Functional studies for evaluating genetic pathogenicity

Functional mutation pathogenicity studies are designed to understand the cellular, biochemical and structural pathways affected in association with information obtained by extensive genomic analysis and to be able to relate them to the biochemical and clinical phenotype.
The lab actively adapts assays developed in the research laboratory into validated clinical assays and makes them widely available for clinical use.

As an example, refers to the Blue Native-PAGE technique, which is used to evaluate the relative amount of fully assembled Mitochondrial Respiratory Chain (oxidative phosphorylation) complexes, being an essential tool to detect various defects in the assembly state of mitochondrial complexes. Depending on the clinical case under study, other techniques can be applied. By expanding the functional assays, we have been able to increase the diagnostic yield in fibroblasts.

• The functional studies to be carried out are suitable for a specific gene, evaluating which assays are most relevant for each case.

They can be subcategorized into:

• Functional Studies - bioenergetics
  Assessment of oxygen consumption and medium acidification rates by real-time extracellular flow analysis;
  Bioenergetics in galactose medium with real-time extracellular flow analysis;
  Mitochondrial membrane potential and ROS;
  Intracellular ATP levels;
  Evaluation of cell growth including spectrophotometric assay of activity of Mitochondrial Respiratory Chain (in medium with galactose).
• Functional Studies - biomolecular and structural
  Study of assembly state by Blue native-PAGE and western-blotting for the complexes of the Mitochondrial Respiratory Chain;
  Study of protein cellular sublocalization by fluorescence microscopy;
  Specific protein, quantification – Western-blotting studies;
  Specific transcript (mRNA) levels - real-time PCR.