Application of second and third generation sequencing in pharmacogenetics

Abstract

The next generation sequencing technologies have advanced pharmacogenetics by enabling rapid assessment of the entire exome or even whole genome for associations between variants or gene regions with drug response. Third generation sequencing such as nanopore sequencing overcomes the limitations of short read sequencing by offering low capital cost and advantages in deciphering complex genes, detecting structural variants, and providing accurate haplotypes. The first aim of this thesis was to explore nanopore sequencing for pharmacogenetic implementation. The second part of this thesis applied genomic technologies to investigate genetic and epigenetic contributors to a rare but serious adverse drug reaction. At the outset of this thesis, a multiplex assay comprising common and rare genetic variants attributed to response variability to clopidogrel and warfarin was developed and validated on the nanopore sequencing technology. A one-tube multiplex PCR was employed to amplify 15 regions containing 27 variants in seven genes. Use of sample barcodes enabled inclusion of 84 samples in one sequencing run to provide a cost-effective assay. With the relatively error-prone state of the nanopore sequencing device and chemistry at the time of study, accuracy of variant genotyping relied heavily on the choice of bioinformatic tools and was sequence-dependent. Using Albacore v2 as the basecalling tool and nanopolish or Clairvoyante as variant calling tools, accuracy of ≥90% was achieved for all variants except one. Nanopolish resulted in <90% accuracy only for VKORC1 rs9923231 and Clairvoyante resulted in <90% only for CYP2C19*2 rs4244285. Accuracy did not improve by increasing read depth. This illustrated that it was possible to develop multiplexed, targeted genotyping assays on the nanopore platform, although further improvements in the platform may be required to obtain optimal accuracy. Nanopore sequencing offers great potential for genotyping complex genes such as CYP2D6, which is hindered by the very polymorphic nature of the gene, high homology with its pseudogene CYP2D7, and the occurrence of structural variants. Therefore, a second goal of this PhD was to establish and apply an effective nanopore sequencing method for CYP2D6. DNA amplicons encompassing the CYP2D6 gene from seven reference samples of known genotype (Coriell Institute, Camden, NJ, USA), and 25 clinical samples from adverse drug reaction cohorts were sequenced and analysed over two sequencing runs on the nanopore sequencer. All alleles were genotyped and all duplicated alleles were determined accurately. In addition, five novel variants, one novel allele, CYP2D6*127, and seven novel subvariants were identified in this study. Although the method proved efficient and generally effective, it was noteworthy that the presence of a hybrid allele CYP2D6*68 in two of the reference samples was undetected by this method. Although pharmacogenetic studies have been fruitful in elucidating the response variability of a large number of drugs, variation in response to many other drugs remains unresolved. An example is angioedema induced by angiotensin converting enzyme inhibitor (ACEi-A). This potentially fatal reaction occurs in approximately three of 1000 individuals taking the drugs. Genome-wide analysis by whole exome sequencing was performed for five DNA samples from participants having this ADR and whole genome sequencing was performed on another 15 samples. Initial analysis on 15 previously reported variants potentially associated with the ADR did not show any significant enrichment in the ACEi-A group compared to population allele frequency. Analysis on the whole exome and whole genome datasets only resulted in one rare coding variant found to be consistently enriched in both datasets, rs4365 in ACE, with allele frequency of 40% in the ACEi-A cohort compared to 3% in the population. This synonymous variant is predicted to be benign, and the possible significance of this variant to ACEi-A is yet to be elucidated. A number of moderately enriched variants were identified, however, no single rare variant with minor allele frequency of <1% was found in more than seven of the 15 whole genome sequencing datasets. Limited CNV analysis on subsets of samples did not find any variant affecting candidate genes. Further understanding of the genetic make-up of this rare ADR will likely benefit from collection of more cases and application of different approaches including analysis of combined effect of multiple variants, analysis of the ADR as complex trait, or more comprehensive analysis of structural variants. As attempts to find the genetic variants responsible for ACEi-A did not implicate any strong candidates; the potential of epigenetic regulation involvement in this ADR was explored by investigating the effect of ACEi on DNA methylation in clinical samples and cell culture model. Analysis of existing methylation array data from patient samples with and without ACEi consumption showed a putative differentially methylated CpG site on chromosome 10. However, this was not validated in the in vitro experiment. Methylation array data from the cell culture experiment resulted in several potential CpG sites showing differential methylation, however none of these was validated using bisulfite amplicon sequencing. Overall, this work showed little evidence of effect of ACEi on DNA methylation, and it seems unlikely that DNA methylation change by ACEi is involved in the association of angioedema with the drug. The work in this thesis applied various approaches and technologies to advance knowledge in pharmacogenetics. Nanopore sequencing was shown to be beneficial for pharmacogenetic applications and with the continual improvement in accuracy, this technology will continue to be a valuable tool, especially in elucidating complex structural variants and providing accurate haplotype information. Short read sequencing technologies for whole exome or whole genome sequencing are useful for interrogation of rare variants; however other approaches might be needed to elucidate the basis of potentially complex phenotypes such as ADR. Lastly, studies of epigenetic regulation in pharmacogenetics are still limited, and the potential role of epigenetics in ADR merits further exploration.