Abies guatemalensis Rehder, an endangered conifer endemic to Central American highlands, is ecologically vital in upper montane forests. It faces threats from habitat fragmentation, unsustainable logging, and illegal Christmas tree harvesting. While previous genetic studies on mature trees from eighteen populations showed high within-population diversity and limited among-population differentiation, the genetic impact of recent anthropogenic pressures on younger generations has yet to be discovered. Understanding these effects is crucial for developing effective conservation strategies for this vulnerable species. We sampled 170 young trees (< 15 years old) from seven populations across Guatemala. Seven microsatellite markers were used to analyse genetic diversity, population structure, and recent demographic history. Moderate levels of genetic diversity were observed within populations (mean Shannon diversity index = 4.97, mean Simpson's index = 0.51, mean allelic richness = 11.59, mean observed heterozygosity = 0.59). Although genetic structure broadly aligned with mountain corridors, substantial admixture patterns suggest historical connectivity across all populations. Most populations showed evidence of recent bottlenecks (p < 0.05) and inbreeding. The results suggest a potential decline in genetic diversity and increased population structuring (ΦST = 0.274, p < 0.01) over the past decades compared to the previous study on old trees. The observed genetic patterns indicate ongoing impacts of habitat fragmentation and anthropogenic pressures on A. guatemalensis. Conservation efforts should prioritise expanding effective population sizes and facilitating gene flow, particularly for isolated populations. While restoration efforts may be logistically easier within mountain ranges, genetic evidence suggests that increasing overall population connectivity could benefit this species. Management strategies should implement systematic seed collection protocols to maintain genetic diversity in future populations. These findings highlight the urgent need for conservation measures to preserve remaining genetic diversity and promote connectivity among A. guatemalensis populations.
- MeSH
- ekosystém * MeSH
- genetická variace * MeSH
- jedle * genetika MeSH
- mikrosatelitní repetice * genetika MeSH
- ohrožené druhy * MeSH
- populační genetika MeSH
- zachování přírodních zdrojů MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Guatemala MeSH
BACKGROUND: Fuchs endothelial corneal dystrophy (FECD) is the most common repeat-mediated disease in humans. It exclusively affects corneal endothelial cells (CECs), with ≤81% of cases associated with an intronic TCF4 triplet repeat (CTG18.1). Here, we utilise optical genome mapping (OGM) to investigate CTG18.1 tissue-specific instability to gain mechanistic insights. METHODS: We applied OGM to a diverse range of genomic DNAs (gDNAs) from patients with FECD and controls (n = 43); CECs, leukocytes and fibroblasts. A bioinformatics pipeline was developed to robustly interrogate CTG18.1-spanning DNA molecules. All results were compared with conventional polymerase chain reaction-based fragment analysis. FINDINGS: Analysis of bio-samples revealed that expanded CTG18.1 alleles behave dynamically, regardless of cell-type origin. However, clusters of CTG18.1 molecules, encompassing ∼1800-11,900 repeats, were exclusively detected in diseased CECs from expansion-positive cases. Additionally, both progenitor allele size and age were found to influence the level of leukocyte-specific CTG18.1 instability. INTERPRETATION: OGM is a powerful tool for analysing somatic instability of repeat loci and reveals here the extreme levels of CTG18.1 instability occurring within diseased CECs underpinning FECD pathophysiology, opening up new therapeutic avenues for FECD. Furthermore, these findings highlight the broader translational utility of FECD as a model for developing therapeutic strategies for rarer diseases similarly attributed to somatically unstable repeats. FUNDING: UK Research and Innovation, Moorfields Eye Charity, Fight for Sight, Medical Research Council, NIHR BRC at Moorfields Eye Hospital and UCL Institute of Ophthalmology, Grantová Agentura České Republiky, Univerzita Karlova v Praze, the National Brain Appeal's Innovation Fund and Rosetrees Trust.
- MeSH
- alely MeSH
- expanze trinukleotidových repetic MeSH
- Fuchsova endoteliální dystrofie * genetika patologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mapování chromozomů MeSH
- nestabilita genomu MeSH
- orgánová specificita genetika MeSH
- senioři MeSH
- transkripční faktor 4 * genetika metabolismus MeSH
- trinukleotidové repetice genetika MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Echinococcus granulosus (Batsch, 1786), a cestode of the Teniidae family, causes human cystic echinococcosis (CE) also known as hydatid disease. Echinococcus granulosus sensu lato includes the G1, G3, G4, G5, G6/7 and G8/10 genotypes which are known to cause human CE. This study aimed to differentiate genotypes of E. granulosus s.l. complex by employing EmsB, a tandemly repeated multilocus microsatellite, using next-generation sequencing (MIC-NGS). Human and animal histopathology-confirmed hydatid cyst tissue samples and reference DNA samples of E. granulosus G1, G3, G4, G5, G6/7 and G10 underwent MIC-NGS assay with custom primers amplifying a 151 bp EmsB DNA fragment. NGS data were analysed using online Galaxy analysis pipeline, a phylogenetic tree was constructed by MEGA software, and haplotype networking was performed with PopArt 1.7. All sixty samples (49 from animals and 11 from humans) included were successfully identified and genotyped with a 100 % success rate. The study showed improved discrimination power to distinguish all study samples including closely related E. granulosus s.s. genotypes G1-G3. The maximum likelihood tree reaffirmed the monophyly of E. granulosus s.l. The median-joining haplotype networking revealed 12 distinct haplotypes. In conclusion, MIC-NGS assay was shown to be sensitive, specific and simple to apply to clinical samples offering a powerful discriminatory tool for the genotyping of E. granulosus s.l.
- MeSH
- Echinococcus granulosus * genetika MeSH
- echinokokóza * veterinární parazitologie MeSH
- fylogeneze MeSH
- genotyp * MeSH
- genotypizační techniky veterinární MeSH
- lidé MeSH
- mikrosatelitní repetice * MeSH
- vysoce účinné nukleotidové sekvenování * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Fuchs endothelial corneal dystrophy (FECD) is an age-related cause of vision loss, and the most common repeat expansion-mediated disease in humans characterised to date. Up to 80% of European FECD cases have been attributed to expansion of a non-coding CTG repeat element (termed CTG18.1) located within the ubiquitously expressed transcription factor encoding gene, TCF4. The non-coding nature of the repeat and the transcriptomic complexity of TCF4 have made it extremely challenging to experimentally decipher the molecular mechanisms underlying this disease. Here we comprehensively describe CTG18.1 expansion-driven molecular components of disease within primary patient-derived corneal endothelial cells (CECs), generated from a large cohort of individuals with CTG18.1-expanded (Exp+) and CTG 18.1-independent (Exp-) FECD. We employ long-read, short-read, and spatial transcriptomic techniques to interrogate expansion-specific transcriptomic biomarkers. Interrogation of long-read sequencing and alternative splicing analysis of short-read transcriptomic data together reveals the global extent of altered splicing occurring within Exp+ FECD, and unique transcripts associated with CTG18.1-expansions. Similarly, differential gene expression analysis highlights the total transcriptomic consequences of Exp+ FECD within CECs. Furthermore, differential exon usage, pathway enrichment and spatial transcriptomics reveal TCF4 isoform ratio skewing solely in Exp+ FECD with potential downstream functional consequences. Lastly, exome data from 134 Exp- FECD cases identified rare (minor allele frequency <0.005) and potentially deleterious (CADD>15) TCF4 variants in 7/134 FECD Exp- cases, suggesting that TCF4 variants independent of CTG18.1 may increase FECD risk. In summary, our study supports the hypothesis that at least two distinct pathogenic mechanisms, RNA toxicity and TCF4 isoform-specific dysregulation, both underpin the pathophysiology of FECD. We anticipate these data will inform and guide the development of translational interventions for this common triplet-repeat mediated disease.
- MeSH
- alternativní sestřih genetika MeSH
- endoteliální buňky metabolismus MeSH
- expanze trinukleotidových repetic * genetika MeSH
- Fuchsova endoteliální dystrofie * genetika MeSH
- lidé MeSH
- rohovkový endotel metabolismus patologie MeSH
- transkripční faktor 4 * genetika metabolismus MeSH
- transkriptom genetika MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The aim of this study was to describe the ocular phenotype in a case with Kearns-Sayre syndrome (KSS) spectrum and to determine if corneal endothelial cell dysfunction could be attributed to other known distinct genetic causes. Herein, genomic DNA was extracted from blood and exome sequencing was performed. Non-coding gene regions implicated in corneal endothelial dystrophies were screened by Sanger sequencing. In addition, a repeat expansion situated within an intron of TCF4 (termed CTG18.1) was genotyped using the short tandem repeat assay. The diagnosis of KSS spectrum was based on the presence of ptosis, chronic progressive external ophthalmoplegia, pigmentary retinopathy, hearing loss, and muscle weakness, which were further supported by the detection of ~6.5 kb mtDNA deletion. At the age of 33 years, the proband's best corrected visual acuity was reduced to 0.04 in the right eye and 0.2 in the left eye. Rare ocular findings included marked corneal oedema with central corneal thickness of 824 and 844 μm in the right and left eye, respectively. No pathogenic variants in the genes, which are associated with corneal endothelial dystrophies, were identified. Furthermore, the CTG18.1 genotype was 12/33, which exceeds a previously determined critical threshold for toxic RNA foci appearance in corneal endothelial cells.
- MeSH
- dospělí MeSH
- fenotyp MeSH
- Fuchsova endoteliální dystrofie patologie MeSH
- genotyp MeSH
- katarakta genetika MeSH
- Kearnsův-Sayreův syndrom genetika MeSH
- lidé MeSH
- mitochondriální DNA MeSH
- rohovkový endotel patologie patofyziologie MeSH
- sekvenční delece MeSH
- sekvenování exomu MeSH
- transkripční faktor 4 genetika MeSH
- trinukleotidové repetice * MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
Non-canonical forms of nucleic acids represent challenging objects for both structure-determination and investigation of their potential role in living systems. In this work, we uncover a structure adopted by GA repetition locked in a parallel homoduplex by an i-motif. A series of DNA oligonucleotides comprising GAGA segment and C3 clip is analyzed by NMR and CD spectroscopies to understand the sequence-structure-stability relationships. We demonstrate how the relative position of the homopurine GAGA segment and the C3 clip as well as single-base mutations (guanine deamination and cytosine methylation) affect base pairing arrangement of purines, i-motif topology and overall stability. We focus on oligonucleotides C3GAGA and methylated GAGAC3 exhibiting the highest stability and structural uniformity which allowed determination of high-resolution structures further analyzed by unbiased molecular dynamics simulation. We describe sequence-specific supramolecular interactions on the junction between homoduplex and i-motif blocks that contribute to the overall stability of the structures. The results show that the distinct structural motifs can not only coexist in the tight neighborhood within the same molecule but even mutually support their formation. Our findings are expected to have general validity and could serve as guides in future structure and stability investigations of nucleic acids.
With the expansion of molecular techniques, the historical collections have become widely used. The last boom started with using next- and second-generation sequencing in which massive parallel sequencing replaced targeted sequencing and third-generation technology involves single molecule technology. Studying plant DNA using these modern molecular techniques plays an important role in understanding evolutionary relationships, identification through DNA barcoding, conservation status, and many other aspects of plant biology. Enormous herbarium collections are an important source of material especially for taxonomic long-standing issues, specimens from areas difficult to access or from taxa that are now extinct. The ability to utilize these specimens greatly enhances the research. However, the process of extracting DNA from herbarium specimens is often fraught with difficulty related to such variables as plant chemistry, drying method of the specimen, and chemical treatment of the specimen. The result of these applications is often fragmented DNA. The reason new sequencing approaches have been so successful is that the template DNA needs to be fragmented for proper library building, and herbarium DNA is exactly that. Although many methods have been developed for extraction of DNA from herbarium specimens, the most frequently used are modified CTAB and DNeasy Plant Mini Kit protocols. Nine selected protocols in this chapter have been successfully used for high-quality DNA extraction from different kinds of plant herbarium tissues. These methods differ primarily with respect to their requirements for input material (from algae to vascular plants), type of the plant tissue (leaves with incrustations, sclerenchyma strands, mucilaginous tissues, needles, seeds), and further possible applications (PCR-based methods, microsatellites, AFLP or next-generation sequencing).
- MeSH
- analýza polymorfismu délky amplifikovaných restrikčních fragmentů MeSH
- chemická frakcionace metody MeSH
- DNA rostlinná genetika izolace a purifikace MeSH
- listy rostlin genetika MeSH
- mikrosatelitní repetice MeSH
- orgánová specificita MeSH
- polymerázová řetězová reakce MeSH
- reagenční diagnostické soupravy MeSH
- rostliny klasifikace genetika MeSH
- sekvenční analýza DNA MeSH
- taxonomické DNA čárové kódování metody MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Opium poppy (Papaver somniferum L.) is a versatile plant exploited by the pharmaceutical and food industries. Unfortunately, it is also infamously known as a source of highly addictive narcotics, primarily heroin. Drug abuse has devastating consequences for users and also has many direct or indirect negative impacts on human society as a whole. Therefore, developing a molecular genetic tool for the individualization of opium poppy, raw opium or heroin samples could help in the fight against the drug trade by retrieving more information about the source of narcotics and linking isolated criminal cases. Bioinformatic analysis provided insight into the distribution, density and other characteristics of roughly 150 thousand microsatellite loci within the poppy genome and indicated underrepresentation of microsatellites with the desired attributes. Despite this fact, 27 polymorphic STR markers, divided into three multiplexed assays, were developed in this work. Internal validation confirmed species-specific amplification, showed that the optimal amount of DNA is within the range of 0.625-1.25 ng per reaction, and indicate relatively well balanced assays according to the metrics used. Moreover, the stutter ratio (mean + 3 SD 2.28-15.59%) and allele-specific stutters were described. The analysis of 187 individual samples led to the identification of 158 alleles in total, with a mean of 5.85 alleles and a range of 3-14 alleles per locus. Most of the alleles (151) were sequenced by the Sanger method, which enabled us to propose standardized nomenclature and create three allelic ladders. The OpiumPlex system discriminates most of the varieties from each other and pharmaceutical varieties from the others (culinary, dual and ornamental).
Cieľ: Cieľom práce bola genetická charakterizácia súboru prípadov suspektných z parciálnej moly hydatidózy na základe nejednoznačného morfologického obrazu placentárneho tkaniva. Súbor a metodika: Práca predkladá výsledky genetickej analýzy súboru 10 pacientok s rôznymi klinickými prejavmi reprodukčných strát, u ktorých bola na základe histopatologického vyšetrenia suponovaná parciálna mola hydatidóza. Kompozícia genómu produktu koncepcie bola určená genotypizáciou krátkych tandemových opakovaní (STR – short tandem repeats) použitím komerčnej súpravy „Devyser Compact v3“ (Devyser). Výsledky a závery: Z 10 vyšetrených prípadov bol v piatich zistený diandrický monogynický triploidný genóm charakteristický pre parciálnu molu. V štyroch prípadoch boli vylúčené aneuploidie chromozómov 13, 18, 21, X a Y, v jednom prípade bol diagnostikovaný Patauov syndróm. Pri nejednoznačnom histopatologickom obraze môže patológom v diferenciálnej diagnostike parciálnej moly významne pomôcť konzultačná DNA analýza (ideálne STR genotypizácia). Histopatologický obraz parciálnej moly hydatidózy môže byť v skorých štádiách gravidity v niektorých prípadoch neúplný a nejednoznačný, čo môže viesť k falošne negatívnemu výsledku vyšetrenia. Na druhej strane, mnohé iné patológie, napr. aneuploidie alebo digynická triploidia, môžu mať histopatologický obraz podobný parciálnej mole, čo naopak vedie k falošnej pozitivite vyšetrenia. Presná diagnostika parciálnej moly hydatidózy použitím molekulárnych genetických metód prispeje k stanoveniu adekvátnej dispenzárnej starostlivosti.
Objective: The aim of the study was the genetic characterization of a set of cases with an unclear morphological profile of the placental tissue suspected of a partial hydatidiform mole. Patients and methods: This work presents the results of a genetic analysis of a group of 10 patients with various clinical manifestations of reproductive loss, where a partial hydatidiform mole was suspected on the basis of a histopathological examination. The composition of the genome of the products of conception was determined by short tandem repeats (STR) genotyping using a commercial kit “Devyser Compact v3 (Devyser) “. Results and conclusions: Out of 10 analyzed cases, five had diandric monogynic triploid genome, characteristic for a partial mole. Aneuploidies of chromosomes 13, 18, 21, X and Y were excluded in four cases and Patau‘s syndrome was diagnosed in one case. In the case of an unclear histopathological profile, consultative DNA analysis (ideally STR genotyping) can significantly help the pathologist in the differential diagnosis of a partial mole. The histopathological profile of a partial hydatidiform mole may be in some cases incomplete and unclear, especially in the early weeks of gestation, which can lead to false negativity of the examination. On the other hand, other pathologies, for example aneuploides or digynic triploidy, may produce a histopathological profile similar to a partial mole, which leads to false positivity. Accurate diagnosis of a partial hydatidiform mole using molecular genetic methods contributes to the determination of adequate dispensary care for patients.
Hybridogenesis is a reproductive tool for sexual parasitism. Hybridogenetic hybrids use gametes from their sexual host for their own reproduction, but sexual species gain no benefit from such matings as their genome is later eliminated. Here, we examine the presence of sexual parasitism in water frogs through crossing experiments and genome-wide data. We specifically focus on the famous Central-European populations where Pelophylax esculentus males (hybrids of P. ridibundus and P. lessonae) live with P. ridibundus. We identified a system where the hybrids commonly produce two types of clonal gametes (hybrid amphispermy). The haploid lessonae genome is clonally inherited from generation to generation and assures the maintenance of hybrids through a process, in which lessonae sperm fertilize P. ridibundus eggs. The haploid ridibundus genome in hybrids received from P. ridibundus a generation ago, is perpetuated as clonal ridibundus sperm and used to fertilize P. ridibundus eggs, yielding female P. ridibundus progeny. These results imply animal reproduction in which hybridogenetic taxa are not only sexual parasites, but also participate in the formation of a sexual taxon in a remarkable way. This occurs through a process by which sexual gametes are being captured, converted to clones, and returned to sexual populations in one generation.
- MeSH
- analýza hlavních komponent MeSH
- genetické lokusy MeSH
- genom * MeSH
- haploidie MeSH
- mikrosatelitní repetice genetika MeSH
- Rana esculenta genetika MeSH
- Rana ridibunda genetika MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
