CYP2D6 Haplotype Determination Using Long Range Allele-Specific Amplification  Andrea Gaedigk, Amanda K. Riffel, J. Steven Leeder  The Journal of Molecular Diagnostics  Volume 17, Issue 6, Pages 740-748 (November 2015) DOI: 10.1016/j.jmoldx.2015.06.007 Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Principle and optimization of allele-specific amplification combined with long-range (ASXL)-PCR. A: The sequence context of the -1584C>G single-nucleotide polymorphism (SNP) (black; SNP is in bold). Primer sequences amplifying either -1584C or -1584G are shown in gray, with matching and mismatching nucleotides at their respective -3′ ends in black and bold. The arrow indicates that a perfectly matching primer is extended; primers with mismatches are not extended, as indicated by an X in the arrow. B: Summary of SNP positions that can be utilized to discriminate CYP2D6*1, *2A, and *4 alleles. SNP positions are shown in reference to AY545216 (+1 = A in the ATG translation start codon). The nucleotide for which AS amplification was achieved is in bold. Gray boxes denote the primers designed based on sequence information for that allele and the alleles that will be amplified with a given AS primer. For example, the CYP2D6*1 and *4 alleles can be separated utilizing positions -2609 (amplifies *4), -2523 (amplifies *1), -2421 (amplifies *4), -2178 (amplifies *1), -1426 (amplifies *1), and -1000 (amplifies *1). CYP2D6*1/*1 and *2A/*2A gDNA samples were amplified with primer -1584C>G (C). The -1584C>G primer amplified a 6358-bp fragment from the CYP2D6*1 (-1584C), but not the CYP2D6*2A (-1584G) allele. D: Real-time ASXL-PCR with primer -740C>T amplifies from the CYP2D6*4 allele (-740C) but not the *1 allele (-740T). Nucleotides providing AS amplification are underlined. var, variant; wt, wild type. The Journal of Molecular Diagnostics 2015 17, 740-748DOI: (10.1016/j.jmoldx.2015.06.007) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Pedigree of a family with initial genotype discord. The genotypes of subjects 6 and 9, mother and daughter, shown with bold circles, were discordant when originally tested. The son's (subject 8) genotype was initially not discordant. Revised genotypes for subjects 6, 8, and 9 containing a CYP2D6*59 allele are indicated with an arrow. Alleles revised are shown in bold and larger font. The Journal of Molecular Diagnostics 2015 17, 740-748DOI: (10.1016/j.jmoldx.2015.06.007) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 TaqMan (Life Technologies Corporation, Carlsbad, CA) cluster plots with the original (C__27102425_10) and alternate (C__27102425_50) assays for 2850C>T (rs16947). The x and y axes show relative fluorescence for 2850T (variant) and 2850C (wild type), respectively. Clusters are shown with trajectory lines. Pedigree subject 9 clusters with other homozygous wild-type samples (2850C/C) with the initial assay (C__27102425_10) while clustering with heterozygous samples (2850C/T) when run with the alternate assay (C__27102425_50). The homozygous variant cluster (2850T/T) is also shown. gDNA, genomic DNA; XL-PCR, long-range PCR. The Journal of Molecular Diagnostics 2015 17, 740-748DOI: (10.1016/j.jmoldx.2015.06.007) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Normalized high-resolution melt curve analysis of the CYP2D6*59 2939G>A tag single-nucleotide polymorphism. Curves represent individuals with 2939G/G, 2939G/A, and 2939A/A genotypes, as indicated. The assay was performed on long-range PCR templates. The Journal of Molecular Diagnostics 2015 17, 740-748DOI: (10.1016/j.jmoldx.2015.06.007) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Overview of a complex CYP2D6 genotype that was resolved using allele-specific long-range PCR. The graph displays the two allelic variants of the subject. CYP2D6, 2D7, and 2D8 are shown in different gray shades. REP denotes repetitive sequences located downstream of CYP2D6 and 2D7. REP-DUP refers to the REP sequence found downstream of a duplicated gene. REP7 is interrupted from common downstream sequences (dark-gray boxes) by a 1.6-Kb region, referred to as spacer. Extra-long PCR fragments are shown as lines under the genes that support their amplification; the X indicates that this fragment was not generated from this gene copy. The hatched line for fragment D indicates that amplification from the CYP2D6*4 allele was poor due to more efficient amplification from the CYP2D6*2 allele. Exon 1 (ex1) and intron 6 (in6) denote the regions amplified by quantitative PCR to determine gene copy number (regions in intron 5 and exon 9 that were also assessed are not shown). The Journal of Molecular Diagnostics 2015 17, 740-748DOI: (10.1016/j.jmoldx.2015.06.007) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions