Project 1. Seeking the biochemical basis of Type III 3-methylglutaconic aciduria through zebrafish models.? ? Background: Type III 3-methylglutaconic aciduria (MGA-III) 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 MGA-III. 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. Depletion of zebrafish Opa3 with antisense morpholino oligonucleotides (MOs) causes the signature increase in 3MGA, but also leads to a more severe eye defect than seen in MGA-III patients. We have gone on to obtain zebrafish with a disruption in the opa3 gene and we are characterizing the associated phenotype. ? ? Project 2. Functional analysis in zebrafish of human alleles associated with holoprosencephaly.? ? Background: Our collaborators identified a large set of SIX3 mutations in holoprosencephaly patients. SIX3 is an attractive gene for functional testing in zebrafish embryos, because it has well-defined biological effects, and also because it acts in two signaling pathways, BMP and WNT, that are important for gastrulation, the main focus of my laboratory (see Z01 HG200309-05). We accordingly established WNT-specific and BMP-specific in vivo assays in whole zebrafish embryos and functionally characterized 42 SIX3 mutations. A large fraction of these alleles displayed partial losses of function. This data adds to the diagnostic markers available to family members wishing to track their risk of having a child with holoprosencephaly. In addition, certain SIX3 alleles showing losses of function in one assay, but not the other may provide molecular clues towards understanding which structural elements of the Six3 molecule interact with which (BMP or WNT) pathway.? ? We have similarly developed zebrafish-based assays to test the activity of holoprosencephaly-associated mutant FOXH1 alleles.

National Institute of Health (NIH)
National Human Genome Research Institute (NHGRI)
Intramural Research (Z01)
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National Human Genome Research Institute
United States
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