The CYP2D6 enzyme is involved in the metabolism of many commonly prescribed drugs. The presence of polymorphisms in the
Cytochrome P450 2D6 (
Class list of major CYP2D6 isoenzyme substrates [
CYP2D6 isoenzyme major substrates |
---|
Tricyclic antidepressants |
Tetracyclic antidepressants |
Selective serotonin reuptake inhibitors |
Serotonin-norepinephrine reuptake inhibitors |
Opioids |
Antipsychotics |
Selective estrogen receptor modulator |
|
Antihypertensives |
Antiarrhythmics |
Antitussives |
Antiemetics |
Antidiabetics |
Histamine antagonists |
Dopamine antagonists |
Antimitotics |
In our previous work [
For this reason, we aimed to create a primary
The choice of the population to be studied may be critical. Isolated populations, such as that found in Sardinia [
Genomic DNA extracted from blood samples of 250 healthy Sardinian individuals were furnished by Professor Francesco Cucca, INN-CNR Cagliari Director. All participating individuals provided informed consent to genetic test.
Selective amplification of the
List of primers used in
Nucleotides position | Name | 5′ 10-mer tag | 5′-sequence-3′ | Direction |
---|---|---|---|---|
From −1780 to −1758 | P–1780 | ACGTTGGATG | GTCCTCCTGTCCTCAGTGGAT | Forward |
From −1584 to −1559 | P–1584_WT | ACGTTGGATG | CAGCCTGGACAACTTGGAAGAAGCC | Forward |
From −1584 to −1559 | P–1584_MUT | ACGTTGGATG | CAGCCTGGACAACTTGGAAGAAGCG | Forward |
From 4706 to 4728 | 2D6–R [ |
ACGTTGGATG | ACTGAGCCCTGGGAGGTAGGTA | Reverse |
For sequencing reactions, we used primers designed by both the Assay Designer Suite [
List of primers used in
Nucleotides position | Forward primers | Reverse primers | ||
---|---|---|---|---|
Name | 5′-sequence-3′ | Name | 5′-sequence-3′ | |
From −1780 to −1426 | P–1780 | GTCCTCCTGTCCTCAGTGGAT | –1426C>T | GTGTGCCACCACGTCTAGCTTTTT |
From −1584 to −1287 | –1584C>G | CTGGACAACTTGGAAGAACC | CYP2D6_02_R | AGACGGAGATTTCCTCTTGTT |
From −1584 to −1000 | –1000G>A | ACATCCTCCCGGGCTGCCTGAGGGT | ||
From −1201 to −740 | CYP2D6_01_F | GGTGCCTGTAGTCCCAGCTA | –740C>T | ACAGACTCACACTGACACTTAG |
From −750 to −327 | –750_–749delGA | TGTGACTGGTGTGTGTGAGAGA | CYP2D6_03_R | TGGGATAACCAGGGTTTCAG |
From −320 to 137 | 2D6–F [ |
CCAGAAGGCTTTGCAGGCTTCA | 137_138insT | GAAGTCCACATGCAGCA |
From 19 to 481 | 19G>A | CTGGGGCTAGAAGCACTG | CYP2D6_04_R | CGTGATTTAAACGGCACTCA |
From 479 to 883 | CYP2D6_02_F | GCTGACACCAGAAGGAAAGG | 883G>C | TCCCCGAAGCGGCGCCGCAA |
From 843 to 1388 | 843T>G | ACTAGGACCTGTAGTCTGGGG | P2x2R [ |
ACCGGATTCCAGCTGGGAAATG |
From 1294 to 1707 | CYP2D6_03_F | ACCTGCACTAGGGAGGTGTG | 1707delT | GCGGCCTCCTCGGTCACCC |
From 1513 to 1979 | 1513C>T | AGCTGGACAGAGCCAGGGACTG | 1979T>C | GGACAGCCCGACTCCTCCTTC |
From 1863 to 2291 | 1863_1864insTTTCGCCCC | GACGCCCCTTTCGCCCC | 2291G>A | CACTCGCCAAGTGCCAGCCTCCAC |
From 2127 to 2616 | CYP2D6_04_F | TTTGGGAAAGGACATTCCAG | 2615_2617delAAG | GGCAGCCACTCTCACCT |
From 2540 to 2988 | 2539_2542delAACT | ACAGCTGGATGAGCTGCT | 2988G>A | CATGTGCCCCCGCCTGTACCCTT |
From 2850 to 3288 | 2850C>T | AGCTTCAATGATGAGAACCTG | 3288G>A | GATGTCATATGGGTCACAC |
From 3030 to 3494 | CYP2D6_07_F | ATGAACTTTGCTGGGACACC | CYP2D6_05_R | CCAGCCCTGCCTATACTCTG |
From 3466 to 3853 | CYP2D6_05_F | TAGTCCTCAATGCCACCACA | 3853G>A | GAAGCGGAAGGGCTTCT |
From 3790 to 4180 | 3790C>T | CCACTCTCACCCTGCATCT | 4180G>C | AGCTCATAGGGGGATGGG |
From 4115 to 4401 | 4115C>T | ACCTCCCTGCTGCAGCACTT | 4401C>T | TAACTGACATCTGCTCAGCCTCAAC |
From 4265 to 4728 | CYP2D6_06_F | TCCCTAGCCAGAGGCTCTAA | 2D6–R [ |
ACTGAGCCCTGGGAGGTAGGTA |
The presence of
We used the Maximum-Likelihood Estimation (MLE), based on the Expectation-Maximization (EM) algorithm [
To verify the reliability of the MLE/EM algorithm and to verify the presence of novel SNPs haplotype associations in samples presenting heterozygous status for –1584G>C SNP, it was possible to modify the Long Range PCR protocol and apply it to new sequencing analysis. We modified the primer −1584G>C (Table
A sample of 250 unrelated healthy Sardinian individuals was analyzed and their haplotype phases were defined. A
List of novel SNPs and new haplotypes found in Sardinian DNA samples by Sequencing Analysis. SNPs are shown in bold characters. Novel SNPs are between horizontal lines. “Sardinian haplotype 1” (SH1) and “Sardinian haplotype 3” (SH3) were found in both homozygous and heterozygous statuses in 37 and 5 samples, respectively. “Sardinian haplotype 2” (SH2) was found in only two samples, one associated to
SNP |
|
|
SH1 |
SH2 |
SH3 |
SH4 |
Position | Variation |
---|---|---|---|---|---|---|---|---|
–1584C>G | C | C | C | C |
|
|
5′-UTR | |
–1237_–1236insAA |
|
|
Not investigated | Not investigated | Not investigated | Not investigated | 5′-UTR | |
–1235A>G |
|
|
|
|
|
|
5′-UTR | |
| ||||||||
–948C>A | C | C | C |
|
C | C | 5′-UTR | |
| ||||||||
–750_–749delGA |
|
GA | GA | GA | GA | GA | 5′-UTR | |
–740C>T |
|
|
|
|
|
|
5′-UTR | |
–678G>A |
|
|
|
|
|
|
5′-UTR | |
|
|
|
|
|
|
|
Intron 1 | |
310G>T |
|
G |
|
|
|
|
Intron 1 | |
746C>G |
|
C |
|
|
|
|
Intron 1 | |
843T>G |
|
T |
|
|
|
|
Intron 1 | |
1661G>C |
|
C |
|
|
|
|
Exon 3 | |
2850C>T |
|
|
|
|
|
|
Exon 6 | R296C |
2988G>A | G |
|
|
|
G | G | Intron 6 | splicing defect |
| ||||||||
3176C>T | C | C | C |
|
C | C | Exon 7 | |
| ||||||||
3384A>C |
|
A |
|
|
|
|
Intron 7 | |
3584G>A |
|
G |
|
|
|
|
Intron 7 | |
3790C>T |
|
C |
|
|
|
|
Intron 7 | |
| ||||||||
3948T>G | T | T | T | T | T |
|
Intron 8 | |
| ||||||||
4180G>C |
|
|
|
|
|
|
Exon 9 | S486T |
4481G>A |
|
G |
|
|
|
|
3′-UTR |
Electropherograms detecting the presence of three novel SNPs. (a) −948C/A; (b) 3176C/T; (c) 3948T/G. The three samples presenting novel SNPs in heterozygous status were submitted to
Allele frequencies, detected from the 250 Sardinian individuals (Table For
Variant allele | Number of chromosomes | Frequency |
Correlated enzymatic activity [ |
---|---|---|---|
*
|
148 | 29.6 |
|
*
|
5 | 1.0 |
|
*
|
4 | 0.8 |
|
*
|
75 | 15.0 |
|
*
|
11 | 2.2 |
|
*
|
8 | 1.6 |
|
|
7 | 1.4 | Not known |
|
1 | 0.2 | Not known |
*
|
11 | 2.2 |
|
*
|
84 | 16.8 |
|
*
|
5 | 1.0 |
|
*
|
1 | 0.2 |
|
*
|
1 | 0.2 |
|
*
|
27 | 5.4 |
|
*
|
3 | 0.6 |
|
*
|
1 | 0.2 |
|
*
|
4 | 0.8 | Not known |
*
|
5 | 1.0 |
|
*
|
46 | 9.2 |
|
|
41 | 8.2 | Not known |
|
2 | 0.4 | Not known |
*
|
4 | 0.8 |
|
*
|
6 | 1.2 |
|
Total chromosomes number = 500. SH1: Sardinian haplotype 1; SH2: Sardinian haplotype 2; SH3: Sardinian haplotype 3; SH4: Sardinian haplotype 4.
Variant allele | Sardinian frequencies (%) |
Sardinian frequencies (%) |
Sardinian frequencies (%) |
Mediterranean frequencies (%) |
German frequencies (%) |
White subjects frequencies (%) |
European frequencies (%) |
Caucasian frequencies (%) [ |
Correlated enzymatic activity [ |
---|---|---|---|---|---|---|---|---|---|
*
|
31.4 | 28.6 | 36.5 | 40.8 |
36.4 | 36.4 | 33.4 | nd |
|
*
|
20.4 | 35.7 | 40.6 | 37.1 |
32.4 | 17.5 | 32.9 | nd |
|
*
|
2.2 | 1.8 | 3.1 | 1.5 | 2.0 | nd | 1.8 | nd |
|
*
|
16.8 | 21.4 | 12.5 | 16.0 |
20.7 | nd | 18.9 | 12–21 |
|
*
|
1.0 | 1.8 | 1.0 | nd | 2.0 | nd | 7.3 | 4–6 |
|
*
|
0.2 | — | — | 0.8 | 0.9 | nd | 1.4 | nd |
|
*
|
0.2 | — | — | 0.6 | 1.8 | 2.1 | 2.6 | nd |
|
*
|
5.4 | 5.4 | 4.2 | 2.9 | 1.5 | 1.8 | 1.4 | 1-2 |
|
*
|
0.6 | nd | nd | nd | 0.1 | nd | nd | nd |
|
*
|
0.2 | nd | nd | nd | nd | nd | nd | nd |
|
*
|
0.8 | nd | nd | nd | nd | nd | nd | nd | Not known |
*
|
1.0 | nd | nd | nd | nd | 5.2 | nd | nd |
|
*
|
17.8 | 3.6 | nd | nd | nd | 8.4 | nd | nd |
|
*
|
0.8 | — | — | nd | 0.5 | — | nd | nd |
|
*
|
1.2 | 1.8 | 2.1 | nd | 1.3 | 1.1 | nd | 1–5 |
|
—: allele not found in the study; nd: allele not detected in reference study; aincluding duplications/multiduplications of haplotype.
Deletions and duplications/multiplications were analyzed using the Applied Biosystems CNV Assay.
Sardinian genotype frequencies for the
Genotype | Observed frequency (%) | Predicted frequency (%) | Predicted enzymatic activity [ |
---|---|---|---|
*
|
9.6 | 9.9 |
|
*
|
13.2 | 12.9 |
|
*
|
1.6 | 1.7 |
|
*
|
10.0 | 10.7 |
|
*
|
0.8 | 0.6 |
|
*
|
0.4 | 0.2 |
|
*
|
0.4 | 0.2 |
|
*
|
3.2 | 3.5 |
|
*
|
0.4 | 0.5 |
|
*
|
0.4 | 0.2 |
|
*
|
0.4 | 0.5 |
|
*
|
0.8 | 0.6 |
|
*
|
10.4 | 10.9 |
|
*
|
0.8 | 0.6 |
|
*
|
0.8 | 0.6 |
|
*
|
4.0 | 4.3 |
|
*
|
0.8 | 1.1 |
|
*
|
6.8 | 6.9 |
|
*
|
0.4 | 0.5 |
|
*
|
2.4 | 2.2 |
|
*
|
0.4 | 0.5 |
|
*
|
0.4 | 0.5 |
|
*
|
7.6 | 7.1 |
|
*
|
0.4 | 0.5 |
|
*
|
0.4 | 0.5 |
|
*
|
0.8 | 0.9 |
|
*
|
0.4 | 0.3 |
|
*
|
0.8 | 0.3 |
|
*
|
2.8 | 2.9 |
|
*
|
0.4 | 0.3 |
|
*
|
2.0 | 1.8 |
|
*
|
0.4 | 0.3 |
|
*
|
0.4 | 0.3 |
|
*
|
0.4 | 0.3 |
|
*
|
6.0 | 5.8 |
|
*
|
0.4 | 0.3 |
|
*
|
0.4 | 0.3 |
|
*
|
0.4 | 0.3 |
|
*
|
0.4 | 0.3 |
|
*
|
2.0 | 1.9 |
|
*
|
0.4 | 0.2 |
|
*
|
0.4 | 0.3 |
|
*
|
0.4 | 0.3 |
|
*
|
3.2 | 3.0 |
|
*
|
0.4 | 0.3 |
|
*
|
0.4 | 0.3 |
|
|
— | 1.6 | |
|
a
Predicted enzymatic activity | Sardinian frequencies (%) |
German frequencies (%) |
White subjects frequencies (%) |
Caucasian frequencies (%) |
---|---|---|---|---|
|
29.6 | 53.9 | 37.3 | 56.7 |
|
62.0 | 36.3 | 45.5 | 27.4 |
|
4.4 | 7.2 | 15.9 | 15.9 |
|
2.4 | 2.6 | 1.3 | — |
|
1.6 | — | — | — |
Differences in drug responses could be due to genetic factors. Knowledge of individual genetic profiling is clinically important and provides benefits for future medical care by predicting the drug response or developing DNA based tests. Substantial interindividual variability in response to specific therapies might be caused by the presence of polymorphisms in genes encoding components of drug metabolism pathways, such as the CYP450 family genes. The CYP2D6 isoenzyme is involved in the metabolism of drugs such as antipsychotics, antidepressants,
In this study, we have developed a primary
This way, we have established the frequencies of most of the
The authors declare that there is no conflict of interests.
The authors gratefully acknowledge Professor Francesco Cucca, INN-CNR Cagliari Director, for gently furnishing Sardinian DNAs; Dr. Maristella Pitzalis and Dr. Francesca Deidda for technical assistance in DNA Sequencing; Dr. Luisella Saba, Dr. Elena Congeddu and Dr. Enrico Sorisio, PharmaNess Sole Director, for useful information provided. A special thanks goes to Professor Annalisa Marchi who has kindly allowed the completion of this paper in her laboratories.