Dr. István Balogh, Dr. János Kappelmayer, Dr. József Tőzsér (2011)
University of Debrecen
The deleterious effect of the vast majority of pathogenic mutations are expressed via the translated protein encoded by the gene. The genetic code consists of 3 letters and each position (Figure 2.1.) can be occupied by any of the 4 nucleotides, therefore in theory there are 64 possibilities, which is more than enough to encode the 20 amino acids.
The genetic code is degenerated because every amino acid is encoded by more than one (normally 3) codon. Some amino acids (leucine, serine, arginine) are encoded by 6 codons, while others are significantly less well-represented. The third nucleotide of the codon can often wobble, which means that whichever nucleotide occupies the position, it will result in the same amino acid. The genetic code is almost universal but there are some exceptions. Chromosomal AGA and AGG codons that encode arginine, in the mitochrondium encode stop codons. ATA (isoleucine) is methionine in the mithocondrium, while TGA (stop) is tryptophane. TAA and TAG codons encode stop signal in both the mitochondrial and the nuclear genome.
Mutations that affect the coding region of the gene are most frequently missense mutations. It means that an amino acid coding codon will be replaced by another, but still amino acid coding codon. In Figure 2.2., CAT is replaced by CCT, which will result in the replacement of the originally coded histidine amino acid by proline. Approximately 70% of the single nucleotide replacements are missense mutations. The proportion of splicing mutations and mutations resulting in premature stop codons is less than 30%, while mutations in regulatory elements represent approximately 1%.
The scope of the possible consequences of missense mutations is extremely wide. Very frequently, especially if the amino acid residue is solvent-exposed or if the amino acid is replaced by another amino acid with similar chemical characteristics, the effect is not crucial with respect to the stucture and function of the protein. Conversely, the effect is much more severe when an amino acid that is involved in the maintenance of the structure of the protein or cysteine residue (which frequently forms disulphide bridge) is mutated. The change of one amino acid might completely destabilize a protein composed of a hundred or a thousand amino acids, resulting in intracellular degradation. The consequences of a missense mutation might be severe, for example a mutation in the active site of an enzyme. Unlike truncating and frameshift-causing mutations, predicting the effect of missense mutations is very difficult. Recombinant systems are often used for testing the effect of missense mutations. In this case, the coding mRNA is reverse transcribed into cDNA is and that is cloned into a system which enables the recombinant protein production. This is usually done by cloning into an expression plasmid and the mutation is introduced using site-directed mutagenesis and the recombinant mutant protein is expressed. There can be many different kinds of expression systems. If the protein is small, post-transcriptionally not modified and does not contain disulphide bridges, prokaryotic (most frequently E. coli-based) system can be the primary choice. In the case of proteins with more complex structure, eukaryotic (yeast or mammalian) systems can be used. Using these systems, it is possible to test the stability and structure-function relationships of the mutant proteins, which is why they are a valuable means of analysing the pathogenicity of the detected mutation.
Contrary to missense mutations, usually no experimental proof is needed if the genetic alteration results in the generation of premature stop codon. When an amino acid – coding triplet is mutated into TGA, TAA or TAG codon, the result is nonsense mutation. The formation of stop codon usually results in truncated protein (if any protein is synthesized from such an RNA template). Premature stop codons are almost always pathogenic resulting in the encoded protein losing its function. When nonsense mutation occurs, often the mRNA will not be able to serve as a template for protein expression; instead, it is later degraded (see nonsense mediated mRNA decay). It means, that the cell possesses defense mechanisms at different levels (ie. mRNA and protein) in order to protect itself from a synthesis of a potentially deleterious mutant protein product.
Figure 2.3. shows a nonsense mutation, where the originally present CAG codon is replaced by a TAG codon, which will results in the finalization of the protein synthesis in this position.