SIX3 mutations in Holoprosencephaly (HPE) are loss-of-function alleles.

SABINA DOMENE; KENIA EL-JAICK; ERICH ROESSLER; FELICITAS LACBAWAN; BEN FELDMAN; MAXIMILIAN MUENKE

San Diego, California, USA

Congreso; American Society of Human Genetics (ASHG) Meeting; 2007

American Society of Human Genetics (ASHG)

Holoprosencephaly (HPE) is the most common structural anomaly of human forebrain development, with a prevalence of ~1 in 250 conceptuses and ~1 in 16,000 at birth. Mutations in at least eight different genes have been identified in human HPE patients. We have previously shown that SIX3, a transcription factor known to be involved in midline forebrain and eye formation during early development in the mouse, is associated with HPE in humans. No functional studies have been performed to date. It consists of two highly conserved domains: a SIX domain needed for interaction with other proteins and a DNA-binding homeodomain. SIX3 interacts with groucho corepressor proteins through two eh1-like motifs located within the SIX domain. This interaction is required both for the autorepression of six3 itself and for the regulation of other early developmental genes.    In addition to 18 previously reported SIX3 mutations we describe here 29 novel mutations. The total of 47 mutations are located throughout the entire SIX3 gene and include 33 missense, 5 nonsense, 8 frameshift mutations and 1 in frame deletion. To demonstrate the function of these mutations we established several complementary approaches using the zebrafish as a model system: 1) overexpression of SIX3, 2) morpholino (MO) knockdown and rescue assay and 3) detection of marker changes using in situ hybridization. With these assays we have functionally characterized these SIX3 mutations for the first time as significant loss-of-function alleles. For example, single point mutations in the eh1-like motif result in loss of function suggesting that interaction with groucho is essential for SIX3 activity. In addition, several nonsense mutations located in the SIX domain and homeodomain which result in early termination of the protein result in loss of function. Our data elucidate how SIX3 functions during development and increase our understanding of its role in the pathogenesis of HPE. Furthermore, these results are crucial for genetic counselling of families with children with HPE.