CYP2D6 Caller

The CYP2D6 Caller is capable of genotyping the CYP2D6 gene from whole-genome sequencing (WGS) data and is derived from the method implemented in Cyrius¹. Due to high sequence similarity with its pseudogene paralog CYP2D7 and a wide variety of common structural variants (SVs), a specialized caller is necessary to resolve variants and identify likely star allele haplotypes.

The CYP2D6 Caller performs the following steps:

1. Determines total CYP2D6 and CYP2D7 copy number from read depth.

2. Determines CYP2D6-derived copy number at CYP2D6/CYP2D7 differentiating sites.

3. Detects SV breakpoints by calculating the changes in CYP2D6-derived copy number along the CYP2D6 gene.

4. Calls small variants in CYP2D6 copies.

5. Identifies star alleles from the detected SV breakpoints and small variants.

6. Identifies the most likely genotype for the called star alleles.

Total CYP2D6 and CYP2D7 Copy Number

The first step of CYP2D6 calling is to determine the combined copy number of CYP2D6 and CYP2D7. Reads aligned to regions in either CYP2D6 or CYP2D7 are counted. The counts in each region are corrected for GC-bias, and then normalized to a diploid baseline. The GC-bias correction and normalization factors are determined from read counts in 3000 preselected 2 kb regions across the genome. These 3000 normalization regions were randomly selected from the portion of the reference genome having stable coverage across population samples. The combined CYP2D6 and CYP2D7 copy number is then calculated from the average sequencing depth across the CYP2D6 and CYP2D7 regions.

Differentiating Sites

The CYP2D6-derived copy number is calculated at 117 predefined differentiating sites across the CYP2D6 gene. The differentiating sites are selected at positions with sequence differences in CYP2D6 and CYP2D7 where calling the CYP2D6-derived copy number shows an accuracy of greater than 98% based on sequencing data from the 1000 Genomes Project.

For each differentiating site, CYP2D6-specific and CYP2D7-specific alleles are counted in reads mapping to either CYP2D6 or the homologous region in CYP2D7. The CYP2D6-derived copy number is then calculated from the two gene-specific allele counts using the total CYP2D6 and CYP2D7 copy number calculated from the previous step.

Structural Variant Calling

The CYP2D6-derived copy number along the CYP2D6 gene is used to identify known population structural variants (SVs), including whole gene deletions and duplications as well as certain gene conversions and gene fusions. The following fusion variants are detected:

Fusion Breakpoint	Hybrid Gene Structure	Star-Allele Designation
exon 9	2D6-2D7	*4.013, *36, *57, *83
exon 9	2D7-2D6	*13
intron 4	2D7-2D6	*13
intron 1	2D7-2D6	*13
intron 1	2D6-2D7	*68

In addition to the exon 9 fusion breakpoints, exon 9 can participate in CYP2D7 gene conversion resulting in an embedded CYP2D7 sequence instead of a true hybrid. The structural variant caller also detects exon 9 gene conversions. Because only changes in CYP2D6-derived copy number yield structural variant calls, there might be rare cases where two hybrid copies result in no structural variant calls. For example, when both *36 and *13 with fusion breakpoint in exon 9 are present. However, the structural variant caller is capable of detecting multiple copies of the same fusion type (eg, *36x2) or cases where both an exon 9 gene conversion copy and an exon 9 2D6-2D7 hybrid are present.

Small Variant Calling

118 small variants that define various star alleles are detected from the read alignments. 96 of these variants are in unique (nonhomologous) regions of CYP2D6 with high mapping quality. Only reads mapping to CYP2D6 are used for calling variants in nonhomologous regions. The other 22 variants occur in homologous regions of CYP2D6 where reads mapping to either CYP2D6 or CYP2D7 are used for variant calling.

For each variant, reads containing either the variant allele or the nonvariant alleles are counted. A binomial model that incorporates the sequencing errors is then used to determine the most likely variant copy number (0 for nonvariant). A strand bias filter is applied to a small subset of variants that would otherwise tend to have false positive calls in the population.

Samples with poor sequencing quality or greater than five copies of CYP2D6 will have allele counts with higher variance. This elevated variance increases the chance that the most likely variant copy number is wrong. To handle these cases, the small variant caller also indicates alternate, less likely variant copy numbers.

Star Allele Identification

The called SVs and small variant genotypes are matched against the definitions of 128 different star alleles. This might result in different sets of star alleles matching the called variant genotypes, such as with *1, *46 and *43, *45 where both sets of star alleles contain the same 4 small variants. When the small variant caller emits alternate, less likely variant copy numbers in addition to the most likely variant copy numbers this might result in different sets of star alleles being identified, since these alternate sets of variant copy numbers are also matched to the star allele definitions. The number of matched star alleles must match the number of CYP2D6-derived gene copies determined from previous steps. When there are fewer than two CYP2D6-derived gene copies, then one or more *5 deletion haplotypes are included in the output set of star alleles. If all variant genotypes cannot be matched to a set of star alleles, the CYP2D6 Caller returns a no call during the genotyping step with filter value No_call.

Genotyping

Given a possible set of star alleles, the genotyping step attempts to identify the two likely haplotypes that contain all star alleles in the set. The deletion haplotype (*5) is considered as a possible haplotype during this process. The likelihood of any given genotype is determined from a table of population frequencies determined from the 1000 Genomes Project and the genotype with the highest population frequency is selected. When two or more possible genotypes are identified with similar population frequencies, then all genotypes are emitted. This results in a call with filter value More_than_one_possible_genotype.

CYP2D6 Output File

The CYP2D6 Caller prints out its calls in the targeted caller output file, <output-file-prefix>.targeted.json that also contains calls from other targets (see Targeted JSON File). An example of the CYP2D6 caller content in the output is as follows:

{
    "cyp2d6": {
        "genotype": "*17/*2",
        "genotypeFilter": "PASS",
        "phenotypeDatabaseAnnotation": "Normal Metabolizer"
    }
}

Fields in JSON	Explanation	Type and Possible Values
genotype	called star allele genotype	string (semi-colon delimited list of possible genotypes with haplotypes separated by /)
genotypeFilter	The filter status for the genotype call	string (The value can include: PASS, No_call, or More_than_one_possible_genotype)
phenotypeDatabaseAnnotation	The metabolism status corresponding to the genotype, mapped from phenotypeDatabaseSources	string

Each CYP2D6 genotype contains two haplotypes separated by a slash (eg *1/*2). Each haplotype consists of one or more star alleles separated by a plus sign (eg *10+*36). When a haplotype contains more than one copy of the same star allele, that star allele only appears once and is followed by a multiplication sign, and then the number of copies (eg *1x2 for two copies of *1).

¹Chen X, Shen F, Gonzaludo N, et al. Cyrius: accurate CYP2D6 genotyping using whole-genome sequencing data. The Pharmacogenomics Journal. 2021;21(2):251-261. doi:10.1038/s41397-020-00205-5