Resumen de Caracterización molecular de CNVs, mutaciones sin sentido y de splicing en enfermedades metabólicas hereditarias investigación en terapias personalizadas
In this work, we have characterized copy number variations (CNVs) and nonsense mutations identified in patients diagnosed with propionic acidemia (PA), one of the most frequent organic acidemias inherited in autosomal recessive fashion as well as diverse splicing mutations identified in different genes involved in several inherited metabolic diseases (IMD). Our findings lead to the investigation in specific mutation therapeutic approaches.
We have employed multiplex ligation probe amplification (MLPA) and long PCR in some cases to screen for genomic deletions in the PCCA gene in 20 patients in whom standard mutation detection techniques had failed to complete genotype analysis. We have identified eight different exonic deletions corresponding to a frequency of 21.3% of the total PCCA alleles genotyped in the laboratory. Two of them were frequent, one involving exons 3 and 4 where two different chromosomal breakpoints were identified and another one involving exon 23. The high frequency of large genomic deletions in the PCCA gene could be due to the characteristics of the gene structure and its abundance in intronic repetitive elements. These data underscore the need of using MLPA analysis to complete routine genetic analysis in PCCA patients.
We have also identified 12 different nonsense mutations in a cohort of 190 PA patients, seven of them novel, accounting for 10% of the total mutant alleles. We have established the proof of principle that nonsense mutations in PA can be partially suppressed in vitro by aminoglycosides with different efficiencies depending on the nonsense mutation and the sequence context. To correct the metabolic defect, the incorporated aminoacid should support protein function, most of them retaining partial activity as was evaluated by in silico and in vitro expression analysis of the predicted missense changes. In patient¿s fibroblasts treated with readthrough compounds we observed a 40¿50 fold increase in enzymatic activity, reaching up to 10¿ 15% levels of treated control cells. These results point to readthrough therapy as a potential or supplementary treatment for a number of PA patients encouraging further clinical trials with newly developed readthrough compounds without toxic effects.
Several splicing defects that overall account for 15¿30% of the mutant alleles in most IMD were also analyzed. We have characterized mutations found at the conserved splice sites and at deep intronic sequences that activate or create new splice sites resulting in the aberrant inclusion of pseudoexons in the mRNA. We have performed in silico and functional analysis using minigenes to analyze the individual effect of mutations on the splicing process to understand the precise molecular mechanism involved in the splicing defect and in the patient¿s phenotypic expression, as well as to investigate in splicing¿directed therapies. In this work we have employed the overexpression of splicing factors and U1snRNAs with extended base¿pairing with the mutated 5¿ splice site to modulate the aberrant pre¿mRNA splicing resulting from different mutations. The antisense therapy has efficiently restored normal splicing affected by pseudoexon activation due to deep intronic mutations with a functional recovery close to normal levels even for a heterozygous mutation.
