One of the greatest challenges in the field of human genetics is to assess genetic variants in regard to their functional effect on the translated protein. The entire genome contains several thousand 'mutations', which ultimately make up the genetic diversity of the different individuals. However, the majority of these 'mutations' are not considered pathogenic, instead they are responsible for the genetic singularity of every human being. These non-pathogenic 'mutations' are referred to as genetic variants and occur in a relatively large percentage within the global population.
The difficulty is to evaluate now variants that are present only in a small percentage of the human population and may therefore be pathogenic, i.e. disease-causing. These are mostly so-called missense variants, in which one nucleotide in a specific DNA sequence is exchanged for another. This can lead to an amino acid exchange in the resulting protein. Whether this has an effect on the proper protein function, thus being potentially pathogenic, depends on e.g. the similarity of the substituted amino acids. To some extent, it is possible to simulate and assess these events using mathematical algorithms, but the reliability of these predictions is still very limited so far.
The most reliable method to analyse the effect of an amino acid exchange on the protein function is therefore the reconstruction of the respective variant in vitro. For this purpose, a corresponding missense variant can be generated by a so-called mutagenesis PCR, introduced into a vector, and heterologously expressed in human cell lines. These transfected cells can be used for a variety of experiments. A common example is the luciferase assay, in which the ability of a transcription factor to bind target DNA is assessed. Furthermore, Western blots can be performed to determine protein expression levels or interacting proteins. Immunofluorescence or immunohistochemical staining represent another method of functional analysis, which is used to analyse the localisation of certain proteins in cells or tissues.