Pharmacogenetic testing revisited: 5′ nuclease real-time polymerase chain reaction test panels for genotyping CYP2D6 and CYP2C19

Figures & data

Table 1 CYP2D6 alleles identified by the 5′nuclease assays compared to those of the Luminex XTag CYP2D6 v.3 and Roche AmpliChip P450 kit

CYP2D6 Alleles	*2	*3	*4	*6	*7	*8	*9	*10	*11	*14A	*14B	*15	*17	*19	*20	*25	*26	*29	*30	*31	*35	*36	*40	*41
2850C>T			+K
2549delA
1846G>A
1707delT
2935A>C
1758G>T
2615-2617delAAG
100C>T			÷M
883G>C
137-138insT
1023C>T
2539_2542delAACT
1973_1974insG
3198C>G
3277T>C
3183G>A
1863_1864Ins TTTCGCCCC
4180G>C			÷J ÷M	+C
1661G>C	÷D		÷B
1659G>A
2988G>A
−1584C>G	+A
31G>A
Allele frequency danish Caucasians.^Citation37	22,4	3,1	19,1	1,6	0	0	3,6	0,9	0	–	–	0	0	–	–	–	–	–	–	–	–	–	–	6,9
Reported allele frequencies in Caucasians.^Citation50	10,5–16,5	0,5 –3,0	12,2 – 21,0	0,5– 2,6	0,0– 0,3	0	0,7– 7,1	1,6– 4,4	0	0	0	0	0– 0,3	0	0–0,5	0	0	0–0,3	0	0	4,4–7,3	0	0	6,3–9,8

Notes: Comparison between SNPs/mutations detected by the 5′ nuclease assays, the AmpliChip P450, and Luminex XTag CYP2D6 kit. Top row shows the list of CYP2D6 alleles identified by the Roche AmpliChip Cytochrome P450 kit (excluding duplications), with their enzymatic activity indicated by color codes: green – normal, yellow – decreased, red – none, and gray – activity unknown. Proposed SNPs/mutations used for the identification of all of the alleles identified by the AmpliChip method are listed on the left side of the table, with the ones included in the 5′ nuclease assay highlighted in bold. The presence of each SNP/mutation in alleles is marked by colored squares. Light green indicate mutations only identified by the AmpliChip kit, while blue indicate mutations detected by both the AmpliChip and the Luminex XTag kit. Subtype of an allele that does not contain a given mutation is marked with a “÷” and their letter (e.g., the CYP2D6*2D is lacking the 1661G>C SNP reported in other CYP2D6*2 alleles). (http://www.cypalleles.ki.se),⁵⁹ Rasmussen et al,³⁷ and Dodgen et al.⁵⁰

Abbreviation: SNP, single-nucleotide polymorphism

Table 2 Validation of 5′ nuclease test panels

Allele	Assay target	Wildtype	Heterozygotes	Homozygotes	Failed PCR	n
CYP2D6
*2	2850C>T	16/16	14/14	18/18	0	48
*3	2549delA	21/21	23/23	1/1	3	48
*4	1846G>A	16/16	16/16	16/16	0	48
*6	1707delT	24/24	22/22	1/1	0	47
*9	2615–2617delAAG	16/16	26/26	5/3	2	47
*10	100C>T	20/19	21/19	20/20	0	61
*17	1023C>T	32/32	2/2	–	0	34
*41	2988G>A	16/16	16/13	16/7	0	48
CYP2C19
*2	19154G>A	16/16	16/15	16/16	1	48
*3	17948G>A	47/47	1/1	–	0	48
*4	1A>G	43/43	1/1	–	5	49

Notes: This table presents the number of samples tested and the result of the individual validation of each of the 11 5′ nuclease assays. Figures to the left are the number of samples used for validating the assay, and right are the number of samples in agreement between the original test and the 5′ nuclease assay.

Abbreviation: PCR, polymerase chain reaction.

Table 3 Specificity of primer and probe sequences with optimized reaction concentrations

Notes: Specificity of sequences and optimized concentrations of primers and probes for each of the 11 assays presented in this paper. Wildtype probes were labeled 5′ with FAM and 3′ with BHQ-1, while mutant probes were labeled 5′ with CalFlour 540 (a TET analog), and 3′ with BHQ-1. The probes used for CYP2C19*4 were synthesized using phosphorothioate bases (u, c). Amplicon size generated by the primers is shown on the right. Sequence color codes: light gray – CYP2D6 nucleotides deviating from CYP2D8; medium gray – CYP2D6 nucleotides deviating from CYP2D7; reverse black - bases deviating from both CYP2D7 and CYP2D8, including inserted bases (CYP2C9 for the CYP2C19 assays). Underlined bold indicates target SNPs/mutations in either the wildtype or the mutant probe. – indicates one-base insert present in pseudogene or mutant target.

Abbreviations: FAM, carboxyfluorescein; BHQ-1; black hole quencher-1; TET, tetrachlorofluorescein; SNP, single-nucleotide polymorphism.

Figure 1 (A) Allelic discrimination plot of the CYP2D*41 assay, showing the 48 samples used for validating the test; X-axis: FAM fluorescence – wildtype probe; Y-axis: CalFluor gold 540 – mutant probe. Ambiguous sample is marked with a red circle, while black square represents “no template” control (NTC). (B) Sequence of the probe region (blue area) of the ambiguous sample. Green arrow marks the 2988G>A SNP, while the red arrow shows the location of the 2980C>T SNP.

Abbreviations: FAM, carboxyfluorescein; SNP, single-nucleotide polymorphism.

Table 4 Retest of CYP2D6*41 alleles based on the presence of the 2988G>A SNP

Original genotyping result	Total *41 alleles retested/no. of samples	Failed no. of alleles in retest	Frequency of failed retest
*1/*41	4	1	0.25
*2/*41	34	0	0.00
*4/*41	35	7	0.20
*5/*41	6	1	0.17
*6/*41	1	0	0.00
*17/*41	1	1	1.00
*41/*41	24	11	0.42
2×*41/*1	2	0	0.00
2×*41/*41	9/3	7	0.78

Notes: Retesting of samples originally tested as either heterozygote or homozygote carriers of CYP2D6 allele *41, based on the presence of the 2850C>T and lack of the −1584C>G SNP. Failed number of alleles are alleles not found to harbor the 2988G>A SNP in the retest. For samples with copy variations, the number of alleles has been set as either 2 (2×*41/*1) or 3 (2×*41/*41).

Abbreviation: SNP, single-nucleotide polymorphism.

RasmussenJOChristensenMSvendsenJMSkausigOHansenELNielsenKACYP2D6 gene test in psychiatric patients and healthy volunteersScand J Clin Lab Invest200666212913616537246

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DodgenTMHochfeldWEFicklHIntroduction of the AmpliChip CYP450 Test to a South African cohort: a platform comparative prospective cohort studyBMC Med Genet2013142023356658

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The Human Cytochrome P450 (CYP) Allele Nomenclature Database Available from: http://www.cypalleles.ki.se/cyp2d6.htmAccessed March 13, 2017

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