Currently, Jonathan’s research is geared towards identifying potential biomarkers for Mucolipidosis Type IV (MLIV). MLIV is a genetic autosomal recessive neurodegenerative disease that is clinically diagnosed with characteristics such as retinal degeneration, motor retardation and cognitive deficits that present themselves during early stages of life. Previous studies with genetic sequencing have revealed an association between mutations or a deletion in the Mucolipin-1 (MCOLN1) gene and the incidence of the disease. Studies indicate that the presence of specific mutations or a deletion in the MCOLN1 gene ultimately give rise to expression of nonfunctional transient receptor potential mucolipin protein-1 (TRPML1), which reveals itself with symptoms that are innate of MLIV. The prevalence of such mutations thus serve as a primary biomarker for determining the onset of the disease. For this reason, the pursuit towards identifying novel biomarkers that can be utilized to reveal underlying mechanisms of the disease and to ultimately develop potential gene-therapeutics holds great concern for Jonathan’s current research.
Recent advances in computing power have granted researchers with the ability to conduct next-generation sequencing (NGS) experiments in lengths that have been reduced dramatically, when compared to traditional sequencing experiments. Additionally, NGS experiments vary wildly and can be accomplished in great depth using relatively limited oversight. Jonathan and his team have taken advantage of these advancements to conduct high throughput cDNA sequencing (RNA-seq) experiments, which utilize NGS techniques to quantify relative gene expression on an immense scope for a given sample. Specifically, Jonathan has utilized previously sequenced cDNA reads from the cortical tissue of a wild-type and knockout mouse model for the MCOLN1 gene to construct transcriptome assemblies for each respective mouse condition. In this case, a NGS software known as DNAStar Lasergene Suite (Lasergene) that includes assembly-construction tools (ArrayStar) was used to assemble the initial transcriptomes for both the wild-type and knockout mouse data. Since the knockout mouse for this experiment was generated by third-party researchers that performed a homologous recombination using a recombinase enzyme to remove exons 3 and 4 of the MCOLN1 gene from the mouse genome, the preliminary steps of the analysis were geared towards verifying that the selected software could produce an output that remained consistent with the expected expression for exons within the knockout mouse model. To do this, Jonathan constructed a merged assembly using the transcriptome assemblies for the wild-type and knockout mouse models. This merged assembly quantified relative gene expression for each condition (wild-type or knockout) and generated histograms that represented the coverage (relative expression) of individual exons within the MCOLN1 gene of each respective condition.
The results of this merged assembly remained consistent with the expected outcome, in that, exons 3 and 4 had a noticeable reduction in coverage for the knockout mouse model when compared to its wild-type counterpart. Additionally, there was also an apparent decline in the observed expression for the remaining exons of the Mucolipin-1 transcript within the knockout mouse model. For this reason, Jonathan also has current plans to investigate the potential presence of hidden mRNA decay mechanisms that present themselves during the pre-diseased or diseased state. It is through these experiments and the knowledge obtained from them, that Jonathan aims to expand the current understanding of MLIV and advance research geared towards developing potential therapeutics.
