Moon, Irene

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.

Objective CYP2C9 and CYP2C19 are highly polymorphic pharmacogenes that encodes enzymes that metabolize a large number of clinically relevant drugs. While the majority of variability in drug metabolism can be explained by a few common alleles, identification of gene sequence variation that alters enzyme activity and incorporation of that information into pharmacogenomic rules for clinical decision support might lead to improved therapy. Using sequence data derived from the Right Drug, Right Dose, Right Time: Using Genomic Data to Individualize Treatment (RIGHT Protocol), we sought to identify variants of unknown significance (VUSs) for CYP2C9 and CYP2C19, and to characterize the function of the encoded enzymes. Methods Targeted next‐generation sequencing for 84 pharmacogenes using the PGRN‐Seq v1.0 capture reagent was performed with DNA samples from 1013 RIGHT Protocol participants. Plasmids containing VUSs were created using site directed mutagenesis and were transiently expressed in COS‐1 cells. Initial determination of enzyme activity was performed using fluorogenic substrates. Enzyme kinetic parameters (Km and Vmax) for each of the variant allozymes were determined using liquid chromatography mass spectrometry with tolbutamide (CYP2C9) and mephenytoin (CYP2C19) as prototypic substrates. We also compared our results with in silico functional predictions by SIFT and Polyphen. Results Seven VUSs were identified for each gene, none of which had been functionally characterized. There were significant positive correlations between protein content and fluorometric enzyme activity for all variants for both CYP2C9 (P<0.05) and CYP2C19 (P<0.005). Notably, CYP2C9 V237L and CYP2C19 A173V activity was much lower than predicted based on protein content. The substrate intrinsic clearance of CYP2C9 variants P73L , S115R , V237L [6.23E‐05±1.08E‐05; 2.55E‐05±4.1E‐06; 7.28E‐05±2.4E‐05 (uL/mg protein/min)], and CYP2C19 variants V113I , A173V , R186C and R186H [2.49E‐04±2.78E‐05; 1.80E‐05±2.19E‐06; 2.33E‐05±9.2E‐06;1.45E‐04±1.2E‐05 (uL/mg protein/min)] were less than 25% of their respective WT enzyme . Conclusion Sequencing of CYP2C9 and CYP2C19 from 1013 subjects identified low frequency variants that had not been functionally characterized. In silico predictions which showed variable results, can be helpful but are not always accurate, hence functional validation is necessary for VUSs requiring protein expression and determination of kinetic parameters. Efforts to identify variation in diverse populations is resulting in a very large number of VUSs, so there is a need for the application of high throughput mutagenesis approaches and functional characterization methods to help pharmacogenomics contribute to precision medicine. Support or Funding Information NIH RO1 GM28157 and the Mayo Center for Individualized Medicine Pharmacogenomics Program.

Abstract The human angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) proteins play key roles in the cellular internalization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the coronavirus responsible for the coronavirus disease of 2019 (COVID-19) pandemic. We set out to functionally characterize the ACE2 and TMPRSS2 protein abundance for variant alleles encoding these proteins that contained non-synonymous single-nucleotide polymorphisms (nsSNPs) in their open reading frames (ORFs). Specifically, a high-throughput assay, deep mutational scanning (DMS), was employed to test the functional implications of nsSNPs, which are variants of uncertain significance in these two genes. Specifically, we used a ‘landing pad’ system designed to quantify the protein expression for 433 nsSNPs that have been observed in the ACE2 and TMPRSS2 ORFs and found that 8 of 127 ACE2, 19 of 157 TMPRSS2 isoform 1 and 13 of 149 TMPRSS2 isoform 2 variant proteins displayed less than ~25% of the wild-type protein expression, whereas 4 ACE2 variants displayed 25% or greater increases in protein expression. As a result, we concluded that nsSNPs in genes encoding ACE2 and TMPRSS2 might potentially influence SARS-CoV-2 infectivity. These results can now be applied to DNA sequence data for patients infected with SARS-CoV-2 to determine the possible impact of patient-based DNA sequence variation on the clinical course of SARS-CoV-2 infection.

Single nucleotide variants in the open reading frames (ORFs) of pharmacogenes are important causes of interindividual variability in drug response. Because of the very large number of missense variants in ORFs, it is difficult to link the genotypes of these variants to their phenotypes using “one at a time” expression systems. Therefore, the functional characterization of variants of unknown significance in clinically important pharmacogenes remains a major challenge in pharmacogenomics. Deep mutational scanning (DMS) is a high‐throughput technique that make it possible to analyze hundreds of different missense variants in a parallel and scalable fashion. Engineered “landing pad” HEK 293T cells were used as a platform to integrate pooled variant libraries, resulting in one variant per cell for further assessment. We adapted this DMS system to analyze the function of missense variants in the ORFs of CYP2C9 and CYP2C19 . The ORFs of CYP2C9 and CYP2C19 were fused to green fluorescent protein, and individual variants with different fluorescence intensities were used to indicate different levels of protein expression. Pooled variant libraries were generated by nicking mutagenesis, and we then used recombinase to integrate the libraries into landing pad cells, one per cell. Multiplexed functional selection performed with Fluorescence Activated Cell Sorting separated cells into different bins by fluorescence readout at the single–cell level. Amplicon sequencing of DNA collected in each bin, followed by computational analysis of the frequency of variants appearing in each bin, was used to determine levels of protein expression. To identify potentially severely damaging variants for these two important pharmacogenes, we studied 243 missense variants in the CYP2C9 and CYP2C19 ORFs that had been identified by the Exome Aggregation Consortium Project and by the Mayo Clinic RIGHT 1K Study. We found that 13 of 115 CYP2C9 and 25 of 128 CYP2C19 missense variants displayed less than approximately 25% of the wildtype protein expression, a level that may have clinical relevance for drug metabolism. Variant calling by the DMS method for the severely damaging variants was compared with the results of individual variant assay by western blot and with the predictions of computational algorithms such as Polyphen and SIFT. Our results suggest that DMS is an efficient method for the high‐throughput identification of damaging ORF variants that might have potential clinical application in pharmacogenomics. Support or Funding Information Funded by NIH grant U19 GM61388 (The Pharmacogenomics Research Network), R01 GM28157, R01 GM125633, T32 GM08685 and by the Mayo Clinic Center for Individualized Medicine This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Single nucleotide variants in the open reading frames (ORFs) of pharmacogenes are important causes of interindividual variability in drug response. The functional characterization of variants of unknown significance within ORFs remains a major challenge for pharmacogenomics. Deep mutational scanning (DMS) is a high‐throughput technique that makes it possible to analyze the functional effect of hundreds of variants in a parallel and scalable fashion. We adapted a “landing pad” DMS system to study the function of missense variants in the ORFs of cytochrome P450 family 2 subfamily C member 9 ( CYP2C9 ) and cytochrome P450 family 2 subfamily C member 19 ( CYP2C19 ). We studied 230 observed missense variants in the CYP2C9 and CYP2C19 ORFs and found that 19 of 109 CYP2C9 and 36 of 121 CYP2C19 variants displayed less than ~ 25% of the wild‐type protein expression, a level that may have clinical relevance. Our results support DMS as an efficient method for the identification of damaging ORF variants that might have potential clinical pharmacogenomic application.

Abstract The FKBP5 gene encodes the FKBP51 immunophilin, a negative regulator of AKT. Variability in level of FKBP5 expression is a major factor contributing to variation in response to several classes of chemotherapeutic agents including gemcitabine, first line treatment for pancreatic cancer. Genetic variation of FKBP5 could influence its expression, function, and, ultimately, treatment response of pancreatic cancer. We set out to comprehensively study the role of genetic variation in FKBP5 identified by Next Generation DNA resequencing in response to gemcitabine treatment of pancreatic cancer by utilizing both tumor and germline DNA samples from 43 pancreatic cancer patients, including 19 paired tumor-normal samples. Genotype-phenotype association studies with overall survival (in tumor only) as well as with FKBP5 gene expression were performed using the same samples in which Next Generation DNA resequencing had been performed, followed by functional genomic studies of top selected single nucleotide polymorphisms (SNPs). In-depth resequencing identified 404 FKBP5 SNPs in both normal and tumor DNA patient samples. SNPs with the smallest p-values for association with FKBP5 survival or expression were subjected to functional genomic studies. Electromobility shift assay (EMSA) showed that the rs73748206 “T” allele altered DNA-protein binding patterns, consistent with a significantly increased reporter gene activity. The effect of rs73748206 was also confirmed by mRNA expression studies using lymphoblastoid cell lines (LCL) derived from the same patients from whom DNA used for resequencing had been obtained. This comprehensive FKBP5 resequencing study provides insight into the role of inheritance in variation in response to gemcitabine therapy of pancreatic cancer. Citation Format: Katarzyna A. Ellsworth, Bruce W. Eckloff, Liang Li, Irene Moon, Brooke L. Fridley, Greg D. Jenkins, Erin Carlson, Abra Brisbin, Ryan Abo, William Bamlet, Gloria Petersen, Eric D. Wieben, Liewei Wang. Contribution of FKBP5 genetic variation to gemcitabine treatment and survival in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2209. doi:10.1158/1538-7445.AM2013-2209

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.

Abstract Aromatase (CYP19) is a critical enzyme in estrogen biosynthesis and aromatase inhibitors (AI) are employed widely for endocrine therapy in postmenopausal women with breast cancer. We hypothesized that single nucleotide polymorphisms (SNPs) in the CYP19 gene may alter the effectiveness of AI therapy in the neoadjuvant setting. Genomic DNA was obtained for sequencing from 52 women pre-AI and post-AI treatment in this setting. Additionally, genomic DNA obtained from 82 samples of breast cancer and 19 samples of normal breast tissue was subjected to resequencing. No differences in CYP19 sequence were observed between tumor and germ-line DNA in the same patient. A total of 48 SNPs were identified including 4 novel SNPs when compared with previous resequencing data. For genotype-phenotype association studies, we determined the levels of aromatase activity, estrone, estradiol, and tumor size in patients pre-AI and post-AI treatment. We defined two tightly linked SNPs (rs6493497 and rs7176005 in the 5'-flanking region of CYP19 exon 1.1) that were significantly associated with a greater change in aromatase activity after AI treatment. In a follow-up study of 200 women with early-stage breast cancer who were treated with adjuvant anastrozole, these same two SNPs were also associated with higher plasma estradiol levels in patients pre-AI and post-AI treatment. Electrophoretic mobility shift and reporter gene assays confirmed likely functional effects of these two SNPs on transcription of CYP19. Our findings indicate that two common genetic polymorphisms in the aromatase gene CYP19 vary the response of breast cancer patients to aromatase inhibitors. Cancer Res; 70(1); 319–28