Pharmacogenomics
References 1 Qiao, W, Yang, Y, Sebra, R, Mendiratta, G, Gaedigk, A, Desnick, RJ and Scott, SA (2016). Long-Read Single Molecule Real-Time Full Gene Sequencing of Cytochrome P450-2D6. Hum. Mutat., 37:315-323. doi: 10.1002/humu.22936.
http://onlinelibrary.wiley.com/ doi/10.1002/humu.22936/ abstract. 2 Buermans, HPJ, Vossen, RHAM, Anvar, SY, Allard, WG, Guchelaar, H-J, White, SJ, den Dunnen, JT, Swen, JJ and van der Straaten, T (2017). Flexible and Scalable Full-Length CYP2D6 Long Amplicon PacBio Sequencing. Hum. Mutat., 38: 310-316. doi: 10.1002/humu.23166.
http://onlinelibrary.wiley.com/d oi/10.1002/humu.23166/full. 3 Seo, J, Rhie, A, Kim, J, Lee, S et al (13 October 2016). De novo assembly and phasing of a Korean human genome. Nature 538, 243–247, doi:10.1038/nature20098.
http://www.nature.com/nature/ journal/v538/n7624/full/nature2
0098.html. 4 Moya, G, Dorado, P, Ferreiro, V, Naranjo, MEG, Peñas-Lledó, EM and LLerena, A (12 April 2016). High frequency of CYP2D6 ultrarapid metabolizer genotypes in an Ashkenazi Jewish population from Argentina. The
Pharmacogenomics Journal. doi:10.1038/tpj.2016.27.
http://www.nature.com/tpj/jour nal/vaop/ncurrent/full/tpj20162
7a.html. 5 Jittikoon, J, Mahasirimongkol, S, Charoenyingwattana, A, Chaikledkaew, U et al (2016). Comparison of genetic variation in drug ADME- related genes in Thais with Caucasian, African and Asian HapMap populations. Journal of Human Genetics 61, 119- 127; doi:10.1038/jhg.2015.115.
http://www.nature.com/jhg/jou rnal/v61/n2/abs/jhg2015115a. html.
variations. Sanger sequencing is laborious and costly, making it an unrealistic option for the high- throughput demands of drug discovery. NGS offers a higher-capacity means of interrogating the gene and requires no a priori knowledge, but most tools generate reads only a few hundred bases long. These snippets get stitched together, but with the
complexity and repetition of CYP2D6
sequence and its pseudogene, many reads get con- flated or misaligned. The end result is an error- prone alignment that makes it all but impossible to call a CYP2D6 profile with accuracy or to iden- tify novel CYP2D6 alleles.
These limitations have prevented scientists from discovering the full universe of natural CYP2D6 variation, and therefore from identifying drug metabolism profiles for all individuals. Having this information is essential for removing confounding variables from pharmacogenomic studies and other projects designed to understand a drug’s activity in each person.
A new approach Recently, scientists have reported results from eval- uating SMRT sequencing to call CYP2D6 profiles. With its multi-kilobase-long reads, this technology can fully span the gene and its copies in a single amplicon. Read length is important for phasing, too, making it possible to link distant SNPs or other types of variation. At the Icahn School of Medicine at Mount Sinai, a team led by Stuart Scott, Wanqiong Qiao and Yao Yang conducted the first evaluation of SMRT sequencing for the CYP2D6 gene1. They used long-range PCR to build 5kb amplicons cov- ering the entire gene as well as any upstream or downstream copies.
The approach was first validated on a set of Coriell DNA samples that had been previously classified with other CYP2D6 profiling tools. Results from the SMRT sequencing pipeline were concordant with known profiles for all 10 samples, and in some cases showed variation that had not been found with the other technologies. With that information, scientists were able to spot novel alle- les and allele-specific duplication while refining genotype calls for some samples.
Continued on page 28 26
Next, the team moved on to samples that had been profiled before but had yielded ambiguous results. With SMRT sequencing, they were able to resolve the full amplicon sequence without any need for assembly, eliminating the troublesome step that had caused some of the previously incon- clusive findings for those 14 samples. The new long-read data offered clear explanations for the
discrepancies seen earlier, providing improved res- olution and structural variant detection. Perhaps the most important result of this inves- tigation was the discovery that existing CYP2D6 profiling methods misclassify an individual’s geno- type more often than expected. While the sample numbers in the study were small, each testing series led to the revision of genotype calls for some 20% of samples. Many of these changes involved report- ing novel alleles, while others reclassified samples to rare alleles not covered by common CYP2D6 genotyping platforms. The scientists were not looking for novel alleles, but found three of them anyway. For a region as well-characterised as CYP2D6, this was an unexpected finding that sug- gests there is far more variation in this gene that has been missed due to technical limitations. Since that original study, the Mount Sinai team has improved on the method to make it robust for routine lab use. They incorporated barcoding and a higher-throughput platform for SMRT sequenc- ing to allow for multiplexing as many as 384 sam- ples in a run, generating better than 100-fold cov- erage of CYP2D6 for each sample. In a separate project, Henk Buermans and Tahar van der Straaten led a team at Leiden University Medical Center to assess the performance of SMRT sequencing for CYP2D6 profiling2. Like the Mount Sinai approach, these scientists used long-range PCR and barcoded amplicons to cover the gene and its copies. However, they used a two-step barcoding system and a longer amplicon (6.6kb) to generate more information in a streamlined protocol. The Leiden scientists sequenced 24 samples, comparing long-read
results to data generated
from a standard CYP2D6 genotyping platform. The SMRT sequencing approach produced full- length CYP2D6 sequences for all individuals, and results were in agreement with the genotyping calls. Of the variants detected, nearly 10% were unique and many were novel, including SNPs, insertions and deletions. The team was able to refine genotype calls based on the long-read data and add to the community’s understanding of nat- ural variation in the gene.
Population studies
As the catalogue of possible CYP2D6 alleles has been fleshed out, it has become increasingly possi- ble to associate some specific metaboliser profiles with certain populations where those genotypes are most common. The rise of population-focused genome projects has also contributed insight by finding variants that appear to exist only in specif- ic groups.
Drug Discovery World Summer 2017
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72