Molecular inversion probe (MIP)-based multiplexed target capture for pharmacogenomic analysis

Abstract

As the target capture platform for the pharmacogenomics analysis, we designed a panel of molecular inversion probes to cover 80 genes which can affect exposure and response to drugs. By stepwise rebalancing, probes were optimized to capture targets with high coverage (96%) and accuracy (99.8%). The panel features the efficient probe preparation as initial microarray-based synthesis of duplex MIPs (microDuMIPs) is applied. Target capture and next-generation sequencing (NGS) of 191 individuals with this panel identified a number of unreported low-frequency genetic variants in targeted genes. In silico prediction showed genetic variants at low frequency enriched for ones with deleterious functional impacts. By further in vitro investigation, selected rare or low-frequency variants were validated to alter functions in influx/efflux transporters (SLC22A1/OCT1, SLCO1B1/OATP1B1, ABCB1/MDR1 and a metabolizing enzyme (CYP2C19). This implicates genetic variants at low frequency, which might have been overlooked in past pharmacogenetic studies, may bear relevance to the inter-individual difference in response to drugs. As a follow-up, we applied the platform to capture DNA sequences of 90 participants in phase 1 clinical trials for Tacrolimus, an immunosuppressive drug known for large inter-individual variance in pharmacokinetics (PK). Besides CYP3A5*3, an established index variant for the PK variability, POR*28 and rare variants in UGT1A4 were revealed to be the account for the part of the PK variability. We verified that the POR*28 allele decreased exposure to drug, distinctively in CYP3A5 full expressers. Rare variants in UGT1A4 may cause variable extent of glucuronidation as phase II metabolism of secondary importance, given that this genetic effect is conspicuous in phenotypic extremes with impaired CYP3A5 function (*1/*3 and *3/*3). To the best of our knowledge, this is the first time to implicate the association of genetic variations in UGT1A4 with variable responses to Tacrolimus. Collectively, POR*28 and rare variants in UGT1A4 can explain the PK variance approximately 6% more than solely by CYP3A5*3. These results suggest that not only common but also rare variants are worth being considered for pharmacogenomic analysis and testing. Our platform also presents a proof of concept that NGS-based pharmacogenomic testing at earlier phase of clinical trials can provide the foundation for understanding metabolic pathways of drugs by extensive genetic screening including rare variants scan. Moreover, this shows that MIP-based multiplexed target capture can be one of effective tools to investigate individualized metabolic pathways of drugs.