2 Materials and methods
2.3 Methods
2.3.2 Genotyping methods
2.3.2.4 Genotyping of CYP2D6 mutations
For the detection of the most functionally important alleles *1,*3,*4,*5,*6,*10 and the gene duplication of CYP2D6, combined PCR-RFLP tests were used, as described by Sachse et al.
(Sac97). The first step was the amplification of a 4681-bp genomic DNA fragment, which contained all nine CYP2D6 exons, using the Expand Long Template PCR SystemTM. Figure 11 shows consecutive steps of CYP2D6 genotyping.
9
Figure 11: Design of the PCR tests for CYP2D6 mutations.
The bold numbers indicate the RCP reactions which are described in the text.
Table 12: PCR-RFLP tests for CYP2D6 genotyping.
PCR-RFLP assay RFLP-fragment patterns [bp] PCR
2 188C>T P113/P121 433 HphI 362/71 262/100/71
3 1795T>Del
PCR No.1 Amplification of the entire CYP2D6 gene.
The amplification of the whole CYP2D6 gene (4681-bp fragment) was performed according to Sachse et al. (Sac97). A 25-µl PCR mix contained 2.5 µl PCR-buffer, 4.5 µl 2 mmol/l dNTPs, 0.5 µl of each of the primers P100 and P200 (all primers are given in Table 13), 1.25 U Taq-polymerase, 16.75 µl of H2O, and 80-100 ng of genomic DNA. Thermocycling conditions were as follows: initial denaturation at 94°C for 2 min, 35 cycles of denaturation at 96°C for 10 s, annealing at 57°C for 20 s, and extension at 68°C for 5 min. The terminal elongation was performed at 68°C for 7 min. If the PCR was successful (checked by 1% agarose gel electrophoresis), 15 µl of the PCR product were diluted with 5 volumes of distilled water and stored at 4°C.
PCR No. 2 Detection of the mutation 188C>T (alleles *4 and *10).
Nested PCR was performed using the PCR product of reaction No. 1. A 433-bp fragment was amplified at 94°C for 2 min, 25 cycles at 95°C for 30 s, at 58°C for 10 s, at 72°C for 1 min, and terminal extension at 72°C for 7 min. A 25-µl PCR mix contained 2.5 µl PCR-buffer, 2.5 µl 2 mmol/l dNTPs, 1.25 µl 25 mmol/l MgCl , 0.5 µl of each of the primers P113 and P121, 1.25
U Taq-polymerase, 17.5 µl H2O, and 1 µl of the diluted 4681 PCR-product. The PCR-product was digested with enzyme HphI at 37°C overnight.
M 1 2 3 M
362 bp 262 bp 100 bp
1: 188TT; 262, 100, 71 bp 2: 188CT; 362, 262, 100, 71 bp 3: 188CC; 362, 71 bp.
M 1 2 3 M
362 bp 262 bp 100 bp
1: 188TT; 262, 100, 71 bp 2: 188CT; 362, 262, 100, 71 bp 3: 188CC; 362, 71 bp.
Figure 12: HphI digest of a 433-bp fragment containing position 188 of the CYP2D6 gene.
PCR No. 3 Detection of mutations 1795 T>Del and 1934G>A (alleles *4 and *6).
PCR No. 4 Detection of mutations 2637A>Del (allele *3).
In the subsequent nested PCRs, conditions were as follows: initial denaturation at 94°C for 2 min, 25 cycles at 95°C for 10 s, at 60°C for 10 s, at 72°C for 1 min, and terminal extension at 72°C for 7 min. A 25-µl PCR mix contained 2.5 µl PCR- buffer, 2.5 µl 2 mmol/l dNTPs, 1.25 µl 25 mmol/l MgCl2, 0.5 µl of each of the primers, 1.25 U Taq-polymerase, 17.5 µl H2O and 1 µl of the diluted 4681 PCR-product. After amplification, the products of nested PCRs were analyzed by an 1% agarose gel electrophoresis and digested with the respective restriction endonucleases. The enzymes and restriction-fragment lengths are given in Table 12. The digestion products were analyzed on a 3% agarose gel, together with a 100-bp DNA weight marker.
353 bp 190 bp 163 bp 139 bp
1: 1795TT, 1934GG; 190, 163 bp 2: 1795TT, 1934GA; 353, 190, 163 bp 3: 1795TT, 1934AA; 353 bp
4: 1795TDel, 1934GG; 190, 163, 139, 23 bp 1 M 2 3 4
353 bp 190 bp 163 bp 139 bp
1: 1795TT, 1934GG; 190, 163 bp 2: 1795TT, 1934GA; 353, 190, 163 bp 3: 1795TT, 1934AA; 353 bp
4: 1795TDel, 1934GG; 190, 163, 139, 23 bp 1 M 2 3 4
Figure 13: BstNI digest of a 353-bp fragment containing the mutations 1795T>Del and 1934G>A of the CYP2D6 gene.
201 bp
Figure 14: BsaAI digests of a 201-bp fragment for the detection of the mutation 2637A>Del.
PCR No. 5 Detection of CYP2D6 deletion (allele *5).
1: allele *5 positive, 3500 bp 2: allele *5 negative, 4500 bp 4500 bp
3500 bp
1 2 M
1: allele *5 positive, 3500 bp 2: allele *5 negative, 4500 bp 4500 bp
3500 bp
1 2 M
Figure 15: Electrophoresis of the PCR-product from reaction No. 5 for detection of the CYP2D6 deletion.
PCR No. 6 Detection of CYP2D6 duplication (allele *MxN).
For the detection of the CYP2D6 deletion (allele *5) and duplication (*MxN), PCR methods were established from literature. The CYP2D6 deletion detecting assay (Ste95) was improved, according to Sachse et al. (Sac97), by adding an internal standard primer. This primer enables genotype independent amplification of an internal standard fragment, as in the CYP2D6 duplication assay of Løvlie et al. (Lov96). These internal standard fragments ensure that false negative results are not possible in these two assays. In both PCRs, a standard fragment, and – if the deletion or duplication allele occurs – a second fragment are amplified from genomic DNA.
After having transferred the product of the enzymatic digestion in 1% agarose gel, it was submitted to electrophoresis, and the results were documented by a digital video system. Long distance PCR technique was applied using the same concentrations of PCR compounds and PCR conditions as described above for the PCR reaction amplifying the entire CYP2D6.
1 2 M
5200 bp
3600 bp 1: duplication*MxN-negative; 5200 bp 2: duplication*MxN-positive; 5200, 3600 bp 1 2 M
5200 bp
3600 bp 1: duplication*MxN-negative; 5200 bp 2: duplication*MxN-positive; 5200, 3600 bp
Figure 16: Electrophoresis of the PCR-product from reaction No. 6 for detection of the CYP2D6 duplication.
PCR No. 7 Detection of CYP2D6 duplication (allele *MxN).
In 3 cases of allele duplication and genotype *1/*4, PCR No. 7 was established according to Johansson et al., 1996 for the detection of duplicated allele. The PCR conditions were as follows: an initial denaturation at 94°C for 2 min, 10 cycles at 96°C for 10 s, at 57°C for 20 s, at 68°C for 12 min, 20 cycles at 96°C for 10 s, at 57°C for 20 s, at 68°C for 12 min + 15 s per cycle and terminal extension at 68°C for 7 min. A 25-µl PCR mix contained 2.5 µl PCR-buffer, 6.25 µl 2 mmol/l dNTPs, 0.75 µl of each of the primers P2x2f and P2xr, 1.85 µl of enzyme mix (Taq and Pwo polymerases), 11.5 µl H2O and 80-100 ng of genomic DNA. The PCR resulted in a 10000-bp fragment, which is a fragment between exon 9 of allele 1 and intron 2 of allele 2.
PCR No. 8 Detection of CYP2D6 duplication (allele *4xN).
PCR No. 9 Detection of CYP2D6 duplication (allele *1xN).
To distinguish between different types of allele duplication, two additional PCR-RFLP tests were developed, both as nested PCR from the diluted product of reaction 7. A PCR-RFLP test detecting the *2-related 4268G>C mutation (reaction 8) showed whether *1 or *2 was duplicated (in *1x2/*2 or *2x2/*1 constellation). The primers P2x2f and P92 were used to amplify a 264-bp product (PCR conditions as for reaction 3), which was digested using BanII.
Performing reaction 9 (same conditions as in reactions 3 and 8) using the 10-kb gene duplication product as a target, the detection of the *4-associated 188C>T mutation by HphI digestion served as an indirect proof of a duplicated *4 allele in cases of a questionable *2x2/*4 or *4x2/*2 constellation.
Table 13: Sequences and locations of primers used in PCR reactions.
Primer Sequence Orientation and position* Specificity P100 5'-GGCCTACCCTGGGTAAGGGCCTGGAGCAGGA f -180 -150
P200 5'-CTCAGCCTCAACGTACCCCTGTCTCAAATGCG r +92 +123
whole CYP2D6 gene
P*3 5'-CCTGGGCAAGAAGTCGCTGGACCAG f 1770 1794
P2 5'-GAGACTCCTCGGTCTCTCG r 2104 2122 alleles *4 and *6
P51 5'-GCTGGGGCCTGAGACTT f 2457 2473
D2 5'-GGCTGGGTCCCAGGTCATAC r 2638 2657 allele *3
P13 5'-ACCGGGCACCTGTACTCCTCA f (2D7) +1619 +1639
P24 5'-GCATGAGCTAAGGCACCCAGAC r +3444 +3465
P81 5'-CGTCTAGTGGGGAGACAAAC f 3621 3640
allele *5
P17 5'-TCCCCCACTGACCCAACTCT f (2D7) +155 +174 P32 5'-CACGTGCAGGGCACCTAGAT r (2D7) +5470 +5489
P2x2f 5'-GCCACCATGGTGTCTTTGCTTTC f 4238 4260
P2x2r 5'-ACCGGATTCCAGCTGGGAAATG r 1384 1405
CYP2D6
duplication (allele
*MxN)
P113 5'-TCAACACAGCAGGTTCA f -82 -66
P121 5'-CTGTGGTTTCACCCACC r 335 351 alleles *10, *4xN
P92 5'-CTCAGCCTCAACGTACCCCT r +104 +123 allele *1x2 Underlined nucleotides refer to mismatched bases
*Position according to Kimura et al. (Kim89)
2.3.2.5 Genotyping of NAT2 mutations