Search Results (651)

The flavivirus NS1 protein is a component of the viral replication complex and plays diverse, yet poorly understood, roles in the viral life cycle. To enable real-time visualization of the developing replication organelle and biochemical analysis of tagged NS1 and its interacting partners, we engineered a replication-competent yellow fever virus (YFV) replicon encoding a C-terminal fusion of NS1 with green fluorescent protein (NS1–GFP). The initial variant was non-viable in the absence of trans-complementation with wild-type NS1; however, viability was partially restored through the introduction of co-adaptive mutations in GFP (Q204R/A206V) and NS4A (M108L). Subsequent cell culture adaptation generated a 17-nucleotide frameshift within the NS1–GFP linker, resulting in a more flexible and less hydrophobic linker sequence. The optimized genome, in the form of a replicon, replicates in packaging cells that produce YFV structural proteins, as well as in naive BHK-21 cells. In the packaging cells, the adapted NS1–GFP replicon produces titers of infectious particles of approximately 10^6 FFU/mL and is genetically stable over five passages. The expressed NS1–GFP fusion protein localizes to the endoplasmic reticulum and co-fractionates with detergent-resistant heavy membranes, a hallmark of flavivirus replication organelles. This NS1–GFP replicon provides a novel platform for studying NS1 functions and can be further adapted for proximity-labeling strategies aimed at identifying the still-unknown protease responsible for NS1–NS2A cleavage. Full article

The neurofibromin 1 (NF1) splice-site mutation c.61-2A>G (rs1131691100) is a rare, pathogenic, autosomal dominant variant that disrupts NF1 tumor-suppressor function, causing neurofibromatosis type 1 (NF1). Its pathogenic mechanism is poorly understood, and the potential for personalized therapeutic genome editing remains unknown due to the absence of a standard framework for investigating splicing disorders. Here, we performed a comprehensive multi-omics analysis of a de novo c.61-2A>G case from South Korea, integrating short- and long-read whole genome sequencing, whole transcriptome sequencing, and methylation profiling. We confirm that c.61-2A>G abolishes the canonical splice acceptor site, activating a cryptic splice acceptor 16 nucleotides downstream in exon 2. This splicing shift generates a 16-nucleotide deletion, causing a frameshift and premature stop codon that truncates the protein’s N-terminal region. Long-read sequencing further reveals that the mutation creates a novel CpG dinucleotide, which is methylated in the majority of reads. Finally, we assessed therapeutic correction strategies, revealing that CRISPR-Cas9 prime editing is the only viable approach for in vivo correction. This study provides the first comprehensive multi-omics characterization of the NF1 c.61-2A>G mutation and establishes a minimal framework for precision therapeutic development in silico in monogenic splicing disorders. Full article

Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancers with adenocarcinoma being the most common subtype. Patients with stage IV NSCLC typically have poor prognosis. In these patients, identification of actionable genomic alterations allows for the selection of targeted therapy rather than chemotherapy or chemo-immunotherapy. EGFR mutations are a common oncogenic driver in NSCLC and are targetable by tyrosine kinase inhibitors (TKIs). However, most of the studies primarily focus on common mutations, which are exon 19 deletions (Ex19del) and exon 21 (L858R) point mutations, and there is inconsistent data on efficacy in the treatment of patients with uncommon EGFR mutations. Currently, the first-line treatment for patients with common EGFR mutations involves a third-generation TKI, typically osimertinib. This case describes a 66-year-old gentleman with two uncommon EGFR frameshift deletions (E701fs and L702fs). His tumor staging was denoted as cT3N2M1b, stage IVA. The patient demonstrated a radiological and biochemical response to osimertinib as part of the OCELOT clinical trial (supported by a grant from AstraZeneca), with evidence of tumor marker decline and radiographic improvement within two months of osimertinib treatment initiation. This response has been durable with continued radiological stability and biochemical improvement at 11 months and ongoing. This case will help guide management for patients with this uncommon EGFR mutations and contribute to the scarce literature of EGFR frameshift deletions in advanced NSCLC patients. Full article

Background/Objectives: Advances in the genetic understanding of pheochromocytoma–paraganglioma (PPGL) have considerably refined personalized approaches to diagnosis and management. This study aims to present our institutional experience on the diagnostic characteristics, clinical course, and genetic background of patients with PPGL, in the context of the current literature. Methods: This retrospective analysis included 35 patients diagnosed with PPGL between years 2020 and 2024, all of whom underwent surgical resection and next-generation sequencing for germline mutations in major PPGL susceptibility genes. Clinical presentation, biochemical profile, pathological findings, and follow-up outcomes were compared between mutation-positive and mutation-negative cases. Results: Of the 35 patients with PPGL, germline mutations were identified in 6 patients (17%): 2 in Cluster 1A genes (SDHA, SDHB), 2 in Cluster 1B (VHL), and 2 in Cluster 2 (NF1). Consistent with existing literature, pathogenic germline variants—particularly SDHB and VHL—were identified in our cohort exclusively in patients younger than 30 years (ages 17, 20, and 25). Mutation-positive patients more frequently exhibited noradrenergic or non-secretory profiles (p = 0.01). Among the three non-secretory tumors in the cohort, two harbored genetic mutations (SDHA, NF1). Interestingly, both NF1-positive patients were normotensive—one (c.3496G > A) with a non-secretory tumor and the other (c.2329T > A) presenting at an unusually late age (63 years)—a strikingly atypical spectrum that underscores the phenotypic variability of NF1-associated PPGL. Bilateral disease was observed exclusively in VHL carriers (p = 0.03). Importantly, we identified a rare VHL c.369delG frameshift variant, not previously reported in association with PPGLs, in a patient with PPGL. No significant difference was observed between SDHB loss (p = 0.1) and proliferative indices (mitotic count, Ki-67) (p = 0.07, p = 0.6) between the two groups. During a median follow-up of 24 months (IQR: 18–36), one SDHB-positive patient had a recurrence, while no distant metastases were detected in the remaining mutation carriers. Conclusions: These findings support characteristic clinical patterns among mutation-positive PPGL and underscore the importance of systematic germline testing in all cases—irrespective of age, family history, or biochemical profile—to guide individualized management and enable cascade screening. The identification of a rare VHL c.369delG variant, previously unreported in association with PPGL, within a characteristic VHL-related clinical phenotype highlights the importance of this association. Similarly, atypical NF1 cases emphasize phenotypic variability and reinforce the importance of germline testing even in clinically silent presentations. Full article

Genome editing of late myogenic regulators provides a way to dissect the mechanisms through which transcriptional programs and growth-related signaling pathways shape muscle gene expression programs in farmed fish. This study disrupted myogenic regulatory factor 4 (MRF4) in Nile tilapia using CRISPR/Cas9 to examine downstream transcriptional changes in fast skeletal muscle across the trunk, belly, and head regions. Adult F0 crispants carried a frameshift mutation that truncated the basic helix–loop–helix domain and showed an approximate 80–85% reduction in MRF4 mRNA across the trunk, belly, and head muscles. The expression of 23 genes representing myogenic regulatory factors, MEF2 paralogs, structural and contractile components, non-myotomal regulators, cell adhesion and fusion-related transcripts, and growth-related genes within the GH–IGF–MSTN axis was quantified and compared between wild-type and MRF4-crispants. Expressions of major structural genes remained unchanged despite MRF4 depletion, whereas MyoG and MyoD were upregulated together with MEF2B and MEF2D, indicating strong transcriptional compensation. Twist1, ID1, PLAU, CDH15, CHRNG, NCAM1, MYMK, GHR, and FGF6 were also significantly elevated, while IGF1 was reduced, and MSTN remained stable. Together, these results show that MRF4 loss is associated with coordinated transcriptional changes in regulatory and growth-related pathways, while major fast-muscle structural and contractile transcript levels remain stable, thereby highlighting candidate transcriptional targets for future studies that will evaluate links to muscle phenotype and growth performance in Nile tilapia. Full article

Background: Coffin–Siris syndrome 12 (CSS12) is a recently described neurodevelopmental disorder caused by heterozygous pathogenic variants in BICRA, a gene encoding a core subunit of the non-canonical BAF (ncBAF) chromatin-remodeling complex. The condition is characterized by developmental delay, hypotonia, hypertrichosis, and joint laxity. However, long-term data remain limited, and systemic manifestations are incompletely defined. Case Description: We report a 22-year-old male with a de novo BICRA frameshift variant, c.2479_2480delinsA (p.Ala827Thrfs*15), previously included in the original cohort reported by Barish et al. Longitudinal follow-up revealed an expanded phenotype extending beyond neurodevelopmental features. Early findings included global developmental delay, growth hormone deficiency, short stature, and joint hypermobility. In adolescence and adulthood, he developed severe intestinal dysmotility requiring total colectomy, recurrent spontaneous pneumothoraces from bilateral apical bullous disease, and portal-vein thrombosis, representing visceral and vascular complications not previously emphasized in BICRA-related disorders. The identified BICRA variant truncates the coiled-coil domain critical for BRD9/BRD4 interaction, consistent with a loss-of-function mechanism. The patient’s systemic features suggest that BICRA haploinsufficiency affects not only neurodevelopmental pathways but also smooth-muscle and connective-tissue integrity. Conclusions: This case expands the phenotypic spectrum of BICRA-related CSS12, demonstrating that visceral and vascular involvement can occur alongside neurodevelopmental and connective-tissue features. Recognition of these broader manifestations underscores the need for lifelong multidisciplinary surveillance and contributes to understanding the diverse biological roles of the ncBAF complex in human development. Full article

Background: Advances in genomic technologies combined with tandem mass newborn screening have enabled early detection and management of several common inborn errors of metabolism. Fructose-1,6-bisphosphatase deficiency, an autosomal recessive treatable disorder reported in around 150 patients worldwide, remains underdiagnosed despite an excellent prognosis with early detection. Although common in highly consanguineous populations, diagnosis is often delayed due to the non-specific clinical and biochemical profile. Methods: This report explores the diagnostic pathway using first-tier next-generation sequencing of three novel cases of fructose-1,6-bisphosphatase deficiency in a tertiary care center in Lebanon. Results: Two patients were diagnosed with first-tier exome sequencing within one month of presentation and had an excellent outcome at 6 years of follow-up. The third patient, undiagnosed for 10 years, suffered from neurological sequalae. The molecular profile was remarkable in two patients for exon 2 deletion in the FBP1 gene, a founder mutation reported in Turkish and Armenian patients, and a rare frameshift mutation in the third case. Conclusions: The use of next-generation sequencing as as a first-tier test for FBP deficiency is a non-invasive and rapid method for early diagnosis and management of this rare yet treatable disorder. It can detect both disease-causing variants and large deletions, founder mutations as well, delineating the molecular profile in populations where this disorder is highly prevalent. Full article

Neurodevelopmental disorders (NDDs) with ataxia are genetically heterogeneous and remain a diagnostic challenge. Recent advances in genomic technologies have facilitated the identification of rare, potentially causative variants in genes not traditionally associated with classic NDD phenotypes. The DNAH14 gene, encoding a dynein axonemal heavy chain involved in ciliary motility, has recently emerged as a novel candidate in neurological syndromes. Here, we report two Turkish siblings presenting with late-onset balance disorder, progressive ataxia, and cognitive impairment. Initial genetic analysis revealed that both siblings also harbor FXN GAA repeat expansions consistent with pathogenic Friedreich’s ataxia (FRDA). To elucidate the molecular basis of the patients’ cognitive impairment, whole-exome sequencing was performed. This analysis identified a novel homozygous frameshift variant in the DNAH14 gene, located within the conserved linker domain upstream of the motor core, which is critical for ATP hydrolysis and microtubule interactions. The variant is absent from population databases, predicted to be deleterious by multiple in silico algorithms, and segregates in the family in a manner consistent with autosomal recessive inheritance. The coexistence of FRDA expansions and a truncating DNAH14 variant suggests a potential dual genetic contribution to the observed phenotype, in which FRDA-associated pathology likely underlies the ataxia, while DNAH14 disruption may contribute to additional neurodevelopmental features. This is the first report describing the co-occurrence of FRDA and a homozygous truncating DNAH14 variant in the same individuals, broadening our understanding of overlapping neurogenetic mechanisms. Our findings expand the phenotypic spectrum of DNAH14-related disorders and highlight the importance of considering multilocus pathogenic variants in patients with complex or atypical ataxia presentations. Full article

Variants of the MYO5B gene, which encodes the molecular motor protein myosin-Vb, have gained prominence as a causative factor in familial intrahepatic cholestasis (FIC). Understanding the disease mechanism is pivotal for therapy development and clinical decision-making. The prevailing theory for the mechanism underlying MYO5B-associated cholestasis implicates faulty trafficking of the ABCB11-encoded bile salt export pump (BSEP) in hepatocytes due to dysfunctional myosin-Vb. This is supported by cell and mouse studies. However, while BSEP localization was abnormal in some patients’ liver biopsies, BSEP appeared normally localized in others, raising questions with regard to the role of BSEP in MYO5B-associated FIC. We present a focused systematic narrative review of all cases of MYO5B variant-associated isolated FIC reported in the MEDLINE database. We assembled a comprehensive patient dataset and assessed clinical features of MYO5B-associated FIC, their relationship with MYO5B genotype, the clinical value and significance of BSEP abnormalities, and the relationship of MYO5B-associated FIC to ABCB11 variant-associated FIC. Our review revealed that aberrant BSEP localization correlated with the absence of one MYO5B allele carrying a truncating nonsense or frameshift variant. Notably, biochemical and clinical parameters including treatment outcome were indistinguishable between patients presenting with normal and aberrant BSEP localization. Further, myosin-Vb and BSEP deficiency-associated FIC patient cohorts showed distinct biochemical and clinical phenotypes, indicating different underlying mechanisms. This suggests that whether or not BSEP localization was abnormal depended on the MYO5B genotype without a predictable effect on clinical parameters and treatment response. Treatment decisions should be guided by clinical parameters rather than by genotype or immunohistochemistry findings. Full article

Monogenic forms of severe early-onset obesity often involve genetic disruptions in the hypothalamic leptin-melanocortin pathway. Pathogenic variants in the SIM1 gene, a key transcription factor required for the development of the paraventricular nucleus, are a known cause of Prader–Willi-like syndrome, characterized by hyperphagia, severe obesity, and developmental delay. We performed targeted next-generation sequencing of 52 obesity-associated genes on a cohort of pediatric patients with severe early-onset obesity. Identified variants were analyzed for population frequency and predicted pathogenicity using in silico tools. The structural impact of the novel missense variants was assessed using protein domain modeling with AlphaFold3. We identified five rare SIM1 variants in eleven patients. Four were heterozygous nonsynonymous variants: one frameshift in the bHLH domain (p.Ser18Ter), one frameshift in the Per-ARNT-Sim domain (p.His143Ter), and two missense variants, p.Pro30Ala and p.Ser663Leu. Structural modeling suggested that the missense variants are likely to disrupt critical protein–protein interactions. The fifth variant was a synonymous change, c.1173G>A, p.(Ser391Ser), which was detected in five unrelated patients. Bioinformatic analysis predicted that this variant could alter splicing. Structural modeling suggested that the missense variants interfere with SIM1 function. This study expands the mutational spectrum of SIM1-linked monogenic obesity, reporting novel likely pathogenic frameshift variants, a missense variant, and a recurrent synonymous variant with a potential splice-site effect. The majority of the variants are predicted to affect the SIM1 protein. Our findings strengthen the critical role of the SIM1 gene in hypothalamic development and energy homeostasis. The results underscore the importance of including the SIM1 gene in genetic testing panels for children with severe obesity and hyperphagia, enabling precise diagnosis and potential future personalized management. Functional in vitro or in vivo validation of these variants is required to confirm their pathogenicity. Full article

Despite years of study, the biological role of the human proinsulin connecting peptide (C-peptide) remains poorly understood. Nevertheless, the C-peptide exhibits subdomains including conserved residues that are thought to have co-evolved with the insulin moiety of proinsulin. Genome-wide association studies in humans suggest that alterations of glycemic control may exhibit a possible linkage with the presence of certain C-peptide variants other than frame-shifts, stop codons, alternative splice variants, or the addition of an extra unpaired Cys residue. Although the C-peptide is ultimately excised from insulin, here, we have bioengineered missense mutations in the amino-terminal portion of the C-peptide (especially involving or near preproinsulin residues Q62,V63) that we find impair proinsulin folding and trafficking efficiency and, in this way, impair insulin biogenesis. We show that proinsulin bearing a C-peptide missense variant can also physically interact with co-expressed wildtype proinsulin, affecting the trafficking behavior of both proinsulin proteins in a manner that is directly related to the relative expression ratio of the variant and wildtype gene products. We conclude that in addition to other possible functions, the amino-terminal portion of the C-peptide influences proinsulin folding and trafficking and, in this way, affects human insulin production. Full article

Background/Objectives: Familial adenomatous polyposis (FAP) is an autosomal dominant disorder caused by pathogenic variants in the adenomatous polyposis coli (APC) gene. Its attenuated form (AFAP) is characterized by fewer colorectal polyps and later onset of colorectal cancer. We aimed to characterize the molecular effects of a novel APC gene variant (NM_000038.6: c.1620_1624delinsT) identified in a patient with AFAP. Methods: A 56-year-old man with the AFAP phenotype underwent germline testing via a multigene NGS panel, which identified a novel APC gene variant (NM_000038.6: c.1620_1624delinsT). In silico analyses predicted disruption of the canonical donor splice site and a frameshift followed by the introduction of a premature stop codon. The transcriptional impact of the identified APC gene variant was investigated by mRNA analysis. Results: mRNA analysis revealed two distinct APC transcripts: the first transcript led to a truncated protein (p.Leu540PhefsTer8), and the second transcript lacked exon 12, resulting in an in-frame 26 amino acid deletion of APC protein (p.Ala517_Gly542del). The transcript lacking exon 12 was more abundant than the transcript with a premature stop codon, likely due to degradation through nonsense-mediated decay. Conclusions: The APC gene variant (NM_000038.6: c.1620_1624delinsT) exhibits a dual transcriptional effect, revealing its pathogenic role in AFAP. This study highlights the diagnostic value of combined DNA–RNA germline testing for improving the clinical classification of novel APC gene variants and their genotype–phenotype correlations in FAP. Full article

Small-grained rice varieties are highly valued in hybrid seed production and food markets because of their unique advantages in mechanized seed production and cooking qualities. Developing new varieties combining small grain size with high nutritional and cooking quality represents an important breeding objective. The OsVIN2 gene has been identified as a key regulator involved in carbohydrate metabolism and grain development in rice. In this study, a CRISPR/Cas9-mediated gene-editing approach was employed to precisely modify the OsVIN2 gene in the restorer line MH86 of high-quality rice varieties. Compared with the wild-type plants, the frameshift mutant seeds showed a significant reduction in length and width by 19.9% and 15.2%, respectively, leading to a 39.2% decrease in thousand-grain weight. Notably, the mutant exhibited improved quality traits, including a decrease of 16.6% in amylose content, an increase of 11.0% protein, and a 77.5% and 84.7% decrease in chalkiness rate and chalkiness degree, respectively. These results demonstrate that targeted editing of OsVIN2 is a promising approach for creating novel small-grained rice germplasm with superior quality attributes. Full article

Bacteriophages are powerful drivers of microbial evolution and are increasingly explored as alternatives to antibiotics against multidrug-resistant pathogens such as Pseudomonas aeruginosa. Here, we describe the isolation, phenotypic characterization and genomic, structural and evolutionary analysis of Pseudomonas phage Adele, a lytic myovirus representing a novel species within the genus Pakpunavirus (family Vandenendeviridae). Phage Adele exhibits a short latent period of 20 min, a burst size of 59 ± 11 virions per infected cell and a high virulence index, efficiently lysing non-O11 Pseudomonas aeruginosa strains and reducing biofilm biomass. In vivo, Adele confers marked protection in a Galleria mellonella infection model. Phylogenetic reconstruction, synteny analysis and structural modeling demonstrate the relatedness of Vandenendeviridae to phages of the Andersonviridae and Vequintavirinae clades, pointing to a stable, ancestral virion architecture that has undergone lineage-specific elaborations, including the duplication and divergence of tail tube proteins. The tail assembly chaperone gene employs a conserved −1 programmed ribosomal frameshift. Phage Adele encodes an elaborate set of metabolic reprogramming and anti-defense systems, reflecting extensive horizontal gene transfer. The combination of a conserved structural architecture and mosaic genome establishes Adele as an exemplary system for studying modular evolution in phages, alongside its demonstrated therapeutic efficacy. Full article

Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) is an RNA-binding protein known to play critical roles in metabolism, cell proliferation, and tumorigenesis. Although its involvement in muscle development has been documented in several species, the function of goose IGF2BP2 remains largely unexplored. In this study, we cloned and characterized the full-length cDNA and genomic DNA sequences of goose IGF2BP2. The cDNA is 2957 bp in length and contains a 1662 bp open reading frame encoding a 553-amino acid protein with five conserved RNA-binding domains. The genomic sequence spans 12,183 bp and consists of 12 exons and 11 introns. A total of 60 genetic variants were identified, including a deletion of a G base at position 2299 (g.2299delG) that results in a frameshift mutation. Expression analysis revealed high levels of IGF2BP2 mRNA in the liver, heart, and muscle tissues of female geese across embryonic (E25d), growing (A70d), and laying (L270d) stages, consistent with a potential role in muscle development (p < 0.05). Functionally, overexpression of IGF2BP2 in skeletal muscle satellite cells (SMSCs) was associated with significant changes in the expression of several genes linked to muscle development and signaling pathways, including upregulation of IGF1, EGFR, FGF19, BMP6, BMP2, ACVR1C and WNT5A and downregulation of MYBPC3, NODAL, HOXD13, TNXB, and ADD2 (Padj < 0.01). Furthermore, protein–protein interaction (PPI) network analysis of these genes suggests that IGF2BP2 may coordinate key genes, contributing to its potential role in skeletal muscle development in geese. Full article