Search Results (335)

Pathogenic germline variants in the ATM gene are associated with a 20–30% lifetime risk of breast cancer. Crucially, a relevant fraction of loss-of-function variants in breast cancer susceptibility genes disrupts pre-mRNA splicing. We aimed to perform splicing analysis of ATM splice-site variants identified in the large-scale sequencing project BRIDGES (Breast Cancer After Diagnostic Gene Sequencing). To this end, we bioinformatically selected 47 splice-site variants across 17 exons that were genetically engineered into three minigenes and assayed in MCF-7 cells. Aberrant splicing was observed in 38 variants. Of these, 30 variants, including 7 missense, yielded no or negligible expression of the minigene full-length (mgFL) transcript. A total of 69 different transcripts were characterized, 48 of which harboured a premature termination codon. Some variants, such as c.2922-1G>A, generated complex patterns with up to 10 different transcripts. Alternative 3′ or 5′ splice-site usage was the predominant event. Integration of ATM minigene read-outs into the ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based specifications for the ATM gene enabled the classification of 30 ATM variants as pathogenic or likely pathogenic and 9 as likely benign. Overall, splicing assays provide key information for variant interpretation and the clinical management of patients. Full article

Precocious-fruiting Amomum villosum Lour. is characterized by early fruit set, rapid yield formation, and shortened economic return cycles, indicating strong cultivation potential. However, the molecular mechanisms underlying its flowering transition remain unclear. To elucidate the flowering mechanism of A. villosum, we used the Illumina NovaSeq X Plus platform to compare gene expression profiles in three tissues (Rhizomes, R; Stems, S; Leaves, L) during the vegetative stage and three tissues (Rhizomes and Inflorescences, R&I; Stems, S; Leaves, L) during the flowering stage of individual plants: VS-R vs. FS-R&I, VS-S vs. FS-S, and VS-L vs. FS-L. We obtained 52.5 Gb clean data and 789 million reads, and identified 2963 novel genes. The 3061 differentially expressed genes (DEGs, FDR ≤ 0.05 and |log2FC| ≥ 1) identified in the three comparison groups included six overlapping genes. The DEGs were enriched primarily in GO terms related to cellular process, metabolic process, binding, catalytic activity, and cellular anatomical entity, as well as multiple terms associated with development and reproduction. KEGG enrichment analysis revealed enrichment primarily in metabolic pathways, including global and overview maps, energy metabolism, and carbohydrate metabolism. Moreover, the most significantly enriched core pathways included metabolic pathways, photosynthesis, and carbon assimilation. Among all alternative splicing (AS) events, skipped exons (SEs) accounted for the largest proportion (59.5%), followed by retained introns (RI, 19.4%), alternative 3′ splice sites (A3SS, 10.7%), alternative 5′ splice sites (A5SS, 6.8%), and mutually exclusive exons (MXE, 3.6%). A preliminary set of 43 key DEGs was predicted, displaying spatiotemporal expression specificity and strong interactions among certain genes. Nine genes were further selected for RT-qPCR validation to confirm the reliability of the RNA-seq results. This study established a foundational framework for elucidating the flowering mechanism of precocious-fruiting A. villosum. Full article

MET exon 14 (METex14) skipping mutations differ from other non-small cell lung cancer (NSCLC) genomic biomarkers as they result in aberrantly spliced MET transcripts and increased MET-signaling. However, the most accurate method for their detection remains debated. We conducted a retrospective study of previously identified METex14 skipping NSCLC samples by using different, commercially available, diagnostic targeted DNA- /RNA-Next-Generation Sequencing (NGS) panels. We primarily used small DNA-NGS panels covering the 5′ splice site of METex14 and supplemented by targeted RNA sequencing for selected cases. Using this approach, we identified <0.2% patients with METex14 mutations. Due to this low frequency, we validated and introduced complementary NGS testing using combined DNA/RNA-panels. This resulted in an increased number of METex14-positive patients (3.5%) and allowed us to identify METex14 skipping transcripts. Collectively, data from our cohort (n = 34) demonstrated that optimal diagnostics of METex14 variants require a complementary DNA-NGS performed with targeted panels covering both METex14 splice sites, and RNA-NGS. Consequently, we propose a new workflow for interpretation of concordant and discordant findings in METex14 detection. Finally, the potential of DNA-identified METex14 variants to cause aberrant splicing was in silico assessed by the MaxEntScan tool, providing a quantitative approach to splicing disruption. Interestingly, we also identified two novel variants located inside METex14, which also produced the METex14 skipping transcript despite being located outside the canonical splice sites. The altered binding site resulting from these exonic mutations was in silico determined by SpliceTransformer. Full article

Background/Objectives: This study reports on findings from the first preconception screening performed in Russia and provides a comprehensive discussion of the significant results and challenges faced during the implementation of the project. Methods: Using a targeted sequencing panel of 33 genes (associated with 29 autosomal recessive and 4 X-linked diseases), we analyzed 165 couples considering pregnancy. The screening design also included analysis of the frequent pathogenic variants in the SMN1, DMD, CFTR, and CYP21A2 genes that may not be detected through the next-generation sequencing approach. The sequential screening protocol, wherein the female partner was tested first, was used. Results: The results revealed that 35.8% of women (n = 59) were carriers of at least one pathogenic or likely pathogenic (P/LP) variant, with 7.9% of women (n = 13) carrying variants in two or more genes. Notably, the analysis identified 5 deletions of exon 7 in the SMN1 gene, 1 deletion of the CYP21A2 gene, and 1 large duplication in the DMD gene in female participants. The most frequently identified pathogenic variants occurred in the CYP21A2, GJB2, SERPINA1, and ATP7B genes. The screening identified six couples (3.6% of the cohort) at high risk of having a child with an autosomal recessive or X-linked genetic disorder. Conclusions: This pilot study confirms the high clinical utility of the gene panel, effectively evaluating reproductive risk in couples without a known family history of monogenic diseases. The findings indicate that the observed frequencies of identified gene variants differ from those theoretically expected, with a notable percentage of identified couples being at relatively high risk. Furthermore, these results highlight the indispensable role of comprehensive genetic counseling both before and after testing to ensure an appropriate preconception testing algorithm and informed reproductive decision-making. Full article

Background: Pathogenic variants in interphotoreceptor matrix proteoglycan 1 (IMPG1) have been associated with autosomal dominant and recessive retinitis pigmentosa (RP) and autosomal dominant adult vitelliform macular dystrophy (AVMD). Monoallelic pathogenic variants in IMPG2 have been linked to maculopathy and biallelic variants to RP with early onset macular atrophy. Herein we characterise the phenotypic and genotypic features of patients with IMPG1/IMPG2 retinopathy and report novel variants. Methods: Patients with IMPG1 and IMPG2 variants and compatible phenotypes were retrospectively identified. Clinical data were obtained from reviewing the medical records. Phenotypic data included visual acuity, imaging included ultra-widefield pseudo-colour, fundus autofluorescence, and optical coherence tomography (OCT). Genetic testing was performed using next generation sequencing (NGS). Variant pathogenicity was investigated using in silico analysis (SIFT, PolyPhen-2, mutation taster, SpliceAI). The evolutionary conservation of novel missense variants was also investigated. Results: A total of 13 unrelated patients were identified: 2 (1 male; 1 female) with IMPG1 retinopathy and 11 (7 male; 4 female) with IMPG2 retinopathy. Both IMPG1 retinopathy patients were monoallelic: one patient had adult vitelliform macular dystrophy (AVMD) with drusenoid changes while the other had pattern dystrophy (PD), and they presented to clinic at age 81 and 72 years, respectively. There were 5 monoallelic IMPG2 retinopathy patients with a maculopathy phenotype, of whom 1 had PD and 4 had AVMD. The mean age of symptom onset of this group was 54.2 ± 11.8 years, mean age at presentation was 54.8 ± 11.5 years, and mean BCVAs were 0.15 ± 0.12 logMAR OD and −0.01 ± 0.12 logMAR OS. Six biallelic IMPG2 patients had RP with maculopathy, where the mean age of onset symptom onset was 18.4 years, mean age at examination was 68.7 years, and mean BCVAs were 1.90 logMAR OD and 1.82 logMAR OS. Variants in IMPG1 included one missense and one exon deletion. A total of 11 different IMPG2 variants were identified (4 missense, 7 truncating). A splicing defect was predicted for the c.871C>A p.(Arg291Ser) missense IMPG2 variant. One IMPG1 and five IMPG2 variants were novel. Conclusions: This study describes the phenotypic spectrum of IMPG1/IMPG2 retinopathy and six novel variants are reported. The phenotypes of PD and AVMD in monoallelic IMPG2 patients may result from haploinsufficiency, supported by the presence of truncating variants in both monoallelic and biallelic cases. The identification of novel variants expands the known genetic landscape of IMPG1 and IMPG2 retinopathies. These findings contribute to diagnostic accuracy, informed patient counselling regarding inheritance pattern, and may help guide recruitment for future therapeutic interventions. Full article

Next-generation sequencing (NGS) has revolutionized genetic research, enabling the massive, rapid, and relatively inexpensive analysis of the genomes, transcriptomes, and epigenomes of various organisms, including maize. Therefore, this paper uses NGS, association mapping, and physical mapping to identify candidate genes associated with yield structure traits and yield in maize (Zea mays L.). Furthermore, expression analysis of selected candidate genes was performed to confirm their contribution to yield formation. The plant material used for the study was 186 F1 hybrids and 20 reference genotypes (high-yielding and low-yielding). Field experiments were conducted simultaneously in two locations (in Smolice and Kobierzyce). NGS yielded a total of 45,876 molecular markers (24,437 SilicoDArT markers and 21,439 SNP markers) relevant to yield and crop structure. The largest number of markers in both localities (Smolice and Kobierzyce) was related to: the number of grain rows (6960), dry matter content after harvest (6616), the number of grains in a row (6721), mass of grain from the cob (6616), and cob length (6564). The smallest number of markers in both localities was related to yield (t ha⁻¹) (1114) and yield from the plot (1237). To narrow down the number of markers for physical mapping, ten were selected from all the significant ones associated with the same traits in both localities (Kobierzyce and Smolice). Significant markers included eight silicoDArT markers (459199, 2447305, 4768759, 4579916, 4764335, 2448946, 2492509, 4774802) and two SNP markers (9692004, 5587791). These markers were used for physical mapping. These markers are located on chromosomes 7, 8, and 10. Some of these markers are located at a considerable distance from characterized genes or within uncharacterized genes. Two markers caught our attention: SNP 5587791 and silicoDArT 4774802. The first one is located on chromosome 8 inside exon 5 of the LOC100383455 U-box domain-containing protein 7 gene, the second marker is also located on chromosome 8 near (300 bp) the LOC103635953 putative WUSCHEL-related homeobox 2 protein gene. Our own research and literature reports indicate the usefulness of next-generation sequencing, association mapping, and physical mapping for identifying candidate genes associated with economically important traits in maize. Furthermore, two genes characterized in detail in the publication, LOC100383455 U-box domain-containing protein 7 gene and LOC103635953 putative WUSCHEL-related homeobox 2 protein gene, may be involved in processes related to maize yield. Full article

This study investigated the influence of maternal nutrient restriction and dietary melatonin supplementation on DNA methylation and gene expression in bovine placental cotyledons, with a focus on sex-specific changes. On day 160 of gestation, 29 Brangus heifers (bred to a single sire by AI) were subjected to a 2 × 2 factorial design: adequately fed (ADQ-CON, n = 7), nutrient-restricted (RES-CON, n = 7), and adequately fed or nutrient-restricted supplemented with 20 mg/d of melatonin (ADQ-MEL, n = 7; RES-MEL, n = 8). Cotyledons were collected at day 240 from 12 female and 17 male conceptuses for Methyl MiniSeq-GWBS and RNA-Seq. In RES-CON vs. ADQ-CON, 93 hypomethylated and 143 hypermethylated DMRs were identified, primarily in exonic, intronic, and promoter regions. Melatonin altered the methylation patterns of male and female cotyledons, respectively, with 203 and 460 DMRs associated with axon guidance, RHOC GTPase cycle, and BDNF signaling pathways. RES-MEL showed higher expression of the UBOX5 gene compared with RES-CON. Moreover, 15 DEGs (5 upregulated and 10 downregulated) were observed in the male vs. female comparison. In melatonin-treated males, PIGX, ATP11C, snoRNA U2-19, ZNF82 genes were upregulated. Thus, melatonin may modulate conceptus growth and development in a sex-specific manner. Full article

Accurate detection of oncogenic gene fusions is becoming increasingly important given the availability of highly effective targeted therapies. However, their identification in clinical practice remains challenging due to the rarity of individual events, diversity of partner genes, and variability of breakpoint locations. Conventional approaches such as immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) lack multiplexing capacity and demonstrate variable sensitivity and specificity, while direct identification of fusion transcripts in whole-transcriptome sequencing (RNA-seq) profiles provides broader applicability but limited sensitivity, as fusion junctions are frequently supported by a minimal number of reads or even no reads at all. In this study, a novel approach was employed to accurately detect clinically actionable RET (REarranged during Transfection) fusions. This approach entailed the measurement of the imbalance in RNA-seq read coverage of potential fusion oncogenes at their 3′ and 5′ exons. A total of 1327 experimental solid tumor RNA-seq profiles were screened, including 154 non-small cell lung cancer and 221 thyroid cancer samples. The RET status was validated in 78 selected cases by targeted NGS and Sanger sequencing. An analysis of the coverage imbalance was conducted, which enabled the accurate discrimination between true and false positive RET fusions. This approach outperformed other methods and yielded 100% sensitivity and specificity with optimized thresholds. The findings were validated using an independent cohort of 79 thyroid cancer cases, confirming the reliability of the results. Among the 18 RET fusion-positive samples, one was identified as an extremely rare case (RUFY3::RET), and two were determined to be novel fusions (FN1::RET, PPP1R21::RET). The findings of this study demonstrate that exon coverage imbalance analysis serves as a robust complement to computational RNA-seq analysis pipelines for the detection of clinically relevant RET fusions. Full article

CDKL5 deficiency disorder (CDD) is a rare developmental epileptic encephalopathy (DEE) caused by mutations in cyclin-dependent kinase-like 5 (CDKL5). The clinical manifestations include early and severe epilepsy, intellectual disability, motor abnormalities, and cortical visual impairments. The pathophysiological mechanisms underlying CDD are not fully understood, and current treatments are limited to symptomatic management and do not target the underlying cause. Characterizing the downstream molecular pathways that are disrupted by CDKL5 deficiency may provide a more complete understanding of the underlying molecular mechanisms and yield therapeutic strategies. Previous studies have focused on mapping the differential expression of protein-coding genes and post-translational modifications of CDKL5 targets, but the role of non-coding RNAs (ncRNAs) in CDD is unknown. Here we performed small RNA sequencing to define the short non-coding RNA landscape in the hippocampus of mice in the Cdkl5 exon 6 deletion mouse model (12-week-old heterozygous mice). Our findings catalog extensive bi-directional alterations in the expression of multiple ncRNA species including microRNAs, tRNAs, piwi-RNAs, snoRNAs, and snRNAs. We further validated two dysregulated miRNAs, namely, miRNA-200c-3p and miRNA-384-3p, in CDD mice. The findings reveal that the loss of this single gene has an extensive impact on the non-coding transcriptional landscape in CDD. Such dysregulated ncRNAs may hold potential as biomarkers and could provide valuable insights into underlying disease mechanisms. Full article

Circular RNAs (circRNAs) are small, non-coding RNAs in which the 5′ and 3′ ends are linked covalently by back-splicing of exons from a single pre-mRNA. More and more scientific evidence is gathered for their wide distribution in the animal world, playing the role of regulators for biological processes, being cell- and tissue-specific. They can influence cellular physiology by various molecular mechanisms, finally modulating gene expression. CircRNAs are believed nowadays to be expressed in both receptive and cancerous endometrium. Due to their abundant expression in the endometrial tissue and their small size and stability, they have been considered potential diagnostic markers and treatment targets for endometrial-related diseases. The regulation of proliferation and differentiation is essential for the formation of receptive endometrium and for endometrial cancer emergence and progression. The receptive endometrium can be regarded as the most highly differentiated state of the endometrium. In contrast, the cancerous endometrium is characterized by a high level of proliferation and the lowest degree of differentiation. These endometria could be conditionally considered opposites. We are investigating the circRNA–miRNA–mRNA regulatory networks that can promote or suppress the proliferation and differentiation of endometrial cells by activating specific signaling pathways in both receptive and cancerous endometria. It could be worth knowing whether there are universal endometrial switches responsible for proliferation and differentiation processes that can alter the balance between them. We are interested in their clinical application as biomarkers and therapeutic targets for both endometrial receptivity issues and EC cases, particularly in diagnosis, progression assessment, and outcome prediction. Full article

Background: Two Shiba Inu littermates presented for investigation of marked and persistent elevation of creatine kinase activities. Method and Results: Histopathology of muscle biopsy samples revealed a dystrophic phenotype and immunostaining confirmed an absence of dystrophin protein in both cases. Whole genome sequencing of one affected dog revealed a complex deletion in the DMD gene encompassing exon 5. Screening of 27 related dogs confirmed an X-linked inheritance. The variant was identified in three related male dogs. One littermate died from cardiac arrest and the other littermate had no clinical myopathic signs at the time of the manuscript’s preparation. An additional related male dog reportedly died suddenly during grooming. Conclusion: This study adds a new breed to the canine dystrophinopathy spectrum having a ~17 kb deletion that encompasses exon 5 of DMD. This same exon 5 deletion has been identified in human dystrophin-deficient muscular dystrophy patients. Full article

Aromatic coconut is a special variety of coconut. Its unique “pandan-like” aroma has won it great popularity among consumers, endowing it with considerable market potential. In our previous study, 2-acetyl-1-pyrroline (2AP), which serves as the main source of the “pandan-like” aroma in aromatic coconut, was found to exhibit significant variation among distinct aromatic coconut individuals. Now, the regulatory mechanism of 2AP has been clarified in fragrant rice, and the ProDH gene is the key gene for 2AP regulation. To further understand the regulation mechanism of 2AP content in aromatic coconut, we cloned and identified the CnProDH gene, the key gene of 2AP regulation in aromatic coconut. The results showed that the CnProDH gene had the typical ProDH structural domain, and its full-length sequence is 23,667 bp, containing 5 exons and a coding sequence (CDS) of 1599 bp. The CnProDH gene encodes a protein that possesses a β₈α₈ barrel structure, consisting of 532 amino acids (aa), with a molecular mass of 58,076.63 kDa and an isoelectric point of 7.11. To further understand the regulatory mechanism of CnProDH in aromatic coconut, we also constructed a yeast one-hybrid (Y1H) library for aromatic coconut. Through the Y1H experiment, combined with the prediction and analysis of cis-acting elements in the promoter of the CnProDH gene, three possible regulatory factors, including CnYABBY2, CnSAP8, and CnBRD3, were identified. These findings provide a molecular basis for clarifying and solving the problem of variations in 2AP content across different aromatic coconuts. Full article

Modern maize breeding worldwide relies on a broad range of molecular genetics research techniques. These technologies allow us to identify genomic regions associated with various phenotypic traits, including resistance to fungi of the genus Fusarium. Therefore, the aim of this publication was to identify new molecular markers linked to candidate genes that confer maize resistance to Fusarium fungi, using next-generation sequencing, association mapping, and physical mapping. In the study, a total of 5714 significant molecular markers related to maize plant resistance to Fusarium fungi were identified. Of these, 10 markers were selected that were significantly associated (with the highest LOD values) with the disease. These markers were identified on chromosomes 5, 6, 7, 8, and 9. The authors were particularly interested in two markers: SNP 4583014 and SilicoDArT 4579116. The SNP marker is located on chromosome 5, in exon 8 of the gene encoding alpha-mannosidase I MNS5. The SilicoDArT marker is located 240 bp from the gene for peroxisomal carrier protein on chromosome 8. Our own research and the presented literature review indicate that both these genes may be involved in biochemical reactions triggered by the stress caused by plant infection with Fusarium fungal spores. Molecular analyses indicated their role in resistance processes, as resistant varieties responded with an increase in the expression level of these genes at various time points after plant inoculation with Fusarium fungal spores. In the negative control, which was susceptible to Fusarium, no significant fluctuations in the expression levels of either gene were observed. Analyses concerning the identification of Fusarium fungi showed that the most abundant fungi on the infected maize kernels were Fusarium poae and Fusarium culmorum. Individual samples were very sparsely colonized by Fusarium or not at all. By using various molecular technologies, we identified genomic regions associated with maize resistance to Fusarium fungi, which is of fundamental importance for understanding these regions and potentially manipulating them. Full article

Background: The use of next-generation sequencing (NGS) in liquid biopsy allows for a comprehensive molecular assessment of circulating tumor DNA (ctDNA) in patients with advanced malignancies. This approach facilitates the detection of clinically relevant mutations linked to prognosis and enables a personalized therapeutic strategy. The objective of this study was to assess the mutational landscape of ctDNA using NGS in patients with advanced non-small cell lung cancer (NSCLC), examine its relationship with clinical and molecular variables, and explore its association with overall survival (OS). Methods: We performed a retrospective observational study including 78 individuals with metastatic NSCLC treated at Arnau de Vilanova Hospital between 2019 and 2021. Plasma samples were analyzed using the AVENIO NGS platform, which targets the exons of 77 genes. Statistical analyses were conducted using SPSS version 25, applying a 95% confidence level. Results: A total of 143 genomic alterations were identified in the study population. NGS-directed therapies were initiated in eight patients (10.25%), including EGFR (n = 5), KRAS (n = 2), and BRCA1 (n = 1). The concordance rate between tissue and plasma NGS for EGFR alterations was 57.02%, with mutation frequencies of 11.4% in tissue and 6.5% in plasma. No BRAF mutations were detected by plasma analysis, despite being present in 4.3% of tissue samples. Patients receiving NGS-informed targeted therapy showed a numerically improved OS compared to those who did not, although this difference did not reach statistical significance (p = 0.34). Conclusions: Liquid biopsy based on NGS represents a reliable and minimally invasive approach for the genomic characterization of advanced NSCLC. In this real-world cohort, ctDNA profiling enabled the identification of actionable alterations in a relevant proportion of patients, supporting its integration into routine clinical practice for therapeutic decision-making. Full article

Background/Objectives: North African catfish (Clarias gariepinus) and bighead catfish (Clarias macrocephalus) play crucial roles in Thai aquaculture. Although significant growth disparities exist among these species, the genetic factors underlying these differences are still unknown. This study aimed to identify GH1 gene polymorphisms, in North African and bighead catfish populations across Thailand and Laos. Methods: Sequencing, phylogenetic, and clustering analyses were performed to assess genetic diversity, selection patterns, and lineage differentiation of catfish partial GH1 fragment. Results: Six alleles of the studied fragment of GH1 gene were identified; they differed at 33 variable sites within intron 2, located between the conserved regions at the 3′ end of exon 2 and the 5′ end of exon 3. At the population-level, GH1 exhibited low heterozygosity (mean H_o = 0.043 ± 0.023; H_e = 0.059 ± 0.028). Bayesian clustering analyses identified two distinct genetic clusters, corresponding to North African and bighead catfish, apart from the bighead population in Laos, indicating their distinct genetic origins. Evidence of purifying selection was observed in both species. Phylogenetic analysis indicated the presence of lineage-specific alleles in the GH1 gene. Conclusions: These findings provide valuable insights into GH1 polymorphisms in commercially important catfish species and may help to develop future breeding programs aimed at enhancing aquaculture productivity. Full article