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Deciphering how human genetic variants affect conserved metabolic enzymes is essential for understanding their evolutionary and clinical significance. Here, we combine sequence analyses of temporally stratified human genomes with a quantitative Saccharomyces cerevisiae complementation assay in a strain lacking SER2, the yeast gene required for the final step of L-serine biosynthesis, to examine functional differences among human phosphoserine phosphatase (PSPH) variants. Population-genomic comparisons between ancient hunter-gatherers and present-day humans identified two PSPH exons with elevated differences in nucleotide diversity, guiding the selection of two ancient-genome-prioritized variants (R27S and Q83H) for functional testing. To place their effects in functional context, we expressed each variant individually and compared complementation with the modern PSPH allele and two disease-associated alleles (D32N and A35T) across multiple environmental conditions. Human PSPH enhanced growth of the SER2 deletion mutant and revealed reproducible quantitative differences among alleles. The modern allele generally conferred the strongest complementation, while the ancient genome variants supported measurable but more condition-dependent rescue, and the disease-associated alleles showed the weakest complementation. These differences were broadly consistent across conditions, while specific environmental perturbations revealed context-dependent shifts in effect size. Together, our results establish a scalable framework that links evolutionary genomics with experimental functional assays to identify and evaluate human metabolic enzyme variants with measurable in vivo effects.

Understanding the evolutionary distribution and functional roles of toxins across diverse taxa remains a fundamental challenge in fungal biology. Aerolysin-like beta-pore-forming toxins are widely distributed across multiple kingdoms of life, yet their specific occurrence and structural diversity within the fungal kingdom remain poorly characterized. In our current study, we address this gap by investigating candidate aerolysin-like proteins in the basidiomycete Armillaria ostoyae using an integrated framework combining structural modeling, comparative genomics, and transcriptomic datasets spanning multiple developmental stages. Our results demonstrate that these candidate proteins are actively transcribed throughout the fungal life cycle, with consistent expression maintained in mature fruiting-body tissues. Notably, we show that the specific gene ARMOST_18480 undergoes significant upregulation under plant-invasive conditions, strongly supporting its role as a putative pathogenicity-associated factor. Structural characterization revealed a modular architecture with deeply conserved pore-forming domains including Alanine-Glycine-Isoleucine-Proline (AGIP)-like loop variants homologous to vertebrate natterins from Thalassophryne nattereri, despite low overall sequence identity. Importantly, phylogenetic inference robustly resolves these Armillaria proteins within distinct fungal lineages well-separated from their vertebrate counterparts. Together, these findings significantly expand the known evolutionary distribution of the aerolysin superfamily and identify key candidates for future functional validation regarding pore-forming activity, plant pathogenicity, and mushroom-associated bioactivity.

Missense variants in breast cancer remain diagnostically challenging due to their functional diversity and complex genomic contexts. Conventional laboratory assays for evaluating pathogenicity are labor-intensive, costly, and often impractical for large-scale screening, creating a pressing need for accurate, scalable, and clinically interpretable computational approaches. In this study, we present a novel deep learning framework for predicting the pathogenicity of breast cancer missense variants, integrating comprehensive preprocessing, advanced imputation, rigorous model benchmarking, and explainability. Genetic variants were curated from multiple genomic databases, annotated using the Ensembl Variant Effect Predictor (VEP), and processed with Variational Autoencoders (VAE) for missing-value imputation. Seven deep learning models, MLP, CNN, DNN, RNN, LSTM, GRU, and Transformer, were trained and evaluated across 11 performance metrics. To quantify performance stability, each model was trained across five random seeds; mean AUC ± SD across seeds is reported as the primary performance estimate, with the best-seed run used only for LIME and PMI interpretability analyses. Recursive feature elimination, permutation importance (PMI), and Local Interpretable Model-Agnostic Explanations (LIME) were employed to enhance transparency. Statistical analyses, including Z-tests, ANOVA, and calibration assessments, validated performance consistency and inter-model differences. GRU achieved the highest internal AUC (0.9956 [95% CI 0.9936-0.9972]; mean across five seeds 0.9941 ± 0.0011), with precision 0.9967 and calibration ECE 0.0095. Externally, LSTM led with AUC 0.9457, exceeding all eleven standalone predictors benchmarked on the same set. Models showed strong alignment with conservation signals such as phyloP470way and Eigen-PC scores. Notably, the pipeline provides performance metrics with 95% confidence intervals and incorporates case-level LIME visualizations for true positive, true negative, false positive, and false negative predictions, bolstering interpretability and clinical relevance. This work delivers one of the most comprehensive evaluations of deep learning in breast cancer variant classification to date. By combining high-performance sequential models with interpretable AI tools, the proposed framework provides a reproducible, transparent benchmark for variant pathogenicity prediction and a foundation for future research use and translation in cancer genomics.

Chimeric antigen receptor T-cell (CAR-T) therapy is an established treatment for several hematological malignancies, with peripheral blood mononuclear cells (PBMCs) serving as the starting material for manufacturing. Cryopreservation of PBMCs may offer logistical flexibility, although its influence on manufacturing outcomes remains incompletely defined. This review aimed to compare the effect of fresh versus cryopreserved PBMC starting material on CAR-T cell manufacturing outcomes, including viability, fold expansion, and transduction efficiency. A systematic review was conducted following PRISMA guidelines. PubMed and Google Scholar were searched from inception through March 2026 for original studies comparing fresh and cryopreserved PBMCs in human CAR-T cell manufacturing. Methodological quality was assessed using the design-appropriate quality-appraisal tools, and findings were synthesized narratively due to heterogeneity in study design and protocols. Five studies published between 2019 and 2025 met the inclusion criteria, comprising two clinical and three experimental analyses. Post-thaw viability and recovery of cryopreserved PBMCs ranged between 77% and 97%, slightly lower than fresh material. Fold expansion, transduction efficiency, and cytotoxic activity were generally comparable between groups, although some studies reported transient early differences including prolonged doubling times, mitochondrial dysfunction signals, and increased TIM-3 expression in cryopreserved-derived products. Cryopreservation can be considered a feasible approach in CAR-T manufacturing, with generally comparable outcomes despite early post-thaw cellular changes. These differences do not seem to consistently compromise the overall manufacturing performance. However, the current evidence remains limited and heterogeneous, and further studies are required to increase confidence in our initial findings.

Acute confusional migraine (ACM) is an uncommon pediatric migraine variant characterized by transient confusion and agitation, nonspecific electroencephalographic changes, and normal bloodwork and neuroimaging. We present the first case of ACM in a child with Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome (OMIM: 617140), a rare multisystem disorder caused by a pathogenic variant in the SON gene, clinically characterized by global developmental delay, intellectual disability, hypotonia, distinctive craniofacial dysmorphic features, and structural brain anomalies. Emerging evidence suggests that SON haploinsufficiency may contribute to a broader neuroinflammatory phenotype, potentially increasing susceptibility to cortical excitability, which is one of the proposed causes of ACM. We hypothesize a direct mechanistic link whereby SON-related splicing defects lower the cortical excitability threshold, predisposing individuals to episodic hyperexcitability syndromes such as ACM. This report expands the known neurological phenotype of ZTTK syndrome. Given the nature of the communication impairments among patients with ZTTK syndrome, ACM symptoms may be underreported or mistaken for epileptic events. Future studies are needed to validate this association and elucidate the underlying molecular pathways.

Oncolytic virotherapy (OVT) represents a promising approach for cancer treatment, employing oncolytic viruses (OVs) that selectively infect and lyse tumor cells while promoting an antitumor immune microenvironment. Vaccinia virus (VACV) serves as an attractive oncolytic vector due to its favorable safety profile, ease of genetic modification, and inherent tumor selectivity. To enhance both safety and tumor-targeting capability, we constructed a recombinant vaccinia virus, VV-dTF/EE, by deleting the viral J2R and F4L genes and inserting the human neutrophil elastase (ELANE) gene, which exhibits tumor-killing activity. Mechanistic studies evaluated with virus replication selectivity, cell apoptosis, genomic damage, immunogenic cell death, alongside analysis of the immune microenvironment. Efficacy was tested in multiple tumor cell models in vitro and lung cancer models in vivo. In tumor cell lines and mouse tumor models, VV-dTF/EE demonstrated tumor-restricted replication, potent oncolytic effects, and induction of immunogenic cell death. Furthermore, VV-dTF/EE augmented VACV-induced antitumor immunity by increasing CD8+ T cell infiltration and suppressing M2-like macrophage polarization. This VV-dTF/EE revealed tumor-selective replication and killing ability while modifying the tumor immune microenvironment to elicit immunogenic cell death. Our findings highlight a novel strategy for safe and effective tumor immunotherapy through dual-gene deletion and ELANE expression in an oncolytic vaccinia platform.

Alcohol drinking is a major risk factor for oropharyngeal cancer, yet the nature of risk differences across drinking patterns remains unclear. We leveraged data from large-scale genome-wide association studies to conduct Mendelian randomization analyses, assessing the effects of alcohol intake with meals, intake frequency, maximum habitual intake, and drinks per week on risks of oral cavity cancer, pharyngeal cancer, stomatitis, lichen planus, and oral leucoplakia. Multiple Mendelian randomization methods were applied to model pleiotropic effects, and multivariable analysis was performed to evaluate the interplay between alcohol consumption and household income. Alcohol intake frequency was associated with increased risk of oral and pharyngeal cancer. In contrast, alcohol intake with meals showed an apparent protective association with these outcomes, which was nullified after adjusting for household income in the multivariable analysis-indicating that the observed benefit was explained by higher socioeconomic status. The frequency-risk relationship remained significant after adjustment, highlighting intake frequency as an independent risk factor under MR assumptions. The number of drinks per week was linked to increased stomatitis risk, and this association remained significant after accounting for household income, suggesting an independent effect of alcohol consumption on stomatitis risk under MR assumptions. Cancer prevention strategies should target harmful alcohol consumption patterns with attention to drinking frequency and socioeconomic context, rather than treating alcohol exposure as monolithic.

A significant proportion of gastric diffuse large B-cell lymphoma with mucosa-associated lymphoid tissue [DLBCL(MALT)] and without MALT ('pure' DLBCL) can be resolved by Helicobacter pylori eradication (HPE). Gastric MALT lymphoma is an indolent lymphoma derived from memory B cells in the marginal zone. In the present study, we aimed to explore the origin of large cells in HPE-responsive gastric DLBCLs (complete remission after HPE). We investigated gastric lymphoma biopsies from 31 patients with HPE-responsive DLBCLs [15 'pure' DLBCLs, 16 DLBCL(MALT)s]. We used the Hans algorithm (CD10, BCL-6, and MUM1) to define the origins of germinal center B cell (GCB) and non-GCB. To further ascertain the cellular origin, 11 'pure' DLBCLs were examined using an Agilent whole-human genome microarray. Eleven DLBCLs [eight with 'pure' DLBCL and three with DLBCL(MALT)] were also assessed using Lymph2Cx. Specific GCB markers, including BACH2, AID, and BCL2 rearrangement and enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) codon 641 mutations, were evaluated in 31 patients with HPE-responsive gastric DLBCLs. According to the Hans algorithm, 53% (8/15) of gastric 'pure' DLBCLs and 50% (8/16) of DLBCL(MALT)s were of the GCB phenotype. Gene expression assays revealed that five of six patients with 'Hans' GCB had GCB genetic signatures, whereas four of five patients with 'Hans' non-GCB had activated B-cell genetic signatures. The Lymph2Cx assay revealed the GCB subtype in seven of eight patients with 'Hans' GCB. The expression patterns of BACH2 (p = 0.005) and AID (p = 0.038) closely correlated with the 'Hans' GCB phenotype. BCL2 rearrangements and EZH2 codon 641 mutations were detected in 44% (7/16) and 13% (2/16) of patients with 'Hans' GCB, respectively. In another cohort of 29 HPE-unresponsive gastric DLBCLs [19 'pure' DLBCLs and 10 DLBCL(MALT)s], we found a close association between the 'Hans' GCB subtype and the GCB subtype as determined by the Agilent whole-human genome microarray and Lymph2Cx in lymphoma cells of these patients. In conclusion, more than half of HPE-responsive large cell lymphoma cases in the stomach were of GCB origin. © 2026 The Pathological Society of Great Britain and Ireland.

Some individuals with airflow obstruction later show normalized spirometric values, but the clinical implications of this phenomenon in the general population are unclear. Using data from the Korean Genome and Epidemiology Study (KoGES), we examined the characteristics and prognosis of individuals with normalized airflow obstruction (NO), defined as FEV1/FVC <0.7 at baseline that normalized to &#x2265;0.7 during follow-up. Between 2000 and 2006, 4,807 participants were classified as NO (n=242), fixed obstruction (FO; persistent FEV1/FVC <0.7; n=277), or normal (FEV1/FVC &#x2265;0.7 throughout; n=4,288). Over eight years of follow-up (2007-2014), the NO group contained more men, current smokers, and individuals with lower body mass index than the normal group and showed intermediate lung function between FO and normal groups. Annual FEV1 decline was similar across groups (-48.8 mL/year normal; -48.3 mL/year NO; -45.8 mL/year FO). However, by the end of the 12-year follow-up period, 37.2% of participants in the NO group developed airflow obstruction, with a 4.22-fold higher risk compared with the normal group (95% CI 3.26-5.46). Findings were consistent in a sensitivity analysis excluding individuals with excessive lung function variability. (HR 7.41, 95% CI 5.45-10.07). These findings indicate that NO may represent a distinct and dynamic spirometric phenotype associated with increased risk of future airflow obstruction and highlight the need for early identification and preventive strategies in at-risk populations.

Phytophthora cinnamomi, the causal agent of phytophthora root rot (PRR), is one of the most destructive pathogens affecting avocados worldwide. Recent global population analyses of P. cinnamomi identified two panglobal clonal lineages named PcG1-A2 and PcG2-A2, both of A2 mating type. In California avocado orchards, P. cinnamomi populations were previously classified into two clades, A2 clade I and A2 clade II, with the latter likely introduced from Mexico. In this study, we conducted the genetic characterization of 125 isolates collected from avocado-producing regions of the United States and investigated their genetic relationships with previous populations from California and Mexico and with the two panglobal A2 clonal lineages using whole-genome resequencing. All isolates were estimated to be triploid based on nQuire. Phylogenetic reconstruction and fastSTRUCTURE analyses based on single nucleotide polymorphisms showed that the PcG1-A2 reference isolate clustered with the previously described California A2 clade I and with most U.S. isolates. In contrast, the PcG2-A2 reference isolate clustered with the California A2 clade II and with most Mexican isolates, supporting a Mexican origin for this lineage. Discriminant analysis of principal components, principal coordinate analysis, and minimum spanning networks further resolved two distinct groups of A2 isolates, reinforcing the presence of two clonal lineages in avocado orchards across the United States. Isolates were assigned to multilocus genotypes (MLGs), including one widespread MLG containing isolates from all sampled avocado-growing regions. Mexico exhibited the highest genetic diversity, although unique MLGs were detected at each location. These results clarify the genetic relationships among P. cinnamomi populations in avocado orchards across the United States and link the previously described California clades to the two panglobal lineages currently recognized. Our findings support a hypothesis of pathogen movement among avocado-producing regions and reveal localized subpopulations that may pose risks to the sustainability of the avocado industry. Continued monitoring of P. cinnamomi populations will be essential for developing effective PRR management strategies to increase avocado production and enhance the national industry's profitability, competitivity, and marketability.

B-cell precursor acute lymphoblastic leukemia (preB-ALL) is characterized by pathogenic variants currently used in precision oncology. However, the mutational landscape of Mexican children with preB-ALL has not yet been thoroughly explored and defined in terms of the clinical significance. We used a custom-designed next-generation sequencing exome panel, along with high-resolution chromosome microarrays and gene fusion-targeted assays, to characterize the mutational landscape of 73 Mexican children with preB-ALL, exploring the profile of clinically significant variants. We classified the variants following the AMP-ASCO-CAP 2017 and ACMG-CG 2019 guidelines recommendations. The mutational landscape includes a broad molecular spectrum of variants affecting cell cycle regulation, B-cell development, kinase signaling, and epigenetic regulation genes. Tier 1 diagnostic variants allowed the identification of pre-B ALL genetic subtypes, diminishing the preB-ALL NOS group from 63% to 37%. Furthermore, 37% of cases presented Tier 1 variants conferring intermediate to adverse prognoses (primarily involving CRLF2 gene fusions, as well as PAX5, IKZF1, and TP53 inactivating mutations). Notably, these patients exhibited high-risk clinical features and a lower event free survival rate than patients without these variants (60% versus 41.2%; p&#x2009;=&#x2009;0.046; 95% CI). Between 19% and 42% of patients had Tier 2 variants targetable with JAK-STAT or RAS-MAPK signaling inhibitors. These patients showed a lower overall survival rate than patients without these variants (64% versus 90%; p&#x2009;=&#x2009;0.048; 95% CI). Tier 1 potential germline variants in cancer predisposition genes (mainly BRCA1/2 and CHEK2) were observed in 10% of patients, some of whom had a family history of cancer. This highlights the importance of genetic counseling for patients and their families. Finally, 33% of patients had Tier 3 variants that were predicted to be deleterious and potentially upgraded to pathogenic with plausible clinical relevance. In conclusion, the mutational landscape analysis revealed variants useful for oncologic management and genetic counseling of Mexican children with preB-ALL.

Canine epitheliotropic T-cell lymphoma (CETL) is an aggressive and generally incurable disease in dogs. CCNU (lomustine) is commonly used for management, even though it carries a significant risk of adverse effects. Several reports describe oclacitinib producing clinical improvement in affected dogs, yet comparative data between the two treatments are lacking. To compare the clinical outcomes in dogs with CETL treated with oclacitinib or CCNU, focussing on tumour reduction (response), survival time and time-to-relapse. This retrospective review (2008-2024) included 23 client-owned dogs with confirmed CETL treated with either CCNU (n&#x2009;=&#x2009;11) or oclacitinib (n&#x2009;=&#x2009;12). Outcomes included response (reduction in measurable tumour burden), survival (time from histopathological diagnosis to death) and time-to-relapse (for dogs with complete or good responses). Clinical presentation and adverse effects also were recorded. Additionally, sex, age and weight were compared between treatment groups to investigate potential confounding factors. No statistically significant differences were identified between oclacitinib and CCNU for response, survival or time-to-relapse (p&#x2009;>&#x2009;0.05 for all). Kaplan-Meier survival analysis showed no significant difference in overall survival (log-rank test, p&#x2009;=&#x2009;0.20). Oclacitinib was associated with fewer adverse effects and reduced monitoring intensity. These findings suggest oclacitinib may offer outcomes comparable to CCNU for CETL, with fewer adverse effects and less intensive monitoring. The small, retrospective sample limits firm conclusions, yet these results indicate that oclacitinib could be a reasonable, possibly safer palliative option warranting further prospective study.

[This corrects the article DOI: 10.3389/fmicb.2026.1842801.].

Genetic studies have significantly advanced our understanding of tauopathies, yet the genetic aetiology of Pick's disease, a rare 3-Repeat tauopathy, remains unclear. The MAPT p.A152T variant has been identified as a risk factor for Alzheimer's disease and progressive supranuclear palsy, but its role in Pick's disease is unknown. In this study, we examined the prevalence of MAPT p.A152T in the largest series of neuropathologically confirmed Pick's disease cases to date (n = 401). Through genotyping, we identified a single mutation carrier in the Pick's disease cohort (minor allele frequency = 0.12%). We previously reported MAPT p.A152T at a 0.20% frequency in healthy controls (n = 2456), suggesting that it does not associate with 3-Repeat tauopathy risk. To further investigate the effect of the variant on MAPT transcript expression, we used bulk RNA sequencing in Alzheimer's disease and progressive supranuclear palsy A152T mutation carriers. We did not detect significant differences in 4-Repeat tau levels, though preliminary trends may indicate more nuanced effects that need to be examined with long-read sequencing in a larger series. Overall, our study suggests that MAPT p.A152T does not increase Pick's disease risk and may instead be linked to 4-Repeat or mixed tau pathologies, warranting further functional investigation.

Chickpea is one of the most consumed legumes due to its high nutritional value and accessibility to low-income populations. However, due to climate change, chickpea cultivation is exposed to various environmental stresses affecting its production and productivity. This study evaluates the agronomic performance of 168 MAGIC subset population across two Mediterranean environments, in Marchouch (Morocco) and Terbol (Lebanon). Genome-wide association studies (GWAS) were conducted to identify potential marker-trait associations (MTAs) using the general linear model (GLM), the mixed linear model (MLM), and the fixed- and random-effect circulant probability unification (FarmCPU), with kinship and principal components used as covariates. The results revealed high genetic variation among the genotypes tested, with significant genotype-by-environment interactions for most traits studied. Genotypes with good agronomic performance (M-1407, M-2038, M-2079, M-242, M-2551, and M-987) were identified under both environments. Early flowering and maturation resulted in a significant increase in grain yield of around 66%. Grain yield varied from 151.85 to 882.7&#xa0;g m-2 and from 183.59 to 365.76&#xa0;g m-2 under Marchouch and Terbol conditions, respectively, showing higher genetic variation under Marchouch than under Terbol. Correlation analysis revealed strong, significant correlations among the studied traits. GWAS revealed clear genetic variation across environments, with Marchouch showing stronger and more consistent association signals than Terbol. In total, 72 reliable MTAs were detected for phenological traits, 38 for plant height, 18 for grain yield, and 197 for hundred-seed weight across both sites. A major genomic hotspot on chromosome 4 (11.97-13.63 Mbp) harbored stable and pleiotropic MTAs. Functional annotation of regions surrounding significant SNPs revealed 59 putative candidate genes, highlighting potential biological processes related to growth, signaling, and stress responses that require further validation. These results provide a foundation for further research on marker-assisted selection to improve chickpea productivity and yield stability in stressed environments.

Atypical Brucella spp., which include Brucella inopinata and related lineages, have emerged as unconventional pathogens with an expanding host range, particularly among exotic amphibians. A captive waxy monkey treefrog (Phyllomedusa bicolor) presented with a focal ulcerative skin lesion above its right eye. Cytology revealed mixed inflammation associated with a bacterial infection, and 16S rRNA sequencing identified the isolate as a member of the Brucella genus. Whole-genome sequencing using hybrid PacBio-Illumina platforms generated a high-quality draft genome. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values demonstrated the highest similarity to B. inopinata (98.08% ANI; 83.50% dDDH). Core genome phylogenetic analysis further confirmed close clustering with atypical Brucella spp., which are clearly distinct from classical zoonotic lineages. This case represents the first detection of an atypical B. inopinata-like Brucella strain in an amphibian in the Republic of Korea.

The WUSCHEL-related homeobox (WOX) gene family encodes plant-specific transcription factors that play pivotal roles in meristem maintenance, organogenesis, regeneration, and developmental phase transitions. Despite their importance, the pangenome-scale composition, expansion dynamics, and evolutionary constraints of WOX genes in alfalfa (Medicago sativa) remain poorly understood. Here, we performed an integrated pangenome and pan-transcriptome analysis of the WOX gene family using 24 high-quality alfalfa genomes. A total of 432 WOX genes were identified and clustered into 24 orthologous gene groups (OGGs). Pangenome profiling revealed that the WOX family is highly conserved across alfalfa accessions, with members of the Ancient clade showing greater conservation than those of the Intermediate and WUS clades. Duplication pattern analysis indicated that dispersed duplication was the primary force driving WOX family expansion, whereas whole-genome duplication (WGD) events predominantly contributed to the long-term retention of core and softcore members. Pairwise Ka/Ks analysis indicated strong purifying selection on most WOX gene pairs, whereas wild-germplasm genes and Intermediate-clade members showed elevated selective pressures. Notably, MsWOX9-related homologs had exceptionally high Ka/Ks values, suggesting potential functional divergence linked to agronomically relevant traits. Furthermore, pan-transcriptome analysis revealed that WOX genes could be classified into three distinct expression patterns during leaf senescence. Collectively, this study presents the first comprehensive pangenome- and pan-transcriptome-based characterization of the WOX family in alfalfa, providing new insights into its evolutionary dynamics and expression divergence.

Muscular dystrophies (MDs) are a genetically heterogeneous group of disorders, posing significant diagnostic challenges, especially in populations with high consanguinity. Despite advances in genetic testing, a substantial proportion of patients remain undiagnosed. Whole-exome sequencing (WES) has emerged as a powerful tool for identifying causal variants in such unresolved cases. To identify the genetic basis of nondystrophinopathic MDs in Iranian families with inconclusive prior genetic testing and to evaluate the diagnostic yield and mutational spectrum in this population. We performed WES on one affected individual from each of 10 unrelated Iranian families with clinically diagnosed MD. Candidate variants were prioritized based on in silico prediction tools (SIFT, PolyPhen-2, CADD, SpliceAI), population frequency databases (gnomAD, 1000 Genomes, EVS), and ACMG/AMP guidelines. Findings were validated by Sanger sequencing, MLPA, and STR haplotype analysis. Segregation analysis was performed in available family members. WES led to a diagnostic yield of 69.2% (9/13 variants in 10 families) after segregation analysis and ACMG-based reclassification. We identified 13 candidate variants in 10 known MD-associated genes, including DYSF, SGCA, TK2, MAP3K20, LMNA, COL6A1, COL6A2, ITGA7, MICU1, and SGCB. Among these, seven variants (54%) were novel. The majority of cases (84.6%) followed an autosomal recessive pattern, consistent with high parental consanguinity (70%). Notably, a de novo splice-site variant in COL6A2 (c.1053+1G>T) was identified in a sporadic case, confirming an autosomal dominant inheritance. Challenges in variant interpretation were observed in families with variants in tightly linked genes (COL6A1 and COL6A2), highlighting the role of linkage disequilibrium in founder populations. WES is a highly effective diagnostic strategy for genetically heterogeneous MDs, particularly in consanguineous populations. Our study expands the mutational spectrum of MDs in Iran and provides critical data for genetic counseling, prenatal diagnosis, and future therapeutic development. The high rate of novel variants underscores the importance of population-specific genomic studies.

Influenza D virus (IDV) is an emerging orthomyxovirus with cattle as its principal reservoir, and D/Yama2019-lineage viruses have become dominant in East Asia. Although IDV has been detected in Korean cattle, the genomic identity, phylogenetic placement, and regional evolutionary relationships of circulating Korean strains have not been defined. To address these gaps, nasal swabs were collected from 578 cattle with mild respiratory signs on 157 farms across eight provinces in South Korea during 2022-2023 and screened by RT-qPCR targeting the PB1 gene. Positive samples underwent complete genome sequencing of all seven segments, followed by maximum-likelihood and Bayesian phylogenetic analyses, discrete phylogeographic inference using a Bayesian stochastic search variable selection (BSSVS) model, and positive selection analyses. Six samples from three farms were IDV-positive (sample-level positivity: 1.04%; farm-level positivity: 1.9%), all from 8-to-10-month-old calves. Phylogenetic analysis of all seven genomic segments placed all six Korean strains within the D/Yama2019 lineage with strong bootstrap support (99%-100%), forming a monophyletic cluster more closely related to Chinese than to Japanese D/Yama2019 reference strains. No phylogenetic evidence of reassortment was detected. Bayesian time-scaled analysis estimated the most recent common ancestor of the Korean strains at ~2018.1-2020.1 across all seven segments. HEF-based BSSVS analysis suggested a China-to-South Korea transition within the sampled dataset (posterior probability (PP)&#x2009;=&#x2009;0.982; Bayes factor (BF)&#x2009;=&#x2009;163.67), although this result should be interpreted in light of the small number of Korean sequences and the single-segment basis of the phylogeographic analysis. Positive selection analyses revealed limited, method-dependent signals without support from the fixed effects likelihood model, and Korean-associated amino acid substitutions in PB1, P3, NS1, and NS2 were interpreted as lineage-associated molecular signatures rather than evidence of adaptive evolution. These findings provide a whole-genome baseline for IDV surveillance in South Korea and support continued longitudinal monitoring to clarify the persistence and transmission dynamics of D/Yama2019-lineage viruses in the region.