Second-generation (2G) bioethanol from lignocellulosic feedstocks is a sustainable alternative to fossil fuels. However, its production is constrained by the poor performance of industrial microbes in hydrolysates that are generated during biomass pretreatment. Scheffersomyces stipitis is a native xylose fermenting yeast and a promising platform for 2G bioethanol production, and adaptive evolution under hydrolysate stress has yielded strains with enhanced performance. However, the chromosomal basis of this adaptation is unknown. Here, we demonstrate that chromosome scale structural variation, rather than point mutations, underlies the improved phenotype of the evolved strains. By integrating long- and short-read genome sequencing, we identify two major chromosomal rearrangements in the top performing isolate: a reciprocal translocation between chromosomes 1 and 2 that disrupts the NUDIX hydrolase gene YSA1, and the formation of a mitotically stable 175 kb minichromosome derived from chromosome 5. Functional analyses show that disruption of YSA1 enhances xylose utilisation and ethanol yield, while the minichromosome contributes to improved performance in hydrolysate conditions. These findings provide direct evidence that balanced rearrangements and minichromosome formation can be selected during prolonged stress and can generate adaptive phenotypes. Taken together, our study establishes genome reorganisation as a key driver of adaptation in S. stipitis.
A male-biased sex ratio (MBS) is a widespread phenomenon in nature, which contributes to inbreeding avoidance, more efficient resource allocation, and the regulation of population dynamics. The factors driving MBS primarily involve genetic and chromosomal mechanisms, environmental conditions, and the influence of endosymbiotic microorganisms. However, the underlying molecular mechanism remains largely elusive. This study aims to investigate the role of Heterochromatin protein 1 (HP1) in driving an extreme MBS in the parasitic wasp Trichogramma dendrolimi (Hymenoptera: Trichogrammatidae). An extremely male-biased T. dendrolimi line was identified from field collections, with a male offspring proportion of 80–100
The genus Passiflora L. comprises approximately 650 species, and exhibits high diversity in the Neotropics, especially the subgenus Passiflora. Several species of this subgenus have an abundant repetitive fraction, consistent with their increased genome sizes. However, the repetitive DNA fraction of Passiflora foetida (n = 10), sister to the other species of the subgenus (n = 9) and the species with the smallest genome, has not yet been described in detail. In addition, this species comprises a complex of morphological varieties with a wide geographical distribution. The aim of this study was to characterize its repeatome variability in comparison to other species in section Dysosmia and other sections in the same subgenus, and to investigate whether it may reflect the high variability reported for this species complex. The results showed that P. foetida has a proportion of repetitive DNA fraction in part consistent with its small genome size, but with marked intraspecific variation. The LTR Ty1/copia retrotransposons were the most abundant, as in the other species of the subgenus, but Ty3/gypsy represented a much smaller fraction compared to the others. The variation observed within P. foetida was greater than that detected among Dysosmia species. Satellite DNAs were partly unique and partly similar to species from different subgenera, supporting its phylogenetic position. In addition, one Dysosmia-specific satellite showed a proximal chromosomal location, making it the first candidate of a centromeric repeat for the genus, despite its variation in abundance among chromosomes.
Aneuploidy accounts for over 57% of spontaneous abortions and occurs even in women of young reproductive age. Despite its major role in pregnancy loss, the genetic basis of fetal aneuploidy remains poorly characterized. This study evaluated the association of MAD1L1 and MAD2L1, two key spindle assembly checkpoint genes, with fetal aneuploidy in women younger than 36 years. Forty aborted fetuses with approved aneuploidy using array comparative genomic hybridization were enrolled in genotyping. The rs121908981 and rs121908982 pathogenic single nucleotide variants (SNVs) of exons 4 and 18 of MAD1L1 and all exons of MAD2L1 were studied in aneuploid fetuses using Sanger sequencing. Identified variants were classified based on ACMG guideline and their frequencies were compared with ethnically matched Iranome database. The effect of SNVs was predicted using the PredictSNP Webtool and Mobidetails database. No targeted pathogenic SNVs were identified in MAD1L1; however, nine other SNVs were detected. Among these, the rare Iranome variants rs1639921 and rs1481591257 were observed in the homozygous state in four and one POCs, respectively, and showed significantly different frequencies compared with Iranome (P < 0.005). The frequency of the deleterious MAD2L1 variant rs903147 did not differ from Iranome, although 15 POCs were homozygous for this variant. In addition, a novel 60-bp deletion in intron 4 of MAD1L1 was identified for the first time. Although more frequent in POCs, its frequency did not differ significantly from that in karyotypically normal controls. Collectively, the rare variants rs1639921 and rs1481591257 may be associated with aneuploidy in POCs.
Meiotic telomeres play a critical role in chromosome dynamics, facilitating homolog pairing and synapsis through attachment to the nuclear envelope (NE). While these mechanisms are well-documented for standard A-set chromosomes, the molecular composition of supernumerary (or B) chromosome telomeres remains poorly understood. In this study, for the first time we investigated the distribution of key telomeric and NE-attachment proteins in Bs. Using immunocytochemistry and immuno-FISH on spermatocytes of the narrow-headed vole, Lasiopodomys gregalis (Rodentia, Mammalia), we identified the presence of the shelterin components RAP1, POT1 and TIN2; the LINC complex proteins SUN1 and KASH5; a major component of the nuclear lamina, Lamin B1; the regulatory kinase CDK2; and telomeric DNA specifically at the termini of meiotic supernumeraries. Our results demonstrate that despite their univalent nature and involvement in meiotic inactivation, Bs of this mammalian species possess a telomeric protein architecture that is highly similar, if even not identical, to that of A-set and sex chromosomes. These findings suggest that Bs are integrated into the meiotic machinery, utilizing standard protective and anchoring mechanisms to ensure genomic stability during prophase I.
Ring chromosomes (RCs) are rare structural variants usually formed by fusion of both chromosome arm extremities, frequently associated with terminal deletions. RCs are investigated through karyotype, fluorescence in situ hybridization, chromosomal microarray analysis, and, more recently, optical genome mapping (OGM), which enables genome-wide structural variant detection; however, its accuracy depends on alignment to a reference genome and proper filtering of SVs. Here, two patients with previously characterized ring chromosomes 3 and 18, each with a terminal deletion in only one chromosome arm (3p and 18q), were analyzed by OGM after aligning the data against two reference genomes (GRCh38/hg38 and T2T-CHM13) and compared with long-read sequencing (LRS) results stemming from a parallel study that included both patients. Patient 1's RC3 was misinterpreted as a translocation between chromosomes 3 and 19 when analyzed with GRCh38/hg38. Reanalysis with T2T-CHM13 correctly identified the RC3 and demonstrated retention of the full 3q arm, including telomeric-associated regions, which was confirmed by LRS. Patient 2’s ring chromosome 18 was identified with GRCh38/hg38 only after lowering the confidence score filter, whereas T2T-CHM13 detected the RC18 immediately without involvement of 18p telomeric-associated regions. LRS diverged from this analysis by showing the presence of these regions in the RC18 due to its nucleotide-level resolution. Only eight out of 3,000 RCs have been reported in the literature as resolved using OGM, and here we add two more cases analyzed with different reference genomes and highlight the techniques’ advantages and limitations for RC characterization. Since the T2T-CHM13 analysis outperformed GRCh38/hg38 because it resolves previously inaccessible telomeric regions, we included an investigation into which other chromosomes could also benefit more from this approach.
The CST complex protects the ss-overhang of telomeres. In animals, it is composed of CTC1, STN1 and TEN1. In Schizosaccharomyces pombe, CTC1 is absent, while in Saccharomyces cerevisiae it is substituted by Cdc13. Pot1 is the only Shelterin protein that binds ssDNA, and is absent in Saccharomyces cerevisiae. In this study, I looked for homologues of CTC1, Cdc13 and Pot1 in Fungi. I found that CTC1 has been lost in Dikarya (yeasts and mushrooms), with its function probably being taken by Pot1. Furthermore, my analyses show that Cdc13 probably evolved from Pot1, by the loss of its third domain, which is responsible for binding Tpz1. In this way, Est3 (the orthologue of Tpz1 in Saccharomyces cerevisiae) became part of the telomerase complex. Cdc13 further evolved by gene duplication in some budding yeasts, giving rise to two paralogues in Serinales, Ascoideales, Phaffomycetales, Saccharomycetales and Saccharomycodales: in these latter two orders, the two paralogues fused to give rise to a “long” Cdc13. Beyond their evolutionary interest, these results could be useful for researchers using new species of yeasts as model organisms.
We present two illustrative cases highlighting diagnostic, surveillance and management complexities of TP53 pathogenic variants (PVs). Case 1 describes a 24-year-old female with early-onset breast cancer and a somatic mosaic TP53 PV with a variant allele frequency of 19% in blood, initially missed by panel sequencing. Case 2 concerns a 59-year-old female with multiple primary tumours and two identical TP53 variants detected in two different tissues which initially suggested somatic mosaicism but were consistent with a myelodysplastic syndrome-related clone secondary to homozygous germline ERCC6L2-associated bone marrow failure. These cases highlight the importance of accurately interpreting TP53 variants for correct clinical decision-making. Contextual factors such as age, phenotype, family history and tissue testing must guide diagnosis, treatment and surveillance.
Epithelial cell adhesion molecule (EPCAM)-associated Lynch syndrome arises from deletions at the 3'-end of EPCAM that disrupt transcriptional termination, generate read-through transcripts and cause epigenetic silencing of MSH2 in EPCAM-expressing tissues. However, the clinical significance of deletions confined to the EPCAM-MSH2 intergenic region remains uncertain without in-depth investigation.We investigated a family with a strong history of Lynch syndrome-related cancers in whom diagnostic testing by short-read sequencing identified a heterozygous deletion spanning the EPCAM-MSH2 intergenic region that was initially classified as a variant of uncertain significance. The variant was further characterised using long-read Oxford Nanopore sequencing with adaptive sampling and methylation profiling.Long-read sequencing defined precise breakpoints, and tumour analysis demonstrated MSH2 promoter hypermethylation with complete loss of MSH2 protein expression in the absence of germline promoter methylation. The molecular phenotype closely mirrored the recognised mechanism for 3'-end EPCAM deletions, whereby aberrant EPCAM transcription interferes with MSH2 promoter regulation in a tissue-specific manner.These findings support reclassification of this variant to likely pathogenic and establish a diagnosis of EPCAM-associated Lynch syndrome. This report provides the first evidence that intergenic EPCAM-MSH2 deletions are associated with MSH2 epimutations and highlights the diagnostic utility of long-read sequencing for noncoding structural variants.
Precise breast cancer risk assessment (BCR) is essential for personalised prevention in women with a family history of hereditary breast and ovarian cancer (HBOC). The CanRisk model integrates monogenic variants with reproductive, lifestyle and familial factors and can be extended by Polygenic Risk Scores (PRS). We evaluated the impact of PRS-BC313 in three groups: (1) healthy carriers of (likely) pathogenic variants (LP/P), (2) affected LP/P carriers and (3) healthy female relatives from variant-negative HBOC families.In healthy LP/P carriers, median 10-year breast cancer (BC) risk remained stable, while individual estimates ranged from+25% to -16% compared with calculations without PRS. Among affected LP/P carriers, contralateral BC risk shifted by+24% to -11%, indicating PRS effects even in high-risk individuals.In healthy relatives, applying the recently introduced German threshold of an 8% BC risk between ages 40 and 50 years resulted in escalation to intensified surveillance in ~6% (5/86) (2.4-14%, Wilson binomial CI with Yates' correction) and de-escalation in ~1% (1/86) (0.061-7.2%).Here, we provide new evidence of PRS clinical impact under the updated German threshold.
BACKGROUND:Familial acute myeloid leukaemia (AML) with germline CEBPA (CCAAT/enhancer-binding protein alpha) variants is a distinct hereditary entity, yet clinically meaningful genotype-phenotype correlations remain incompletely defined. METHODS:We integrated cases from our institution with all published pedigrees of germline CEBPA-mutated AML and analysed 56 affected patients from 40 families, including one newly identified pedigree harbouring a novel TAD2 (transactivation domain 2) variant (p.C133Ter). RESULTS:Variant localisation significantly influenced disease phenotype and outcome. Compared with non-TAD2 variants, TAD2 variants were associated with older age at AML onset (40 years vs 21 years, p<0.001), a higher burden of cooperating somatic variants (64.3% vs 27.3%, p=0.023) and lower complete remission rates after induction therapy (69.2% vs 95.3%, p=0.022). TAD2 involvement conferred inferior overall survival (p<0.001) and progression-free survival (p=0.025). In univariate analyses, TAD2 variants emerged as adverse prognostic factors for 2-year overall survival (OR=0.06, 95% CI 0.01 to 0.34, p=0.002) and progression-free survival (OR=0.07, 95% CI 0.01 to 0.35, p=0.003). CONCLUSIONS:Although germline CEBPA-mutated AML is generally regarded as favourable-risk, TAD2-involving variants may define a biologically and clinically high-risk subset, supporting variant topography as a clinically actionable prognostic marker.
Background Steroid-resistant nephrotic syndrome (SRNS) is a severe paediatric kidney disease and a leading cause of end-stage kidney disease in children, with a high genetic contribution. While over 80 monogenic causes of SRNS have been identified, a significant proportion of affected patients still lack a clear genetic diagnosis, indicating that additional causative genes remain to be discovered.Methods Through whole-exome sequencing of a paediatric SRNS cohort, we identified three probands carrying biallelic FLNB pathogenic variants. Sanger sequencing was performed for familial cosegregation verification and ACMG classification. Expression of Filamin B, Nephrin and Synaptopodin in renal tissues was assessed by immunohistochemistry/immunofluorescence. Wild-type and patient-derived variant FLNB plasmids were constructed and transfected into HEK293T cells and immortalised human podocytes (HPCs). The effects of these variants on protein expression, localisation and cytoskeletal organisation were assessed by western blotting and immunofluorescence. FLNB expression in HPCs was silenced using shRNA to evaluate the impact on podocyte marker proteins, cytoskeletal integrity and migratory capacity. A zebrafish flnb knockdown model was employed to validate its effects on renal development.Results All three probands presented with isolated SRNS without skeletal developmental abnormalities, and renal tissues showed significantly reduced Filamin B protein expression. In vitro, p.L117P and p.M1803L variants led to markedly reduced protein expression, while p.R470L and p.K2586R induced perinuclear aggregation of Filamin B accompanied by F-actin rearrangement. FLNB silencing led to downregulation of Nephrin and Synaptopodin, cytoskeletal disorganisation and impaired cell migration. Zebrafish flnb knockdown exhibited pericardial oedema, defective nephron development and abnormal podocyte foot processes.Conclusion We report for the first time that biallelic FLNB pathogenic variants are associated with paediatric SRNS by disrupting Filamin B expression, cytoskeletal integrity and podocyte function, providing evidence that FLNB is a novel monogenic cause of SRNS.
Background Since 2020, the UK National Institute for Health and Care Excellence (NICE) recommends screening for Lynch syndrome in all people newly diagnosed with endometrial cancer. Screening involves tumour testing for loss of the mismatch repair (MMR) proteins using immunohistochemistry (IHC), MLH1 methylation testing and germline sequencing for Lynch syndrome according to a diagnostic algorithm. Here we review adherence to NICE guidance at a gynaecological cancer centre in North-West England.Methods We conducted a prospective audit of Lynch syndrome screening for consecutive patients newly diagnosed with endometrial cancer discussed at the gynaecological oncology multidisciplinary (MDT) meeting. We recorded adherence with the NICE recommended diagnostic algorithm, testing turnaround times and the impact of gynaecology-led genetic testing (mainstreaming) on diagnostic intervals.Results Between November 2021 and November 2023, 421 new endometrial cancer patients were discussed at MDT. Overall, 96.9% (408/421) underwent IHC and 26.0% (106/408) were MMR deficient, mostly due to MLH1 hypermethylation (17.4%, 71/408). In total, 7.1% (29/408) had MMR deficiency not due to MLH1 hypermethylation (25/408 had MSH2/MSH6/PMS2 loss and 4/408 were non-hypermethylated) that required germline testing for Lynch syndrome, and 3.2% (13/408) had Lynch syndrome. Only 19/27 (70.4%) with positive tumour triage underwent Lynch syndrome testing (four declined and four died before testing). The median time from tumour MMR IHC results to germline test result was 121 days (IQR 103, 133) with gynaecology-led testing compared with 278 days (IQR 183, 476) with testing organised by clinical genetics.Conclusion The prevalence of Lynch syndrome in our unselected endometrial cancer population was 3.2% but not everyone at risk was tested. Some declined germline Lynch syndrome testing due to a lack of at-risk family members and delay getting their genetic appointment. A significant minority died before they could be offered or receive Lynch syndrome testing. We recommend gynaecology-led germline testing and referral to clinical genetics only patients with confirmed Lynch syndrome. This approach would ease the burden of an already overstretched genetics service while promptly identifying Lynch syndrome in high-risk patients. This in turn enables timely colorectal cancer surveillance and cascade testing of at-risk family members.
Expansion microscopy (ExM) enlarges biological samples by embedding them in a swellable hydrogel, enabling nanoscale imaging of subcellular structures with standard light microscopes. This offers an accessible alternative to super-resolution methods. ExM has yet to be combined with the Oligopaint DNA fluorescence in situ hybridization (FISH) technology. We present an optimized ExM workflow for simultaneous Oligopaint DNA FISH and immunofluorescence (IF) in intact Drosophila ovaries. The protocol incorporates nucleic-acid anchoring, reliable protein retention, and digestion conditions that preserve chromatin while maintaining probe accessibility. Our approach achieves 5 × expansion and strong signal retention, enabling high-resolution studies of nuclear organization.
This methodological study outlines effective and standardized procedures for obtaining high-quality chromosomal preparations in birds, which display complex karyotypes with a large number of microchromosomes. We describe every stage from sample obtaining and culture setup to the successful acquisition of metaphase chromosomes appropriate for in-depth analysis by combining three main methodologies (fibroblast, lymphocyte, and bone marrow cultures) into a coherent and methodical workflow. Furthermore, we offer thorough troubleshooting instructions to tackle typical problems, such as low mitotic indices, poor chromosome spreading, contamination, and suboptimal chromosomal morphology. We also focused on repeatability and adaptation across several bird taxa, taking into consideration species-specific differences in their body sizes, cell development, and chromosomal behavior, extending beyond standard procedure improvement. The incorporation of diverse methodologies into a unified framework offers avian cytogenetics researchers a readily available and practical workflow. Ultimately, our approach enhances the integration of cytogenetic data with novel genomic resources, facilitates the re-evaluation of chromosomal counts, and enables more precise karyotype characterization. This integration is crucial for advancing our understanding of chromosomal evolution, avian genome architecture, and the broader mechanisms underlying avian speciation and diversification.
Mosaic neurofibromatosis type 1 (NF1) poses a significant diagnostic challenge due to low-level mosaicism and the confinement of pathogenic variants to neuroectodermal lineages, frequently resulting in false-negative findings when testing is limited to peripheral blood. At present, molecular confirmation of NF1 often relies on the detection of pathogenic variants in tumour tissue. However, many young individuals present exclusively with benign pigmentary manifestations, such as café-au-lait macules and freckling, and therefore lack accessible tumour material. Molecular analysis of pigmentary lesions is technically challenging because skin biopsies contain only a small proportion of melanocytes, which particularly hampers the detection of low-level copy number and structural variants using conventional diagnostic approaches. We report a young woman referred for reproductive counselling who presented with unilateral pigmentary lesions suggestive of mosaic NF1, in whom a combined approach using melanocyte culture and optical genome mapping identified a reciprocal balanced translocation disrupting the NF1 locus, t(15;17)(q26.1;q11.2), representing the molecular predisposing event. To our knowledge, this is the first report of a reciprocal translocation in mosaic NF1, highlighting the role of structural genomic rearrangements in this disorder and underscoring the diagnostic utility of optical genome mapping in suspected cases. Molecular confirmation of mosaic NF1 is critical for accurate diagnosis, clinical management, and genetic counselling.
Genetic testing for cancer susceptibility underpins precision cancer prevention and care. Gaps in the healthcare providers’ genetic literacy and an ambiguous lexicon for variant description may hinder proper delivery and clinical application of consistently trustworthy test results. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) international consortium supports controlled terminology and recommends a framework for reporting germline variants in cancer susceptibility genes, using breast cancer as an exemplar. Moving forward towards terminological coherence across disciplines and borders, the ENIGMA Clinical Working Group launched a multinational effort to release consortium-approved translations of the published recommendations. The herein reported Vocabulary Translation Project offered an opportunity to reappraise and align the reference text to the recent BRCA1 and BRCA2 specifications to the American College of Medical Genetics and Genomics/Association for Molecular Pathology rules by the ENIGMA Variant Curation Expert Panel and to highlight country-specific differences in breast cancer risk assessment and management. The updated recommendations and their 20 translations are now provided as easy to handle documents, covering 11 of the most widely spoken languages in the world. They will contribute to minimised erroneous inferences, more informed decision-making, improved health outcomes and equity in the use of genetic testing for cancer predisposition and in translational oncology.
Developmental epileptic encephalopathy (DEE) comprises neurodevelopmental disorders with early-onset seizures and developmental impairment. Despite >900 implicated genes, many patients remain undiagnosed after short-read sequencing (SRS). We assessed long-read genome sequencing (LR-GS) in 38 previously unsolved infantile-onset DEE probands (10 singletons, 28 trios). Variant detection included single nucleotide variants (SNVs), structural variants, copy number variants and short tandem repeats in established repeat expansion disease genes. LR-GS identified candidate variants in 8 out of 38 probands (21%) missed by SRS: five large deletions, one SNV in a low-mappability region of NSF, one case resolved via haplotype phasing of compound heterozygous SNVs without parental samples and one case where LR-GS detected an allele missed due to coverage gaps. An additional eight probands (21%) harboured variants technically detectable by SRS but were missed due to newly associated genes, synonymous variants lacking splicing evaluation or prior analytic pipelines. LR-GS substantially increases diagnostic yield in unsolved infantile-onset DEE, supporting its incorporation into clinical workflows as a second-tier genetic test for otherwise unsolved neurodevelopmental disorders.
The Neotropical family Serrasalmidae (pacus and piranhas) exhibits remarkable karyotype conservation, yet cytogenetic data remain limited for several genera, particularly Myloplus. Here we present the first integrative cytogenomic characterization of Myloplus tiete, an endemic and near-threatened species from the upper Paraná River basin, combining classical cytogenetics with satellitome analysis. Karyotyping of 12 specimens collected from Rio Grande, Frutal—MG, Brazil, revealed a consistent diploid number of 2n = 58 chromosomes with karyotype formula 16m + 20sm + 22a, while C-banding identified heterochromatic blocks predominantly in pericentromeric and subtelomeric regions. Genome sequencing and iterative satDNA mining identified 32 satellite DNA families, with repeat lengths ranging from 24 to 2,265 bp and a predominance of AT-rich sequences. Comparative analysis with the Colossomatinae species Colossoma macropomum and Piaractus mesopotamicus revealed 12 conserved satDNAs across 40 million years of divergence, with moderate consensus turnover rates. FISH mapping of the ten most abundant satDNAs revealed diverse chromosomal distributions, including pericentromeric, subtelomeric, and dispersed patterns, highlighting the diverse genomic integration of conserved repeats. These findings provide evidence for long-term evolutionary conservation of both karyotype architecture and satDNA repertoires across Serrasalmidae subfamilies and suggest that structural or functional constraints may contribute to shaping the evolution of repetitive DNA in this ecologically and aquaculturally important fish group.
Non-coding DNA, long considered “junk”, is now recognized as a central regulator of genome architecture. Highly repetitive satellite DNA sequences shape heterochromatin and are essential for chromosome stability, segregation, and gene regulation. Pericentromeric heterochromatic variants, or chromosomal heteromorphisms (CHs), have emerged as modulators of human fertility, potentially affecting gametogenesis and early embryonic development. Despite their ubiquity, the functional and clinical significance of CHs remains largely enigmatic. Molecular reference genomes fail to fully capture these repetitive domains, and cytogenetic assessment has shown critical inconsistencies due to the lack of standardized evaluation criteria. To address this gap, we proposed a comparison-based scoring system to reliably identify and characterize CHs. By applying this framework to 300 individuals with idiopathic reproductive disorders and 155 fertile controls, we observed a significantly higher CH frequency in the infertile cohort (2.4-fold increase; p < 0.001). Chromosome 9 variants were the most prevalent (5.3
