functional activity quantification of mutations in the human six3 Gene in holoprosencephaly

JORGE I VELEZ; SABINA DOMENE; ERICH ROESSLER; BENJAMIN FELDMAN; MAXIMILIAN MUENKE

National Institutes of Health, Bethesda, Maryland, USA

Simposio; Sixth Annual NIH Graduate Student Research Symposium; 2010

National Human Genome Research Institute, NIH

Holoprosencephaly is the most common, heterogeneous and etiologically complex malformation ofthe human forebrain. Consequently, its genetics is still incompletely understood. Mutations in SIX3,a transcription factor gene expressed in the anterior forebrain and eyes during early vertebratedevelopment, have been frequently detected in patients with HPE. However, only a few mutationshave been investigated with limited functional studies that would confirm a role in HPE pathogenesis.Because of the biological implications, the characterization of mutations in the human SIX3 gene aseither loss-of-function (LOF) or gain-of-function (GOF) has always been of interest, but no standardway to accomplish this had been available until now.After developing a set of robust and sensitive assays using the zebrafish as an animal model, we alsodeveloped the statistical and graphical representation methods necessary for the characterization of46 distinct mutations located throughout the entire SIX3 gene. These methods involving computer-intensive procedures allows us to (1) propose and validate the Wild Type Adjust Activity (WTAA)index, which compares the functional activity of any HPE-related mutation in the SIX3 gene with itsWild Type equivalent; (2) confirm that 89 % of these putative deleterious mutations are significantLOF alleles; (3) compare the developed set of assays in terms of result concordance and (4) ageneralization of the WTAA index and its estimation in real time by developing the appropriatecomputational frame-work.