Missense Research Articles

Novel Loss-of-function Variants of ZP3 Associated with Premature Ovarian Insufficiency.

Premature ovarian insufficiency (POI) is one of the leading causes of female infertility. To date, the genetic etiology of POI has been elucidated in approximately 20-25% of the total cases. The human zona pellucida (ZP) plays an important role in the organization and differentiation of granulosa cells, follicle formation, and sperm recognition. Mutations in ZP1, ZP2, and ZP3 have been reported to cause female infertility due to oocyte degeneration, empty follicle, or in vitro fertilization failure. In this study, we identified three novel missense mutations in ZP3 (NM_001110354.2): c.643G > A (p.Asp215Asn), c.215C > T (p.Thr72Ile), and c.152T > C (p.Leu51Pro) in three sporadic Han Chinese POI patients through whole-exome sequencing. These variants are absent from population databases and were predicted to be deleterious by multiple in silico tools. Structure prediction analysis showed that the affected amino acid altered the ZP3 protein structure. Western blot further confirmed that these ZP3 variants reduced the expression and secretion of ZP components. In summary, this study reports three novel deleterious variants in ZP3 associated with POI, thereby broadening the mutation spectrum of ZP3 in POI patients.

Dominant myosin storage myopathy mutations disrupt striated muscles in Drosophila and the myosin tail-tail interactome of human cardiac thick filaments.

Myosin storage myopathy (MSM) is a rare skeletal muscle disorder caused by mutations in the slow muscle/β-cardiac myosin heavy chain (MHC) gene. MSM missense mutations frequently disrupt the tail's stabilizing heptad repeat motif. Disease hallmarks include subsarcolemmal hyaline-like β-MHC aggregates, muscle weakness and, occasionally, cardiomyopathy. We generated transgenic, heterozygous Drosophila to examine the dominant physiological and structural effects of the L1793P, R1845W, and E1883K MHC MSM mutations on diverse muscles. The MHC variants reduced lifespan and flight and jump abilities. Moreover, confocal and electron microscopy revealed that they provoked indirect flight muscle breaks and myofibrillar disarray/degeneration with filamentous inclusions. Incorporation of GFP-myosin enabled in situ determination of thick filament lengths, which were significantly reduced in all mutants. Semi-automated heartbeat analysis uncovered aberrant cardiac function, which worsened with age. Thus, our fly models phenocopied traits observed among MSM patients. We additionally mapped the mutations onto a recently-determined, 6Å resolution, cryo-EM structure of the human cardiac thick filament. The R1845W mutation replaces a basic arginine with a polar-neutral, bulkier tryptophan, while E1883K reverses charge at critical filament loci. Both would be expected to disrupt the core and the outer shell of the backbone structure. Replacing L1793 with a proline, a potent breaker of alpha-helices, could disturb the coiled-coil of the myosin rod and alter the tail-tail interactome. Hence, all mutations likely destabilize and weaken the filament backbone. This may trigger disease in humans, while potentially analogous perturbations are likely to yield the observed thick filament and muscle disruption in our fly models.

Polo-like kinase 2 targeting as novel strategy to sensitize mutant p53-expressing tumor cells to anticancer treatments.

Polo-like kinase 2 (Plk2) belongs to a family of serine/threonine kinases, and it is involved in tumorigenesis of diverse kind of tissues. We previously reported that Plk2 gene was a transcriptional target of the mutant p53/NF-Y oncogenic complex. Plk2 protein can bind to and phosphorylate mutant p53 triggering an oncogenic autoregulatory feedback loop involved in cancer cell proliferation and chemoresistance. In this study, we aimed to assess whether the specific inhibition of Plk2 kinase activity by the selective TC-S 7005 inhibitor could decrease cell proliferation and migration inhibiting mutant p53 phosphorylation, thus disarming its oncogenic potential. We found that the Plk2 inhibitor treatment sensitized the cells to the irradiation and chemotherapy drugs, thereby overcoming the mutant p53-dependent chemoresistance. Taken together, we provided results that Plk2 could be considered a tractable pharmacological target for cancers expressing mutant p53 proteins. The combined treatment with conventional chemotherapeutic drugs and Plk2 inhibitors may represent a new candidate intervention approach, which may be considered for improving tumor cell sensitivity to DNA damaging drugs. KEY MESSAGES : Missense mutations are present in the TP53 gene in about half of all human cancers and correlate with poor patient outcome. Mutant p53 proteins exert gain of function (GOF) activities in tumor cells such as increased proliferation, genomic instability and resistance to therapies. Polo-like kinase 2 (PLK2) binds and phosphorylates mutant p53 protein strengthening its GOF activities. Pharmacologically targeting PLK2 weakens mutant p53 proteins and sensitizes tumor cells to therapeutic treatments.

Molecular and Clinicopathologic Characterization of HER2-overexpressed Squamous Cell Carcinoma of the Cervix.

HER2 amplification in cervical cancer has been associated with worse clinical prognosis and a potential favorable response to HER2 inhibitors. Immunohistochemistry for the HER2 receptor is a universally accepted surrogate test for HER2 amplification, but no standardized scoring system currently exists for cervical carcinomas. In this study, we investigated HER2 overexpression in cervical squamous cell carcinoma and correlated it with HER2 amplification using fluorescence in situ hybridization (FISH) and molecular methods. Seventy-two cases of human papillomavirus-associated cervical cancer were retrospectively reviewed, and at least 2 representative tumor sections were stained for HER2. HER2 scoring was performed using the 2018 American Society of Clinical Oncology/College of American Pathologist breast cancer criteria, and cases with equivocal (2+) to positive (3+) expression were analyzed for HER2 amplification using FISH and next-generation sequencing. The average patient age was 50yrs (range: 27-85yr), with most patients being African American (73.6%) and diagnosed at FIGO stage I (65.3%). Nineteen (26.4%) had equivocal HER2 expression and 4 (5.5%) showed positive expression. Three of the 4 cases with positive expression had enough tumors for FISH, and all 3 were amplified. Three cases with equivocal expression showed HER2 polysomy on FISH, and none showed HER2 amplification. Late clinical stage, high tumor grade, and regional lymph node metastasis were significantly correlated with HER2 overexpression and HER2 amplification. Next-generation sequencing of the 3 HER2-amplified tumors showed amplification of various genes, including CD274, JAK2, BIRC3, and ERBB2, and a PIK3CA missense mutation. In summary, HER2 immunohistochemistry is a reliable predictive marker of HER2 amplification in cervical cancer.

A novel root hair mutant, srh1, affects root hair elongation and reactive oxygen species levels in wheat

BackgroundRoot hairs are single-celled projections on root surfaces, critical for water and nutrient uptake. Here, we describe the first short root hair mutant in wheat (Triticum aestivum L.), identified in a mutagenized population and termed here short root hair 1 (srh1).ResultsWhile the srh1 mutant can initiate root hair bulges, lack of subsequent extension results in very short root hairs. Due to its semi-dominant nature, heterozygous lines displayed intermediate root hair lengths compared to wild-type. Bulked segregant analysis in a BC1F3 segregating population genotyped via exome capture sequencing localized the genetic control of this mutant to a region on the long arm of chromosome 3A. Via RNA sequencing and bioinformatic analysis, we identified two promising candidate genes. The first was a respiratory burst oxidase homolog (RBOH) encoding gene TaNOX3-A, orthologous to RBOH genes controlling root hair elongation in rice (OsNOX3) and maize (ZmRTH5), that carries a missense mutation in a conserved region of the predicted protein. RBOHs are membrane bound proteins that produce reactive oxygen species (ROS) which trigger cell wall extensibility, allowing subsequent root hair elongation. Notably, reduced ROS levels were observed in srh1 root hair bulges compared to wild-type. The second candidate was the calreticulin-3 encoding gene TaCRT3-A, located within the wider srh1 interval and whose expression was significantly downregulated in srh1 root tissues.ConclusionsThe identification of a major effect gene controlling wheat root hair morphology provides an entry point for future optimization of root hair architecture best suited to future agricultural environments.

Discovery and prioritization of genetic determinants of kidney function in 297,355 individuals from Taiwan and Japan

Current genome-wide association studies (GWAS) for kidney function lack ancestral diversity, limiting the applicability to broader populations. The East-Asian population is especially under-represented, despite having the highest global burden of end-stage kidney disease. We conducted a meta-analysis of multiple GWASs (n = 244,952) on estimated glomerular filtration rate and a replication dataset (n = 27,058) from Taiwan and Japan. This study identified 111 lead SNPs in 97 genomic risk loci. Functional enrichment analyses revealed that variants associated with F12 gene and a missense mutation in ABCG2 may contribute to chronic kidney disease (CKD) through influencing inflammation, coagulation, and urate metabolism pathways. In independent cohorts from Taiwan (n = 25,345) and the United Kingdom (n = 260,245), polygenic risk scores (PRSs) for CKD significantly stratified the risk of CKD (p < 0.0001). Further research is required to evaluate the clinical effectiveness of PRSCKD in the early prevention of kidney disease.

Extracellular vesicles characterization in patients with hypertrophic cardiomyopathy

Abstract Background Hypertrophic cardiomyopathy (HCM) is a genetic disorder, diagnosed according to the presence of phenotypical traits of the myocardium. A specific biomarker which can associate with the severity of HCM is lacking. Extracellular Vesicles (EVs), nanoparticles released by cells into biological fluids, hold promise as accessible diagnostic tools, as their abundance and molecular composition can reflect the severity of cardiac diseases (Rizzuto 2024). We aim at characterising plasma-derived EVs isolated from 28 HCM patients and 18 healthy volunteers (CTR). Methods The whole cohort underwent transthoracic echocardiography, whereas magnetic resonance and exome sequencing was performed on HCM patients. EVs were isolated via ultracentrifugation from plasma of both groups. Quantitative and qualitative assessments of EVs were performed by nanoparticle tracking analysis, transmission electron microscopy, Western blot, targeted (Olink) and untargeted (nLC-MS/MS) proteomics and FACS analysis. Plasma-derived EV subpopulations were characterised according to their cellular origin. Data are expressed as median and interquartile range (25th, 75th). Results Most patients were male (68% HCM and 66% CTR) with a median age of 58 (49-69) years (HCM) and 47 (44-52) (CTR). Among HCM patients, missense mutations in the MYH7 gene were the most identified. The median maximum wall thickness (WTMax) in HCM was 16 mm (15-19) vs 8 mm (7-9) in CTR. The isolated EVs expressed tetraspanins (CD9, CD63 and CD81), Alix and β-integrin and showed an intact phospholipid bilayer of 100 nm in size. EV concentration was reduced in HCM vs CTRL, respectively 2.8*109 EV/ml/cell count (2*109-4*109) and 4.6*10⁹ EV/ml/cell count (3*109-6*109). Among 15 plasma-derived EV subpopulations studied, those released from platelets (CD41a) and activated platelets (CD62P and CD40L) were increased in HCM patients vs CTR by 1.7 fold. Negative associations were found between E/e’, parameter of diastolic function, and cardiomyocyte-derived EVs (r= -0.2658), and between left ventricle ejection fraction and platelet-derived EVs (r=-0.2189); a positive correlation was found between WTMax and EVs derived from activated endothelial cells (r=0.2330). The proteomic analysis of EVs shows an enrichment in proteins related to platelets adhesion and inflammation in HCM patients. Conclusions HCM patients present a peculiar phenotypic pattern of EVs that may associate with diagnostic clinical features of HCM.

Effects of the restrictive cardiomyopathy-associated cardiac troponin I K178E mutation on cAMP signalling at the myofilament

Abstract Background Catecholaminergic stimulation of cardiac β-adrenergic receptors (β-ARs) and subsequent cAMP signalling control cardiac inotropy and lusitropy. cAMP signalling is known to be compartmentalised to optimise sympathetic control by producing heterogeneous cAMP signals and protein kinase A (PKA) activation at distinct subcellular sites. This signalling may be disrupted in diseases such as restrictive cardiomyopathy (RCM) characterised by diastolic dysfunction, which is associated with certain mutations including the K178E missense mutation in cardiac troponin I (TPNI). The impaired ventricular relaxation associated with this mutation is thought to arise from myofilament calcium hypersensitivity, which can be modulated by PKA-dependent TPNI phosphorylation. However, a detailed understanding of the pathological effects mediated by the K178E-TPNI mutation is lacking. Purpose This study aims to determine whether the K178E-TPNI mutation alters local cAMP/PKA signalling at TPNI, potentially affecting myofilament calcium sensitivity and cardiac myocyte relaxation. Methods Real-time imaging of cAMP was carried out using the Fluorescence Resonance Energy Transfer (FRET)-based sensor CUTie (cAMP Universal Tag for imaging experiments) [1] fused to wild type (WT)- or K178E-TPNI. The sensors were expressed by transfection in neonatal rat ventricular myocytes (NRVMs) and FRET-changes were recorded at the myofilament on application of the β-AR stimulant isoproterenol (ISO). We further assessed the involvement of cAMP hydrolysing phosphodiesterases (PDEs) in the regulation of cAMP levels selectively at TPNI. Results The peak FRET-change and FRET-change after 5mins on ISO application were significantly higher in NRVMs expressing the mutant compared to the WT sensor, as was the rate of cAMP accumulation. A strong trend showed that the rise in FRET following PDE4 inhibition was larger in NRVMs expressing the WT compared to the mutant sensor, suggesting the involvement of this isoform in degrading cAMP locally at TPNI and reduced PDE4 activity in the presence of the K178E mutation. Co-immunoprecipitation analysis in HEK293 cells demonstrated interaction between WT-TPNI and a PDE4D isoform, and a weaker interaction of this isoform with the K178E-TPNI mutant. Conclusions This study shows that the K178E-TPNI mutation attenuates the TPNI/PDE4D interaction and alters the magnitude and kinetics of the local cAMP response at the myofilament. This mechanistic insight may aid the development of therapeutics targeting the TPNI/PDE4D interaction to treat RCM-associated diastolic dysfunction.The TPNI-CUTie SensorFRET Response Trace

Novel bi-allelic DNAH3 variants cause oligoasthenoteratozoospermia

BackgroundOligoasthenoteratozoospermia (OAT) is a widespread cause of male infertility. One of the usual clinical manifestations of OAT is multiple morphological abnormalities of the sperm flagella (MMAF), which are frequently associated with mutations and defects in the dynein family. However, the relationship between the newly identified Dynein Axonemal Heavy Chain 3 (DNAH3) mutation and oligonasthenospermia in humans has not yet been established.MethodsWhole exome sequencing, pathogenicity analysis, and species conservation analysis of mutation sites were conducted on two patients from different unrelated families with DNAH3 mutations. We identified representative mutation sites and predicted the protein structure following these mutations. The sperm characteristics of the two patients with DNAH3 mutations were verified using Papanicolaou staining, scanning electron microscopy, and transmission electron microscopy. Additionally, mRNA and protein levels were assessed through RT-qPCR and Western blotting.ResultsThe biallelic mutations in the first progenitor included a heterozygous deletion and insertion, c.6535_6536 delinsAC (to infect mutation (p.Asp2179Thr), and stop codon premutation, c.3249G &amp;gt; A (p.Trp1083Ter). In Family II, the patient (P2) harbored a DNAH3 heterozygous missense mutation, c. 10439G&amp;gt; A(p.Arg3480Gln), along with a stop codon premutation, (c.10260G &amp;gt; A; p.Trp3420Ter). Patients with premature termination of transcription or translation due to DNAH3 mutations exhibit OAT phenotypes, including fibrous sheath dysplasia and multiple tail malformations. We identified the representative sites after mutation, predicted the protein structure, and assessed changes in the protein levels of DNAH3 and related genes following mutations. Notably,a significant reduction in DNAH3 protein expression was validated in these patients. We may explore in the future how DNAH3 affects sperm motility and quality through regulatory mechanisms involving protein structural changes.ConclusionNovel biallelic mutations in DNAH3, especially those resulting in a premature stop codon, may alter protein expression, structure, and active site, leading to spermatogenic failure and potentially inducing OAT. The discovery of new mutations in DNAH3 may be the key to the diagnosis and treatment of OAT.

Unraveling the molecular determinants of a rare human mitochondrial disorder caused by the P144L mutation of FDX2.

Episodic mitochondrial myopathy with or without optic atrophy and reversible leukoencephalopathy (MEOAL) is a rare, orphan autosomal recessive disorder caused by mutations in ferredoxin-2 (FDX2), which is a [2Fe-2S] cluster-binding protein participating in the formation of iron-sulfur clusters in mitochondria. In this biosynthetic pathway, FDX2 works as electron donor to promote the assembly of both [2Fe-2S] and [4Fe-4S] clusters. A recently identified missense mutation of MEOAL is the homozygous mutation c.431C>T (p.P144L) described in six patients from two unrelated families. This mutation alters a highly conserved proline residue located in a loop of FDX2 that is distant from the [2Fe-2S] cluster. How this Pro to Leu substitution damages iron-sulfur cluster biosynthesis is unknown. In this work, we have first compared the structural, dynamic, cluster binding and redox properties of WT and P144L [2Fe-2S] FDX2 to have clues on how the pathogenic P144L mutation can perturb the FDX2 function. Then, we have investigated the interaction of both WT and P144L [2Fe-2S] FDX2 with its physiological electron donor, ferredoxin reductase FDXR, comparing their electron transfer efficiency and protein-protein recognition patterns. Overall, the data indicate that the pathogenic P144L mutation negatively affects the FDXR-dependent electron transfer pathway from NADPH to FDX2, thereby reducing the capacity of FDX2 in assembling both [2Fe-2S] and [4Fe-4S] clusters. Our study also provided solid molecular evidences on the functional role of the C-terminal tail of FDX2 in the electron transfer between FDX2 and FDXR.

Identification of Two Novel Missense Variants in BNC1 in Han Chinese Patients With Non-syndromic Premature Ovarian Insufficiency.

Two novel heterozygous missense mutations in BNC1 (NM_001717): c.1000A>G (p.Arg334Gly) and c.1535C>T (p.Pro512Leu) were identified through whole-exome sequencing in two Han Chinese POI patients, expanding the spectrum of BNC1 variants in non-syndromic POI diseases.

In silico identification and characterisation of pathogenic genetic variants (nsSNPs) in the eukaryotic initiation factors eIF2 and eIF2B affecting miRNA binding sites

BackgroundThe eukaryotic translation initiation factors (eIF2 and eIF2B) are known to play a regulatory role in translation initiation. Studies have indicated that several missense mutations in both eIF2 and eIF2B subunits can lead to severe neurological diseases and cancer. In the current study, we have attempted to identify and characterise the single-nucleotide polymorphisms (SNPs) in the said subunits and their correlation with various diseases.ResultsInterestingly, we could identify SNPs only in 3′ untranslated regions (3′UTR) from EIF2 (EIF2S1 and S3) and EIF2B (EIF2B1, B2 and B5 subunits). Of which, two SNPs, one in each EIF2B1 (rs1050448) and EIF2B2 (rs4556), are observed to be affecting miRNA binding sites. The gene ontology (GO) analysis of identified miRNAs indicates their association with central nervous system development, various stress responses, growth factors, and immune system signalling pathways. Furthermore, molecular docking studies also confirm that the identified miRNAs have an excellent binding ability with corresponding wild-type/mutant dsDNA and mRNA with HADDOCK binding scores in the range of − 38.78 to − 3.99 kcal/mol and − 86.47 to − 23.78 kcal/mol, respectively.ConclusionWe conclude that the identified miRNAs may play a regulatory role in the symptomatic progression of neurological disorders and cancer and the same is validated by existing experimental evidences. Overall, the identified miRNAs serve as potential candidates for carrying out clinical investigations.Graphical abstract

Unveiling genetic anchors in saccharomyces cerevisiae: QTL mapping identifies IRA2 as a key player in ethanol tolerance and beyond.

Ethanol stress in Saccharomyces cerevisiae is a well-studied phenomenon, but pinpointing specific genes or polymorphisms governing ethanol tolerance remains a subject of ongoing debate. Naturally found in sugar-rich environments, this yeast has evolved to withstand high ethanol concentrations, primarily produced during fermentation in the presence of suitable oxygen or sugar levels. Originally a defense mechanism against competing microorganisms, yeast-produced ethanol is now a cornerstone of brewing and bioethanol industries, where customized yeasts require high ethanol resistance for economic viability. However, yeast strains exhibit varying degrees of ethanol tolerance, ranging from 8 to 20%, making the genetic architecture of this trait complex and challenging to decipher. In this study, we introduce a novel QTL mapping pipeline to investigate the genetic markers underlying ethanol tolerance in an industrial bioethanol S. cerevisiae strain. By calculating missense mutation frequency in an allele located in a prominent QTL region within a population of 1011 S. cerevisiae strains, we uncovered rare occurrences in gene IRA2. Following molecular validation, we confirmed the significant contribution of this gene to ethanol tolerance, particularly in concentrations exceeding 12% of ethanol. IRA2 pivotal role in stress tolerance due to its participation in the Ras-cAMP pathway was further supported by its involvement in other tolerance responses, including thermotolerance, low pH tolerance, and resistance to acetic acid. Understanding the genetic basis of ethanol stress in S. cerevisiae holds promise for developing robust yeast strains tailored for industrial applications.

Endothelial KLF11 is a novel protector against diabetic atherosclerosis

BackgroundAtherosclerotic cardiovascular diseases remain the leading cause of mortality in diabetic patients, with endothelial cell (EC) dysfunction serving as the initiating step of atherosclerosis, which is exacerbated in diabetes. Krüppel-like factor 11 (KLF11), known for its missense mutations leading to the development of diabetes in humans, has also been identified as a novel protector of vascular homeostasis. However, its role in diabetic atherosclerosis remains unexplored.MethodsDiabetic atherosclerosis was induced in both EC-specific KLF11 transgenic and knockout mice in the Ldlr−/− background by feeding a diabetogenic diet with cholesterol (DDC). Single-cell RNA sequencing (scRNA-seq) was utilized to profile EC dysfunction in diabetic atherosclerosis. Additionally, gain- and loss-of-function experiments were conducted to investigate the role of KLF11 in hyperglycemia-induced endothelial cell dysfunction.ResultsWe found that endothelial KLF11 deficiency significantly accelerates atherogenesis under diabetic conditions, whereas KLF11 overexpression remarkably inhibits it. scRNA-seq profiling demonstrates that loss of KLF11 increases endothelial-to-mesenchymal transition (EndMT) during atherogenesis under diabetic conditions. Utilizing gain- and loss-of-function approaches, our in vitro study reveals that KLF11 significantly inhibits EC inflammatory activation and TXNIP-induced EC oxidative stress, as well as Notch1/Snail-mediated EndMT under high glucose exposure.ConclusionOur study demonstrates that endothelial KLF11 is an endogenous protective factor against diabetic atherosclerosis. These findings indicate that manipulating KLF11 could be a promising approach for developing novel therapies for diabetes-related cardiovascular complications.

The Saccharomyces cerevisiae cell lysis mutant cly8 is a temperature-sensitive allele of ESP1.

The Saccharomyces cerevisiae mutants designated cly ( c ell ly sis) cause cell lysis at elevated temperatures. The cly8 mutation, previously localized to an 80 kilobase region between GCD2 and SPT6 on the right arm of chromosome VII, has been used for mutation mapping and in recombination assays, but its genetic identity has remained unknown. Whole genome sequencing of cly8 mutant and CLY8 wild-type strains revealed four missense mutations specific to the cly8 mutant strain in the GCD2 - SPT6 interval, three of these missense mutations affected essential genes. The esp1-G543E mutation, but not the two other missense mutations, recapitulated the temperature-sensitive growth, the cell lysis phenotype, and recessive nature of the cly8 mutation. The ESP1 gene encodes S. cerevisiae separase and is required for normal chromosome segregation during mitosis. Shifting cells containing the esp1-G543E mutation to the non-permissive temperature caused chromosome missegregation based on the analysis of DNA content by flow cytometry analysis. Taken together, these results indicate that the temperature-sensitive cly8 mutation is an allele of the essential ESP1 gene.

Bystander base editing interferes with visual function restoration in Leber congenital amaurosis.

Base editors (BEs) have emerged as a powerful tool for gene correction with high activity. However, bystander base editing, a byproduct of BEs, presents challenges for precise editing. Here, we investigated the effects of bystander edits on phenotypic restoration in the context of Leber congenital amaurosis (LCA), a hereditary retinal disorder, as a therapeutic model. We observed that in rd12 of LCA model mice, the highest editing activity version of an adenine base editors (ABEs), ABE8e, generated substantial bystander editing, resulting in missense mutations despite RPE65 expression, preventing restoration of visual function. Through AlphaFold-based mutational scanning and molecular dynamics simulations, we identified that the ABE8e-driven L43P mutation disrupts RPE65 structure and function. Our findings underscore the need for more stringent requirements in developing precise BEs for future clinical applications.

The NIPBL-gene mutation of a Cornelia de Lange Syndrome patient causes deficits in the hepatocyte differentiation of induced Pluripotent Stem Cells via altered chromatin-accessibility

The Cornelia de Lange syndrome (CdLS) is a rare genetic disease, which is characterized by a cohesinopathy. Mutations of the NIPBL gene are observed in 65% of CdLS patients. A novel iPSC (induced Pluripotent Stem Cell) line was reprogrammed from the leukocytes of a CdLS patient carrying a missense mutation of the NIPBL gene. A mutation-corrected isogenic iPSC-line and two iPSC-lines generated from the healthy parents were used as controls. The iPSC lines were differentiated along the hepatocyte-lineage. Comparative immunofluorescence, RNA-seq and ATAC-seq analyses were performed on undifferentiated and differentiated iPSCs. In addition, chromatin organization was studied by ChIP-Seq analysis on the patient derived iPSCs as well as the respective controls. Relative to the mutation-corrected and the healthy-parents iPSCs, the patient-derived counterparts are defective in terms of differentiation along the hepatocyte-lineage. One-third of the genes selectively up-regulated in CdLS-derived iPSCs and hepatic cells are non-protein-coding genes. By converse, most of the selectively down-regulated genes code for transcription factors and proteins regulating neural differentiation. Some of the transcriptionally silenced loci, such as the DPP6 gene on chromosome 7q36.2 and the ZNF gene cluster on chromosome 19p12, are located in closed-chromatin regions. Relative to the corresponding controls, the global transcriptomic differences observed in CdLS undifferentiated iPSCs are associated with altered chromatin accessibility, which was confirmed by ChIP-Seq analysis. Thus, the deficits in the differentiation along the hepatocyte lineage observed in our CdLS patient is likely to be due to a transcriptional dysregulation resulting from a cohesin-dependent alteration of chromatin accessibility.

Insilico Identification of Association between Nicotine Dependence and Pathogenic Missense Mutations of Catechol-O-Methyltransferase Gene

Introduction: Oral cancers are known to be strongly associated with the usage of tobacco, which is majorly composed of nicotine. Genetic studies evidenced the nicotine addiction as hereditary. AIM: The aim of the present study is to analyse any genetically functional missense mutations in Catechol-O-methyl-transferase (COMT) gene and derive an association with nicotine dependence that might influence tobacco habit initiation, addiction, and cessation. Methodology: The study involved an insilico analysis about the association between COMT gene and nicotine dependence. This was done with the help of different databases available in website. Initially, database called Ensembl (https://asia.ensembl.org) was used to pool the genetic data on missense mutations of the human COMT gene. The collected data was then fed into computational tools such as SIFT, PolyPhen, PROVEAN, I-Mutant, MutPred to discover the pathogenic mutations if exists. Results: Among 78 missense mutations collected, 74 were identified as damaging variants in which 8 variants were found to cause increased protein stability and 66 were found to cause decreased protein stability. Among those 66 missense variants causing decreased protein stability, 25 variants were identified as highly pathogenic while 19 variants were identified as pathogenic. Conclusion: The in-silico analysis discovered the existence of highly pathogenic mutations in COMT gene which might have a strong association with nicotine dependence that can influence the tobacco habit initiation, addiction, and cessation.

Comparative targeted genome profiling between solid and liquid biopsies in gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs): a proof-of-concept pilot study.

Clinical presentation and genetic profile of gastro-entero-pancreatic neuroendocrine tumors (GEP-NETs) are highly variable, hampering their management. Sequencing of circulating tumor DNA (ctDNA) from liquid biopsy (LB) has been proposed as a less invasive alternative to solid biopsy (SB). Our aim is to compare the mutational profile (MP) provided by LB with that deriving from SB in GEP-NETs. SB and LB derived simultaneously from 6 GEP-NETs patients. A comparative targeted Next Generation Sequencing (NGS) analysis was performed on DNA from SB and LB to evaluate the mutational status of 11 genes (MEN1, DAXX, ATRX, MUTYH, SETD2, DEPDC5, TSC2, ARID1A, CHECK2, MTOR, PTEN). Patients (M:F =2:1; median age 64 yrs) included 3 with pancreatic and 3 with ileal NETs. NGS detected a median number of 55 variants/sample in SB and 66.5 variants/sample in LB specimens (mutational burden: 0.2-1.9 and 0.3-1.8 mut/Mb, respectively). Missense and nonsense mutations were prevalent in both, mainly represented by C&gt;T transitions. ARID1A, MTOR, and ATRX were consistently mutated in SB and ARID1A, TSC2, MEN1, PTEN, SETD2, and MUTYH were consistently mutated in LB. DAXX mutations were absent in LB. 17 recurrent mutations were shared between SB and LB; in particular, MTOR single nucleotide variants (SNVs) c.G4731A and c.C2997T were shared by 5 out of 6 patients. Hierarchical clustering supported genetic similarity between SB and LB. This pilot study explores the applicability of LB in GEP-NETs MP evaluation. Further studies with larger cohorts are needed to validate LB and to define the clinical impact.

Intramuscular hemangioma capillary type with HRAS mutation: Expanding the molecular genetic spectrum with an emphasis on overlap with arteriovenous malformations and distinct from infantile hemangioma

AimsIntramuscular hemangioma capillary type (IHCT) is a rare entity that refers to fast-flow vascular lesions. This study aims to elucidate the relationships between clinicopathological, radiological, and molecular characteristics in IHCT patients. Methods and resultsWe reviewed all IHCT cases which were treated surgically in our pathology database from 2014 to 2023. Ten cases were analyzed via next-generation sequencing (NGS) and Sanger sequencing. The cohort consisted of 10 patients (6 males, 4 females) with a median age of 18 years (range: 1–37). Disease lesions were located in the trunk (n = 4), upper extremity (n = 2), lower extremity (n = 2), shoulder (n = 1), and neck (n = 1). IHCT is most commonly a progressively increasing painless mass. Histopathologically, all lesions exhibited aggregates, lobules, and anastomosing cords of capillary-type vessels separating or infiltrating the skeletal muscles. Four cases exhibited irregularly dilated vessels with thick walls, such as arteriovenous malformations (AVM) in the lesion's periphery. MRI findings commonly demonstrated a well-delineated, homogeneous mass. Somatic mutations were detected in seven of the ten IHCT cases. Four cases harbored mutations in MAP2K1 (p.Q58_E62del, p.K57_G61del, p.K57 N), two cases harbored mutations in KRAS (p.Q61R and p.L56V, p.G13R), and one case harbored a mutation in HRAS (p.D69_Q70insRWYSAMRD). Mutant allele frequencies detected by sequencing ranged from 9.98% to 15.97%. ConclusionsThe hemodynamic and molecular genetic phenotypes of IHCT closely resemble those observed in AVMs. Newly identified KRAS missense mutations, including cases with coexisting mutation types, and HRAS insertion mutations offer valuable insights into the genetic basis of vascular anomalies. These findings may also present potential targets for the development of novel pharmacotherapeutic interventions.