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Browsing by discipline "Translational Medicine"

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  • C21orf57 is an ybey-homolog in mammalian mitochondria 
    Duarte, Daniel Pereira (2016)
    Mitochondrion is an essential organelle capable to produce high amounts of energy from oxidative phosphorylation (OXPHOS). The organelle counts on its own set of mitoribosomes and quality control (QC) factors to efficiently translate the OXPHOS components encoded by the mitochondrial DNA. Organelle dysfunction leads to development of mitochondrial diseases, which show a wide variety of symptoms and poor prognosis. ybeY is a highly conserved gene amongst prokaryotes that acts as factor for maturation and QC of bacterial ribosome. The ybeY-homolog C21orf57 is found remarkably conserved in eukaryotes, but its role in mammalian cells is completely unknown. In this study we characterised the mouse gene C21orf57 (mYBEY) in mammalian cells. mYBEY is transcribed at low levels in mouse tissues, where nervous tissue, skeletal and cardiac muscle show relatively higher amounts of mYBEY transcript. Endogenous mYbey protein could not be detected immunoblot due to its low expression level and/or rapid protein turnover. In parallel, mYBEY was cloned and successfully expressed in mammalian cells using transient and stable expression methodologies. We discovered that mYbey is imported into mitochondria and not covalently associated with mitoribosomes. Interestingly, protein translation stress and mitoribosome decay promoted by actinonin treatment is accompanied by a decrease in mYbey protein. In addition, depletion of mYbey using iRNA promotes the accumulation of mitoribosome proteins. We hypothesise a model in which mYbey is part of a QC mechanism for mitoribosome, and possibly involved with mitoribosome turnover. Although we successfully deleted mYBEY gene in mouse embryonic fibroblasts using the CRISPR/Cas9 approach, the isolation of viable mYBEY knock-out cells was not possible due to the possible deleterious and/or suppressive effects. In conclusion, we verified for the first time that mYbey is located within mammalian mitochondria and may be involved in a possible QC mechanism as a factor recruited for mitoribosome turnover. Further studies are necessary to fully elucidate the role of mYbey in the mitochondrial context.
  • Identification of disease mutations in early-onset neuropathies by whole exome sequencing 
    Pöyhönen, Julia Rosanna Hellin (2013)
    Charcot-Marie-Tooth (CMT) neuropathy is phenotypically and genetically a very heterogeneous disease. It can be inherited as an autosomal recessive, dominant or X-linked trait. CMT is characterized by distal muscle weakness, atrophy and deformity of the feet as well as clumsiness of gait. The onset of CMT varies and also the symptoms of the disease can vary even among the members of a single family. So far more than 40 genes have been identified for CMT and the list is estimated to grow by 30-50 genes. Whole exome sequencing (WES) is a new next generation sequencing technique, which targets the protein-coding area of the genome. Through WES analysis it is possible to search for disease causing mutations with all kinds of inheritance patterns. Patients suffering from CMT are good candidates for WES analysis because of the genetic heterogeneity of their disease. WES can be used for diagnosing Mendelian disorders with atypical symptoms as well as diseases, which are difficult to confirm using clinical criteria alone and which require costly evaluation, e.g. CMT. In this master study new disease causing mutations for early-onset neuropathies are identified by whole exome sequencing (WES). The aims of this study include using WES for the molecular diagnosis of four patients suffering from early-onset axonal neuropathies, the functional analysis of possible causative variants and improving and developing the process of analyzing variants from whole exome sequencing data, especially the analyzing steps of insertion and deletion variants. Finding causative variants among the insertion and deletion variants has previously been often left out from the WES analysis because of the lack of systematic analysis technique. As a result of the WES data analysis a new candidate disease gene, tripartite motif containing 2 (TRIM2) was identified. A missense mutation c.761T>A (p.E254V) and a deletion c.1779delA (p.K594Rfs7X) were found in patient 2, who suffers from severe CMT type 2. The carrier frequency was analysed to see whether the variants are present in the general population or not. The functional analysis of TRIM2 was started by preparing constructs carrying the missense mutation and the deletion and by setting up conditions for western blotting.

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