Explore the vast range of titles available.

Variable number tandem repeats (VNTRs) remain among the most challenging regions of the human genome to characterize, due to their repetitive structure and high sequence variability. Short-read sequencing lacks the resolution to span these regions, and even long-read variant callers often fail to resolve true allelic variation due to alignment ambiguity and motif heterogeneity. We present a novel bioinformatics workflow for the analysis of VNTRs from nanopore sequencing data. To our knowledge, it is the first to offer fully automated variant detection, classification of loss-of-function (LoF) variants, and integrated quality control using nanopore sequencing technology. The pipeline separates reads into alleles, constructs reference-free consensus sequences, and aligns motif structures for visualization. LoF variants are identified and reported at their exact position within the repeat. The method was validated using PCR amplicons from reference genomes HG001–HG004 for two genes harboring VNTRs ( ACAN and MUC1 ), as well as four clinical control samples containing known frameshift mutations in the MUC1 VNTR. We further demonstrate its applicability to whole-genome nanopore datasets. Using our pipeline, it is now possible to completely analyze and characterize VNTRs as well as detect disease-causing variants in a cost and time-efficient manner using amplicon-based nanopore sequencing.

Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily caused by mutations in the X-linked MECP2 gene. Patient-derived fibroblasts serve as a practical system to study systemic aspects of RTT, however, their limited proliferative capacity due to cellular senescence poses a significant challenge. In this study, we establish a robust workflow to isolate and immortalize clonal fibroblast lines from female RTT patients heterozygous for two distinct MECP2 mutations (c.705delG and 1155del32). By employing single-cell cloning prior to hTERT-mediated immortalization, we generated stable, proliferative fibroblast clones with verified clonality and severely skewed X-chromosome inactivation. Wildtype clones exclusively expressed full-length MeCP2 protein, whereas mutant clones exhibit truncated or absent MeCP2. Immortalized lines retained elevated hTERT expression and sustained proliferation even at late passages. Notably, mutant clones recapitulated key molecular features of RTT, including histone hyperacetylation, dysregulation of oxidative stress markers, and aberrant expression of key signaling genes. Our approach provides a scalable and renewable in vitro model that faithfully captures critical aspects of RTT pathology and offers a complementary platform to existing animal and iPSC based systems. Moreover, the approach is adaptable for studying other X-linked genetic disorders and supports applications in mechanistic research and preclinical drug screening.

Polyomaviruses have the potential to cause significant morbidity not only in transplant medicine, but also in other forms of disease or variants of immunosuppression. In kidney transplant recipients or recipients of human stem cell transplants, the BK-Virus is the major proponent of manifestations such as BKPyV-associated nephropathy or hemorrhagic cystitis. As no polyomavirus-specific drug with proven in vivo effects has been developed so far, methods to screen for such drugs are important. This work describes the establishment of a virus-secreting cell line. By infecting a pre-established monkey kidney cell line (COS-1) with a non-rearranged human BK polyomavirus isolated from a kidney transplant patient suffering from BKPyV-associated nephropathy, a continuously replicating cell type with consistent virus secretion could be established and was termed COSSA. Measurements of BKPyV replication, virion production, and secretion were performed both intracellularly and in the cell supernatant. Viral proteins such as VP1 and LTAg were accurately tracked by confocal microscopy, as well as by immunoblot and qPCR. An intracellular flow cytometry (FACS) assay detecting VP1 protein was established and revealed an expanded range of positive intracellular signals. The viruses produced proved to be infectious in human tubular epithelial cell lines. Long-range sequencing of the COSSA genome using Oxford Nanopore Technology revealed a total of five distinct BKPyV integration events. One integration of a partial BKPyV genome was located upstream of the epidermal growth factor receptor gene. The second and third, both truncated forms of integration, were close to histocompatibility gene locuses, while the fourth was characterized by a ninefold and the fifth by a fourfold tandem repeat of the BKPyV genome. From both of the repeat forms, virus replicates were derived showing deletions/duplications on early and late genes and inversions within the non-coding control region (NCCR). This pattern of repetitive viral genome integration is a potential key driver of enhanced viral replication and increased virion assembly, ultimately supporting efficient virus egress. Quantitative PCR analysis confirmed the release of approximately 108/mL viral units per 48 h from 2 × 105 COSSA cells into the culture supernatant. Notably, the NCCR region of the most frequent copies of circular virus and the integrated tetrameric tandem repeat exhibited a rearranged configuration, which may contribute to the observed high replication dynamics. The establishment of a consistent methodology to generate and secrete BKPyV from a cell line is expected to significantly facilitate antiviral drug development.

Background Congenital myasthenic syndromes (CMS) are a heterogeneous group of disorders caused by genetic defects resulting in impaired neuromuscular transmission. Although effective treatments are available, CMS is probably underdiagnosed, and systematic clinico-genetic investigations are warranted. Methods We used a nationwide approach to collect Austrian patients with genetically confirmed CMS. We provide a clinical and molecular characterization of this cohort and aimed to ascertain the current frequency of CMS in Austria. Results Twenty-eight cases with genetically confirmed CMS were identified, corresponding to an overall prevalence of 3.1 per million (95% CI 2.0–4.3) in Austria. The most frequent genetic etiology was CHRNE ( n  = 13), accounting for 46.4% of the cohort. Within this subgroup, the variant c.1327del, p.(Glu443Lysfs*64) was detected in nine individuals. Moreover, causative variants were found in DOK7 ( n  = 4), RAPSN ( n  = 3), COLQ ( n  = 2), GMPPB ( n  = 2), CHAT ( n  = 1), COL13A1 ( n  = 1), MUSK ( n  = 1) and AGRN ( n  = 1). Clinical onset within the first year of life was reported in one half of the patients. Across all subtypes, the most common symptoms were ptosis (85.7%), lower limb (67.9%), upper limb (60.7%) and facial weakness (60.7%). The majority of patients (96.4%) received specific treatment, including acetylcholinesterase inhibitors in 20, adrenergic agonists in 11 and 3,4-diaminopyridine in nine patients. Conclusions Our study presents the first systematic characterization of individuals with CMS in Austria, providing prevalence estimates and genotype–phenotype correlations that may help to improve the diagnostic approach and patient management.

Exome sequencing has been increasingly implemented in prenatal genetic testing for fetuses with morphological abnormalities but normal rapid aneuploidy detection and microarray analysis. We present a retrospective study of 90 fetuses with different abnormal ultrasound findings, in which we employed the singleton exome sequencing (sES; 75 fetuses) or to a lesser extent (15 fetuses) a multigene panel analysis of 6713 genes as a primary tool for the detection of monogenic diseases. The detection rate of pathogenic or likely pathogenic variants in this study was 34.4%. The highest diagnostic rate of 56% was in fetuses with multiple anomalies, followed by cases with skeletal or renal abnormalities (diagnostic rate of 50%, respectively). We report 20 novel disease-causing variants in different known disease-associated genes and new genotype–phenotype associations for the genes KMT2D, MN1, CDK10, and EXOC3L2. Based on our data, we postulate that sES of fetal index cases with a concurrent sampling of parental probes for targeted testing of the origin of detected fetal variants could be a suitable tool to obtain reliable and rapid prenatal results, particularly in situations where a trio analysis is not possible.

The transcriptional regulator Methyl-CpG-binding protein 2 (MeCP2) is an intrinsically disordered protein, mutations in which, are implicated in the onset of Rett Syndrome, a severe and debilitating neurodevelopmental disorder. Delivery of this protein fused to the cell-penetrating peptide TAT could allow for the intracellular replenishment of functional MeCP2 and hence potentially serve as a prospective Rett Syndrome therapy. This work outlines the expression, purification and characterization of various TAT-MeCP2 constructs as well as their full-length and shortened eGFP fusion variants. The latter two constructs were used for intracellular uptake studies with subsequent analysis via western blotting and live-cell imaging. All purified MeCP2 samples exhibited high degree of stability and very little aggregation propensity. Full length and minimal TAT-MeCP2-eGFP were found to efficiently transduce into human dermal and murine fibroblasts and localize to cell nuclei. These findings clearly support the utility of MeCP2-based protein replacement therapy as a potential Rett Syndrome treatment option.

Motivation Facioscapulohumeral Muscular Dystrophy (FSHD) is an autosomal dominant form of muscular dystrophy caused by genetic or epigenetic changes within the D4Z4-repeat at the DUX4-gene, on chromosome 4q. Genetic analysis is challenging due to a nearly identical region on chromosome 10, multiple haplotypes, long and short repeat subtypes, and complex rearrangements such as translocations and duplications. So far, no single method detects all known causes of FSHD. Results We have developed an integrated approach combining an optimized wet-lab protocol with an automated bioinformatics workflow, called DUCKS4. It enables read-level resolution of the D4Z4 array for FSHD1 repeat sizing, variant detection for FSHD2, and detection of methylation patterns. Using NCBI BLAST, it assigns reads to chromosomes and haplotypes, supporting robust filtering and analysis. Our approach also includes a novel adaptive sampling workflow for human high molecular-weight DNA. With long-read Nanopore sequencing technology, our tool enables precise determination of D4Z4 array size, individual haplotype assignment, methylation profiling, and complex allele analysis. It also allows for the detection of mosaicism and structural variation like interchromosomal translocations, providing a comprehensive, single-method solution for FSHD analysis. Availability and implementation DUCKS4 is implemented within a Docker environment. Source code and supplementary materials are accessible at https://github.com/tamara-nano/ducks4 , https://github.com/tamara-nano/ducks4_wovar (light version without variant calling) and archived at https://figshare.com/projects/DUCKS4-FSHD/260306

Since 2008, when angiotensin II type I receptor blockade with losartan was introduced in the prevention of cardiovascular manifestation of Marfan syndrome (MFS), a specific treatment to address the cardiovascular lesions became available. The present study aimed to compare the response of such in an unselected cohort of patients with genotyped MFS. At a tertiary university children's hospital, 20 pediatric and adolescent patients aged 1.7 to 21.6 years with genetically proven MFS were enrolled in a prospective treatment study of losartan for evaluation of the aortic dimensions and elasticity indexes. The mean follow-up period was 33 ± 11 months. A significant reduction in the normalized aortic dimensions with losartan was observed in the valve, root, sinotubular junction, and ascending aortic segments (p = 0.008, p <0.001, p = 0.012, and p = 0.001, respectively). No correlation between elasticity behavior and the decrease in the aortic dimension with losartan therapy was detectable. A significant correlation between stronger improvement and younger age at onset (r = 0.643, p = 0.002) and a longer therapy duration (r = −0.532, p = 0.016) was verifiable. However, no correlation between improvement with therapy and the type of mutation or presentation of clinical forms was remarkable. Elasticity also seemed to improve but not significantly. In conclusion, in our cohort of young patients with MFS, a significant improvement with losartan monotherapy was proved in all affected proximal aortic segments, with a better response to therapy when started at an earlier age and with a longer therapy duration.

Background Epilepsy occurs in up to 90 % of all individuals with tuberous sclerosis complex (TSC). In 67 % disease onset is during childhood. In ≥ 50 % seizures are refractory to currently available treatment options. The mTOR-Inhibitor Everolimus (Votubia®) was approved for the treatment of subependymal giant cell astrocytoma (SEGA) and renal angiomyolipoma (AML) in Europe in 2011. It’s anticonvulsive/antiepileptic properties are promising, but evidence is still limited. Study aim was to evaluate the efficacy and safety of Everolimus in children and adolescents with TSC-associated epilepsies. Methods Inclusion-criteria of this investigator-initiated, single-center, open, prospective study were: 1) the ascertained diagnosis of TSC; 2) age ≤ 18 years; 3) treatment indication for Votubia® according to the European Commission guidelines; 4) drug-resistant TSC-associated epilepsy, 5) prospective continuous follow-up for at least 6 months after treatment initiation and 6) informed consent to participate. Votubia® was orally administered once/day, starting with 4.5 mg/m 2 and titrated to achieve blood trough concentrations between 5 and 15 ng/ml. Primary endpoint was the reduction in seizure frequency of ≥ 50 % compared to baseline. Results Fifteen patients (nine male) with a median age of six (range; 1–18) years fulfilled the inclusion criteria. 26 % (4/15) had TSC1, 66 % (10/15) had TSC2 mutations. In one patient no mutation was found. Time of observation after treatment initiation was median 22 (range; 6–50) months. At last observation, 80 % (12/15) of the patients were responders, 58 % of them (7/12) were seizure free. The overall reduction in seizure frequency was 60 % in focal seizures, 80 % in generalized tonic clonic seizures and 87 % in drop attacks. The effect of Everolimus was seen already at low doses, early after treatment initiation. Loss of efficacy over time was not observed. Transient side effects were seen in 93 % (14/15) of the patients. In no case the drug had to be withdrawn. Conclusion Everolimus seems to be an effective treatment option not only for SEGA and AML, but also for TSC-related epilepsies. Although there are potential serious side effects, treatment was tolerated well by the majority of patients, provided that patients are under close surveillance of epileptologists who are familiar with immunosuppressive agents.

Background In this study we aimed to describe the morphological and pathogenetic differences between tracheal agenesis and tracheal atresia, which are not clearly distinguished from each other in the literature, and to contribute thereby to the understanding and management of these conditions. Both tracheal agenesis and tracheal atresia represent rare disorders of still unknown aetiology that cannot be detected by prenatal ultrasound. If the affected foetuses survive until birth these conditions result in respiratory failure and in futile attempts to rescue the infant’s life. Results Autopsies and genetic analyses, including singleton or trio exome sequencing, were performed on five neonates/foetuses with tracheal agenesis and three foetuses with tracheal atresia. Tracheal agenesis was characterized by absence of the sublaryngeal trachea and presence of a bronchooesophageal fistula and by pulmonary isomerism and occurred as an isolated malformation complex or as part of a VACTERL association. Special findings were an additional so-called ‘pig bronchus’ and a first case of tracheal agenesis with sirenomelia. Tracheal atresia presenting with partial obliteration of its lumen and persistence of a fibromuscular streak resulted in CHAOS. This condition was associated with normal lung lobulation and single, non-VACTERL type malformations. Trio ES revealed a novel variant of MAPK11 in one tracheal agenesis case. Its involvement in tracheooesophageal malformation is herein discussed, but remains hypothetical. Conclusion Tracheal agenesis and tracheal atresia represent different disease entities in terms of morphology, pathogenesis and accompanying anomalies due to a primary developmental and secondary disruptive possibly vascular disturbance, respectively.