Transposable elements drive plant genome size variation and structural diversification, but their recent dynamics remain poorly characterized in medicinal plants. We analyzed 95 medicinal plant genomes to evaluate repeatome divergence, recent transposable element composition and long terminal repeat retrotransposon dynamics. Genome-wide transposable element proportion varied substantially among species and was strongly positively associated with genome size, indicating that repeat accumulation is a major axis of genome-size divergence. Recent transposable element fractions, operationally defined by sequence identity ≥ 85%, were dominated by long terminal repeat retrotransposons in most genomes. Higher recent transposable element fractions were negatively associated with Shannon diversity and Pielou's evenness, suggesting compositional concentration rather than balanced expansion across major classes. Phylogenetically informed Bayesian models revealed class-specific differences in the magnitude and uncertainty of estimated recent transposable element proportions across taxonomic orders. Insertion-time analyses showed heterogeneous recent accumulation patterns, whereas phylogenetic reconstruction of intact long terminal repeat retrotransposons with paired long terminal repeat identity ≥ 95% revealed contrasting lineage composition between Copia and Gypsy. Copia elements were concentrated mainly in two dominant lineages, whereas Gypsy elements were distributed across a broader set of lineages. An exploratory 2-kb gene-centered analysis of predicted biosynthetic gene clusters showed that 62.5% of 31,162 unique genes had at least one proximal long terminal repeat retrotransposon annotation, usually together with other transposable element classes. These results indicate that medicinal plant repeatome evolution is associated with genome-wide transposable element burden, recent compositional concentration and lineage-level long terminal repeat retrotransposon dynamics.
Ceratonia siliqua is a nutritionally and economically important Mediterranean tree species whose natural stands in Morocco are increasingly threatened by habitat degradation and genetic erosion. However, the spatial organisation of genetic diversity across its natural range in Morocco remains insufficiently resolved. To support conservation and germplasm management initiatives, interpopulation genetic differentiation among Moroccan carobs populations was assessed using a pooled DNA sample and SSR markers analysed through three genotyping workflows: high-resolution melting (HRM), conventional PCR, and capillary electrophoresis. Genetic relationships among populations were evaluated using Neighbour-Joining (NJ) analysis, principal component analysis (PCA), and K-means clustering. Among the evaluated approaches, HRM provided the highest discriminatory resolution and generated the most informative polymorphism profiles across loci. All analytical frameworks consistently revealed substantial interpopulation differentiation and broad geographic grouping patterns. Southern populations formed genetically cohesive groups, whereas northern and central populations displayed more complex patterns of genetic similarity. K-means clustering supported the presence of four major population groups, while PCA identified a subset of highly discriminative SSR loci contributing to regional differentiation. Overall, the results reveal a genetically diverse and spatially differentiated Moroccan carob germplasm and demonstrate that SSR-HRM is a robust and cost-effective approach for population-level genetic screening. These findings provide useful genetic resources for future conservation, germplasm management, and breeding programmes in Moroccan carob.
Background: Primary ciliary dyskinesia (PCD) is a rare and genetically heterogeneous disorder that remains underdiagnosed in low- and middle-income countries, largely due to limited access to specialized diagnostic tests. Genetic analysis has become an essential component of PCD diagnosis, particularly where functional and ultrastructural evaluations are unavailable. Methods: We conducted an investigational study including children and adolescents with clinical suspicion of PCD followed at a Brazilian tertiary center. Clinical characterization included detailed phenotyping and calculation of the PICADAR score. Molecular investigation was performed using exome sequencing as a frontline diagnostic approach. Results: Among 27 individuals evaluated, 10 (37%) received a confirmed molecular diagnosis of PCD. An additional 6 (22%) individuals had inconclusive molecular findings, mainly due to variants of uncertain significance (VUS), and were classified as likely PCD based on combined clinical and molecular evidence. Higher PICADAR scores were more frequently observed among individuals with confirmed or likely molecular diagnosis, with 9 of 10 confirmed cases presenting a score above 5. Beyond PCD-associated findings, exome sequencing also enabled the identification of clinically relevant additional diagnoses, including cystic fibrosis, FGFR3-related hypochondroplasia, and ACMG-reportable secondary finding involving BRCA2. Some unresolved cases may also reflect inherent technical limitations of exome sequencing, including restricted sensitivity for copy-number variants, suboptimal coverage of highly homologous or GC-rich regions, and limited detection of deep intronic and other variants. Additional factors include challenges in variant interpretation and incomplete knowledge of disease-associated genes. Conclusions: Frontline exome sequencing is a valuable diagnostic tool for PCD, particularly when integrated with robust clinical phenotyping. Clinical scoring systems such as PICADAR may help prioritize individuals for genetic testing and optimize diagnostic yield in resource-limited settings.
Skeletal muscle is crucial for glucose regulation and amino acid storage, significantly influencing overall metabolic balance. Its function is tightly regulated by complex mechanisms, with histone acetylation as a key epigenetic control point. Our previous work identified eIF6 as a key regulator of muscle energy homeostasis and demonstrated its role in modulating histone acetylation in the liver. However, whether similar epigenetic mechanisms underpin eIF6's effects in muscle remains undetermined. To investigate this, we measured H3K9 acetylation levels and HDAC activity both in vivo, using eIF6+/- mice, and in vitro, following eIF6 depletion. Our findings demonstrate that eIF6 downregulation in C2C12 myoblasts drives an increase in histone acetylation, a pattern also evident in heterozygous eIF6 primary satellite cells, both in their undifferentiated state and following differentiation. In vivo, eIF6+/- mice show pronounced histone hyperacetylation, especially in younger animals, which correlates with a specific decrease in class II HDACs, particularly HDAC4 and HDAC5. This trend is further supported by in vitro data and findings from Drosophila eIF6+/- mutants, both of which exhibit decreased HDAC activity. Importantly, the reduction in HDAC4 and HDAC5 activity appears to result from decreased protein levels, driven by eIF6-dependent translational regulation of their mRNAs. All together, these findings establish a link between mRNA translation and histone acetylation in muscle, underpinning translational control as a master regulator of histone acetylation.
This scientometric and bibliometric study analyzes global research trends on polyhydroxyalkanoate (PHA)-producing microorganisms and their applications in bio-based packaging between 2014 and 2025. Using the Scopus database and advanced tools such as Bibliometrix in RStudio and VOSviewer, scientific output and international collaboration networks were evaluated. The results demonstrate exponential growth in publications, driven by the urgent need to mitigate the petrochemical plastics crisis and develop biodegradable alternatives within the circular economy. Multidisciplinary analysis reveals a thematic shift from basic physiological and taxonomic studies towards complex applications in metabolic engineering, synthetic biology, the optimization of low-cost substrates such as industrial effluents, multi-omics tools, gene editing with CRISPR-Cas9, and, as an emerging exploratory approach, quantum modeling to optimize cell performance. Despite significant progress, critical technological gaps were identified related to challenges in downstream processing, the management of mixed microbial communities, and insufficient funding for the characterization of physicochemical and biocompatibility properties. It is concluded that, to ensure the commercial scalability and sustainability of PHAs, future research must prioritize overcoming these economic and technological bottlenecks, fostering strategic collaboration between academia and the biotechnology industry.
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A major goal in agriculture is to engineer crops to maintain yield with less nitrogen (N) fertilizer. Important regulators of plant N-responses include HRS1 HOMOLOG (HHO) transcription factors (TFs); yet, their redundant repressive mode of action hinders functional analyses. Here, we highlight HHO5 as a unique HHO TF based on its phylogenetic position, phloem specific expression, and dual role in regulating responses to inorganic and organic N-dose signals. Our results support a model whereby HHO5 mediates feedback repression of inorganic nitrate uptake under organic N satiety: (i) Meta-analyses revealed HHO5 expression is repressed by inorganic N, but induced by organic N treatments. (ii) HHO5 directly binds and represses nitrate response genes, but indirectly induces organic N-response genes. (iii) HHO5 indirect target gene induction occurs via WRKY partner TFs, validated using a cell-based DoubleTARGET TF co-perturbation assay. (iv) HHO5 initiates a validated gene regulatory network path which encompasses ∼12% of the N-dose response genes in planta. (v) Phenotypically, single hho5 T-DNA mutants show reduced N-dose dependent growth, and decreased seed N content. (vi) HHO5 represses nitrate uptake but induces root growth responses to Glu in planta. Overall, our findings support a model wherein HHO5 orchestrates dose dependent feedback regulation of organic versus inorganic N-signaling in Arabidopsis.
Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by epithelial barrier dysfunction, immune dysregulation, and marked clinical heterogeneity. Although sex hormones and gut microbiota have independently been implicated in AD, their potential interactions remain incompletely understood. This narrative review integrates evidence from clinical, experimental, and preclinical studies examining the interplay among progesterone, testosterone, gut microbiota, and AD, with emphasis on immune regulation, epithelial barrier function, and gut-skin communication. Overall, the available evidence suggests that progesterone is predominantly associated with type 2 immune responses, alterations in epithelial barrier homeostasis, and context-dependent microbial remodeling. In contrast, physiological testosterone is generally associated with immunoregulatory effects and microbial profiles enriched in short-chain fatty acid-producing bacteria, whereas local androgen metabolism may contribute to skin barrier dysfunction. Current evidence also supports a bidirectional relationship between gut microbiota and sex hormones, whereby hormonal fluctuations influence microbial composition while microbial metabolism may modify steroid hormone bioavailability. Collectively, these findings support an integrated endocrine-microbial-immune framework that may contribute to AD beyond cutaneous inflammation. Although direct evidence simultaneously evaluating these components remains limited, this framework may help explain sex-specific differences in disease susceptibility, severity, and clinical course while guiding future mechanistic studies of the hormone-gut microbiota-skin axis.
Baroque horses represent a distinctive group of breeds that emerged mainly between the 16th and 18th centuries, shaped by the demands of European royalty and their courts. Although recognised as separate breeds, they share common ancestry, breeding goals, and historical gene flow. This study aimed to evaluate their genomic diversity, population structure, and admixture patterns. We analysed 1 160 individuals from eight breeds - Lipizzan, Old Kladruby Horse, Pura Raza Española, Lusitano, Friesian, Criollo, Peruvian Paso, and Puerto Rican Paso Fino - using 34 026 single-nucleotide polymorphisms, with the Croatian Arab horse included as an outgroup. The Lipizzan was further divided into national subpopulations, the two Old Kladruby subpopulations defined by colour (black and grey), and within the Pura Raza Española, the Carthusian strain was treated as a distinct subpopulation. Observed genetic diversity, measured by heterozygosity and haplotype richness, was broadly comparable across breeds, although clear differences were observed between breeds with the lowest values (Friesian) and those with the highest values (Lusitano and Criollo). Genomic inbreeding coefficients, estimated using runs of homozygosity (FROH>2Mb) and homozygosity-by-descent segments (FHBD), ranged from ∼0.06 to ∼0.29 and ∼0.14 to ∼0.27, respectively. These values indicate relatively high levels of inbreeding, which may reflect the closed breeding structure of the populations and their relatively limited effective population sizes. In contrast, most individual inbreeding coefficient (FIS) values were negative, reflecting deliberate avoidance of close-relative matings. Estimates of contemporary effective population size (NeLD) ranged from ∼50 to ∼240 across breeds, depending on the method. Historical NeLD trajectories revealed two distinct demographic decline patterns: one occurring around 12 generations ago and the other around 22 generations ago. Evidence of recent between-breed gene flow was detected among Iberian breeds and between Croatian Arab and Lusitano, whereas shared historical ancestry among Baroque breeds was evident across populations. These results provide new insights into the genomic architecture of Baroque horses and offer valuable guidance for managing their genetic diversity.
Alport syndrome (AS) is one of the most common causes of inherited chronic kidney disease. Timely diagnosis and treatment of this condition can significantly influence its natural course. The evaluation of at-risk family members allows for the identification of new patients and the initiation of renoprotective measures that help prevent the progression of kidney disease. The aim of this study was to describe the implementation and results of cascade screening among at-risk relatives of a cohort of patients with AS followed at our unit. This is a prospective, single-center study conducted at Nephrology Department, Health Local Unit Gaia/Espinho. We provided patients with letters intended for their first-degree relatives, highlighting the benefits of a referral for renal impairment assessment and genetic counseling. A total of 93 at-risk relatives underwent evaluation through biochemical testing (kidney function and urinalysis abnormalities) and were offered molecular screening for the familial COL4 variant following genetic counseling. Clinical, genetic, and laboratory data were systematically collected. Seventy-six (81.7%) at-risk relatives underwent molecular evaluation. A positive molecular test was achieved in 52 (68.4%) relatives, whereas the familial variant was excluded in 24 (31.6%) individuals. 9 (9.6%) individuals declined to proceed with screening and 8 (8.6%) experienced delay in obtaining their results. Among those with a positive molecular screening, 36.5% (n = 19) had urinary protein-creatinine ratio > 0.1 g/g creatinine and 13.5% (n = 7) had glomerular filtration rate (by the CKD-EPI [Chronic Kidney Disease Epidemiology Collaboration] 2021 equation) < 60 mL/min/1.73 m2. 30 (57.7%) relatives initiated treatment with a renin-angiotensin-aldosterone system inhibitor. These individuals were older, had higher levels of proteinuria, and had a lower glomerular filtration rate at baseline. The relatively small size of this series from a single unit. The cascade screening based on a patient-led approach may not extent to all the at-risk relatives. Cascade screening of relatives of patients with AS is an effective strategy for identifying individuals with ongoing kidney disease or those at risk of future renal impairment. This approach enables the early initiation of nephroprotective measures and ensures timely access to appropriate genetic counseling. Alport syndrome (AS) is a leading cause of inherited chronic kidney disease. Timely diagnosis and treatment of this condition can significantly influence its natural course. The evaluation of at-risk family members enables the identification of new patients and the early initiation of renoprotective measures to prevent the progression of kidney disease. We implemented a patient-led cascade screening of at-risk relatives of patients with AS. More than half of the evaluated relatives were diagnosed with AS, and most of them started pharmacologic interventions to delay progression of the disease. These findings underscore the importance of cascade screening in identifying individuals with AS and enabling early therapeutic intervention to slow the progression of kidney impairment.
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Recent evidence suggests that cardiovascular diseases and cancer are linked through circulating factors, mainly proteins with oncogenic properties found in heart failure (HF) patients. However, the role of long non-coding RNAs (lncRNAs) as potential factors in cancer progression associated with HF remains unknown. This study aims to determine the role of HF-related lncRNAs in cancer progression. Using public RNA-sequencing datasets from the left ventricle and whole blood of HF patients, as well as colon cancer tumors, we identified HF-related lncRNAs with predicted competing endogenous RNA (ceRNA) interactions. These were integrated with co-expression analysis and upregulated colon cancer protein-coding genes to construct an HF-related regulatory network associated with colon cancer and epithelial-mesenchymal transition (EMT)-associated genes. LINC00670-overexpressing DLD-1 colon cancer cells showed increased migration and expression of Vimentin, along with N-cadherin, ZEB1, ESRP1, and E-cadherin transcript levels. Long-read nanopore sequencing of LINC00670-overexpressing DLD-1 cells revealed positively correlated genes, including type I interferon signaling pathway genes (STAT1 and SLC7A11) and epithelial-mesenchymal transition (EMT)-associated migratory genes (DDIT4 and PABPC1), as genes associated with the response to HF-related LINC00670 overexpression. Our findings reveal LINC00670 as an HF-related lncRNA that promotes partial EMT-transcriptional changes associated with type I interferon-associated genes, suggesting that LINC00670 plays a key role in inducing mesenchymal traits in the cardio-oncological context.
Over the past decade, the use of nanomaterials and nanomedical devices has been increasingly explored for cancer treatment. Although the outcomes of conventional therapies have improved patient survival, these approaches still present important limitations for some types of cancer and metastasis. Challenges such as poor drug accumulation in solid tumors and lack of specificity and selectivity can be addressed through alternative nanomedicine-based treatments. Among the wide range of nanoplatforms whose composition and shape have been designed for cancer treatment, this review focuses specifically on those based on natural proteins, including advanced carriers and engineered proteins bearing active targeting and/or therapeutic agents. The objective of this review is to provide a comparative and translational analysis of protein-based nanomaterials for cancer therapy, highlighting their unique characteristics, such as biocompatibility, biodegradability, and the ability to integrate bioactive peptides that can trigger or respond to tumor-specific or altered physiological stimuli. Several protein-based nanomedical devices have been developed for theranostic applications, demonstrating enhanced performance in tumor imaging and cancer treatment. This review introduces a structured analytical framework that classifies protein-based nanomaterials according to their biological origin, functional design, and clinical readiness, enabling systematic evaluation across platforms. Rather than providing a descriptive overview, this work offers a structured comparative analysis of protein-based nanomaterials, highlighting design trade-offs, translational challenges, and factors influencing clinical applicability.
Small ruminant lentivirus (SRLV) infects goats and sheep of all breeds and ages worldwide. There are no current records regarding the three-dimensional structure or antigenic capacity of the nucleocapsid protein p14 of FESC-752 Mexican strain. The antigenic structure of p14 protein of a B1 genotype was predicted. cDNA from FESC-752 was used to overexpress the recombinant SRLV-rp14 protein. Then, its antigenicity was verified in vitro by evaluating plasma samples from goats and sheep naturally infected with SRLV. Antigenicity prediction showed a "horseshoe"-type structure shared by different lentiviruses and five epitopes distributed throughout the p14 surface regions where they coincide suggesting conserved epitopes in the zinc-finger structures of the nucleoproteins of the SRLV, Human Immunodeficiency Virus (HIV-1), and Feline Immunodeficiency virus (FIV) retroviruses. Multi-species molecular docking showed a notable structural convergence where caprine, bovine, murine, and human immunoglobulins target a predictive 23 amino acid epitope (residues 41-64) within the core zinc-finger region. Furthermore, CABS docking simulations predicted that p14-derived peptides preferentially bind within the antigen-presenting cleft of both caprine and bovine major histocompatibility complex class I (MHC-I) molecules. The stability of these immunological complexes is mediated by dense networks of hydrophobic interactions and highly conserved aromatic anchoring residues. Antigenicity analysis revealed that 78.7% of samples from naturally infected goats showed immunoreactivity toward SRLV-rp14 and the predictive evidence that p14 can simultaneously stimulate both humoral and cellular pathways makes it a strategic candidate for the design of next-generation vaccines aimed at controlling lentiviruses in small ruminants.
El objetivo de este trabajo es analizar el desarrollo conceptual del modelo secuencial de carcinogénesis gástrica propuesto por el profesor Pelayo Correa, y destacar su integración transdisciplinaria y su relevancia para comprender la variación poblacional del riesgo y las oportunidades de prevención en América Latina. El modelo de Correa se originó en observaciones epidemiológicas en Colombia y evolucionó hacia un marco integrador que combina historia natural, patología, biología molecular, genética del huésped y virulencia bacteriana. La cascada precancerosa -gastritis crónica, atrofia, metaplasia, displasia y carcinoma- se confirmó como un proceso dinámico y potencialmente reversible, modulada por la persistencia de Helicobacter pylori. El ensayo clínico de quimioprevención en Nariño demostró que la erradicación de H. pylori acelera la regresión de lesiones y modifica la trayectoria de la cascada. Un aporte central del modelo es la incorporación de la coevolución huésped-patógeno: la incompatibilidad entre linajes humanos y cepas de H. pylori de distinto origen explica diferencias marcadas en la progresión de lesiones precursoras entre poblaciones cercanas. Esta articulación conceptual sustenta estrategias de prevención primaria y vigilancia basadas en riesgo. La obra del profesor Correa muestra que las enfermedades crónicas requieren marcos que integren múltiples niveles biológicos y analíticos, e identifica la erradicación de H. pylori como punto crítico de intervención. En América Latina, este enfoque transdisciplinario orienta estrategias costo-efectivas de prevención y vigilancia adaptadas a contextos de alta vulnerabilidad y heterogeneidad poblacional.
Multidrug-resistant (MDR) Klebsiella pneumoniae is a critical cause of nosocomial infections associated with high mortality. Of particular concern is the genomic convergence of multidrug resistance and hypervirulence (MDR-hvKP), which represents a severe public health threat. This study characterised the resistome, virulome, and population structure of seven clinical MDR K. pneumoniae isolates collected between 2024 and 2025 across four Peruvian regions (Moquegua, Ucayali, Loreto, and Lima) using whole-genome sequencing (WGS). Genomic analysis identified six distinct sequence types, with the high-risk clone ST307 being the most prevalent (2/7 isolates), consistently associated with the KL102 capsular locus. Remarkably, one isolate from Loreto (oph_54) exhibited a convergent MDR-hvKP genomic profile (ST218, KL57, virulence score 4), carrying yersiniabactin, salmochelin (iro), and aerobactin (iuc) loci alongside multiple resistance determinants. The isolates presented a robust resistome dominated by the extended-spectrum beta-lactamase gene blaCTX-M-15 (5/7) and blaOXA-1, coupled with aac(6')-Ib-cr and fosA6 genes. Plasmid analysis revealed a predominance of IncFIB(K) replicons. Although exploratory, these findings demonstrate the regional presence of high-risk ST307 lineages and the emergence of convergent MDR-hvKP genomic profiles within the analysed Peruvian hospitals. Immediate and continuous genomic surveillance is urgently required to monitor the potential spread of these highly dangerous, MDR bacterial lineages within these healthcare environments.
Animal models of inflammatory pain frequently rely on local administration of irritants that trigger acute inflammation and hypersensitivity. Lipopolysaccharide (LPS) is a well-established activator of innate immune pathways. Subcutaneous (intraplantar) injection of LPS into the hind paw produces a localized inflammatory reaction characterized by swelling, cellular infiltration, and increased mechanical and thermal sensitivity. Acid-sensing ion channels (ASICs) are key contributors to nociceptive signaling; however, their presence and regulation at the level of peripheral terminals, particularly in the skin, remain insufficiently characterized. We previously showed that formalin-induced acute pain increases ASIC1a expression in the central nervous system and peripheral dorsal root ganglia (DRGs). In this study, using the LPS paw model in both male and female mice, we demonstrate a robust upregulation of ASIC1a directly within inflamed paw tissue, together with changes in lumbar dorsal root ganglia (DRGs), the somata of sensory neurons innervating the paw. We further show that ASIC1a upregulation is associated with paw edema, ERK activation, miRNA-dependent regulatory mechanisms, and the development of behavioral hypersensitivity induced by LPS. Importantly, local pharmacological blockade of ASIC1a with subcutaneous PcTx-1 attenuated both mechanical and thermal hypersensitivity. Together, our findings identify inflamed peripheral tissue as an important site of ASIC1a regulation during LPS-induced inflammation and provide new insight into the molecular mechanisms governing ASIC1a expression in inflammatory pain, supporting further exploration of ASIC1a-targeted therapeutic strategies.
Background/Objectives: To evaluate whether the combination of oral eplerenone and half-dose full-fluence photodynamic therapy (HD-FF PDT) provides greater efficacy than HD-FF PDT alone in persistent central serous chorioretinopathy (CSCR). Methods: This monocentric, retrospective, observational study included patients with persistent (>6 months) simple or complex CSCR who had previously undergone either half-dose full-fluence photodynamic therapy (HD-FF PDT) alone or HD-FF PDT combined with oral eplerenone as part of routine clinical practice between September 2024 and March 2025. Functional and morphological data collected at baseline and at 1, 3, and 6 months after treatment were retrospectively reviewed. An artificial intelligence-based algorithm was used to analyze OCT scans, quantifying subretinal and intraretinal fluid volumes (SRFV, IRFV) and assessing ellipsoid zone and external limiting membrane integrity, hyperreflective foci, subfoveal choroidal thickness (SCT), and central macular thickness (CMT). Results: Fifty patients (53 eyes; mean age 52 years) were included, with no significant baseline differences between groups. Best-corrected visual acuity improved more significantly in Group B at 1 and 6 months (p = 0.032 and p = 0.009, respectively). At 6 months, subretinal fluid volume (SRFV), quantified by AI-based OCT analysis, was significantly lower in the combined therapy group (p = 0.014). An AI-defined complete resolution of subretinal fluid (SRFV < 0.010 mm3) was achieved more frequently in Group B than in Group A (77% vs. 22%, p = 0.001). Conclusions: Although HD-FF PDT remains the standard treatment for persistent CSCR, adjunctive therapy with the mineralocorticoid receptor antagonist eplerenone may enhance subretinal fluid reabsorption and improve mid-term anatomical and functional outcomes.
Background/Objectives: The DPYD gene encodes the enzyme dihydropyrimidine dehydrogenase that metabolizes fluoropyrimidines. Genetic variants in DPYD have been associated with altered enzyme activity; therefore, accurate detection and interpretation is critical for individualized fluoropyrimidine therapy. The most common causal variant is c.1129-5923C>G (rs75017182) located in intron 10, which introduces a cryptic splice site. This variant is in high linkage disequilibrium (LD) in the HapB3 haplotype with a benign synonymous variant in exon 11, c.1236G>A (rs56038477). Since c.1129-5923C>G and c.1236G>A have been reported in LD, many commercial kits use c.1236G>A as a proxy for the function-altering intronic variant. Methods: A DPYD genotyping protocol was implemented following the quality regulations that apply to clinical laboratories (EN-ISO9001:2015 and EN-ISO15189:2022). NGS, MLPA and Sanger sequencing were used for validation purposes. Results: Over the last 5 years a total of 2007 patients have been analyzed at our department. The observed DPYD genotype frequencies aligned with those observed in European populations. Importantly, we have identified a patient harboring the c.1236G>A variant, but in the absence of the c.1129-5923C>G variant. This last result supports recently published findings suggesting that these two variants may not be in perfect LD, as previously assumed, and lead to suboptimal dosing for those patients carrying this allele. Finally, low frequency variants (c.496A>G, c.2194G>A, and c.1601G>A), not described in DPYD analysis guidelines recommendations, were found in two patients who required fluoropyrimidines dose adjustment. Conclusions: These findings highlight the limitations of relying on proxy variants for clinical decision-making, as incomplete linkage disequilibrium may lead to misclassification of patients' metabolic capacity. Furthermore, in order to provide safer protocols for DPYD-based personalized treatment genetic panels should expand to include additional rare DPYD variants.
Polyneoptera comprises hemimetabolous insect orders of significant agricultural, ecological, and medical relevance, motivating phylogenetic and molecular research that has nevertheless focused predominantly on canonical mitochondrial markers. Here, we assembled new mitogenomes for Polyneoptera and evaluated the usefulness of genes located in nucleotide-diversity hotspots as markers for species identification and phylogenetic inference. To expand the available mitogenomic resources, raw sequencing data were retrieved from public databases, resulting in the assembly and annotation of 26 complete mitogenomes, all exhibiting the typical insect mitochondrial architecture. These newly assembled genomes were combined with publicly available mitogenomes from Orthoptera, Blattodea, Plecoptera, Mantodea, and Phasmatodea to reconstruct phylogenetic relationships using both complete and reduced datasets comprising nucleotide-diversity hotspot-associated genes. The performance of these hotspot regions was further assessed through barcoding gap analyses and comparisons with the most comprehensive datasets to identify candidate mitochondrial markers for molecular species identification and phylogenetic inference. Across orders, different mitochondrial regions, including the classical markers 16S and COX1, as well as genes from the NADH dehydrogenase complex, emerged as the most informative, although optimal markers varied among lineages. Overall, our findings highlight the value of publicly accessible sequencing data for generating high-quality genomic resources and improving phylogenetic and taxonomic tools.