Allele definition

How to obtain PGx allele definitions from literature

PGx alleles are collected and distributed through various channels

• PGx alleles on JSON-LD format from the PharmGKB API
• PGx alleles on Excel-style formats, also accessible through the PharmGKB API (seems to be hidden from the Swagger documentation, but direct links when searching for haplotype definitions at the pharmgkb.org website)
• PGx alleles for use in PharmCAT are included in their source code. It is reasonable to suspect that these files are parsed from the Excel files.
• PGx alles for use in Aldy are included in their source code. Due to the lack of GRCh37 variant definitions for CYP2D6, Aldy has likely lifted over GRCh38 variants to GRCh37. The Aldy developers have also added new star alleles with (non functional) variants that are often observed together with the main star allele.
• PGx alleles as VCF files from PharmVar

How to chose the correct PGx allele definitions

Chosing an appropriate genomic reference build

PGx allele definitions are given in either GRCh37 or GRCh38 reference coordinates. PharmCAT, CPIC and PharmGKB as a rule use build GRCh38. The process of changing from GRCh37 to GRCh38 for the PharmGKB API seems to be only partially completed. For instance, in the PharmGKB API JSON-LD data, the build is given as "hg38", but the actual coordinates are mostly GRCh37 (hg19). In the PharmGKB and CPIC Excel sheets, the move to GRCh38 is completed. As is evident from liftOver, hg19 is not equal to GRCHh37, nor hg38 to GRCHh38. Are we sure that there are no errors here?

Chosing the correct variants to include in the PGx alleles

The allele definitions from PharmGKB and PharmVar are not always one-to-one, and some background knowledge about why this is, is required.

Additional prefiltering of PharmGKB or PharmVar allele definitions by domain experts may be needed. Prefiltering was performed by the PGx pipeline of the University of Tartu, and a list of the resulting variants were published in the supplementary material to Reisberg et al.. The variants per star allele are, however, not written out explicitly.

An example that illustrates many definition problems is CYP2C19*19:

CYP2C19*19	PharmGKB	PharmVar	PharmCAT	Comment
NC_000010.10(GRCh37)	Source API: g.96522561T(rs17885098), g.96602623G(rs3758581), g.96522613A>G, g.96609568T>C(rs4917623)	Source VCF: g.96521422A>G(rs7902257), g.96522613A>G	Source GitHub: g.96522613A>G(by liftOver)	Disagree on rs4917623(intron), rs7902257(2kb upstream variant). rs17885098 and rs3758581 are included here because PharmGKB assume different reference bases for these positions (seems like a problem caused by change of Major allele between reference builds GRCh37/GRCh38. Note that refSeq also agrees with GRCh38 and not GRCh37)
NC_000010.11(GRCh38)	Source Pharmgkb.org: g.94762856A>G	Source VCF: g.94762804C>T(rs17885098), g.94762856A>G, g.94842866A>G(rs3758581)	Source GitHub: g.94762856A>G	PharmGKB does not agree with itself when reporting GRCh38 variants on the homepage and GRCh37 variants in the API. The differences probably caused by non-standard use of Major/Minor Allele (rs17885098, rs3758581) with respect to dbSNP, and removing intron variants (rs4917623) may be sensible from an exon/protein-coding view.

The main problem with the changes in definitions is that the same patient may be given different PGx-advice depending on the build version of the pipeline (unless of course that the haplotype is always conserved)

How to define PGx alleles for next generation sequencing

As we saw in the previous section, PGx alleles are defined as collections of one or more SNPs, INDELs or structural variants. When a patient is sequenced by next generation sequencing (NGS) technology we typically observe more variants than those which are included in any individual PGx allele definitions.

The 16 possible haplotypes for a four loci, decomposed variant calling

This means that

• Patients may have a large, ambiguous number of matching PGx alleles
• Patients may have additional variants that may modify the effect of a known PGx allele

We illustrate some of the problems that we encountered when trying to match patient haplotypes to the PGx allele definitions, by a four loci PGx gene

The SNP array method

PGx alleles defined as collections of variants, with no requirement on loci that are not part of the definition, will assign the same PGx allele to several different haplotypes

This definition only requires matches for variants explicitly included in PGx allele definitions.

This means that

• Several PGx alleles may match the patient
• But the presence of additional variants will have no effect on reported PGx alleles

The PharmCAT method

This definition requires matches also for variants not explicitly included in PGx allele definitions.

PGx alleles defined as complete haplotypes classifies the patient uniquely

This means that

• Only one PGx allele can exist simultaneously for the same patient
• But whenever we have additional variants, no PGx alleles will be reported

(Note that in practice PharmCAT lets the user decide which allele definitions to use in their NamedAlleleMatcher)

Which definition should we stick to?

Method	Advantages	Disadvantages
SNP array method	Compatible with previous SNP array methods. Assigns PGx alleles to the maximum number of patients	Multiple PGx alleles are possible
PharmCAT method	One PGx allele per patient	Less compatible with previous SNP array methods. Some patients are no longer assigned to a known PGx allele

One partial solution is to use the PharmCAT method only for coding regions (which may be more preserved by evolution, with less variation), possibly together with some selected non-exonic variants.