Molecular genomics of a genetic code alteration

The genetic code is not universal. Alterations to its standard form have been discovered in both prokaryotes and eukaryotes and demolished the dogma of an immutable code. For instance, several Candida species translate the standard leucine CUG codon as serine. In the case of the human pathogen Candida albicans, a serine tRNA (tRNACAGSer) incorporates in vivo 97% of serine and 3% of leucine in proteins at CUG sites. Such ambiguity is flexible and the level of leucine incorporation increases significantly in response to environmental stress. To elucidate the function of such ambiguity and clarify whether the identity of the CUG codon could be reverted from serine back to leucine, we have developed a forced evolution strategy to increase leucine incorporation at CUGs and a fluorescent reporter system to monitor such incorporation in vivo. Leucine misincorporation increased from 3% up to nearly 100%, reverting CUG identity from serine back to leucine. Growth assays showed that increasing leucine incorporation produced impressive arrays of phenotypes of high adaptive potential. In particular, strains with high levels of leucine misincorporation exhibited novel phenotypes and high level of tolerance to antifungals. Whole genome re-sequencing revealed that increasing levels of leucine incorporation were associated with accumulation of single nucleotide polymorphisms (SNPs) and loss of heterozygozity (LOH) in the higher misincorporating strains. SNPs accumulated preferentially in genes involved in cell adhesion, filamentous growth and biofilm formation, indicating that C. albicans uses its natural CUG ambiguity to increase genetic diversity in pathogenesis and drug resistance related processes. The overall data provided evidence for unantecipated flexibility of the C. albicans genetic code and highlighted new roles of codon ambiguity on the evolution of genetic and phenotypic diversity.

Familial amyloid polyneuropathy: TTR sequencing and "in silico" analysis

Familial amyloid polyneuropathy (FAP) or paramiloidosis is an autosomal dominant neurodegenerative disease with onset on adult age that is characterized by mutated protein deposition in the form of amyloid substance. FAP is due to a point alteration in the transthyretin (TTR) gene and until now more than 100 amyloidogenic mutations have been described in TTR gene. FAP shows a wide variation in age-at-onset (AO) (19-82 years, in Portuguese cases) and the V30M mutation often runs through several generation of asymptomatic carriers, before expressing in a proband, but the protective effect disappear in a single generation, with offspring of late-onset cases having early onset. V30M mutation does not explain alone the symptoms and AO variability of the disease observed in the same family. Our aim in this study was to identify genetic factors associated with AO variability and reduced penetrance which can have important clinical implications. To accomplish this we genotyped 230 individuals, using a directautomated sequencing approach in order to identify possible genetic modifiers within the TTR locus. After genotyping, we assessed a putative association of the SNPs found with AO and an intensive in silico analysis was performed in order to understand a possible regulation of gene expression. Although we did not find any significant association between SNPs and AO, we found very interesting and unreported results in the in silico analysis since we observed some alterations in the mechanism of splicing, transcription factors binding and miRNAs binding. All of these mechanisms when altered can lead to dysregulation of gene expression, which can have an impact in AO and phenotypic variability. These putative mechanisms of regulation of gene expression within the TTR gene could be used in the future as potential therapeutical targets, and could improve genetic counselling and follow-up of mutation carriers.