16-P021 Isolation of NOCE, a novel node and notochord-specific enhancer element from the Noto locus

Meckel–Gruber syndrome (MKS) is a rare lethal autosomal recessive disease that is the most common cause of syndromic neural tube defects. There are five causative genes, mapped to six known disease loci, whose products encode proteins involved in formation and function of primary cilia. One of these genes, MKS3 (located on chromosome 8q22.1), is now well-characterized and a number of mutations in this gene have now been identified in MKS patients, and for the allelic condition Joubert syndrome. MKS3 encodes meckelin, a putative Frizzled-like transmembrane receptor that localizes to the ciliary membrane and apical cell surface in ciliated cells. MKS3 mutation screening of MKS patients has revealed a number of missense, in-frame deletion and synonymous sequence variants of unknown pathogenic potential. To assess the pathogenicity of these variants in functional assays, we determined the subcellular localization of mutated forms of HA epitopetagged meckelin, in comparison to a wild-type construct and endogenous meckelin. We introduced patient mutations using a standard site-directed mutagenesis strategy. Ciliated cells were transiently transfected with these constructs, immunostained with anti-HA and anti-calreticulin (an ER marker) and visualized by confocal microscopy. This revealed diverse meckelin localizations, which differ from the wild-type and endogenous, including aberrant ER aggregation. Our results demonstrate that mislocalization of meckelin is the probable pathogenic mechanism for a number of mutations in MKS. On-going work is assessing the consequences of these mutations on non-canonical Wnt signaling.