Approach 1. Seeking the biochemical basis of Costeff syndrome (Type III 3-methylglutaconic aciduria) through zebrafish models. Background: Costeff Syndrome is a rare disorder characterized by early-onset optic atrophy and later-onset spasticity, cerebellar ataxis, cognitive deficit and increased urinary excretion of 3-methylglutaconic acid (3MGC) and 3-methylglutaric acid (3MGA). Genetic mapping and sequencing efforts have identified two familial mutations in the OPA3 gene associated with Costeff Syndrome. The normal function of OPA3 and the biochemical basis of MGA-III remain undetermined, however it is known that 3MGC and 3MGA are derived through leucine catabolism in mitochondria and the levels of these organic acids are also influenced by the mevalonate pathway in peroxisomes. Results: We previously established a zebrafish model of Costeff Syndrome and used affected embryos to demonstrate the likelihood for a 3MGC salvage pathway that might be affected in Costeff syndrome. One of our collaborators has subsequently found evidence that a molecular mimic of 3MGC is elevated in a related syndrome, and we therefore checked for levels of this mimic in Costeff patient in patient plasma samples. Observing an analogous increase, we then obtained skin culture fibroblasts from Costeff syndrome patients and examined the levels of an upstream enzyme. We have evidence that the levels of this enzyme are specifically decreased in skin fibroblast cultures from more severely affected Costeff patients. Approach 2. Functional analysis in zebrafish of human alleles associated with holoprosencephaly. Background: We are attempting to generalize our ability to measure candidate human allele functionality in zebrafish, with the sole requirements being that the gene in question is sufficiently analogous to a zebrafish gene with embryonic activity. For this we are exploring a new platform, Nanostring, to count and correlate the quantity of injected WT and candidate mutant alleles of human RNAs with the output (activation or repression ) of a signature subset of zebrafish genes. We are currently applying this to two test scenarios, both in the sonic hedgehog signaling pathway: human SHH alleles and human GLI2 alleles. Approach 3. We are trying to identify the pathways upon which a candidate human obesity gene acts by examining the set of genes whose expression is altered when a zebrafish orthologue to the human candidate gene is disrupted with an antisense reagent. This experiment was performed and analysis is ongoing.
