Dysfunction of the primary cilium has been associated with human syndromes called the ciliopathies. These include Nephronophthisis (NPHP) and Meckel Syndrome (MKS). Mutations in multiple genes have been identified in NPHP and MKS patients, but in most patients the affected genes are unknown and their function has remained elusive. Surprisingly, NPHP and MKS patients with very distinct disease phenotypes can have similar mutations in shared genes and although NPHP and MKS are recessive disorders, numerous patients have been identified that have only a single heterozygous change. In the absence of functional assessment;however, it is uncertain as to whether these changes are benign or are pathogenic. These findings have led to the hypothesis that phenotypic outcome may be determined through interactions of these heterozygous mutations with additional mutations in other cilia genes in the patients'backgrounds. In support of this model is our data showing that the NPHP and MKS homologs in C. elegans form two distinct complexes that have strong genetic interactions. Mutations affecting both complexes simultaneously cause synergistic effects on the phenotype that are not seen in compound mutations affecting only one of the complexes. Further our data indicate that the NPHP and MKS proteins are collectively required for the formation of a region called the transition zone (TZ). The TZ is a structure located at the base of the cilium that mediates a connection between the microtubule axoneme of the cilium and the overlying membrane. We currently know very few proteins that are directly required for TZ formation. We hypothesize that the NPHP and MKS proteins are key components in the TZ and further that they establish a barrier influencing protein entry, exit and retention in the ciliary compartment. Thus, the NPHP and MKS proteins would have critical roles in allowing the cilium to form a specialized compartment which is distinct from the rest of the cell. In this proposal we will utilize comparative approaches in C. elegans, mouse models, and mammalian cell lines to dissect the role of the NPHP and MKS proteins. We will analyze how genetic interactions between the NPHP and MKS mutations influence disease phenotypes in C. elegans and mouse models, determine the direct role of the NPHP and MKS proteins in TZ formation, maintenance, and its function as a restrictive barrier utilizing high-resolution localization and in vivo imaging approaches. We will also determine the consequence that missense mutations, which have been identified in human patients, have on protein localization, function, and on formation of the NPHP and MKS complexes. Finally, based on the synergistic effects observed in double nphp and mks C. elegans mutants, we propose an innovative mutagenesis screen that will identify additional proteins required for MKS and NPHP complex formation that will be novel candidate loci involved in human NPHP and MKS patients. These data will result in a better understanding of how cilia dysfunction causes the wide spectrum of disease phenotypes in human patients and contribute to eventual treatment strategies.

Public Health Relevance

Nephronophthisis (NPHP) and Meckel Syndrome (MKS) are severe human disorders resulting in perinatal lethality, cystic kidney disease, and blindness that are caused by the dysfunction of a small structure called the cilium. Numerous proteins involved in NPHP and MKS have now been identified;however, we have a poor understanding of what these proteins actually do in a cell and how these mutations affect the function of these proteins. Thus, the overall objective of this proposal is to analyze the activity and interactions between normal and mutant forms of the NPHP and MKS genes and corresponding proteins to provide important molecular and cellular insights into the basis of these disorders, and even more significant, to assess how loss of normal cilia activity can contribute to human disease phenotypes.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Special Emphasis Panel (ZRG1-DKUS-A (11))
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Rasooly, Rebekah S
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University of Alabama Birmingham
Anatomy/Cell Biology
Schools of Medicine
United States
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