Assigning function to sequence variants observed in association with clinical studies remains a key priority in the post-genomic era. Numerous reports over the last decade demonstrate that in many instances, overexpression of functional human alleles are sufficient to rescue loss of function (lof) orthologous knockdowns and knockouts in zebrafish. The inability of polymorphic and variant human alleles to rescue lof zebrafish models is thus an efficient platform to assign functional significance to these alleles. To explore this potential further, our collaborative team is investigating known sequence variants in Tyrosinase (Tyr) and T (Brachyury) and applying what is learned through this study to inform an ongoing study of sequence variants of unknown significance from patients with underlying vertebral malformations. Human ORFs are cloned into pT3TS or pKTol2BA RNA and DNA expression vectors, respectively. ORFs are modified using pcr mediated overlap extension to create variant alleles and cloned similarly. Both human functional and potential lof variant alleles are then tested for their capacity to rescue zebrafish lof phenotypes. Initial studies of two families with likely inherited vertebral malformations have been examined using whole exome sequencing. Resulting sequences have been processed by applying bioinformatics filters for stop gain/loss and non-synonymous coding changes to ensure high relevance of the 30 variant alleles identified and include genes such as Polr1d, Tle4, and Ppib for example. Functional studies of these alleles are commencing following initial technical progress demonstrating that overexpression of human Tyr rescues an orthologous Tyr lof zebrafish model. These results support the use of zebrafish for functionally validating pathogenic sequence variants and ultimately improving clinical relevance of association studies for complex disease.