Rare diseases may affect the quality of life of patients and be life-threatening. Therapeutic opportunities are often limited, in part because of the lack of understanding of the molecular mechanisms underlying these diseases. This can be ascribed to the low prevalence of rare diseases and therefore the lower sample sizes available for research. A way to overcome this is to integrate experimental rare disease data with prior knowledge using network-based methods. Taking this one step further, we hypothesized that combining and analyzing the results from multiple network-based methods could provide data-driven hypotheses of pathogenic mechanisms from multiple perspectives.We analyzed a Huntington's disease transcriptomics dataset using six network-based methods in a collaborative way. These methods either inherently reported enriched annotation terms or their results were fed into enrichment analyses. The resulting significantly enriched Reactome pathways were then summarized using the ontological hierarchy which allowed the integration and interpretation of outputs from multiple methods. Among the resulting enriched pathways, there are pathways that have been shown previously to be involved in Huntington's disease and pathways whose direct contribution to disease pathogenesis remains unclear and requires further investigation.In summary, our study shows that collaborative network analysis approaches are well-suited to study rare diseases, as they provide hypotheses for pathogenic mechanisms from multiple perspectives. Applying different methods to the same case study can uncover different disease mechanisms that would not be apparent with the application of a single method.

• Klíčová slova
• Collaborative analysis, Huntington’s disease, Network analysis, Rare disease,
• MeSH
• genové regulační sítě MeSH
• Huntingtonova nemoc * genetika   MeSH
• lidé MeSH
• stanovení celkové genové exprese * metody   MeSH
• transkriptom * MeSH
• výpočetní biologie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Academic-sponsored trials for rare diseases face many challenges; the present paper identifies hurdles in the set-up of six multinational clinical trials for drug repurposing, as use cases. METHODS: Six academic-sponsored multinational trials aiming to generate knowledge on rare diseases drug repurposing were used as examples to identify problems in their set-up. Coordinating investigators leading these trials provided feedback on hurdles linked to study, country, and site set up, on the basis of pre-identified categories established through the analysis of previous peer-reviewed publications. RESULTS: Administrative burden and lack of harmonization for trial-site agreements were deemed as a major hurdle. Other main identified obstacles included the following: (1) complexity and restriction on the use of public funding, especially in a multinational set up, (2) drug supply, including procurement tendering rules and country-specific requirements for drug stability, and (3) lack of harmonization on regulatory requirements to get trial approvals. CONCLUSION: A better knowledge of the non-commercial clinical research landscape and its challenges and requirements is needed to make drugs-especially those with less commercial gain-accessible to rare diseases patients. Better information about existing resources like research infrastructures, clinical research programs, and counseling mechanisms is needed to support and guide clinicians through the many challenges associated to the set-up of academic-sponsored multinational trials.

• Klíčová slova
• Academic-sponsored, Barriers, Challenges, Drug repurposing, Randomized clinical trials, Rare diseases,
• MeSH
• klinické zkoušky jako téma MeSH
• lidé MeSH
• organizace MeSH
• přehodnocení terapeutických indikací léčivého přípravku * MeSH
• vzácné nemoci * diagnóza   farmakoterapie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

OBJECTIVE: Hereditary spastic paraplegia (HSP) is a highly heterogeneous neurologic disorder characterized by lower-extremity spasticity. Here, we set out to determine the genetic basis of an autosomal dominant, pure, and infantile-onset form of HSP in a cohort of 8 patients with a uniform clinical presentation. METHODS: Trio whole-exome sequencing was used in 5 index patients with infantile-onset pure HSP to determine the genetic cause of disease. The functional impact of identified genetic variants was verified using bioinformatics and complementary cellular and biochemical assays. RESULTS: Distinct heterozygous KPNA3 missense variants were found to segregate with the clinical phenotype in 8 patients; in 4 of them KPNA3 variants had occurred de novo. Mutant karyopherin-α3 proteins exhibited a variable pattern of altered expression level, subcellular distribution, and protein interaction. INTERPRETATION: Our genetic findings implicate heterozygous variants in KPNA3 as a novel cause for autosomal dominant, early-onset, and pure HSP. Mutant karyopherin-α3 proteins display varying deficits in molecular and cellular functions, thus, for the first time, implicating dysfunctional nucleocytoplasmic shuttling as a novel pathomechanism causing HSP. ANN NEUROL 2021;90:738-750.

• MeSH
• alfa karyoferiny genetika   MeSH
• dospělí MeSH
• fenotyp MeSH
• heterozygot MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mutace genetika   MeSH
• předškolní dítě MeSH
• rodokmen MeSH
• sekvenování exomu metody   MeSH
• spastická paraplegie dědičná genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alfa karyoferiny MeSH
• KPNA3 protein, human MeSH Prohlížeč

Reverse transcription quantitative PCR (RT-qPCR) has delivered significant insights in understanding the gene expression landscape. Thanks to its precision, sensitivity, flexibility, and cost effectiveness, RT-qPCR has also found utility in advanced single-cell analysis. Single-cell RT-qPCR now represents a well-established method, suitable for an efficient screening prior to single-cell RNA sequencing (scRNA-Seq) experiments, or, oppositely, for validation of hypotheses formulated from high-throughput approaches. Here, we aim to provide a comprehensive summary of the scRT-qPCR method by discussing the limitations of single-cell collection methods, describing the importance of reverse transcription, providing recommendations for the preamplification and primer design, and summarizing essential data processing steps. With the detailed protocol attached in the appendix, this tutorial provides a set of guidelines that allow any researcher to perform scRT-qPCR measurements of the highest standard.

• Klíčová slova
• RT-qPCR, gene expression, preamplification, quantitative PCR, reverse transcription, sample collection, single cell,
• MeSH
• analýza jednotlivých buněk metody   normy   MeSH
• kvantitativní polymerázová řetězová reakce metody   normy   MeSH
• lidé MeSH
• reverzní transkripce genetika   MeSH
• senzitivita a specificita MeSH
• stanovení celkové genové exprese metody   normy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH