Stilling, Gail A.

Background: The primary role of natriuretic peptide receptor 3 (NPR3) is clearance of natriuretic peptides. Genetic variation in NPR3 has been associated with blood pressure regulation and obesity. Despite its importance, variation in NPR3 characterizing coding single nucleotide polymorphisms (SNPs) has not been addressed by resequencing the gene. Objective: We set out to identify and functionally characterize common variation in NPR3 in 3 ethnic groups. DNA samples from such subjects are studied because common genetic variation needs to be defined initially in “normal” individuals prior to studying drug effect and response. Also, rare variants identified by sequencing are enriched in complex disease traits. Methods and Results: DNA samples from 96 European-American, 96 African-American, and 96 Han Chinese-American healthy subjects were used to resequence NPR3. There were 105 polymorphisms identified of which 50 were novel, including 8 nonsynonymous (ns) SNPs of which 7 were novel. Expression constructs for the nsSNPs were created and HEK-293 cells were transfected with constructs for wild type (WT) and the variants. Recombinant proteins were analyzed by quantitative Western blot analysis. A significant reduction in NPR3 immunoreactive protein (60% or less of WT) was observed for the following variants Cys3, Arg146, Val499, and Ser477+Asp521. The Arg146 novel polymorphism resulted in the least protein expression (20% of WT). After treatment of WT and Arg146 cells with proteasome (MG132) and autophagy (3MA) inhibtors, it was determined that autophagy and not proteasomes plays an important role in the degradation of the Arg 146 variant protein. Modeling based on the NPR3 x-ray crystallography structure confirmed that the Arg146 variant were not compatible with WT conformation and could result in protein misfolding or instability. Conclusions: Multiple novel polymorphisms of NPR3 were identified in 3 ethnic groups. There were 4 variant receptors that displayed a significant decrease in NPR3 protein quantity, Arg146 being the variant with the least expression. Degradation of the variant Arg146 was mediated predominantly by autophagy. This SNP could have a significant effect on the metabolism of natriuretic peptides with clinical implications.

Background— The primary role of natriuretic peptide receptor-3 (NPR3) or NPR-C is in the clearance of natriuretic peptides that play an important role in modulating intravascular volume and vascular tone. Genetic variation in NPR3 has been associated with variation in blood pressure and obesity. Despite the importance of NPR3, sequence variation in the gene has not been addressed using DNA from different ethnic populations. We set out to identify and functionally characterize genetic variation in NPR3 in 3 ethnic groups. Methods and Results— DNA samples from 96 European American, 96 African American, and 96 Han Chinese American healthy subjects were used to resequence NPR3 exons, splice junctions, and flanking regions. We identified 105 polymorphisms, 50 of which were novel, including 8 nonsynonymous single-nucleotide polymorphisms, 7 were novel. Expression constructs were created for the nonsynonymous single-nucleotide polymorphisms. HEK293 cells were transfected with constructs for wild type and variant allozymes; and recombinant proteins were measured by quantitative Western blot analysis. The most significant change in NPR3 protein was observed for the Arg146 variant allozyme, with 20% of wild-type protein, primarily because of autophagy-dependent degradation. NPR3 structural modeling confirmed that the Arg146 variant protein was not compatible with wild-type conformation and could result in protein misfolding or instability. Conclusions— Multiple novel NPR3 genetic polymorphisms were identified in 3 ethnic groups. The Arg146 allozyme displayed a significant decrease in protein quantity because of degradation mediated predominantly by autophagy. This genetic variation could have a significant effect on the metabolism of natriuretic peptides with potential clinical implications.