Abstract SY14-02: From disease genetics to diagnostics to functional biology: High-throughput proteomics and protein interaction network building guides discovery of novel disease genes in ciliopathies, cystic disease, and cancer
Authors: Jackson Peter K. 
Published: June 2012

Abstract
With the advent of deep sequencing, genome-wide association studies, and rapid advances in human genetics, extensive genomic data linking human gene variants to specific diseases, including cancer, neurological disease, and metabolic syndromes, is rapidly accumulating. These disease associations, especially mutations, provide a definitive link between protein function and disease state, and can provide critical diagnostics for the disease. However, in many cases, the disease links are unclear and fail to 1) connect individual mutations to specific biological pathways, 2) provide functional hypotheses for understanding disease mechanism, or 3) identify targets for therapeutic intervention. Bioinformatic analyses can provide key clues to specific experimental tests, but a more extensible methodology is required to build from genomic hits to functional links. We have developed and implemented automated, a recombination-driven “tandem-affinity” proteomic probes, called the g-LAP (Gateway-Location and Affinity Purification) system. This approach is amenable to high-throughput and allows us to purify proteins associated with disease-related “bait” proteins and thus identify protein complexes and networks with high confidence. We have also established similarly high-throughput tools for studying protein localization, protein-protein interaction both in vivo and in vitro, and for testing interactions among biochemical complexes and activities.
To date, we have tagged over 100 new human genetic disease genes, which have lent insight into a number of important pathways, notably in ciliary diseases or ciliopathies, neurological disease, and for novel tumor suppressors. Within the ciliopathies, the identification of assembly and regulatory complexes has provided new insight into the cell biology and signaling pathways associated with primary cilia. In the case of Bardet-Biedl syndrome, patients present with retinal degeneration, olfactory and hearing loss, polydactyly, neurological problems, and obesity linked to hyperphagia. Purifying proteins associated with LAP-tagged BBS proteins, we discovered seven of fourteen known BBS disease genes were present in the highly conserved BBSome complex, important to activate Rab8 vesicles for ciliary membrane formation1. In the tubby/Tubby-like (TULP) family of proteins, we recently purified the Hedgehog regulatory TULP3 protein and found the protein forms a complex together with the complete, six-component intraflagellar transport A (IFT-A) complex, which function together at the base of the cilia to regulate entry of G-protein coupled receptors into cilia2.
A link between cilia and cancer was demonstrated by the link between cilia, Hedgehog signaling, and specific receptor tyrosine kinases, including the PDGF receptor α. Among cancers and proliferative diseases, specific ciliary pathways function in the kidney to limit polycystic kidney disease and renal cell carcinoma. Notably, the Von Hippel-Lindau (VHL) tumor suppressor protein and autosomal dominant polycystic kidney disease genes PKD1 and PKD2 proteins are present in primary cilia. Among ciliopathies, several may control the polarity and function of ductal structures of the kidney, but also of the pancreas, and liver. Do ciliary genes play an important role in cancer progression in these tissues? The molecular mechanism suppressing formation of cysts is poorly understood, but may include changes in the apical-basal polarity of cells, cell cycle, or secretory behavior of cells. Specific changes in ciliary signaling mechanisms may be important for cyst suppression.
Among cystic regulators, nephronophthisis (NPHP), Joubert (JBTS), and Meckel-Gruber (MKS) syndromes are autosomal-recessive ciliopathies presenting with cystic kidneys, retinal degeneration, and cerebellar/neural tube malformation. Nephronophthisis (NPHP) is a rare pediatric autosomal cystic kidney disease caused by mutations of nine known genes, NPHP1-9. NPHP patients also show cerebellar defects, situs inversus, and retinitis pigmentosa (RP). The pediatric Joubert (JBST) and Meckel-Gruber (MKS) syndromes also present nephronophthisis and RP, but are characterized by cerebellar malformation or severe neural tube defects. There are nine JBST and seven MKS genes, with considerable allelism among NPHP, JBST, and MKS genes. We used high-confidence proteomics to identify over 200 interactors copurifying with nine NPHP/JBTS/MKS proteins and discovered three connected modules functioning at the apical cell surface and ciliary “transition zone” (NPHP1/4/8), at the basal body (NPHP5/6), or in a novel Hedgehog pathway (Mks1/Mks6/Tectonic). We employed assays for ciliogenesis and cell polarity defects using 3-D renal cultures to demonstrate that renal cystic disease links primarily to apical organization defects, whereas ciliary and Hedgehog pathway defects appear central to neural malformations. Using 38 interactors as candidates, linkage and sequencing analysis of 250 NPHP patients identified TCTN2 as a new Joubert syndrome disease gene, plus two other novel NPHP human. Our Tctn2 mouse knockout showed neural tube and Hedgehog signaling defects. Our studies demonstrate the power of linking proteomic networks and human genetics to uncover critical disease pathways.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY14-02. doi:10.1158/1538-7445.AM2011-SY14-02