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PubMed2026

Identification and validation of an alternatively spliced novel isoform of maspin that modulate genes involved in inflammatory and apoptotic pathways.

Maspin regulates cellular adhesion, migration, apoptosis, angiogenesis, and tumor suppression in a tissue and context-dependent manner. Its functional diversity is governed largely thorough extracellular matrix interactions, subcellular localization, and the reactive center loop (RCL), although the structural details are not well understood. To examine whether alternative splicing contributes to this heterogeneity, we analysed the SERPINB5 gene using a computational genomics approach and identified a novel 80 bp coding exon upstream of the first coding exon (E1). The alternatively spliced transcript was validated in human skin and esophagus by semi-nested touchdown PCR, quantitative real-time PCR, and Sanger sequencing. Recombinant B5N displayed a red-shifted fluorescence emission spectrum, indicating a more solvent-exposed conformation, which was supported by molecular dynamics simulations showing greater exposure of the nuclear localization signal (NLS) and the reactive center loop. Enzyme kinetic assays demonstrated concentration-dependent enhancement of tissue plasminogen activator (tPA) activity by both isoforms. In HaCaT cells, wildtype maspin produced stronger antiproliferative and anti-migratory effects, whereas B5N was only mildly antiproliferative. Annexin V/7-AAD staining revealed that wildtype maspin induced higher early apoptosis and cell death, while B5N produced lower overall cell death but a greater proportion of late apoptotic cells. RNA-seq of transfected HaCaT cells identified differentially expressed genes enriched in inflammatory, antiviral, and apoptotic pathways, which was validated by qPCR, and several of these were markedly upregulated in SARS-CoV-2 infected A549 cells. Thus, a novel N-terminally extended maspin isoform with differentially regulated gene profile is identified and validated in this study.

Apoptosis↗
PubMed2025

Newborn screening for common genetic variants associated with permanent hearing loss: Implementation in Ontario and review of the first 3 years.

PURPOSE: Early hearing detection and intervention (EHDI) programs using audiometric screening techniques alone have a limited ability to detect noncongenital childhood permanent hearing loss (PHL). In 2019, Ontario launched universal newborn screening (NBS) for PHL risk factors, including congenital cytomegalovirus and 22 common variants in GJB2 and SLC26A4. Here, we describe our experience in screening for genetic risk factors. METHODS: Ontario newborns who participated in universal newborn hearing screening (UNHS) were offered risk factor screening using dried blood spots (DBS) collected for conventional newborn screening. The screening was conducted using a custom MassArray assay, and positive results were confirmed by Sanger sequencing or polymerase chain reaction. Diagnostic audiological assessments were performed for all screen-positive infants. RESULTS: Of the 412,424 infants screened, 93 had 2 variants in GJB2 or SLC26A4. Of these, 72 had confirmed PHL, 20 had normal hearing, and 1 declined follow-up. Thirteen infants with PHL (1 in 31,724; 11.8% of screen positives) were not identified through audiometric testing as they passed (3) or missed (10) the screening. Importantly, among infants who ultimately received cochlear implants, the detection of genetic etiology through NBS led to an accelerated time to diagnosis, assessment, and intervention. CONCLUSION: Genetic screening has strengthened UNHS and care for infants with or at risk of PHL in Ontario. This study is a step toward the broader inclusion of genomic testing in NBS.

Humans↗
PubMed2025

Biallelic null variants in C19orf44 cause a unique late-onset retinal dystrophy phenotype characterized by patchy perifoveal chorioretinal atrophy.

PURPOSE: To identify the genetic cause for disease in individuals affected with inherited retinal disease and to characterize their retinal phenotype and the properties of the underlying gene. METHODS: Participants underwent a comprehensive ophthalmological evaluation, including best-corrected visual acuity, visual field testing, fundus autofluorescence, optical coherence tomography, and electroretinography. Genetic analyses included exome, genome, and Sanger sequencing. Gene expression pattern was analyzed by reverse transcription-polymerase chain reaction. Localization of the encoded protein in cells and in the human retina was examined by immunofluorescence staining. RESULTS: Four different pathogenic variants in C19orf44 were identified in 15 biallelic individuals from 11 unrelated families. The most common variant was c.549_550del p.(Ser185ProfsTer2). Most individuals were affected with a unique clinical phenotype characterized by late-onset patchy perifoveal chorioretinal atrophy and electroretinographic features of rod-cone degeneration. C19orf44 is expressed in various human tissues, including the retina, where it was found in the outer nuclear layer and in the outer plexiform layer. In cultured cells (hTERT RPE-1 and HeLa) and in human primary fibroblasts, C19orf44 is found in the nucleus, and it is downregulated during mitosis. CONCLUSION: Based on our results, C19orf44 is crucial for normal human retinal function, and pathogenic variants in this gene are associated with autosomal recessive inherited retinal disease.

Humans↗
PubMed2025

Rare pathogenic NR2F2 (COUP-TFII) variants as potential etiological causes in pediatric patients with congenital heart diseases (CHDs).

OBJECTIVES: Congenital heart diseases (CHDs) are complex genetic disorders, and their genetic basis is not yet fully understood. Nuclear receptor subfamily 2 group F member 2 (NR2F2 or COUP-TFII) encodes a transcription factor which is expressed at high levels during mammalian development. Few studies have identified heterozygous and rare variants in the NR2F2 gene in individuals with CHD. This study aimed to evaluate the association between pathogenic genetic alterations in NR2F2 with CHD risk. METHODS: A case-control study was conducted on a group of 135 patients (83 boys and 52 girls) with various types of non-hereditary, isolated CHD who were undergoing open-heart surgery. Additionally, 95 matched healthy children without syndromic or isolated heart abnormalities were selected. RESULTS: Using Sanger sequencing, we identified 5 heterozygous single nucleotide variants in exons 2 and 3 of the NR2F2 gene. These variations were novel and not present in any genomic variation databases. Four of the variations were missense mutations (p.Pro159Arg, p.Ser329Phe, p.Qln338Pro, and p.Tyr348Ser) and one was a synonymous variant (p.G361 = ) in the coding region. Importantly, in silico results indicated that the missense variants had pathogenic effects on protein function. Additionally, the missense variants substantially altered the predicted structure of COUP-TFII. CONCLUSION: The results we obtained not only validate the correlation between NR2F2 mutations and CHDs but also have significant potential for guiding new preventive and therapeutic strategies. This could contribute to the advancement of medical interventions in the fields of cardiology and genetics.

Humans↗
PubMed2005

Dual-probe assay for detection of lamivudine-resistance hepatitis B virus by real-time PCR.

A rapid and accurate minor groove binder (MGB) real-time PCR is described for detection lamivudine resistance mutations in hepatitis B virus. The real-time PCR was compared with direct Sanger sequencing in 53 clinical patients samples, the results of the real-time PCR correlate with the nucleotide sequence and the assay has the advantage of detecting a mixture of quasi-species with higher sensitivity than sequencing. As a simple, easy, rapid, accurate and high throughout method, MGB real-time PCR assay should be useful for detecting lamivudine resistance variants during lamivudine therapy in clinical settings.

Antiviral Agents↗
PubMed2005

Comparison of real-time PCR and pyrosequencing for typing Bordetella pertussis toxin subunit 1 variants.

We describe two newly developed methods for rapid typing of the pertussis toxin subunit 1 gene (ptxS1). A real-time PCR assay based on hybridization probes and a Pyrosequencing assay were developed and the specificity, sensitivity, cost, hands-on time and post-assay data processing were compared to Sanger sequencing. Both methods enabled discrimination of all four allelic variants, correctly identified all ptxS1 alleles of 143 strains tested and proved suitable for large-scale screening of B. pertussis strains.

Alleles↗
PubMed2026

Multiplexed CRISPR/Cas9 mediated knockdown of BCH gene in potato enhances beta-carotene to combat vitamin A deficiency.

The inadequate amounts of provitamin A carotenoids in crops contribute to the widespread vitamin A deficiency, leading to malnutrition and blindness in humans. Suppression of the β-carotene hydroxylase (BCH) increases β-carotene levels. In the current study, we utilized the multiplexed CRISPR/Cas9 approach by designing three targets against the BCH gene in a local potato cultivar. Transformation efficiency was recorded as 15%, the successful integration of the CRISPR/Cas9-BCH multiplex construct in potatoes was confirmed through PCR. When analysed using TIDE software, Sanger sequencing revealed the highest indel efficacy of 92.1% in plant 7 and 26.6% in plant 1. qRT-PCR (quantitative real-time PCR) analysis indicated a significant 89-fold reduction in BCH transcript levels in genome-edited potato lines compared to control plants. Spectrophotometry demonstrated a notable increase in beta-carotene levels in genome-edited potato plants, ranging from 0.831 µg/mL FW to 4.236 µg/mL FW, compared to the control plant with the lowest beta-carotene concentration (0.344 µg/mL FW). HPLC analysis further confirmed increased beta-carotene levels in genome-edited potato plants, ranging from 0.11 mg/mL FW to 0.36 mg/mL FW, compared to the unmodified control plant with a minimum beta-carotene value of 0.09 mg/mL. Our results revealed that the multiplexed CRISPR-Cas9 approach targeting the BCH gene results in enhanced beta-carotene contents in potato tubers.

Solanum tuberosum↗
PubMed2026

Prevalence of POMC allele associated with obesity in feline population in Vietnam.

Feline obesity is an increasingly important health problem influenced by both genetic predisposition and husbandry practices. This study investigated the prevalence and phenotypic relevance of the feline proopiomelanocortin (POMC) c.28G > C (p.Gly10Arg) variant in a Vietnamese cat population and evaluated environmental factors associated with obesity. A total of 63 clinically healthy cats were classified as normal weight (body condition score [BCS] 5-6/9; n = 40) or obese (BCS ≥7/9; n = 23). Genotyping was performed using a newly developed PCR-restriction fragment length polymorphism assay and validated by Sanger sequencing. Ventral subcutaneous adipose tissue (VSAT) thickness was measured ultrasonographically as an objective indicator of adiposity. Genotyping revealed a high prevalence of the risk-associated C allele, with 45/63 cats (71.4%) carrying the GC genotype and 18/63 (28.6%) carrying the CC genotype, whereas the GG genotype was not detected, giving a Callele frequency of 64.3%. Obese cats had significantly greater body weight and VSAT thickness than normal-weight cats. Neuter status and ad libitum feeding were significantly associated with obesity, whereas diet type, housing, exercise frequency, and begging behavior were not. Although genotype distribution did not differ significantly between normal and obese cohorts, obese CC cats showed significantly greater VSAT thickness than obese GC cats, indicating an allele-dosage effect on adiposity. These findings suggest that the POMC c.28G > C variant is a useful risk-informative marker and that combining genetic screening with objective fat assessment may support earlier identification and prevention of feline obesity in Vietnam under routine laboratory conditions and guide personalized management strategies in practice.

Animals↗
PubMed2026

miR-6388 regulates granulosa cell function in sheep by targeting GDF9 and modulating the TGF-β signaling pathway.

Litter size is an economically important trait in sheep and is closely associated with ovarian follicular development and granulosa cell (GC) function. This study investigated the association between GDF9 polymorphisms and litter size, and examined the post-transcriptional regulation of GDF9 by miR-6388 in ovine GCs. Variants were initially identified by Sanger sequencing in 20 ewes, and subsequently genotyped in 377 three-year-old ewes, including 231 Sonid (SN) sheep and 146 Ujimqin (UM) sheep for association analysis. Candidate miRNAs targeting litter size-associated variants in the GDF9 3'UTR were predicted, and the miR-6388-GDF9 interaction was evaluated using dual-luciferase reporter assays. RT-qPCR, Western blotting, EdU incorporation, and flow cytometry were used to assess endogenous GDF9 expression and GC function. Twelve single-nucleotide polymorphisms were identified, including the putatively novel variant g.42114076C > G. The linkage disequilibrium block comprising g.42116936C > T, g.42113821T > A, and g.42113962G > A polymorphisms of GDF9 was significantly associated with litter size in both SN and UM sheep, whereas the c.477G > A was associated with litter size only in UM sheep. Reporter assays showed that the GDF9 3'UTR region carrying the G allele of g.42113962G > A was more responsive to miR-6388-mediated repression than the region carrying the A allele. miR-6388 overexpression reduced GDF9 mRNA and GDF9 protein levels, inhibited GC proliferation, altered cell-cycle distribution, and promoted apoptosis, whereas miR-6388 knockdown increased GDF9 expression and GC proliferation and reduced apoptosis. These cellular changes were accompanied by altered expression of cell-cycle and apoptosis-related genes and TGF-β signaling-related components.

Animals↗
PubMed2026

Generation and validation of a Myh11Dre-Spp1Cre intersectional mouse model for lineage tracing of disease-associated smooth muscle cell states.

BACKGROUND: Phenotypic modulation of vascular smooth muscle cells (VSMCs) is a hallmark of vascular remodeling and cardiovascular disease. Recent lineage-tracing and single-cell transcriptomic studies have identified secreted phosphoprotein 1 (SPP1) as a prominent marker associated with disease-associated VSMC states, particularly those linked to fibrotic remodeling and vascular calcification. However, the cellular origins and fate of SPP1-associated VSMC populations remain incompletely understood. METHODS AND RESULTS: We generated a novel Spp1-rSTOPr-Cre (Spp1Cre) knock-in mouse line in which Cre recombinase is expressed from the endogenous Spp1 locus following Dre-mediated excision of a rox-flanked transcriptional STOP cassette. Correct targeting of the knock-in allele was validated by internal, 5' junction, 3' junction, and long-range PCR analyses, as well as Sanger sequencing. To establish an intersectional lineage-tracing strategy, Spp1Cre mice were crossed with Myh11DreERT2 and Rosa26-RSR-LSL-tdTomato-LSL-eGFP reporter mice, enabling permanent labeling of VSMC-derived populations following activation of the endogenous Spp1 locus. Under physiological conditions, eGFP-positive cells were detected at low frequency within the vascular wall and were predominantly negative for the contractile markers ACTA2 and MYH11. As a proof-of-principle application, eGFP-positive cells markedly expanded within atherosclerotic lesions induced by AAV-PCSK9D377Y and high-fat diet feeding. These lineage-traced cells remained largely ACTA2- and MYH11-negative, consistent with a modulated phenotype. Notably, only a minority of eGFP-positive cells expressed SPP1 or fibronectin at the time of analysis, demonstrating the utility of permanent lineage tracing for tracking cells with a history of endogenous Spp1 activation during vascular remodeling. CONCLUSION: We report the generation and validation of a novel Myh11Dre-Spp1Cre intersectional mouse model for lineage tracing of VSMC-derived populations that have activated the endogenous Spp1 locus. This genetic resource provides a valuable platform for investigating the origin, fate, and phenotypic evolution of Spp1-associated VSMC populations during vascular remodeling and cardiovascular disease.

Animals↗
PubMed2024

Genetic and functional analyses of CTBP2 in anorexia nervosa and body weight regulation.

The C-terminal binding protein 2 (CTBP2) gene (translational isoforms: CTBP2-L/S, RIBEYE) had been identified by a cross-trait analysis of genome-wide association studies for anorexia nervosa (AN) and body mass index (BMI). Here, we did a mutation analysis in CTBP2 by performing polymerase chain reactions with subsequent Sanger-sequencing to identify variants relevant for AN and body weight regulation and ensued functional studies. Analysis of the coding regions of CTBP2 in 462 female patients with AN (acute or recovered), 490 children and adolescents with severe obesity, 445 healthy-lean adult individuals and 168 healthy adult individuals with normal body weight detected 24 variants located in the specific exon of RIBEYE. In the initial analysis, three of these were rare non-synonymous variants (NSVs) detected heterozygously in patients with AN (p.Arg72Trp - rs146900874; p.Val289Met -rs375685611 and p.Gly362Arg - rs202010294). Four NSVs and one heterozygous frameshift variant were exclusively detected in children and adolescents with severe obesity (p.Pro53Ser - rs150867595; p.Gln175ArgfsTer45 - rs141864737; p.Leu310Val - rs769811964; p.Pro397Ala - rs76134089 and p.Pro402Ser - rs113477585). Ribeye mRNA was detected in mouse hypothalamus. No effect of fasting or overfeeding on murine hypothalamic Ribeye expression was determined. Yet, increased Ribeye expression was detected in hypothalami of leptin-treated Lepob/ob mice. This increase was not related to reduced food intake and leptin-induced weight loss. We detected rare and frequent variants in the RIBEYE specific exon in both patients with AN and in children and adolescents with severe obesity. Our data suggest RIBEYE as a relevant gene for weight regulation.

Humans↗
PubMed1999

Direct genetic analysis by matrix-assisted laser desorption/ionization mass spectrometry.

An approach to analyzing single-nucleotide polymorphisms (SNPs) found in the human genome has been developed that couples a recently developed invasive cleavage assay for nucleic acids with detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The invasive cleavage assay is a signal amplification method that enables the analysis of SNPs by MALDI-TOF MS directly from human genomic DNA without the need for initial target amplification by PCR. The results presented here show the successful genotyping by this approach of twelve SNPs located randomly throughout the human genome. Conventional Sanger sequencing of these SNP positions confirmed the accuracy of the MALDI-TOF MS analysis results. The ability to unambiguously detect both homozygous and heterozygous genotypes is clearly demonstrated. The elimination of the need for target amplification by PCR, combined with the inherently rapid and accurate nature of detection by MALDI-TOF MS, gives this approach unique and significant advantages in the high-throughput genotyping of large numbers of SNPs, useful for locating, identifying, and characterizing the function of specific genes.

Base Sequence↗
PubMed2026

Association of ERBB4 and SHBG gene polymorphisms with polycystic ovarian syndrome in South Indian women: a case-control genetic analysis.

INTRODUCTION: Polycystic ovary syndrome (PCOS) is a multifactorial endocrinological disorder with a substantial genetic component. However, the role of genes involved in follicular development and androgen regulation remains incompletely understood, particularly in South Indian populations. This study aimed to evaluate how variations in the ERBB4 and SHBG genes affect PCOS risk. METHODOLOGY: A hospital-based case-control study was conducted among 400 South Indian women, comprising 200 women with PCOS and 200 age-matched healthy controls. Genomic DNA was extracted to study SNPs at ERBB4 (rs2178575 and rs1351592) and SHBG (rs1799941 and rs727428) using ARMS-PCR genotyping. The study compared genotype and allele frequencies between cases and controls while assessing their associations with allelic, homozygous, heterozygous, dominant, recessive, and over-dominant genetic models. Genotyping accuracy was confirmed by re-genotyping and Sanger sequencing of a subset of samples. RESULTS: The ERBB4 rs2178575 polymorphism demonstrated a significant association with PCOS, as the AA genotype and A allele combination increased risk across all three genetic models, including homozygous, recessive, and allelic models. The ERBB4 rs1351592 variant was associated with 3-fold higher risk of PCOS in heterozygous and GC carriers. The SHBG rs1799941 polymorphism showed a significant link to PCOS through its effects on heterozygous and allelic states, whereas rs727428 displayed no significant connection due to its monomorphic distribution. CONCLUSION: These findings suggest that polymorphisms in ERBB4 and SHBG may contribute to PCOS susceptibility in South Indian women in a locus- and model-specific manner, revealing the intricate genetic structure that defines this medical condition.

Humans↗
PubMed1994

Termination of DNA synthesis by novel 3'-modified-deoxyribonucleoside 5'-triphosphates.

Eight 3'-modified-dNTPs were synthesized and tested in two different DNA template assays for incorporation activity. From this enzymatic screen, two 3'-O-methyl-dNTPs were shown to terminate DNA syntheses mediated by a number of polymerases and may be used as alternative terminators in Sanger sequencing. 3'-O-(2-Nitrobenzyl)-dATP is a UV sensitive nucleotide and was shown to be incorporated by several thermostable DNA polymerases. Base specific termination and efficient photolytic removal of the 3'-protecting group was demonstrated. Following deprotection, DNA synthesis was reinitiated by the incorporation of natural nucleotides into DNA. The identification of this labile terminator and the demonstration of a one cycle stop-start DNA synthesis are initial steps in the development of a novel sequencing strategy.

Base Sequence↗
PubMed2026

Detection of opportunistic bacterial pathogens with intrinsic amoxicillin- and cephalosporin-resistance in wild koala faecal microbiomes.

Opportunistic bacterial pathogens frequently associated with human clinical infections, including antimicrobial-resistant strains, are infiltrating the microbiomes of wild animals, where they have the potential to negatively impact wildlife health. Bacterial genes conferring resistance to amoxicillin have previously been reported in koala (Phascolarctos cinereus) faecal DNA. Koalas are facing several key threats, including wildfires, and affected individuals may receive amoxicillin therapy to treat burn wounds. This study aimed to identify the species of amoxicillin-resistant bacteria in koala gut microbiomes and determine if they are opportunistic pathogens. Faecal samples collected from 98 wild-caught koalas were cultured using amoxicillin-supplemented media to isolate amoxicillin-resistant Gram-negative enteric bacteria. Isolates were screened using 16S rRNA PCR and Sanger sequencing to identify opportunistic pathogenic species, which then underwent whole-genome sequencing and antimicrobial susceptibility testing. Intrinsically amoxicillin-resistant opportunistic pathogens were obtained from 9.2% (9/98) of koala faecal samples and comprised Klebsiella oxytoca (6/98, 6.1%), Klebsiella pneumoniae (1/98, 1.0%) and Citrobacter spp. (2/98, 2.0%). Seven of nine amoxicillin-resistant opportunistic pathogens also exhibited cephalosporin resistance. Four K. oxytoca isolates belonged to lineages associated with human clinical infections, which also have the potential to cause disease in koalas, including fatal systemic infections in pouch young. The presence of amoxicillin- and cephalosporin-resistant strains may also increase the risk of gut dysbiosis and opportunistic infections when penicillins or cephalosporins are required to treat bacterial infections in koalas, highlighting the importance of good antimicrobial stewardship. The study findings demonstrate the One Health perspective of microbial pathogens and the intertwined microbial ecology between humans and wildlife.

Animals↗
PubMed2026

Molecular characterization of CD36 deficiency in blood donors of Middle Eastern and African origin reveals transcript-level defects beyond genomic variants.

BACKGROUND: The increasing diversity of blood donor populations has created new challenges for transfusion services worldwide. The identification of donors lacking relevant high-prevalence antigens is becoming increasingly important to ensure compatible blood products for alloimmunized patients and to support the development of rare donor registries. CD36 (ISBT 045) is a glycoprotein expressed on platelets, monocytes, and erythroid precursor cells. CD36 deficiency has been reported across multiple populations and is of relevance due to its association with anti-CD36 isoantibodies, which may cause platelet transfusion refractoriness and fetal/neonatal alloimmune thrombocytopenia. STUDY DESIGN AND METHODS: We analyzed CD36 expression in 1250 blood donors of diverse ancestry using flow cytometry. CD36-negative samples underwent molecular characterization using Sanger sequencing and next-generation sequencing of genomic DNA, complemented by cDNA analysis and cloning to investigate transcript-level alterations. RESULTS: We identified CD36 deficiency in 27 donors (2.16%). Genomic sequencing revealed 18 distinct coding variants, including three novel variants, most in the heterozygous state. In one CD36-negative donor, cDNA analysis demonstrated a 52-bp deletion in exon four and complete skipping of exon 9, despite the absence of splice-site variants in genomic DNA. Cloning confirmed coexistence of aberrant and wild-type transcripts in this individual. CONCLUSION: Our findings demonstrate that CD36 deficiency can arise from transcript-level defects in the absence of detectable coding or splice-site variants. These results indicate that genomic sequencing alone may be insufficient to fully resolve CD36-negative phenotypes and highlight the importance of integrating transcriptomic approaches to improve molecular diagnostics and transfusion support in increasingly diverse donor populations.

CD36 deficiency↗
PubMed2024

A rapid and simple clonality assay for bovine leukemia virus-infected cells by amplified fragment length polymorphism (AFLP) analysis.

UNLABELLED: Enzootic bovine leukosis (EBL), although eradicated in some European countries, is still the most common neoplastic disease of cattle, caused by the bovine leukemia virus (BLV). During the progression of EBL, BLV-infected cells clonally expand, and some of which result in tumor onset. The clonality of BLV-infected cells is generally evaluated with NGS or Sanger sequencing. Although these methods clearly distinguish EBL from non-EBL cases, the procedures are complex and not practical for routine veterinary diagnosis. In this study, we developed an amplified fragment length polymorphism (AFLP) analysis for BLV clonality assay (BLV-AFLP). This analysis uses restriction enzyme digestion to amplify the chimeric regions of BLV 3' linear transcribed region (LTR) and host genome through conventional polymerase chain reaction (PCR) and visualizes the results by gel-electrophoresis. The method was established using cattle samples representing different stages of the disease: BLV-uninfected, non-EBL, and EBL cattle. Non-EBL cattle showed smeared bands, indicating polyclonal proliferation, while EBL cattle showed distinct bands, indicating clonal expansion. The results of BLV-AFLP correlated well with those of previously reported methods, suggesting its efficacy in detecting clonal proliferation. The validation using blood samples of non-EBL cattle and tumor samples of EBL cattle confirmed that BLV-AFLP could effectively identify clonal proliferation in EBL samples. Moreover, the emergence of dominant clones in the tumor at later stages was successfully detected before EBL onset in some cattle, highlighting its sensitivity and potential for early detection. Overall, BLV-AFLP is suitable for practical use in the field, improving BLV management strategies and minimizing economic losses. IMPORTANCE: Enzootic bovine leukosis (EBL) is routinely diagnosed based on external manifestations at the farm, such as the presence of tumors and/or general lymph node enlargement. However, due to the nonspecific clinical manifestations of EBL, over half of EBL cases are unrecognized at the farm, with most cases being diagnosed during postmortem inspection at the slaughterhouse. Early detection and monitoring of clonal expansion are necessary for managing EBL and reducing economic losses. In this study, we developed BLV-AFLP that represents a significant advancement in the diagnosis of EBL in cattle. This method can rapidly assess the clonal proliferation of BLV-infected cells, crucial for distinguishing between asymptomatic and EBL cattle. Additionally, tracking clonal dynamics offers insights into the disease's progression, potentially providing strategies for avoiding economic losses. Overall, as BLV-AFLP is a simple and rapid test for detecting EBL, it is feasible and efficient for routine veterinary practice.

Leukemia Virus, Bovine↗
PubMed2026

Scrape Cytology of DEK::AFF2 Fusion-Associated Papillary Squamous Cell Carcinoma of the Sinonasal Tract Masquerading as Schneiderian Papilloma: A Case Report.

INTRODUCTION: DEK::AFF2 fusion-associated papillary squamous cell carcinoma is a recently characterized sinonasal neoplasm that closely mimics Schneiderian papilloma. Although one report has described fine-needle aspiration cytology of a metastatic lymph node, scrape cytological features from the primary site remain undocumented. CASE PRESENTATION: We report the scrape cytology of this tumor in a 26-year-old woman. While certain features (perivascular arrangement and intracytoplasmic nuclear debris-like structures) overlapped with Schneiderian papilloma, several findings diverged: a predominantly discohesive pattern, nuclear enlargement with anisonucleosis, prominent nucleoli, stippled chromatin distinct from the neuroendocrine pattern, and a crackled cytoplasmic appearance. Immunohistochemistry for synaptophysin and chromogranin A was negative, excluding neuroendocrine differentiation. CD163 immunohistochemistry confirmed that the debris-containing cells were not histiocytes. E-cadherin showed heterogeneous downregulation in the initial biopsy, suggesting a candidate molecular basis for the discohesive pattern. The diagnosis was confirmed by AFF2 immunohistochemistry, DEK break-apart fluorescence in situ hybridization, and reverse transcription polymerase chain reaction with Sanger sequencing. Retrospective AFF2 immunohistochemistry of the initial biopsy, originally diagnosed as Schneiderian papilloma, was positive. The patient remained free of disease progression 34 months after completion of chemoradiotherapy for the antecedent lacrimal sac carcinoma. CONCLUSION: These findings provide the first comprehensive cytological documentation of this entity from a primary sinonasal site and delineate features diverging from Schneiderian papilloma that may prompt ancillary investigations.

Case report↗