Molecular genetics of Meckel-Gruber syndrome, and functional characterization of meckelin and MKS1

Neural tube defects (NTDs) occur in 1 per 1,000 births, and cleft lip and palate occurs in 1 per 550 births. NTDs and clefting problems are the most common forms of birth defect. The most common syndromic form of NTD is Meckel-Gruber syndrome (MKS) which is an inherited condition with brain, liver and kidney defects that arise during embryonic development. In addition, cleft lip and palate is a common clinical feature of MKS. Recently, we found that MKS is caused by changes in a unique gene that makes a large, novel protein that we have called meckelin. Meckelin carries developmental signals from the outside to the inside of a cell, controlling how the cell will behave during embryonic development. This regulation is lost if meckelin is defective or absent. Meckelin is a component of primary cilia, which are finger-like projections from cells. Cilia are thought to detect and respond to chemical or mechanical cues, such as fluid flow, during the formation of the neural tube and other tubular structures. We aim to have a deeper understanding of the molecular roles of meckelin and other proteins found at cilia during embryonic development, and how these processes go wrong in human diseases. We hope that the work will also provide key insights into the causes of complex genetic diseases, including NTDs such as spina bifida, and cleft lip and palate.

This proposal requests funding for one Research Associate and one post-doctoral Research Fellow to continue with my groups? autozygosity mapping work to identify new genes in an existing cohort of MKS cases, and to continue the functional characterization of MKS1 and meckelin. An existing project grant is funding yeast two-hybrid (Y2H) assays to identify proteins that interact with either meckelin or MKS1. Since the function of these proteins is unclear, we feel that Y2H assays are an essential first step to identify putative downstream effectors or components in a multisubunit complex. This proposal therefore also requests funding to allow the validation of putative interactions with meckelin and/or MKS1 that will be identified by the existing Y2H screen. To validate interactions we will use both directed ?one-to-one? Y2H assays and independent biochemical methodologies. A further aim is to identify the ligands of meckelin, which is a novel orphan receptor of unknown function. This proposal therefore seeks MRC funding for the following work:
(a) To identify new loci and disease genes for MKS, exploiting our extensive clinical resource of DNA samples from consanguineous MKS families. We will utilize the strategy of autozygosity mapping in these families, using mixed affected samples hybridised to a single Affymetrix 50K SNP chip to identify chromosomal regions that are identical-by-descent (IBD). Candidate genes in the intervals highlighted will be prioritized, in the first instance, by interrogating the primary ciliary proteome followed by direct sequencing to identify putative pathogenic mutations
(b) To confirm any meckelin and MKS1 protein-interacting partners, identified from an existing LexA-based yeast two-hybrid (Y2H) assay by, firstly, performing directed ?one-to-one? Y2H assays and, secondly, a series of biochemical assays to validate interacting protein partners and downstream effectors. These analyses will include coimmunoprecipitations and/or pulldowns of GST fusion proteins etc. that will also create a resource of reagents (antisera, vector constructs, purified GST fusion proteins etc.)
(c) identify ligands of the novel meckelin receptor with expression cloning (?panning?) using a soluble form of meckelin, comprising the extracellular N-terminal domain (residues 1 to 557) that contains a novel cysteine-rich domain (CRD). An alternative strategy will be to use the soluble form of this domain as an affinity-purification reagent for cognate ligands.
(d) target interacting partners of meckelin and/or downstream components of signalling pathways with RNAi-mediated abrogation of gene expression to determine their effect on, for example, centriole migration and ciliogenesis