farms has been applied to achieve enhancements in phenotype, extending from improvements in single root characteristics to the dynamic nature of entire crop systems. Several specific signals, regulatory elements, and mechanisms that regulate the initiation, morphogenesis, and growth of new lateral or adventitious root species have been identified, but much more work remains. Today, phenotyping technology drives the development of root traits. Available models for simulation can support all phenotyping decisions (root trait improvement). The detection and use of markers for quantitative trait loci (QTLs) are effective for enhancing selection efficiency and increasing reproductive genetic gains. Furthermore, QTLs may help wheat breeders select the appropriate roots for efficient nutrient acquisition. Single-nucleotide polymorphisms (SNPs) or alignment of sequences can only be helpful when they are associated with phenotypic variation for root development and elongation. Here, we focus on major root development processes and detail important new insights recently generated regarding the wheat genome. The first part of this review paper discusses the root morphology, apical meristem, transcriptional control, auxin distribution, phenotyping of the root system, and simulation models. In the second part, the molecular genetics of the wheat root system, SNPs, TFs, and QTLs related to root development as well as genome editing (GE) techniques for the improvement of root traits in wheat are discussed. Finally, we address the effect of omics strategies on root biomass production and summarize existing knowledge of the main molecular mechanisms involved in wheat root development and elongation.

Whole-Exome Sequencing in Turkish Patients with Inherited Retinal Dystrophies Reveals Novel Variants in Ten Genes.

Introduction: Inherited retinal dystrophies (IRDs) associated with more than 300 genes are a clinically and genetically heterogeneous group of retinal diseases. This study aimed to identify causative gene variants and molecular basis of Turkish patients with IRD. Methods: Whole-exome sequencing was performed in 28 unrelated patients. The potential pathogenicity of variants was evaluated using the American College of Medical Genetics variant interpretation guidelines, in silico prediction tools, published literature or Human Gene Mutation Database, and compatibility with inheritance patterns or known phenotypes. Results: Causative variants in 21 genes, including MERTK, SNRP200, MYO7A, AIPL1, RDH12, OTX2, ADGRV1, RPGRIP1, SPATA7, USH2A, MFSD8, CDHR1, EYS, CACNA1F, CNGA3, RDH5, TULP1, BBS2, BEST1, RS1, GUCY2D were detected in 26 (92.9%) of 28 patients. The most prevalent causative variants were observed MERTK (10.7% of cases), followed by CDHR1, AIPL1, RDH12, SPATA7, CNGA3, TULP1 (7.1% of cases, each). The most common variant type in this study was missense variants (53%), followed by frameshift (21%), nonsense (20%), and splice (6%). Twelve novel variants, 6 of frameshift and 6 of missense, were detected in ten genes. Retinitis pigmentosa was the most common phenotype followed by Leber congenital amaurosis. Conclusion: This study provides an overview of causative gene variants in Turkish patients with IRD. Variants identified in this study expand the variant spectrum of IRD genes. We believe it is essential to combine molecular and clinical data to diagnose IRD patients, especially with the emergence of therapeutic options.

Discovery and functional investigation of BMP4 as a new causative gene for human congenital heart disease.

OBJECTIVE: Aggregating evidence highlights the strong genetic basis underpinning congenital heart disease (CHD). Here BMP4 was chosen as a prime candidate gene causative of human CHD predominantly because BMP4 was amply expressed in the embryonic hearts and knockout of Bmp4 in mice led to embryonic demise mainly from multiple cardiovascular developmental malformations. The aim of this retrospective investigation was to discover a novel BMP4 mutation underlying human CHD and explore its functional impact. METHODS: A sequencing examination of BMP4 was implemented in 212 index patients suffering from CHD and 236 unrelated non-CHD individuals as well as the family members available from the proband carrying a discovered BMP4 mutation. The impacts of the discovered CHD-causing mutation on the expression of NKX2-5 and TBX20 induced by BMP4 were measured by employing a dual-luciferase analysis system. RESULTS: A new heterozygous BMP4 mutation, NM_001202.6:c.318T>G;p.(Tyr106*), was found in a female proband affected with familial CHD. Genetic research of the mutation carrier's relatives unveiled that the truncating mutation was in co-segregation with CHD in the pedigree. The nonsense mutation was absent from 236 unrelated non-CHD control persons. Quantitative biologic measurement revealed that Tyr106*-mutant BMP4 failed to induce the expression of NKX2-5 and TBX20, two genes whose expression is lost in CHD. CONCLUSION: The current findings indicate BMP4 as a new gene predisposing to human CHD, allowing for improved prenatal genetic counseling along with personalized treatment of CHD patients.

Distinct features of ribonucleotides within genomic DNA in Aicardi-Goutières syndrome ortholog mutants of Saccharomyces cerevisiae.

Ribonucleoside monophosphates (rNMPs) are abundantly found within genomic DNA of cells. The embedded rNMPs alter DNA properties and impact genome stability. Mutations in ribonuclease (RNase) H2, a key enzyme for rNMP removal, are associated with the Aicardi-Goutières syndrome (AGS), a severe neurological disorder. Here, we engineered orthologs of the human RNASEH2A-G37S and RNASEH2C-R69W AGS mutations in yeast Saccharomyces cerevisiae: rnh201-G42S and rnh203-K46W. Using the ribose-seq technique and the Ribose-Map bioinformatics toolkit, we unveiled rNMP abundance, composition, hotspots, and sequence context in these AGS-ortholog mutants. We found a high rNMP presence in the nuclear genome of rnh201-G42S-mutant cells, and an elevated rCMP content in both mutants, reflecting preferential cleavage of RNase H2 at rGMP. We discovered unique rNMP patterns in each mutant, showing differential activity of the AGS mutants on the leading or lagging replication strands. This study guides future research on rNMP characteristics in human genomes with AGS mutations.

Evaluation of Acute Myeloid Leukemia Genomes using Genomic Proximity Mapping.

Background: Cytogenetic analysis encompasses a suite of standard-of-care diagnostic testing methods that is routinely applied in cases of acute myeloid leukemia (AML) to assess chromosomal changes that are clinically relevant for risk classification and treatment decisions. Objective: In this study, we assess the use of Genomic Proximity Mapping (GPM) for cytogenomic analysis of AML diagnostic specimens for detection of cytogenetic risk variants included in the European Leukemia Network (ELN) risk stratification guidelines. Methods: Archival patient samples (N=48) from the Fred Hutchinson Cancer Center leukemia bank with historical clinical cytogenetic data were processed for GPM and analyzed with the CytoTerra® cloud-based analysis platform. Results: GPM showed 100% concordance for all specific variants that have associated impacts on risk stratification as defined by ELN 2022 criteria, and a 72% concordance rate when considering all variants reported by the FH cytogenetic lab. GPM identified 39 additional variants, including variants of known clinical impact, not observed by cytogenetics. Conclusions: GPM is an effective solution for the evaluation of known AML-associated risk variants and a source for biomarker discovery.

Molecular Genetics of Acquired Temporal Lobe Epilepsy.

An epilepsy diagnosis reduces a patient's quality of life tremendously, and it is a fate shared by over 50 million people worldwide. Temporal lobe epilepsy (TLE) is largely considered a nongenetic or acquired form of epilepsy that develops in consequence of neuronal trauma by injury, malformations, inflammation, or a prolonged (febrile) seizure. Although extensive research has been conducted to understand the process of epileptogenesis, a therapeutic approach to stop its manifestation or to reliably cure the disease has yet to be developed. In this review, we briefly summarize the current literature predominately based on data from excitotoxic rodent models on the cellular events proposed to drive epileptogenesis and thoroughly discuss the major molecular pathways involved, with a focus on neurogenesis-related processes and transcription factors. Furthermore, recent investigations emphasized the role of the genetic background for the acquisition of epilepsy, including variants of neurodevelopmental genes. Mutations in associated transcription factors may have the potential to innately increase the vulnerability of the hippocampus to develop epilepsy following an injury-an emerging perspective on the epileptogenic process in acquired forms of epilepsy.