Transcriptomic and functional characterisation of marginal and clinically severe 3-methylcrotonyl-CoA carboxylase deficiency

Abstract

Urinary 3-hydroxyisovaleric acid and 3-methylcrotonylglycine are usually indicative of the possibility of 3-methylcrotonyl-CoA carboxylase (MCC) deficiency. In this study a South African family which presented with these metabolites was investigated. A standard metabolic work-up, analyses of relevant enzyme activity and in vivo loading tests indicated that two of the males in the family might have marginal MCC-deficiency of unknown genetic origin. The standard workup was extended with transcriptome analyses. Affymetrix HuExST1.0 arrays were used to generate the transcriptome from cultured skin fibroblasts of two affected males of the family and then the underlying molecular interactions and functional network analyses were explored. Transcriptomes were also generated from immortalised skin fibroblast cultures of well-documented clinically severe MCC-deficient patients as well as healthy controls. Subsequently, the three transcriptomes (from the South African family, the clinically severe MCC-deficient patients and controls) were compared to further characterise and identify similarities and differences between clinically severe and marginal MCC deficiency. The biochemical phenotype indicative of MCC-deficiency in this South African family suggested an X-linked association. The transcriptomic and functional analyses identified possible candidate genes to further investigate this apparent X-linked association of some MCC-deficient patients, especially the FAAH2 gene. The clinically severe 3-methylcrotonyl-CoA carboxylase deficient skin fibroblast transcriptome had a footprint indicative of mitochondrial dysfunction. The comparison of the transcriptomes and functional analyses from clinically severe and marginal 3-methylcrotonyl-CoA carboxylase deficiency further suggested the presence of aberrant pro-inflammatory cytokine signalling and associated impaired membrane integrity. The data presented in this thesis supports the notion that secondary factors other than the MCC loci might contribute to the presentation of the biochemical phenotype which is usually indicative of MCC-deficiency. The data also suggested that the long-term impact of a 3-methylcrotonyl-CoA carboxylase deficient biochemical phenotype should not be underestimated, especially since aberrant regulation of reactive oxygen species seems to play an intricate role in MCC-deficiency. It is evident that MCC-deficiency is far more complex than what was thought. However, despite the complexity of the functional analyses and the secondary signalling responses observed in the transcriptomes, interesting relationships were revealed that contribute to a better insight into the molecular impact of MCC-deficiency. In summary, it is clear that this dataset has potential to be mined even more. It is however important to keep in mind that the current state of the data is of an explorative nature and any specific implications thereof must be confirmed experimentally. A vast amount of options for possible follow-up experiments are available and should be carefully explored.