Abstract
Abstract only The accumulation of human genetic disease mutations underlies a need to understand protein networks altered in these diseases. We build high‐confidence protein‐protein interaction networks, using unbiased Tandem Affinity Purification‐mass spectrometry (AP‐MS). Using the gLAP Gateway recombination‐based vector system, and Flp‐recombinase enabled “Flip‐in” lines, we construct stable cell lines expressing GFP‐tagged, correctly localized proteins for AP/MS‐based protein identification. We tagged 12 human genetic disease proteins from Nephronophthisis (NPHP), Joubert (JBTS), and Meckel‐Gruber (MKS) syndromes. These autosomal recessive diseases have defects in the primary cilium, a hair‐like signaling structure on cells. These “ciliopathies” show cystic kidneys, retinal degeneration, and neural malformations. We mapped high confidence interacting networks with NPHP/JBTS/MKS proteins and discovered 800 proteins in connected protein modules that function (1) at the ciliary base; (2) at centrosomes; (3) in the pericentriolar material; and (4) around the ciliary axoneme, suggesting how proteins are trafficked in cilia. Using these interactors as candidates, linkage and sequencing analysis of 250 patients identified new ciliopathy disease genes: PDE6D, SCDDAG8, Ataxin 10 and Tectonic 2. A Tectonic 2 knockout mouse shows a similar neural phenotype. Our study illustrates the power of linking proteomic networks and human genetics to uncover critical disease pathways.