Liu, Duan

BACKGROUND Most functionally‐significant single nucleotide polymorphisms (SNPs) are outside of open reading frames. Mechanisms by which these SNPs contribute to gene regulation are poorly understood. Aryl hydrocarbon receptor (AHR) is a nuclear receptor that regulates drug metabolism, xenobiotic response and immune response. Our previous data indicate that SNPs outside response elements can impact transcription factor binding and gene regulation. We hypothesized that SNPs near but outside AHR response elements (AhREs) might impact AHR binding and gene regulation. METHODS We have collected dense SNP‐genotypes across 300 lymphoblastoid cell lines (LCLs) – a powerful tool for generating and testing pharmacogenomic hypotheses. Eight LCLs were used to generate hypotheses about AHR SNP‐dependent transcription utilizing RNA‐ and ChIP‐seq, which were tested in additional LCLs using qRT‐PCR and ChIP after AHR agonist or antagonist treatment. RESULTS 69 genes were highly significantly differentially expressed after agonist or antagonist treatment and 80% of those genes had an AhRE within 50kb. Five SNP‐gene pairs have been functionally validated in additional LCLs (Table 1). CONCLUSION SNPs that are outside of AhREs can alter AHR binding and gene transcription. This is a general phenomenon that may have large‐scale influence on gene transcriptional regulation and pharmacogenomics. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Several antiepileptic drugs (AEDs) have US Food and Drug Administration (FDA) approval for use as mood stabilizers in bipolar disorder (BD), but not all BD patients respond to these AED mood stabilizers (AED‐MSs). To identify genetic polymorphisms that contribute to the variability in AED‐MS response, we performed a discovery genome‐wide association study (GWAS) of 199 BD patients from the Mayo Clinic Bipolar Disorder Biobank. Most of these patients had been treated with the AED‐MS valproate/divalproex and/or lamotrigine. AED‐MS response was assessed using the Alda scale, which quantifies clinical improvement while accounting for potential confounding factors. We identified two genome‐wide significant single‐nucleotide polymorphism (SNP) signals that mapped to the THSD7A (rs78835388, P = 7.1E‐09) and SLC35F3 (rs114872993, P = 3.2E‐08) genes. We also identified two genes with statistically significant gene‐level associations: ABCC1 (P = 6.7E‐07; top SNP rs875740, P = 2.0E‐6), and DISP1 (P = 8.9E‐07; top SNP rs34701716, P = 8.9E‐07). THSD7A SNPs were previously found to be associated with risk for several psychiatric disorders, including BD. Both THSD7A and SLC35F3 are expressed in excitatory/glutamatergic and inhibitory/γ‐aminobutyric acidergic (GABAergic) neurons, which are targets of AED‐MSs. ABCC1 is involved in the transport of valproate and lamotrigine metabolites, and the SNPs in ABCC1 and DISP1 with the strongest evidence of association in our GWAS are strong splicing quantitative trait loci in the human gut, suggesting a possible influence on drug absorption. In conclusion, our pharmacogenomic study identified novel genetic loci that appear to contribute to AED‐MS treatment response, and may facilitate precision medicine in BD.

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.

We previously reported that testis‐specific Y‐encoded‐like protein (TSPYLs) are transcription regulators for CYP3A4, CYP2C9, and CYP2C19. Here, we observed dual roles for TSPYLs in mediating serotonin transport and the metabolism of selective serotonin reuptake inhibitors (SSRIs) in patients with major depressive disorder (MDD). The widely prescribed SSRIs, citalopram, and escitalopram are metabolized mainly by CYP2C19. The TSPYL1 rs3828743 single nucleotide polymorphism (SNP), which decreases its suppression of CYP2C19 expression, was associated with rapid escitalopram metabolism and worse treatment response in the Mayo PGRN‐AMPS clinical trial. We also found that TSPYLs can regulate expression of the serotonin transporter protein, SLC6A4, and, in turn, serotonin transport into cells. The SNPs in tight linkage disequilibrium with the TSPYL1 rs10223646 SNP were significantly correlated with baseline severity of depression in patients with MDD in the Sequenced Treatment Alternatives to Relieve Depression and International SSRI Pharmacogenomics Consortium clinical trials. Our findings suggest that genetic variation in TSPYL genes may be novel indicators for baseline severity of depression and SSRI poor response.

Anastrozole is a widely prescribed aromatase inhibitor for the therapy of estrogen receptor positive (ER+) breast cancer. We performed a genome‐wide association study (GWAS) for plasma anastrozole concentrations in 687 postmenopausal women with ER+ breast cancer. The top single‐nucleotide polymorphism (SNP) signal mapped across SLC38A7 (rs11648166, P = 2.3E‐08), which we showed to encode an anastrozole influx transporter. The second most significant signal (rs28845026, P = 5.4E‐08) mapped near ALPPL2 and displayed epistasis with the SLC38A7 signal. Both of these SNPs were cis expression quantitative trait loci (eQTL)s for these genes, and patients homozygous for variant genotypes for both SNPs had the highest drug concentrations, the highest SLC38A7 expression, and the lowest ALPPL2 expression. In summary, our GWAS identified a novel gene encoding an anastrozole transporter, SLC38A7, as well as epistatic interaction between SNPs in that gene and SNPs near ALPPL2 that influenced both the expression of the transporter and anastrozole plasma concentrations.

The aryl hydrocarbon receptor (AHR) is a nuclear receptor that modulates the response to environmental stimuli. It was recognized historically for its role in toxicology but, in recent decades, it has been increasingly recognized as an important modulator of disease—especially for its role in modulating immune and inflammatory responses. AHR has been implicated in many diseases that are driven by immune/inflammatory processes, including major depressive disorder, multiple sclerosis, rheumatoid arthritis, asthma, and allergic responses, among others. The mechanisms by which AHR has been suggested to impact immune/inflammatory diseases include targeted gene expression and altered immune differentiation. It has been suggested that single nucleotide polymorphisms (SNPs) that are near AHR-regulated genes may contribute to AHR-dependent disease mechanisms/pathways. Further, we have found that SNPs that are outside of nuclear receptor binding sites (i.e., outside of AHR response elements (AHREs)) may contribute to AHR-dependent gene regulation in a SNP- and ligand-dependent manner. This review will discuss the evidence and mechanisms of AHR contributions to immune/inflammatory diseases and will consider the possibility that SNPs that are outside of AHR binding sites might contribute to AHR ligand-dependent inter-individual variation in disease pathophysiology and response to pharmacotherapeutics.

AbstractBackgroundRenal dysfunction occurs commonly after heart transplantation (HTx) with wide inter‐individual variability but whether a genetic predisposition exists in these patients is unknown. Genomewide association studies (GWAS) have not been performed to assess the association of genetic variation with change in renal function after HTx.MethodsClinical and demographic data of patients who underwent HTx and provided blood samples and consent for genetic analysis were included. Genotyping was performed using Illumina Infinium Human CoreExome v1.0 analysis kit. A GWAS utilizing linear regression models was performed with estimated glomerular filtration rate (eGFR) at 1 year as the phenotype after adjusting for baseline eGFR prior to HTx and conversion from calcineurin inhibitor to sirolimus as primary immunosuppression therapy.ResultsA total of 251 HTx recipients were genotyped for 314,903 single nucleotide polymorphisms (SNPs). The mean (SD) age was 50 (12.5) years; most patients were of European origin (n = 243, 96.8%) and males (n = 179, 71.3%). After adjustment for potential confounders, two variants, rs17033285 (P = 4.3 × 10−7) and rs4917601 (P = 6.46 × 10−7), in a long non‐coding RNA (lncRNA) gene LINC01121 and a pseudogene BTBD7P2, were identified to have a significant association with change in GFR at 1 year after HTx.ConclusionsOur first of its kind GWAS demonstrates that genetic variation affects renal function after HTx independent of other risk factors. Agnostic genetic approaches such as these may lead to identification of novel biological pathways such as the role of lncRNAs in the development of renal dysfunction post‐HTx.

The testis‐specific Y‐encoded‐like protein (TSPYL) gene family includes TSPYL1 to TSPYL6. We previously reported that TSPYL5 regulates cytochrome P450 (CYP) 19A1 expression. Here we show that TSPYLs, especially TSPYL 1, 2, and 4, can regulate the expression of many CYP genes, including CYP17A1, a key enzyme in androgen biosynthesis, and CYP3A4, an enzyme that catalyzes the metabolism of abiraterone, a CYP17 inhibitor. Furthermore, a common TSPYL1 single nucleotide polymorphism (SNP), rs3828743 (G/A) (Pro62Ser), abolishes TSPYL1's ability to suppress CYP3A4 expression, resulting in reduced abiraterone concentrations and increased cell proliferation. Data from a prospective clinical trial of 87 metastatic castration‐resistant prostate cancer patients treated with abiraterone acetate/prednisone showed that the variant SNP genotype (A) was significantly associated with worse response and progression‐free survival. In summary, TSPYL genes are novel CYP gene transcription regulators, and genetic alteration within these genes significantly influences response to drug therapy through transcriptional regulation of CYP450 genes.

Abstract Introduction: Small studies have indicated a possible correlation between a HER2 gene polymorphism at codon 655 and trastuzumab-associated cardiotoxicity. Association between a synonymous coding variant rs7853758 within the SLC28A3 gene and anthracycline induced cardiotoxicity has also been reported. This study aimed to validate these correlations and assess for any relationship with prognosis. Methods: Genomic DNA was isolated from 666 patients enrolled in a large trial of adjuvant chemotherapy in HER2 amplified early breast cancer (BCIRG 006). Genotyping was conducted using Sequenom MassARRAY System for HER2 G->A polymorphism at amino acid codon 655 (rs1136201) and variant rs7853758 (L461L) within the SLC28A3 gene. Results: Of the 666 patients analyzed, 216 patients were treated with anthracycline based therapy, 226 with trastuzumab based therapy, and 224 with regimens containing both an anthracycline and trastuzumab. Compared with the overall results of the BCIRG006 study (N=3,222), in the subset of patients genotyped in this analysis, a less robust improvement in disease free survival (DFS) was observed for the trastuzumab arms than control arm (HR, 0.821). When stratified for prognostic features, the hazard ratio in favor of trastuzumab was consistent with that of the overall study (HR, 0.674). Samples from 662 patients were successfully genotyped for rs1136201. Of these, 424 (64%) were AA, 30 (4.5%) were GG, 208 (31%) were AG genotype. Samples from 665 patients were successfully genotyped for rs7853758. Of these, 19 (3%) were AA, 475 (71%) were GG, and 171 (26%) were AG genotype. There was no correlation seen between mean left ventricular ejection fraction (LVEF) and HER2 genotype at codon 655 in patients treated with trastuzumab. Of patients tested for the HER2 polymorphism, cardiac dysfunction [defined as > 10% decline in LVEF or clinical congestive heart failure (CHF)] developed in 16% of patients with AA, 17% of patients with GG and 20% of patients with AG. There was also no correlation between mean LVEF and variant rs7853758 in patients treated with anthracyclines. The percentage of patients who developed cardiac dysfunction was 13%, 17% and 21% in AA, GG, and AG genotypes respectively. No correlation between disease free survival and any of the genotypes was seen. Conclusion: In the largest analysis to date to evaluate for relationship between cardiac toxicity and HER2 polymorphism, we did not find a correlation with rs1136201 HER2 polymorphism and trastuzumab induced cardiac toxicity. Our study also did not show a correlation between variant rs7853758 (L461L) and anthracycline induced cardiotoxicity. Neither polymorphism correlated with prognosis. Citation Format: Peddi PF, Hurvitz SA, Fasching PA, Wang L, Cunningham J, Weinshilboum RM, Liu D, Quinaux E, Fourmanoir H, Robert NJ, Valero V, Crown J, Falkson C, Brufsky A, Pienkowski T, Eiermann W, Martin M, Bee V, Slamon DJ. Genetic polymorphism and correlation with treatment induced cardiotoxicity and prognosis in HER2 amplified early breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-03-09.