Project background
Our project focuses on patients with severe neurological disorders with childhood onset in which a molecular diagnosis was not obtained after thorough genetic diagnostics at AMG, OUS. A monogenic disease is highly likely in the recruited patients, since many of them have one or more of the following characteristics: progressive disease course, metabolic abnormalities, affected siblings and/or consanguineous parents. Non-genetic causes, such as infection, trauma or hypoxia, have been excluded. Many of the patients present with brain abnormalities found on clinical examination and/or detected on cerebral imaging (exemplified in Figure 1).
Genetic diagnostic investigations nowadays use sequencing of the whole genome (WGS), usually analysing data from exons in all genes known to be associated with diseases. This results in a molecular diagnosis in <50% of the patients, highlighting the lack of knowledge about these diseases.
In our project, we expand the bioinformatics analyses to all known genes in the WGS-data, facilitating the identification of “novel disease genes”, i.e. detecting disease causing variants in one of the >15.000 genes not yet known to cause disease when mutated. We also perform transcriptome sequencing (RNA-seq) and combine analyses of RNA-seq and WGS-data to detect putative disease causing variants in introns and extragenic sequences.
We use patient fibroblasts to characterize how the variants detected affect cell physiology. We share potential novel disease genes through GeneMatcher.org to identify unrelated patients with overlapping phenotypes and mutations affecting the same gene. By performing RNA-seq we also elucidate knowledge about the underlying molecular mechanisms causing these severe diseases (exemplified in Figure 2).
Our group has extensive experience with the relevant technologies and has recently identified and characterized several novel disease genes (see selected references 1-4).
Contributions by the student
The student will analyze one promising novel candidate disease gene already identified by comparison of WGS-data from patients and parents. The molecular consequences of the variant will be studied in vitro using patient fibroblasts. Gene expression levels will be measured by qRT PCR or RNA-seq. Protein extracts (total protein, nuclear or cytoplasmic extracts) will also be analyzed by western blot assays to quantify the levels of the proteins of interest in patient cells, and to assess activation of signal transduction pathways. Qualitative and quantitative immunofluorescence (IF) microscopy assays will be used to assess changes in location and abundance of affected proteins. Manipulation of candidate gene expression will be performed by RNAi and/or ectopic expression from cDNA constructs.
The MSc project is carried out at Department of Medical Genetics (AMG) at Oslo University Hospital (OUS)/University of Oslo (UiO), supervised by Prof. E. Frengen and Dr. D. Misceo, in close collaboration with Prof. C. Progida at IBV.
AMG serves half the Norwegian population and has a strong environment in sequencing technologies and bioinformatics.
Information about Frengen’s research group: https://ous-research.no/frengen/
Selected reference
- Str?mme et al. Thyroid 28, 1406-1415, 2018. (DOI 10.1089/thy.2018.0595)
- Epting et al. Hum Mutat 41, 2179-2194, 2020. (DOI 10.1002/humu.24127)
- Sumathipala et al. Brain 145, 2602-2616, 2022. (DOI 10.1093/brain/awac034)
- Misceo et al. Brain 146, 3513-3527, 2023. (DOI 10.1093/brain/awad086)