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 found that the zebrafish orthologue, opa3, is expressed ubiquitously during the first day of embryogenesis (from fertilization through segmentation) and is enriched in the brain from the pharyngula stage (24 hours post fertilization hpf) until at least 120 hpf. We established a genetic model of Costeff Syndrome that recapitulates biochemical, behavioral and morphological aspects of Costeff syndrome. Using this model, we found evidence for a novel biochemical source of elevated 3-MGA and we demonstrated an increased sensitivity of opa3 mutants to inhibitors of the electron transfer chain enzyme complexes in the inner mitochondrial membrane. Approach 2. Functional analysis in zebrafish of human alleles associated with holoprosencephaly and congenital heart disease. Background: Our collaborators identified a number of NODAL gene mutations in patients with holoprosencephaly and patients with congenital pediatric heart conditions that are hypothesized to arise from errors in left-right patterning. NODAL is an attractive gene for functional testing in zebrafish embryos, because it is critical in mesoderm and endoderm formation, the main focus of my laboratory (see Z01 HG200309-05). We accordingly established a NODAL-specific in vivo assay in zebrafish embryos and quantified the functionality of 21 human disease-associated alleles, revealing that 9 were loss of function, 8 moderately reduced in function, and 4 potentially unaffected. This revealed a surprising sensitivity of the NODAL gene to mutations in a domain of NODAL that is removed to create the mature signaling protein.
