# Genotyping ofCYP2D6 Polymorphisms by MALDI-TOF Mass Spectrometry in Sardinian People

Authors: Matteo Falzoi; Luigi Pira; Paolo Lazzari; Luca Pani
Journal: ISRN Genetics
 (2013)

---

## Abstract

TheCYP2D6 enzyme is involved in the metabolism of many commonly prescribed drugs. The presence of CYP2D6 gene SNPs can alter CYP2D6 enzymatic activity with effects ranging considerably within a population. Objectives. In this study, we have developed a genotyping platform able to determine the alleles related to interindividual variability in the CYP2D6 gene. Design and Methods. We used a long PCR strategy coupled to MALDI-TOF mass spectrometry (Sequenom) to develop a SNPs genotyping method. Furthermore, an amplification allele specific was carried out to infer the correct allelic phase. Results. We tested the multiplex platform in 250 DNA Sardinian samples and found it to be 100% concordant with the sequencing results of our previous work. Conclusions. The MALDI-TOF-based multiplexing system allowed simultaneous and efficient genotyping of a set of CYP2D6 SNPs, evidencing its potential use in diagnostic test development to predict drug responses and clinical outcomes.


---

## Body

## 1. Introduction

The presence of polymorphisms in the cytochrome P450 2D6 (CYP2D6) gene may modulate enzyme levels affecting individual responses to pharmacological treatment in drug level, response, and adverse reactions and includes individuals with ultrarapid (UM), extensive (EM), intermediate (IM), and poor (PM) metabolizer status. Furthermore, the presence of multiple functional gene copies or the deletion of the entire gene results in increased or absent drug metabolism, respectively [1, 2]. Genotypic analysis to identify individual polymorphisms has become increasingly important during drug development and for selection of individualized therapies. For this reason, high-throughput technology for rapid, accurate, and efficient genotyping is needed. Several strategies and methods for SNPs genotyping have been developed. Techniques commonly used, such as PCR-RFLP, real-time PCR, and the TaqMan allele-specific assays from Applied Biosystems (CA, USA), are often laborious, and a restricted number of alleles can be simultaneously detected by these techniques. Conversely, the high-throughput oligonucleotide microarray technology, such as the Affymetrix/Roche (CA, USA) AmpliChip CYP450 GeneChip test, has the disadvantage over other assays in that it cannot be customized by the user, and the benefits of this technology are often not compensable due to unfavourable costs [3]. In our previous work [4], we tried to create a complete genotyping platform for the simultaneous analysis of CYP3A4, CYP3A5, CYP2C9, CYP2C19, and CYP2D6 SNPs. The genotyping of the CYP2D6 gene was difficult due to its polymorphic nature, the presence of two flanking pseudogenes and copy number variants. To avoid false genotyping, resulting from nonspecific coamplification of the highly homologous pseudogenes, we developed the analysis of this gene in another way by using long PCR protocol coupled with single allele analysis and followed by sequencing [5]. In this work, we aimed to create a CYP2D6 medium-throughput SNPs screening platform using the Sequenom (CA, USA) matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) [6], a widely used technology that is proving to be a competitive analysis method in SNPs genotyping. Advantages of MALDI-TOF MS over previously described methods include the option for medium-high-throughput automated analysis of SNPs, the relative ease of setup for multiplex assays, and the reduced costs per genotype [3].


## 2. Methods and Materials

SNPs and Sequences Selection. The 69 polymorphisms analyzed in this study were selected using principal SNP web databases such as the Human CYP Allele Nomenclature Committee [7] and the NCBI Single Nucleotide Polymorphism dbSNP [8] websites. Selection criteria depended mainly on the pharmacogenetic effects described for every allele in the Caucasian population [5, 7, 9–16]. Not all selected SNPs are involved in aminoacidic or transcriptional variation (Table 1). Some of these are silent or promoter, leader, trailer, and intronic changes and inserted in our study because they are essential for the reconstruction of haplotype phases. The SNPs’ recombination allowed the reconstruction of 66 among allelic variants and subvariants. List of alleles is summarized in Table 5.Table 1
List of 69 analyzed SNPs and correlation to aminoacid changes or transcriptional modificafications.
CYP2D6 SNPs
Variations
-1584C>G
—
-1426C>T
—
-1235A>G
—
-1000G>A
—
-948C>A
—
-750_-749delGA
—
-740C>T
—
-678G>A
—
19G>A
V7M
31G>A
V11M
77G>A
R26H
82C>T
—
100C>T
P34S
124G>A
G42R
137_138insT
Frameshift
214G>C (*)
—
310G>T
—
746C>G
—
843T>G
—
883G>C
Splicing defect
957C>T
A85V
974C>A
L91M
984A>G
H94R
997C>G
—
1039C>T
—
1513C>T
—
1659G>A
V136I
1661G>C
—
1704C>G
Q151E
1707delT
Frameshift
1724C>T
—
1749A>G
N166D
1757C>T
—
1758G>A
G169R
1758G>T
G169X
1846G>A
Splicing defect
1863_1864insTTTCGCCCC
174_175insFRP
1869T>C
—
1943G>A
R201H
1979T>C
—
2291G>A
—
2483G>T
A237S
2539_2542delAACT
Frameshift
2549delA
Frameshift
2575C>A
—
2587_2590delGACT
Frameshift
2615_2617delAAG
K281del
2850C>T
R296C
2853A>C
I297L
2935A>C
H324P
2939G>A
—
2988G>A
Splicing defect
3176C>T
—
3183G>A
V338M
3198C>G
R343G
3277T>C
I369T
3288G>A
—
3384A>C
—
3582A>G
—
3584G>A
—
3790C>T
—
3828G>A
—
3853G>A
E410K
3877G>A
E418K
3948T>G
—
4115C>T
—
4155C>T (°)
H478Y
4180G>C
S486T
4401C>T
—
4481G>A
—
In MALDI-TOF MS analysis, (*) 214G>C SNP was used to discriminate gene conversion to CYP2D7P in Intron 1, and (°) 4155C>T SNP was used to discriminate gene conversion to CYP2D7P in Exon 9. A = alanine; R = arginine; N = asparagine; D = aspartic acid; C = cysteine; E = glutamic acide; Q = glutamine; G = glycine; H = histidine; I = isoleucine; L = leucine; K = lysine; M = methionine; F = phenylalanine; P = proline; S = serine; T = threonine; W = tryptophan; Y = tyrosine; V = valine. In bold characters newly discovered SNPs in [5, 17, 18, 21].DNA Samples. In this study, we reanalyzed the genomic DNAs studied in [5]. Sardinian DNA samples were gently furnished by Professor Francesco Cucca, INN-CNR Cagliari Director. All participating individuals provided informed consent to genetic test. To genotype these samples, we implemented a CYP2D6 Genotyping Platform based on MALDI-TOF MS. This way, we would be able to compare our genotyping results with a previous sequencing analysis in [5].


### 2.1.CYP2D6 Genotyping Platform

#### 2.1.1. Long Primary PCR

Selective amplification of theCYP2D6 gene was carried out modifying a long PCR protocol implemented in our previous work [5]. Forward primer (P-1780, Table 2) was designed in a highly nonhomologous CYP2D6/CYP2D7P/CYP2D8P 5′ untranslated region [5]. The reverse primer used was 2D6-R (Table 2), as previously described [19]. A 5′ 10-mer tag (5′-ACGTTGGATG-3′) was added to both PCR primers in order to improve PCR efficiency. PCR reactions were performed in a final volume of 5 μL using the QIAGEN (Hilden, Germany) LongRange PCR Kit protocol [20] with the following minor modifications: 20 ng genomic DNA, 400 μM of each PCR primer (Metabion, Martinsried, Germany), 0.2 U QIAGEN LongRange PCR enzyme, 1X QIAGEN LongRange PCR buffer (containing MgCl2 25 mM), and 500 μM Invitrogen (CA, USA) 2′-deoxynucleoside-5′-triphosphate (dNTP) Set PCR Grade. The PCR conditions were as follows: initial denaturation at 93°C for 3 min; 35 cycles at 93°C for 30 s, 61°C for 30 s, and 68°C for 6 min.Table 2
List of primers used inCYP2D6 long PCR protocols. The 5′ 10-mer tag was added to PCR primers, in order to improve PCR efficiency.
Nucleotides position
Name
5′ 10–mer tag
5′-Sequence-3′
Direction
From -1780 to -1758
P-1780 [5]
ACGTTGGATG
GTCCTCCTGTCCTCAGTGGAT
Forward
From -1584 to -1559
P-1584_WT [5]
ACGTTGGATG
CAGCCTGGACAACTTGGAAGAAGCC
Forward
From -1584 to -1559
P-1584_MUT [5]
ACGTTGGATG
CAGCCTGGACAACTTGGAAGAAGCG
Forward
From 4706 to 4728
2D6–R [19]
ACGTTGGATG
ACTGAGCCCTGGGAGGTAGGTA
Reverse


#### 2.1.2. SAP Dephosphorylation

To neutralize unincorporated dNTPs after amplification reactions, 0.3 U of Shrimp Alkaline Phosphatase (SAP) (Sequenom) [26, 27] was used. SAP cleaved a phosphate from the unincorporated dNTPs, converting them to 2′-deoxynucleoside-5′-diphosphate (dNDP) and rendering them unavailable for following reactions. Dephosphorylation conditions were as follows: 37°C for 20 min and 85°C for 5 min.


#### 2.1.3. Assay Design

SNP-specific unextended minisequencing primers (UEPs) and multiplexed UEPs assays were designed using both the Sequenom MassARRAY assay design version 3.1 and the RealSNP assay database [28]. A sequence of 400 base pairs (bp) flanking each selected SNP was downloaded from the corresponding genomic sequence stored in the public NCBI Single Nucleotide Polymorphism dbSNP database [8] or the Ensembl Genome Browser [29] and was analyzed by Vector NTI Suite Software version 5.5 (InforMax, Oxford, UK). Combination of the UEPs into multiplex assays was supported by these software applications to allow the optimization of several different parameters, for example, to avoid the risk of primer-primer interactions and hairpin-loop formations, GC content, molecular mass range, and annealing temperatures. To achieve the highest possible multiplexing levels, we tested many primer combinations, leading us to the final assay design consisting in a total of 69 SNPs successfully assembled in five medium-plex assays (13-, 14-, 13-, 14-, and 15-plex) (Table 3). For some SNPs, Sequenom MassARRAY assay design and RealSNP assay database could not design SNP-specific UEPs because of the presence of proximal SNPs. Other SNPs were excluded during platform validation because of UEPs cross-hybridization in highly homologous PCR template regions or for the presence of primer dimers which created false allele. But the high number of SNPs inserted in our analysis has allowed us to infer the correct alleles for each DNA analysis. List of SNPs not included is summarized in Table 4.Table 3
Sequences and molecular weights of unextended (UEP) and extended (EP) iPLEX primers.
UEPs’  sequences
UD
UM
EP lower mass sequences
ELM
ELC
EP higher mass sequences
EHM
EHC
(1) 13-PLEX
3877G>A
CCTTCCGCTTCCACCCC
F
4978
CCTTCCGCTTCCACCCCA
5249
A
CCTTCCGCTTCCACCCCG
5265
G
3183G>A
CCGCACCTGCCCTATCA
R
5051
CCGCACCTGCCCTATCAC
5299
G
CCGCACCTGCCCTATCAT
5378
A
2615_2617 delAAG
GGCAGCCACTCTCACCT
R
5091
GGCAGCCACTCTCACCTC
5339
del
GGCAGCCACTCTCACCTT
5418
AGA
2483G>T
GCGTAGGACCTTGCCAG
R
5211
GCGTAGGACCTTGCCAGC
5459
G
GCGTAGGACCTTGCCAGA
5483
T
3853G>A
GAAGCGGAAGGGCTTCT
R
5275
GAAGCGGAAGGGCTTCTC
5523
G
GAAGCGGAAGGGCTTCTT
5603
A
3277T>C
TGCAGCGCTTTGGGGACA
F
5556
TGCAGCGCTTTGGGGACAC
5803
C
TGCAGCGCTTTGGGGACAT
5883
T
4180G>C
AGCTCATAGGGGGATGGG
R
5645
AGCTCATAGGGGGATGGGC
5892
G
AGCTCATAGGGGGATGGGG
5932
C
2939G>A
TGGGCTCACGCTGCACATC
R
5766
TGGGCTCACGCTGCACATCC
6013
G
TGGGCTCACGCTGCACATCT
6093
A
3288G>A
GATGTCATATGGGTCACAC
R
5828
GATGTCATATGGGTCACACC
6075
G
GATGTCATATGGGTCACACT
6155
A
3176C>T
GGCCGTGTCCAACAGGAGAT
F
6167
GGCCGTGTCCAACAGGAGATC
6414
C
GGCCGTGTCCAACAGGAGATT
6494
T
2850C>T
AGCTTCAATGATGAGAACCTG
F
6454
AGCTTCAATGATGAGAACCTGC
6701
C
AGCTTCAATGATGAGAACCTGT
6781
T
2935A>C
AGTGCTCACGCTGCACATCCGGA
R
7010
AGTGCTCACGCTGCACATCCGGAG
7297
C
AGTGCTCACGCTGCACATCCGGAT
7337
A
1757C>T
TTGTGCCGCCTTCGCCAACCACTC
F
7201
TTGTGCCGCCTTCGCCAACCACTCC
7448
C
TTGTGCCGCCTTCGCCAACCACTCT
7528
T
(2) 14-PLEX
1661G>C
CAGAGGCGCTTCTCCGT
F
5162
CAGAGGCGCTTCTCCGTC
5410
C
CAGAGGCGCTTCTCCGTG
5450
G
1869T>C
ACGGCTTTGTCCAAGAG
R
5210
ACGGCTTTGTCCAAGAGA
5482
T
ACGGCTTTGTCCAAGAGG
5498
C
1846G>A
GGGGCGAAAGGGGCGTC
R
5342
GGGGCGAAAGGGGCGTCC
5588
G
GGGGCGAAAGGGGCGTCT
5668
A
214G>C
TGGAGGGCGGCAGAGGT
F
5357
TGGAGGGCGGCAGAGGTC
5604
C
TGGAGGGCGGCAGAGGTG
5644
G
19G>A
CTGGGGCTAGAAGCACTG
F
5565
CTGGGGCTAGAAGCACTGA
5836
A
CTGGGGCTAGAAGCACTGG
5852
G
1707delT
GCGGCCTCCTCGGTCACCC
R
5702
GCGGCCTCCTCGGTCACCCC
5949
del
GCGGCCTCCTCGGTCACCCA
5973
T
746C>G
GCGACCCAGCCTCCTGATC
R
5710
GCGACCCAGCCTCCTGATCC
5957
G
GCGACCCAGCCTCCTGATCG
5997
C
997C>G
GAACAGGCGGGCGGTCGGC
R
5920
GAACAGGCGGGCGGTCGGCC
6167
G
GAACAGGCGGGCGGTCGGCG
6207
C
100C>T
AATGCTGGGCTGCACGCTAC
F
6118
AATGCTGGGCTGCACGCTACC
6365
C
AATGCTGGGCTGCACGCTACT
6445
T
1979T>C
GGACAGCCCGACTCCTCCTTC
R
6303
GGACAGCCCGACTCCTCCTTCA
6574
T
GGACAGCCCGACTCCTCCTTCG
6590
C
-948C>A
CAGGCTGGGGCAAGGGCCTTC
F
6488
CAGGCTGGGGCAAGGGCCTTCC
6735
C
CAGGCTGGGGCAAGGGCCTTCA
6759
A
1749A>G
GCCCATCACCCACCGGAGTGGT
R
6681
GCCCATCACCCACCGGAGTGGTC
6929
G
GCCCATCACCCACCGGAGTGGTT
7008
A
77G>A
GTAGCGTGCAGCCCAGCGTTGG
R
6792
GTAGCGTGCAGCCCAGCGTTGGC
7040
G
GTAGCGTGCAGCCCAGCGTTGGT
7120
A
1513C>T
AGCTGGACAGAGCCAGGGACTG
F
6835
AGCTGGACAGAGCCAGGGACTGC
7082
C
AGCTGGACAGAGCCAGGGACTGT
7162
T
(3) 13-PLEX
984A>G
GCGAGTGTCCTCGCCG
R
4874
GCGAGTGTCCTCGCCGC
5121
G
GCGAGTGTCCTCGCCGT
5201
A
1863_1864ins TTTCGCCCC
GACGCCCCTTTCGCCCC
F
5043
GACGCCCCTTTCGCCCCA
5315
–
GACGCCCCTTTCGCCCCT
5370
TTTCGCCCC
1039C>T
ACCCAGATCCTGGGTTT
F
5161
ACCCAGATCCTGGGTTTC
5409
C
ACCCAGATCCTGGGTTTT
5489
T
1943G>A
CCAGCAGCCTGAGGAAG
R
5229
CCAGCAGCCTGAGGAAGC
5477
G
CCAGCAGCCTGAGGAAGT
5557
A
974C>A
CGGCCGTGCGCGAGGCG
F
5253
CGGCCGTGCGCGAGGCGC
5501
C
CGGCCGTGCGCGAGGCGA
5525
A
1704C>G
GGCAAGAAGTCGCTGGAG
F
5614
GGCAAGAAGTCGCTGGAGC
5861
C
GGCAAGAAGTCGCTGGAGG
5901
G
310G>T
TAAATGCCCTTCTCCAGGA
R
5748
TAAATGCCCTTCTCCAGGAC
5995
G
TAAATGCCCTTCTCCAGGAA
6019
T
957C>T
GTCGTGCTCAATGGGCTGG
F
5876
GTCGTGCTCAATGGGCTGGC
6123
C
GTCGTGCTCAATGGGCTGGT
6203
T
883G>C
TCCCCGAAGCGGCGCCGCAA
R
6073
TCCCCGAAGCGGCGCCGCAAC
6320
G
TCCCCGAAGCGGCGCCGCAAG
6360
C
1659G>A
CGCGAGCAGAGGCGCTTCTCC
F
6408
CGCGAGCAGAGGCGCTTCTCCA
6679
A
CGCGAGCAGAGGCGCTTCTCCG
6695
G
31G>A
AGCAGGAAGATGGCCACTATCA
R
6777
AGCAGGAAGATGGCCACTATCAC
7024
G
AGCAGGAAGATGGCCACTATCAT
7104
A
1724C>T
GCGAAGGCGGCACAAAGGCAGGC
R
7158
GCGAAGGCGGCACAAAGGCAGGCA
7429
T
GCGAAGGCGGCACAAAGGCAGGCG
7445
C
124G>A
TCACATGCAGCAGGTTGCCCAGCC
R
7299
TCACATGCAGCAGGTTGCCCAGCCC
7546
G
TCACATGCAGCAGGTTGCCCAGCCT
7626
A
(4) 14-PLEX
3198C>G
ACCCATCTCTGGTCGCC
R
5082
ACCCATCTCTGGTCGCCC
5330
G
ACCCATCTCTGGTCGCCG
5370
C
2549delA
TGGGTCCCAGGTCATCC
R
5162
TGGGTCCCAGGTCATCCG
5450
del
TGGGTCCCAGGTCATCCT
5490
A
3582A>G
GAATGTTGGAGGACCCA
F
5259
GAATGTTGGAGGACCCAA
5531
A
GAATGTTGGAGGACCCAG
5547
G
2539_2542 delAACT
ACAGCTGGATGAGCTGCT
F
5540
ACAGCTGGATGAGCTGCTA
5811
AACT
ACAGCTGGATGAGCTGCTG
5827
del
3790C>T
CCACTCTCACCCTGCATCT
F
5620
CCACTCTCACCCTGCATCTC
5867
C
CCACTCTCACCCTGCATCTT
5947
T
4115C>T
ACCTCCCTGCTGCAGCACTT
F
5989
ACCTCCCTGCTGCAGCACTTC
6236
C
ACCTCCCTGCTGCAGCACTTT
6316
T
4481G>A
GAATCTGACTGCCCAGATTG
F
6117
GAATCTGACTGCCCAGATTGA
6388
A
GAATCTGACTGCCCAGATTGG
6404
G
3828G>A
ACTCATCACCAACCTGTCATC
F
6270
ACTCATCACCAACCTGTCATCA
6541
A
ACTCATCACCAACCTGTCATCG
6557
G
3384A>C
CATGCTGGGGCTATCACCAGG
R
6447
CATGCTGGGGCTATCACCAGGG
6734
C
CATGCTGGGGCTATCACCAGGT
6774
A
2853A>C
GAGAACAGGTCAGCCACCACTA
R
6722
GAGAACAGGTCAGCCACCACTAG
7010
C
GAGAACAGGTCAGCCACCACTAT
7050
A
2988G>A
CATGTGCCCCCGCCTGTACCCTT
R
6888
CATGTGCCCCCGCCTGTACCCTTC
7135
G
CATGTGCCCCCGCCTGTACCCTTT
7215
A
3584G>A
GTCAGAATGTTGGAGGACCCAAC
F
7098
GTCAGAATGTTGGAGGACCCAACA
7369
A
GTCAGAATGTTGGAGGACCCAACG
7385
G
4401C>T
TAACTGACATCTGCTCAGCCTCAAC
R
7546
TAACTGACATCTGCTCAGCCTCAACA
7817
T
TAACTGACATCTGCTCAGCCTCAACG
7833
C
2587_2590 delGACT
ATCAGCTCAGCCCCCCCGAGACCTGA
F
7846
ATCAGCTCAGCCCCCCCGAGACCTGAC
8093
GACT
ATCAGCTCAGCCCCCCCGAGACCTGAG
8133
del
(5) 15-PLEX
3948T>G
TCTCAGCAGGTGCCTG
F
4873
TCTCAGCAGGTGCCTGG
5160
G
TCTCAGCAGGTGCCTT
5200
T
-1235A>G
GCACCACCCAGCCTAAT
R
5084
GCACCACCCAGCCTAATC
5332
G
GCACCACCCAGCCTAATT
5411
A
137_138insT
GAAGTCCACATGCAGCA
R
5188
GAAGTCCACATGCAGCAA
5460
T
GAAGTCCACATGCAGCAG
5476
—
4155C>T
CAGCCCCGGCCCAGCCAC
F
5376
CAGCCCCGGCCCAGCCACC
5623
C
CAGCCCCGGCCCAGCCACT
5703
T
82C>T
GTGTAGCGTGCAGCCCAGC
R
5830
GTGTAGCGTGCAGCCCAGCA
6101
T
GTGTAGCGTGCAGCCCAGCG
6117
C
-1584C>G
CTGGACAACTTGGAAGAACC
F
6135
CTGGACAACTTGGAAGAACCC
6382
C
CTGGACAACTTGGAAGAACCG
6422
G
2575C>A
GACCTGGGACCCAGCCCAGCC
F
6362
GACCTGGGACCCAGCCCAGCCC
6609
C
GACCTGGGACCCAGCCCAGCCA
6633
A
843T>G
ACTAGGACCTGTAGTCTGGGG
F
6502
ACTAGGACCTGTAGTCTGGGGG
6789
G
ACTAGGACCTGTAGTCTGGGGT
6829
T
-740C>T
ACAGACTCACACTGACACTTAG
R
6672
ACAGACTCACACTGACACTTAGA
6944
T
ACAGACTCACACTGACACTTAGG
6960
C
-678G>A
CTTTGTGTGGGTGATTTTCTGC
F
6769
CTTTGTGTGGGTGATTTTCTGCA
7041
A
CTTTGTGTGGGTGATTTTCTGCG
7057
G
-750_-749 delGA
TGTGACTGGTGTGTGTGAGAGA
F
6902
TGTGACTGGTGTGTGTGAGAGAA
7173
del
TGTGACTGGTGTGTGTGAGAGAG
7189
GA
2291G>A
CACTCGCCAAGTGCCAGCCTCCAC
R
7204
CACTCGCCAAGTGCCAGCCTCCACC
7451
G
CACTCGCCAAGTGCCAGCCTCCACT
7531
A
-1426C>T
GTGTGCCACCACGTCTAGCTTTTT
R
7286
GTGTGCCACCACGTCTAGCTTTTTA
7533
T
GTGTGCCACCACGTCTAGCTTTTTG
7573
C
1758G>T>A
TTGTGCCGCCTTCGCCAACCACTCC
F
7490
TTGTGCCGCCTTCGCCAACCACTCCA
7761
A
TTGTGCCGCCTTCGCCAACCACTCCG
7777
G
TTGTGCCGCCTTCGCCAACCACTCCT
7817
T
-1000G>A
ACATCCTCCCGGGCTGCCTGAGGGT
R
7635
ACATCCTCCCGGGCTGCCTGAGGGTC
7882
G
ACATCCTCCCGGGCTGCCTGAGGGTT
7962
A
UD: Unextended mini–sequencing primer Direction; F: Forward UEP direction; R: Reverse UEP direction; UM: Unextended mini–sequencing primers Mass; ELM: Extended primer Lower Mass; EHM: Extended primer Higher Mass; ELC: Extended primer lower Mass nucleotide Called; EHC: Extended primer higher Mass nucleotide Called; All molecular weight masses are expressed in Dalton (Da).Table 4
List of SNPs excluded in assay design validation and correlation to transcriptional variations.
SNPs excluded
Variations
Reason
-1770G>A
—
Cross-hybridization
-1298G>A
—
Primer dimers
-1253A>G
—
Primer dimers
-1237_-1236insAA
—
Proximal SNP -1235A>G Primer dimers
1973_1974insG
Frameshift
Proximal SNP 1979T>C
1976G>A
—
Proximal SNP 1979T>C
1978C>T
—
Proximal SNP 1979T>C
2097A>G
—
Primer dimers
2470T>C
—
Cross hybridization
4042G>A
—
Primer dimersTable 5
CYP2D6 allele frequencies in 250 healthy Sardinian people. Total chromosomes number = 500. Human cytochrome P450 Allele Nomenclature Committee [7] served as reference for variant allele and correlated enzymatic activity.
Variant allele
Number of chromosomes
Frequency (%)
Correlated enzymatic activity
*
1A
148
29.6
EM
*1B
5
1.0
EM
*1D
4
0.8
EM
*1E
—
—
EM
*2A
75
15.0
EM
*2B
—
—
EM
*2D
—
—
EM
*2E
—
—
EM
*2F
—
—
EM
*2G
—
—
EM
*2K
—
—
EM
*2L
11
2.2
EM
*2M
8
1.6
EM
SH3
7
1.4
Not known
SH4
1
0.2
Not known
*3A
—
—
PM
*3B
11
2.2
PM
*4A
84
16.8
PM
*4B
—
—
PM
*4D
—
—
PM
*4K
—
—
PM
*4L
—
—
PM
*4M
—
—
PM
*4N
—
—
PM
*5
5
1.0
PM
*6A
1
0.2
PM
*6C
—
—
PM
*6D
—
—
PM
*7
—
—
PM
*8
—
—
PM
*9
1
0.2
IM
*10A
—
—
IM
*10B
27
5.4
IM
*11
—
—
PM
*12
—
—
PM
*14A
—
—
PM
*14B
—
—
IM
*15
3
0.6
PM
*17
—
—
IM
*19
—
—
PM
*20
1
0.2
PM
*22
—
—
Not known
*23
—
—
Not known
*24
—
—
Not known
*25
—
—
Not known
*26
—
—
Not known
*27
—
—
EM
*28
4
0.8
Not known
*29
—
—
IM
*30
—
—
Not known
*31
—
—
PM
*32
—
—
Not known
*33
—
—
EM
*35A
5
1.0
EM
*36
—
—
IM
*37
—
—
Not known
*38
—
—
PM
*39
—
—
EM
*41
46
9.2
IM
SH1
41
8.2
Not known
SH2
2
0.4
Not known
*43
—
—
Not known
*58
—
—
Not known
*59
—
—
IM
*64
—
—
Not known
*65
—
—
Not known
*1xN
4
0.8
UM
*2xN
6
1.2
UM
SH1,2,3,4 = Sardinian haplotype 1,2,3,4 [5, 22–25]. CYP2D6*5,*1xN; and *2xN alleles were evaluated by The CYP2D6 Applied Biosystems CNV Assay in [5].


#### 2.1.4. iPLEX Reactions

iPLEX reactions were carried out following the Sequenom standard low/medium-plex protocol [6, 26, 27], with minor modifications. Because of the length of the primary PCR fragment and the high GC content, we increased the denaturation time at 94°C from 5 to 30 sec and the annealing temperature from 52 to 56°C. An iPLEX reaction cocktail was added to the amplification products and thermocycled to process the iPLEX reaction, which involved the enzymatic addition of one of the four mass-modified nucleotides, 2′,3′-dideoxynucleoside-5′-triphosphate (ddNTP), into the polymorphic site. During the iPLEX reaction, each primer was extended by one of the ddNTPs which terminated the extension of primers, thus producing allele-specific extension products of different masses. The iPLEX reaction cocktail included 0.222X iPLEX buffer plus, 0.5X iPLEX termination mix, 0.5X iPLEX enzyme, and UEPs, which were divided into four mass groups, according to the position of their respective mass peaks to use the whole spectrum (final concentrations of 0.8–2.0 μM). In the iPLEX termination mix, all four ddNTPs are present at the same concentration. The reactions were performed using a two cycling loop program: initial denaturation at 94°C for 30 s; followed by 40 cycles of 94°C for 30 s, 56°C for 5 s, and 80°C for 5 s. This annealing and extension procedure was repeated four times (to give a total of 200 short cycles), followed by a final extension step of 3 min at 72°C. After desalting by addition of 6 mg clean resin (Sequenom), each 384-well sample was diluted with 16 μL of sterilized H2Odd.Multiplex PCR reactions, SAP dephosphorylation, and iPLEX reactions were performed using Thermo-Fast 384 PCR Plates (ABgene, Epsom, UK) and a DNA Engine Tetrad 2 Peltier Thermal Cycler (Bio-Rad, CA, USA). All pipetting steps were performed using the automatic station Matrix PlateMate 2 × 2 (Sequenom).


#### 2.1.5. MALDI-TOF MS Measurement

An aliquot ranging from 15 to 20 nL of each iPLEX reaction product was loaded in a 384-spot SpectroChip (Sequenom) using the MassArray Nanodispenser (Samsung, Seoul, Repubic of Korea). SpectroChip analysis was performed by MassARRAY Compact System (Sequenom). After laser desorption/ionization, automated spectra acquisition analysis was performed and interpreted using Sequenom MassARRAY RT version 3.3 software. Examples of multiplex mass spectrum and cluster plot distributions are shown in Figures1 and 2.Figure 1
An example of 15–plex mass spectrum is shown (Well 5). List of SNPs investigated in this plex is shown in Table3. The figure shows two examples highlighted in different colours. Unextend primer (UEP) peak is marked by an asterisk and dotted arrow, while the solid arrows indicate the presence of the two different alleles. Dotted vertical lines represent UEPs and extended primers (EPs) expected masses. In blue, an AG heterozygous genotype example is shown. *: −1235A>G UEP; expected mass = 5084 Dalton (Da). G: −1235A>G EP; expected mass = 5332 Da. A: −1235A>G EP; expected mass = 5411 Da. In red, a CC wild type homozygous genotype example is shown. *: 82C>T UEP; expected mass = 5830 Da. T: 82C>T not EP; expected mass = 6101 Da. C: 82C>T EP; expected mass = 6117 Da.In (a) a Cluster plot for 1661G>C SNP is shown. Homozygous wild type G/G samples are displayed in (α), homozygous mutate C/C samples in (β) and heterozygous G/C samples in (γ); in (δ) and (ε) are displayed outlier samples resulting in gene copy number variation [5]; (ζ) negative control (H2Odd). In (b) a Mass Spectra for the detection of 1661G>C in a heterozygous G/C sample, displayed in (a) position (γ). In (c) a Mass Spectra for the detection of 1661G>C in the outlier sample, displayed in (a) position (δ), and resulting in CYP2D6*1×N allele [5]. In (d) Mass Spectra for the detection of 1661G > C in an outlier sample, displayed in (a) position (ε), and resulting in CYP2D6*2×N allele [5].
(a)
(b)
(c)
(d)


### 2.2.CYP2D6 Single Allele Genotyping

Following direction of our previous work [5], we decided to apply the single allele protocol creating a single allele genotyping method MALDI-TOF MS based. For each sample, a double long PCR reaction was carried out using P-1584_WT or P-1584_MUT [5] (Table 2) as forward primers. The reverse primer was 2D6-R [19] for both PCR reactions. In this way, we were able to directly determine a direct and correct chromosome phase in samples presenting with a heterozygous status for −1584G>C SNP. PCR reactions were performed in a final volume of 5 μL using 20 ng genomic DNA, 400 μM of each PCR primer (Metabion), 0.2 U QIAGEN LongRange PCR enzyme, 1X QIAGEN LongRange PCR buffer (containing MgCl2 25 mM), and 800 μM Invitrogen dNTP set PCR grade. The PCR conditions were as follows: initial denaturation at 93°C for 3 min; 10 cycles at 93°C for 30 s, 67°C for 30 s, and 68°C for 3 min; 25 cycles at 93°C for 30 s, 65°C for 30 s, and 68°C for 6 min. SAP dephosphorylation, iPLEX Reactions and MALDI-TOF MS measurement were performed without modifications as indicated in paragraph 1 “CYP2D6 Genotyping Platform”. In multiplexed assay 5, −1584G>C UEP (Table 3) was excluded. Examples of cluster distribution for novel 3176C>T and 3948T>G SNPs [5, 17, 18] are shown in Figure 3.MALDI-TOF MS cluster plot distribution for 3176C>T [17] and 3948G>T [18] newly discovered SNPs [5]. In each cluster, there are visualized 32 samples presenting heterozygous status for −1584G>C SNP and submitted to both types of PCR analysis, LongRange PCR (Section 2, paragraph 1) and the two single allele PCR (Section 2, paragraph 2), for a total of 96 PCR analyses. (a) Cluster plot for 3176C>T SNP. (α) homozygous wild type C/C samples; (β) one out of the two heterozygous C/T samples presenting heterozygous status for −1584G>C SNP also; (γ) the same heterozygous sample analysed in single allele PCR; (ζ) negative control (H2Odd). (b) Cluster plot for 3948T>G SNP. (α) homozygous wild type T/T samples; (β) the only heterozygous T/G sample; (γ) the only heterozygous sample analysed in single allele PCR; (ζ) negative control (H2Odd).
(a)
(b)


## 2.1.CYP2D6 Genotyping Platform

### 2.1.1. Long Primary PCR

Selective amplification of theCYP2D6 gene was carried out modifying a long PCR protocol implemented in our previous work [5]. Forward primer (P-1780, Table 2) was designed in a highly nonhomologous CYP2D6/CYP2D7P/CYP2D8P 5′ untranslated region [5]. The reverse primer used was 2D6-R (Table 2), as previously described [19]. A 5′ 10-mer tag (5′-ACGTTGGATG-3′) was added to both PCR primers in order to improve PCR efficiency. PCR reactions were performed in a final volume of 5 μL using the QIAGEN (Hilden, Germany) LongRange PCR Kit protocol [20] with the following minor modifications: 20 ng genomic DNA, 400 μM of each PCR primer (Metabion, Martinsried, Germany), 0.2 U QIAGEN LongRange PCR enzyme, 1X QIAGEN LongRange PCR buffer (containing MgCl2 25 mM), and 500 μM Invitrogen (CA, USA) 2′-deoxynucleoside-5′-triphosphate (dNTP) Set PCR Grade. The PCR conditions were as follows: initial denaturation at 93°C for 3 min; 35 cycles at 93°C for 30 s, 61°C for 30 s, and 68°C for 6 min.Table 2
List of primers used inCYP2D6 long PCR protocols. The 5′ 10-mer tag was added to PCR primers, in order to improve PCR efficiency.
Nucleotides position
Name
5′ 10–mer tag
5′-Sequence-3′
Direction
From -1780 to -1758
P-1780 [5]
ACGTTGGATG
GTCCTCCTGTCCTCAGTGGAT
Forward
From -1584 to -1559
P-1584_WT [5]
ACGTTGGATG
CAGCCTGGACAACTTGGAAGAAGCC
Forward
From -1584 to -1559
P-1584_MUT [5]
ACGTTGGATG
CAGCCTGGACAACTTGGAAGAAGCG
Forward
From 4706 to 4728
2D6–R [19]
ACGTTGGATG
ACTGAGCCCTGGGAGGTAGGTA
Reverse


### 2.1.2. SAP Dephosphorylation

To neutralize unincorporated dNTPs after amplification reactions, 0.3 U of Shrimp Alkaline Phosphatase (SAP) (Sequenom) [26, 27] was used. SAP cleaved a phosphate from the unincorporated dNTPs, converting them to 2′-deoxynucleoside-5′-diphosphate (dNDP) and rendering them unavailable for following reactions. Dephosphorylation conditions were as follows: 37°C for 20 min and 85°C for 5 min.


### 2.1.3. Assay Design

SNP-specific unextended minisequencing primers (UEPs) and multiplexed UEPs assays were designed using both the Sequenom MassARRAY assay design version 3.1 and the RealSNP assay database [28]. A sequence of 400 base pairs (bp) flanking each selected SNP was downloaded from the corresponding genomic sequence stored in the public NCBI Single Nucleotide Polymorphism dbSNP database [8] or the Ensembl Genome Browser [29] and was analyzed by Vector NTI Suite Software version 5.5 (InforMax, Oxford, UK). Combination of the UEPs into multiplex assays was supported by these software applications to allow the optimization of several different parameters, for example, to avoid the risk of primer-primer interactions and hairpin-loop formations, GC content, molecular mass range, and annealing temperatures. To achieve the highest possible multiplexing levels, we tested many primer combinations, leading us to the final assay design consisting in a total of 69 SNPs successfully assembled in five medium-plex assays (13-, 14-, 13-, 14-, and 15-plex) (Table 3). For some SNPs, Sequenom MassARRAY assay design and RealSNP assay database could not design SNP-specific UEPs because of the presence of proximal SNPs. Other SNPs were excluded during platform validation because of UEPs cross-hybridization in highly homologous PCR template regions or for the presence of primer dimers which created false allele. But the high number of SNPs inserted in our analysis has allowed us to infer the correct alleles for each DNA analysis. List of SNPs not included is summarized in Table 4.Table 3
Sequences and molecular weights of unextended (UEP) and extended (EP) iPLEX primers.
UEPs’  sequences
UD
UM
EP lower mass sequences
ELM
ELC
EP higher mass sequences
EHM
EHC
(1) 13-PLEX
3877G>A
CCTTCCGCTTCCACCCC
F
4978
CCTTCCGCTTCCACCCCA
5249
A
CCTTCCGCTTCCACCCCG
5265
G
3183G>A
CCGCACCTGCCCTATCA
R
5051
CCGCACCTGCCCTATCAC
5299
G
CCGCACCTGCCCTATCAT
5378
A
2615_2617 delAAG
GGCAGCCACTCTCACCT
R
5091
GGCAGCCACTCTCACCTC
5339
del
GGCAGCCACTCTCACCTT
5418
AGA
2483G>T
GCGTAGGACCTTGCCAG
R
5211
GCGTAGGACCTTGCCAGC
5459
G
GCGTAGGACCTTGCCAGA
5483
T
3853G>A
GAAGCGGAAGGGCTTCT
R
5275
GAAGCGGAAGGGCTTCTC
5523
G
GAAGCGGAAGGGCTTCTT
5603
A
3277T>C
TGCAGCGCTTTGGGGACA
F
5556
TGCAGCGCTTTGGGGACAC
5803
C
TGCAGCGCTTTGGGGACAT
5883
T
4180G>C
AGCTCATAGGGGGATGGG
R
5645
AGCTCATAGGGGGATGGGC
5892
G
AGCTCATAGGGGGATGGGG
5932
C
2939G>A
TGGGCTCACGCTGCACATC
R
5766
TGGGCTCACGCTGCACATCC
6013
G
TGGGCTCACGCTGCACATCT
6093
A
3288G>A
GATGTCATATGGGTCACAC
R
5828
GATGTCATATGGGTCACACC
6075
G
GATGTCATATGGGTCACACT
6155
A
3176C>T
GGCCGTGTCCAACAGGAGAT
F
6167
GGCCGTGTCCAACAGGAGATC
6414
C
GGCCGTGTCCAACAGGAGATT
6494
T
2850C>T
AGCTTCAATGATGAGAACCTG
F
6454
AGCTTCAATGATGAGAACCTGC
6701
C
AGCTTCAATGATGAGAACCTGT
6781
T
2935A>C
AGTGCTCACGCTGCACATCCGGA
R
7010
AGTGCTCACGCTGCACATCCGGAG
7297
C
AGTGCTCACGCTGCACATCCGGAT
7337
A
1757C>T
TTGTGCCGCCTTCGCCAACCACTC
F
7201
TTGTGCCGCCTTCGCCAACCACTCC
7448
C
TTGTGCCGCCTTCGCCAACCACTCT
7528
T
(2) 14-PLEX
1661G>C
CAGAGGCGCTTCTCCGT
F
5162
CAGAGGCGCTTCTCCGTC
5410
C
CAGAGGCGCTTCTCCGTG
5450
G
1869T>C
ACGGCTTTGTCCAAGAG
R
5210
ACGGCTTTGTCCAAGAGA
5482
T
ACGGCTTTGTCCAAGAGG
5498
C
1846G>A
GGGGCGAAAGGGGCGTC
R
5342
GGGGCGAAAGGGGCGTCC
5588
G
GGGGCGAAAGGGGCGTCT
5668
A
214G>C
TGGAGGGCGGCAGAGGT
F
5357
TGGAGGGCGGCAGAGGTC
5604
C
TGGAGGGCGGCAGAGGTG
5644
G
19G>A
CTGGGGCTAGAAGCACTG
F
5565
CTGGGGCTAGAAGCACTGA
5836
A
CTGGGGCTAGAAGCACTGG
5852
G
1707delT
GCGGCCTCCTCGGTCACCC
R
5702
GCGGCCTCCTCGGTCACCCC
5949
del
GCGGCCTCCTCGGTCACCCA
5973
T
746C>G
GCGACCCAGCCTCCTGATC
R
5710
GCGACCCAGCCTCCTGATCC
5957
G
GCGACCCAGCCTCCTGATCG
5997
C
997C>G
GAACAGGCGGGCGGTCGGC
R
5920
GAACAGGCGGGCGGTCGGCC
6167
G
GAACAGGCGGGCGGTCGGCG
6207
C
100C>T
AATGCTGGGCTGCACGCTAC
F
6118
AATGCTGGGCTGCACGCTACC
6365
C
AATGCTGGGCTGCACGCTACT
6445
T
1979T>C
GGACAGCCCGACTCCTCCTTC
R
6303
GGACAGCCCGACTCCTCCTTCA
6574
T
GGACAGCCCGACTCCTCCTTCG
6590
C
-948C>A
CAGGCTGGGGCAAGGGCCTTC
F
6488
CAGGCTGGGGCAAGGGCCTTCC
6735
C
CAGGCTGGGGCAAGGGCCTTCA
6759
A
1749A>G
GCCCATCACCCACCGGAGTGGT
R
6681
GCCCATCACCCACCGGAGTGGTC
6929
G
GCCCATCACCCACCGGAGTGGTT
7008
A
77G>A
GTAGCGTGCAGCCCAGCGTTGG
R
6792
GTAGCGTGCAGCCCAGCGTTGGC
7040
G
GTAGCGTGCAGCCCAGCGTTGGT
7120
A
1513C>T
AGCTGGACAGAGCCAGGGACTG
F
6835
AGCTGGACAGAGCCAGGGACTGC
7082
C
AGCTGGACAGAGCCAGGGACTGT
7162
T
(3) 13-PLEX
984A>G
GCGAGTGTCCTCGCCG
R
4874
GCGAGTGTCCTCGCCGC
5121
G
GCGAGTGTCCTCGCCGT
5201
A
1863_1864ins TTTCGCCCC
GACGCCCCTTTCGCCCC
F
5043
GACGCCCCTTTCGCCCCA
5315
–
GACGCCCCTTTCGCCCCT
5370
TTTCGCCCC
1039C>T
ACCCAGATCCTGGGTTT
F
5161
ACCCAGATCCTGGGTTTC
5409
C
ACCCAGATCCTGGGTTTT
5489
T
1943G>A
CCAGCAGCCTGAGGAAG
R
5229
CCAGCAGCCTGAGGAAGC
5477
G
CCAGCAGCCTGAGGAAGT
5557
A
974C>A
CGGCCGTGCGCGAGGCG
F
5253
CGGCCGTGCGCGAGGCGC
5501
C
CGGCCGTGCGCGAGGCGA
5525
A
1704C>G
GGCAAGAAGTCGCTGGAG
F
5614
GGCAAGAAGTCGCTGGAGC
5861
C
GGCAAGAAGTCGCTGGAGG
5901
G
310G>T
TAAATGCCCTTCTCCAGGA
R
5748
TAAATGCCCTTCTCCAGGAC
5995
G
TAAATGCCCTTCTCCAGGAA
6019
T
957C>T
GTCGTGCTCAATGGGCTGG
F
5876
GTCGTGCTCAATGGGCTGGC
6123
C
GTCGTGCTCAATGGGCTGGT
6203
T
883G>C
TCCCCGAAGCGGCGCCGCAA
R
6073
TCCCCGAAGCGGCGCCGCAAC
6320
G
TCCCCGAAGCGGCGCCGCAAG
6360
C
1659G>A
CGCGAGCAGAGGCGCTTCTCC
F
6408
CGCGAGCAGAGGCGCTTCTCCA
6679
A
CGCGAGCAGAGGCGCTTCTCCG
6695
G
31G>A
AGCAGGAAGATGGCCACTATCA
R
6777
AGCAGGAAGATGGCCACTATCAC
7024
G
AGCAGGAAGATGGCCACTATCAT
7104
A
1724C>T
GCGAAGGCGGCACAAAGGCAGGC
R
7158
GCGAAGGCGGCACAAAGGCAGGCA
7429
T
GCGAAGGCGGCACAAAGGCAGGCG
7445
C
124G>A
TCACATGCAGCAGGTTGCCCAGCC
R
7299
TCACATGCAGCAGGTTGCCCAGCCC
7546
G
TCACATGCAGCAGGTTGCCCAGCCT
7626
A
(4) 14-PLEX
3198C>G
ACCCATCTCTGGTCGCC
R
5082
ACCCATCTCTGGTCGCCC
5330
G
ACCCATCTCTGGTCGCCG
5370
C
2549delA
TGGGTCCCAGGTCATCC
R
5162
TGGGTCCCAGGTCATCCG
5450
del
TGGGTCCCAGGTCATCCT
5490
A
3582A>G
GAATGTTGGAGGACCCA
F
5259
GAATGTTGGAGGACCCAA
5531
A
GAATGTTGGAGGACCCAG
5547
G
2539_2542 delAACT
ACAGCTGGATGAGCTGCT
F
5540
ACAGCTGGATGAGCTGCTA
5811
AACT
ACAGCTGGATGAGCTGCTG
5827
del
3790C>T
CCACTCTCACCCTGCATCT
F
5620
CCACTCTCACCCTGCATCTC
5867
C
CCACTCTCACCCTGCATCTT
5947
T
4115C>T
ACCTCCCTGCTGCAGCACTT
F
5989
ACCTCCCTGCTGCAGCACTTC
6236
C
ACCTCCCTGCTGCAGCACTTT
6316
T
4481G>A
GAATCTGACTGCCCAGATTG
F
6117
GAATCTGACTGCCCAGATTGA
6388
A
GAATCTGACTGCCCAGATTGG
6404
G
3828G>A
ACTCATCACCAACCTGTCATC
F
6270
ACTCATCACCAACCTGTCATCA
6541
A
ACTCATCACCAACCTGTCATCG
6557
G
3384A>C
CATGCTGGGGCTATCACCAGG
R
6447
CATGCTGGGGCTATCACCAGGG
6734
C
CATGCTGGGGCTATCACCAGGT
6774
A
2853A>C
GAGAACAGGTCAGCCACCACTA
R
6722
GAGAACAGGTCAGCCACCACTAG
7010
C
GAGAACAGGTCAGCCACCACTAT
7050
A
2988G>A
CATGTGCCCCCGCCTGTACCCTT
R
6888
CATGTGCCCCCGCCTGTACCCTTC
7135
G
CATGTGCCCCCGCCTGTACCCTTT
7215
A
3584G>A
GTCAGAATGTTGGAGGACCCAAC
F
7098
GTCAGAATGTTGGAGGACCCAACA
7369
A
GTCAGAATGTTGGAGGACCCAACG
7385
G
4401C>T
TAACTGACATCTGCTCAGCCTCAAC
R
7546
TAACTGACATCTGCTCAGCCTCAACA
7817
T
TAACTGACATCTGCTCAGCCTCAACG
7833
C
2587_2590 delGACT
ATCAGCTCAGCCCCCCCGAGACCTGA
F
7846
ATCAGCTCAGCCCCCCCGAGACCTGAC
8093
GACT
ATCAGCTCAGCCCCCCCGAGACCTGAG
8133
del
(5) 15-PLEX
3948T>G
TCTCAGCAGGTGCCTG
F
4873
TCTCAGCAGGTGCCTGG
5160
G
TCTCAGCAGGTGCCTT
5200
T
-1235A>G
GCACCACCCAGCCTAAT
R
5084
GCACCACCCAGCCTAATC
5332
G
GCACCACCCAGCCTAATT
5411
A
137_138insT
GAAGTCCACATGCAGCA
R
5188
GAAGTCCACATGCAGCAA
5460
T
GAAGTCCACATGCAGCAG
5476
—
4155C>T
CAGCCCCGGCCCAGCCAC
F
5376
CAGCCCCGGCCCAGCCACC
5623
C
CAGCCCCGGCCCAGCCACT
5703
T
82C>T
GTGTAGCGTGCAGCCCAGC
R
5830
GTGTAGCGTGCAGCCCAGCA
6101
T
GTGTAGCGTGCAGCCCAGCG
6117
C
-1584C>G
CTGGACAACTTGGAAGAACC
F
6135
CTGGACAACTTGGAAGAACCC
6382
C
CTGGACAACTTGGAAGAACCG
6422
G
2575C>A
GACCTGGGACCCAGCCCAGCC
F
6362
GACCTGGGACCCAGCCCAGCCC
6609
C
GACCTGGGACCCAGCCCAGCCA
6633
A
843T>G
ACTAGGACCTGTAGTCTGGGG
F
6502
ACTAGGACCTGTAGTCTGGGGG
6789
G
ACTAGGACCTGTAGTCTGGGGT
6829
T
-740C>T
ACAGACTCACACTGACACTTAG
R
6672
ACAGACTCACACTGACACTTAGA
6944
T
ACAGACTCACACTGACACTTAGG
6960
C
-678G>A
CTTTGTGTGGGTGATTTTCTGC
F
6769
CTTTGTGTGGGTGATTTTCTGCA
7041
A
CTTTGTGTGGGTGATTTTCTGCG
7057
G
-750_-749 delGA
TGTGACTGGTGTGTGTGAGAGA
F
6902
TGTGACTGGTGTGTGTGAGAGAA
7173
del
TGTGACTGGTGTGTGTGAGAGAG
7189
GA
2291G>A
CACTCGCCAAGTGCCAGCCTCCAC
R
7204
CACTCGCCAAGTGCCAGCCTCCACC
7451
G
CACTCGCCAAGTGCCAGCCTCCACT
7531
A
-1426C>T
GTGTGCCACCACGTCTAGCTTTTT
R
7286
GTGTGCCACCACGTCTAGCTTTTTA
7533
T
GTGTGCCACCACGTCTAGCTTTTTG
7573
C
1758G>T>A
TTGTGCCGCCTTCGCCAACCACTCC
F
7490
TTGTGCCGCCTTCGCCAACCACTCCA
7761
A
TTGTGCCGCCTTCGCCAACCACTCCG
7777
G
TTGTGCCGCCTTCGCCAACCACTCCT
7817
T
-1000G>A
ACATCCTCCCGGGCTGCCTGAGGGT
R
7635
ACATCCTCCCGGGCTGCCTGAGGGTC
7882
G
ACATCCTCCCGGGCTGCCTGAGGGTT
7962
A
UD: Unextended mini–sequencing primer Direction; F: Forward UEP direction; R: Reverse UEP direction; UM: Unextended mini–sequencing primers Mass; ELM: Extended primer Lower Mass; EHM: Extended primer Higher Mass; ELC: Extended primer lower Mass nucleotide Called; EHC: Extended primer higher Mass nucleotide Called; All molecular weight masses are expressed in Dalton (Da).Table 4
List of SNPs excluded in assay design validation and correlation to transcriptional variations.
SNPs excluded
Variations
Reason
-1770G>A
—
Cross-hybridization
-1298G>A
—
Primer dimers
-1253A>G
—
Primer dimers
-1237_-1236insAA
—
Proximal SNP -1235A>G Primer dimers
1973_1974insG
Frameshift
Proximal SNP 1979T>C
1976G>A
—
Proximal SNP 1979T>C
1978C>T
—
Proximal SNP 1979T>C
2097A>G
—
Primer dimers
2470T>C
—
Cross hybridization
4042G>A
—
Primer dimersTable 5
CYP2D6 allele frequencies in 250 healthy Sardinian people. Total chromosomes number = 500. Human cytochrome P450 Allele Nomenclature Committee [7] served as reference for variant allele and correlated enzymatic activity.
Variant allele
Number of chromosomes
Frequency (%)
Correlated enzymatic activity
*
1A
148
29.6
EM
*1B
5
1.0
EM
*1D
4
0.8
EM
*1E
—
—
EM
*2A
75
15.0
EM
*2B
—
—
EM
*2D
—
—
EM
*2E
—
—
EM
*2F
—
—
EM
*2G
—
—
EM
*2K
—
—
EM
*2L
11
2.2
EM
*2M
8
1.6
EM
SH3
7
1.4
Not known
SH4
1
0.2
Not known
*3A
—
—
PM
*3B
11
2.2
PM
*4A
84
16.8
PM
*4B
—
—
PM
*4D
—
—
PM
*4K
—
—
PM
*4L
—
—
PM
*4M
—
—
PM
*4N
—
—
PM
*5
5
1.0
PM
*6A
1
0.2
PM
*6C
—
—
PM
*6D
—
—
PM
*7
—
—
PM
*8
—
—
PM
*9
1
0.2
IM
*10A
—
—
IM
*10B
27
5.4
IM
*11
—
—
PM
*12
—
—
PM
*14A
—
—
PM
*14B
—
—
IM
*15
3
0.6
PM
*17
—
—
IM
*19
—
—
PM
*20
1
0.2
PM
*22
—
—
Not known
*23
—
—
Not known
*24
—
—
Not known
*25
—
—
Not known
*26
—
—
Not known
*27
—
—
EM
*28
4
0.8
Not known
*29
—
—
IM
*30
—
—
Not known
*31
—
—
PM
*32
—
—
Not known
*33
—
—
EM
*35A
5
1.0
EM
*36
—
—
IM
*37
—
—
Not known
*38
—
—
PM
*39
—
—
EM
*41
46
9.2
IM
SH1
41
8.2
Not known
SH2
2
0.4
Not known
*43
—
—
Not known
*58
—
—
Not known
*59
—
—
IM
*64
—
—
Not known
*65
—
—
Not known
*1xN
4
0.8
UM
*2xN
6
1.2
UM
SH1,2,3,4 = Sardinian haplotype 1,2,3,4 [5, 22–25]. CYP2D6*5,*1xN; and *2xN alleles were evaluated by The CYP2D6 Applied Biosystems CNV Assay in [5].


### 2.1.4. iPLEX Reactions

iPLEX reactions were carried out following the Sequenom standard low/medium-plex protocol [6, 26, 27], with minor modifications. Because of the length of the primary PCR fragment and the high GC content, we increased the denaturation time at 94°C from 5 to 30 sec and the annealing temperature from 52 to 56°C. An iPLEX reaction cocktail was added to the amplification products and thermocycled to process the iPLEX reaction, which involved the enzymatic addition of one of the four mass-modified nucleotides, 2′,3′-dideoxynucleoside-5′-triphosphate (ddNTP), into the polymorphic site. During the iPLEX reaction, each primer was extended by one of the ddNTPs which terminated the extension of primers, thus producing allele-specific extension products of different masses. The iPLEX reaction cocktail included 0.222X iPLEX buffer plus, 0.5X iPLEX termination mix, 0.5X iPLEX enzyme, and UEPs, which were divided into four mass groups, according to the position of their respective mass peaks to use the whole spectrum (final concentrations of 0.8–2.0 μM). In the iPLEX termination mix, all four ddNTPs are present at the same concentration. The reactions were performed using a two cycling loop program: initial denaturation at 94°C for 30 s; followed by 40 cycles of 94°C for 30 s, 56°C for 5 s, and 80°C for 5 s. This annealing and extension procedure was repeated four times (to give a total of 200 short cycles), followed by a final extension step of 3 min at 72°C. After desalting by addition of 6 mg clean resin (Sequenom), each 384-well sample was diluted with 16 μL of sterilized H2Odd.Multiplex PCR reactions, SAP dephosphorylation, and iPLEX reactions were performed using Thermo-Fast 384 PCR Plates (ABgene, Epsom, UK) and a DNA Engine Tetrad 2 Peltier Thermal Cycler (Bio-Rad, CA, USA). All pipetting steps were performed using the automatic station Matrix PlateMate 2 × 2 (Sequenom).


### 2.1.5. MALDI-TOF MS Measurement

An aliquot ranging from 15 to 20 nL of each iPLEX reaction product was loaded in a 384-spot SpectroChip (Sequenom) using the MassArray Nanodispenser (Samsung, Seoul, Repubic of Korea). SpectroChip analysis was performed by MassARRAY Compact System (Sequenom). After laser desorption/ionization, automated spectra acquisition analysis was performed and interpreted using Sequenom MassARRAY RT version 3.3 software. Examples of multiplex mass spectrum and cluster plot distributions are shown in Figures1 and 2.Figure 1
An example of 15–plex mass spectrum is shown (Well 5). List of SNPs investigated in this plex is shown in Table3. The figure shows two examples highlighted in different colours. Unextend primer (UEP) peak is marked by an asterisk and dotted arrow, while the solid arrows indicate the presence of the two different alleles. Dotted vertical lines represent UEPs and extended primers (EPs) expected masses. In blue, an AG heterozygous genotype example is shown. *: −1235A>G UEP; expected mass = 5084 Dalton (Da). G: −1235A>G EP; expected mass = 5332 Da. A: −1235A>G EP; expected mass = 5411 Da. In red, a CC wild type homozygous genotype example is shown. *: 82C>T UEP; expected mass = 5830 Da. T: 82C>T not EP; expected mass = 6101 Da. C: 82C>T EP; expected mass = 6117 Da.In (a) a Cluster plot for 1661G>C SNP is shown. Homozygous wild type G/G samples are displayed in (α), homozygous mutate C/C samples in (β) and heterozygous G/C samples in (γ); in (δ) and (ε) are displayed outlier samples resulting in gene copy number variation [5]; (ζ) negative control (H2Odd). In (b) a Mass Spectra for the detection of 1661G>C in a heterozygous G/C sample, displayed in (a) position (γ). In (c) a Mass Spectra for the detection of 1661G>C in the outlier sample, displayed in (a) position (δ), and resulting in CYP2D6*1×N allele [5]. In (d) Mass Spectra for the detection of 1661G > C in an outlier sample, displayed in (a) position (ε), and resulting in CYP2D6*2×N allele [5].
(a)
(b)
(c)
(d)


## 2.1.1. Long Primary PCR

Selective amplification of theCYP2D6 gene was carried out modifying a long PCR protocol implemented in our previous work [5]. Forward primer (P-1780, Table 2) was designed in a highly nonhomologous CYP2D6/CYP2D7P/CYP2D8P 5′ untranslated region [5]. The reverse primer used was 2D6-R (Table 2), as previously described [19]. A 5′ 10-mer tag (5′-ACGTTGGATG-3′) was added to both PCR primers in order to improve PCR efficiency. PCR reactions were performed in a final volume of 5 μL using the QIAGEN (Hilden, Germany) LongRange PCR Kit protocol [20] with the following minor modifications: 20 ng genomic DNA, 400 μM of each PCR primer (Metabion, Martinsried, Germany), 0.2 U QIAGEN LongRange PCR enzyme, 1X QIAGEN LongRange PCR buffer (containing MgCl2 25 mM), and 500 μM Invitrogen (CA, USA) 2′-deoxynucleoside-5′-triphosphate (dNTP) Set PCR Grade. The PCR conditions were as follows: initial denaturation at 93°C for 3 min; 35 cycles at 93°C for 30 s, 61°C for 30 s, and 68°C for 6 min.Table 2
List of primers used inCYP2D6 long PCR protocols. The 5′ 10-mer tag was added to PCR primers, in order to improve PCR efficiency.
Nucleotides position
Name
5′ 10–mer tag
5′-Sequence-3′
Direction
From -1780 to -1758
P-1780 [5]
ACGTTGGATG
GTCCTCCTGTCCTCAGTGGAT
Forward
From -1584 to -1559
P-1584_WT [5]
ACGTTGGATG
CAGCCTGGACAACTTGGAAGAAGCC
Forward
From -1584 to -1559
P-1584_MUT [5]
ACGTTGGATG
CAGCCTGGACAACTTGGAAGAAGCG
Forward
From 4706 to 4728
2D6–R [19]
ACGTTGGATG
ACTGAGCCCTGGGAGGTAGGTA
Reverse


## 2.1.2. SAP Dephosphorylation

To neutralize unincorporated dNTPs after amplification reactions, 0.3 U of Shrimp Alkaline Phosphatase (SAP) (Sequenom) [26, 27] was used. SAP cleaved a phosphate from the unincorporated dNTPs, converting them to 2′-deoxynucleoside-5′-diphosphate (dNDP) and rendering them unavailable for following reactions. Dephosphorylation conditions were as follows: 37°C for 20 min and 85°C for 5 min.


## 2.1.3. Assay Design

SNP-specific unextended minisequencing primers (UEPs) and multiplexed UEPs assays were designed using both the Sequenom MassARRAY assay design version 3.1 and the RealSNP assay database [28]. A sequence of 400 base pairs (bp) flanking each selected SNP was downloaded from the corresponding genomic sequence stored in the public NCBI Single Nucleotide Polymorphism dbSNP database [8] or the Ensembl Genome Browser [29] and was analyzed by Vector NTI Suite Software version 5.5 (InforMax, Oxford, UK). Combination of the UEPs into multiplex assays was supported by these software applications to allow the optimization of several different parameters, for example, to avoid the risk of primer-primer interactions and hairpin-loop formations, GC content, molecular mass range, and annealing temperatures. To achieve the highest possible multiplexing levels, we tested many primer combinations, leading us to the final assay design consisting in a total of 69 SNPs successfully assembled in five medium-plex assays (13-, 14-, 13-, 14-, and 15-plex) (Table 3). For some SNPs, Sequenom MassARRAY assay design and RealSNP assay database could not design SNP-specific UEPs because of the presence of proximal SNPs. Other SNPs were excluded during platform validation because of UEPs cross-hybridization in highly homologous PCR template regions or for the presence of primer dimers which created false allele. But the high number of SNPs inserted in our analysis has allowed us to infer the correct alleles for each DNA analysis. List of SNPs not included is summarized in Table 4.Table 3
Sequences and molecular weights of unextended (UEP) and extended (EP) iPLEX primers.
UEPs’  sequences
UD
UM
EP lower mass sequences
ELM
ELC
EP higher mass sequences
EHM
EHC
(1) 13-PLEX
3877G>A
CCTTCCGCTTCCACCCC
F
4978
CCTTCCGCTTCCACCCCA
5249
A
CCTTCCGCTTCCACCCCG
5265
G
3183G>A
CCGCACCTGCCCTATCA
R
5051
CCGCACCTGCCCTATCAC
5299
G
CCGCACCTGCCCTATCAT
5378
A
2615_2617 delAAG
GGCAGCCACTCTCACCT
R
5091
GGCAGCCACTCTCACCTC
5339
del
GGCAGCCACTCTCACCTT
5418
AGA
2483G>T
GCGTAGGACCTTGCCAG
R
5211
GCGTAGGACCTTGCCAGC
5459
G
GCGTAGGACCTTGCCAGA
5483
T
3853G>A
GAAGCGGAAGGGCTTCT
R
5275
GAAGCGGAAGGGCTTCTC
5523
G
GAAGCGGAAGGGCTTCTT
5603
A
3277T>C
TGCAGCGCTTTGGGGACA
F
5556
TGCAGCGCTTTGGGGACAC
5803
C
TGCAGCGCTTTGGGGACAT
5883
T
4180G>C
AGCTCATAGGGGGATGGG
R
5645
AGCTCATAGGGGGATGGGC
5892
G
AGCTCATAGGGGGATGGGG
5932
C
2939G>A
TGGGCTCACGCTGCACATC
R
5766
TGGGCTCACGCTGCACATCC
6013
G
TGGGCTCACGCTGCACATCT
6093
A
3288G>A
GATGTCATATGGGTCACAC
R
5828
GATGTCATATGGGTCACACC
6075
G
GATGTCATATGGGTCACACT
6155
A
3176C>T
GGCCGTGTCCAACAGGAGAT
F
6167
GGCCGTGTCCAACAGGAGATC
6414
C
GGCCGTGTCCAACAGGAGATT
6494
T
2850C>T
AGCTTCAATGATGAGAACCTG
F
6454
AGCTTCAATGATGAGAACCTGC
6701
C
AGCTTCAATGATGAGAACCTGT
6781
T
2935A>C
AGTGCTCACGCTGCACATCCGGA
R
7010
AGTGCTCACGCTGCACATCCGGAG
7297
C
AGTGCTCACGCTGCACATCCGGAT
7337
A
1757C>T
TTGTGCCGCCTTCGCCAACCACTC
F
7201
TTGTGCCGCCTTCGCCAACCACTCC
7448
C
TTGTGCCGCCTTCGCCAACCACTCT
7528
T
(2) 14-PLEX
1661G>C
CAGAGGCGCTTCTCCGT
F
5162
CAGAGGCGCTTCTCCGTC
5410
C
CAGAGGCGCTTCTCCGTG
5450
G
1869T>C
ACGGCTTTGTCCAAGAG
R
5210
ACGGCTTTGTCCAAGAGA
5482
T
ACGGCTTTGTCCAAGAGG
5498
C
1846G>A
GGGGCGAAAGGGGCGTC
R
5342
GGGGCGAAAGGGGCGTCC
5588
G
GGGGCGAAAGGGGCGTCT
5668
A
214G>C
TGGAGGGCGGCAGAGGT
F
5357
TGGAGGGCGGCAGAGGTC
5604
C
TGGAGGGCGGCAGAGGTG
5644
G
19G>A
CTGGGGCTAGAAGCACTG
F
5565
CTGGGGCTAGAAGCACTGA
5836
A
CTGGGGCTAGAAGCACTGG
5852
G
1707delT
GCGGCCTCCTCGGTCACCC
R
5702
GCGGCCTCCTCGGTCACCCC
5949
del
GCGGCCTCCTCGGTCACCCA
5973
T
746C>G
GCGACCCAGCCTCCTGATC
R
5710
GCGACCCAGCCTCCTGATCC
5957
G
GCGACCCAGCCTCCTGATCG
5997
C
997C>G
GAACAGGCGGGCGGTCGGC
R
5920
GAACAGGCGGGCGGTCGGCC
6167
G
GAACAGGCGGGCGGTCGGCG
6207
C
100C>T
AATGCTGGGCTGCACGCTAC
F
6118
AATGCTGGGCTGCACGCTACC
6365
C
AATGCTGGGCTGCACGCTACT
6445
T
1979T>C
GGACAGCCCGACTCCTCCTTC
R
6303
GGACAGCCCGACTCCTCCTTCA
6574
T
GGACAGCCCGACTCCTCCTTCG
6590
C
-948C>A
CAGGCTGGGGCAAGGGCCTTC
F
6488
CAGGCTGGGGCAAGGGCCTTCC
6735
C
CAGGCTGGGGCAAGGGCCTTCA
6759
A
1749A>G
GCCCATCACCCACCGGAGTGGT
R
6681
GCCCATCACCCACCGGAGTGGTC
6929
G
GCCCATCACCCACCGGAGTGGTT
7008
A
77G>A
GTAGCGTGCAGCCCAGCGTTGG
R
6792
GTAGCGTGCAGCCCAGCGTTGGC
7040
G
GTAGCGTGCAGCCCAGCGTTGGT
7120
A
1513C>T
AGCTGGACAGAGCCAGGGACTG
F
6835
AGCTGGACAGAGCCAGGGACTGC
7082
C
AGCTGGACAGAGCCAGGGACTGT
7162
T
(3) 13-PLEX
984A>G
GCGAGTGTCCTCGCCG
R
4874
GCGAGTGTCCTCGCCGC
5121
G
GCGAGTGTCCTCGCCGT
5201
A
1863_1864ins TTTCGCCCC
GACGCCCCTTTCGCCCC
F
5043
GACGCCCCTTTCGCCCCA
5315
–
GACGCCCCTTTCGCCCCT
5370
TTTCGCCCC
1039C>T
ACCCAGATCCTGGGTTT
F
5161
ACCCAGATCCTGGGTTTC
5409
C
ACCCAGATCCTGGGTTTT
5489
T
1943G>A
CCAGCAGCCTGAGGAAG
R
5229
CCAGCAGCCTGAGGAAGC
5477
G
CCAGCAGCCTGAGGAAGT
5557
A
974C>A
CGGCCGTGCGCGAGGCG
F
5253
CGGCCGTGCGCGAGGCGC
5501
C
CGGCCGTGCGCGAGGCGA
5525
A
1704C>G
GGCAAGAAGTCGCTGGAG
F
5614
GGCAAGAAGTCGCTGGAGC
5861
C
GGCAAGAAGTCGCTGGAGG
5901
G
310G>T
TAAATGCCCTTCTCCAGGA
R
5748
TAAATGCCCTTCTCCAGGAC
5995
G
TAAATGCCCTTCTCCAGGAA
6019
T
957C>T
GTCGTGCTCAATGGGCTGG
F
5876
GTCGTGCTCAATGGGCTGGC
6123
C
GTCGTGCTCAATGGGCTGGT
6203
T
883G>C
TCCCCGAAGCGGCGCCGCAA
R
6073
TCCCCGAAGCGGCGCCGCAAC
6320
G
TCCCCGAAGCGGCGCCGCAAG
6360
C
1659G>A
CGCGAGCAGAGGCGCTTCTCC
F
6408
CGCGAGCAGAGGCGCTTCTCCA
6679
A
CGCGAGCAGAGGCGCTTCTCCG
6695
G
31G>A
AGCAGGAAGATGGCCACTATCA
R
6777
AGCAGGAAGATGGCCACTATCAC
7024
G
AGCAGGAAGATGGCCACTATCAT
7104
A
1724C>T
GCGAAGGCGGCACAAAGGCAGGC
R
7158
GCGAAGGCGGCACAAAGGCAGGCA
7429
T
GCGAAGGCGGCACAAAGGCAGGCG
7445
C
124G>A
TCACATGCAGCAGGTTGCCCAGCC
R
7299
TCACATGCAGCAGGTTGCCCAGCCC
7546
G
TCACATGCAGCAGGTTGCCCAGCCT
7626
A
(4) 14-PLEX
3198C>G
ACCCATCTCTGGTCGCC
R
5082
ACCCATCTCTGGTCGCCC
5330
G
ACCCATCTCTGGTCGCCG
5370
C
2549delA
TGGGTCCCAGGTCATCC
R
5162
TGGGTCCCAGGTCATCCG
5450
del
TGGGTCCCAGGTCATCCT
5490
A
3582A>G
GAATGTTGGAGGACCCA
F
5259
GAATGTTGGAGGACCCAA
5531
A
GAATGTTGGAGGACCCAG
5547
G
2539_2542 delAACT
ACAGCTGGATGAGCTGCT
F
5540
ACAGCTGGATGAGCTGCTA
5811
AACT
ACAGCTGGATGAGCTGCTG
5827
del
3790C>T
CCACTCTCACCCTGCATCT
F
5620
CCACTCTCACCCTGCATCTC
5867
C
CCACTCTCACCCTGCATCTT
5947
T
4115C>T
ACCTCCCTGCTGCAGCACTT
F
5989
ACCTCCCTGCTGCAGCACTTC
6236
C
ACCTCCCTGCTGCAGCACTTT
6316
T
4481G>A
GAATCTGACTGCCCAGATTG
F
6117
GAATCTGACTGCCCAGATTGA
6388
A
GAATCTGACTGCCCAGATTGG
6404
G
3828G>A
ACTCATCACCAACCTGTCATC
F
6270
ACTCATCACCAACCTGTCATCA
6541
A
ACTCATCACCAACCTGTCATCG
6557
G
3384A>C
CATGCTGGGGCTATCACCAGG
R
6447
CATGCTGGGGCTATCACCAGGG
6734
C
CATGCTGGGGCTATCACCAGGT
6774
A
2853A>C
GAGAACAGGTCAGCCACCACTA
R
6722
GAGAACAGGTCAGCCACCACTAG
7010
C
GAGAACAGGTCAGCCACCACTAT
7050
A
2988G>A
CATGTGCCCCCGCCTGTACCCTT
R
6888
CATGTGCCCCCGCCTGTACCCTTC
7135
G
CATGTGCCCCCGCCTGTACCCTTT
7215
A
3584G>A
GTCAGAATGTTGGAGGACCCAAC
F
7098
GTCAGAATGTTGGAGGACCCAACA
7369
A
GTCAGAATGTTGGAGGACCCAACG
7385
G
4401C>T
TAACTGACATCTGCTCAGCCTCAAC
R
7546
TAACTGACATCTGCTCAGCCTCAACA
7817
T
TAACTGACATCTGCTCAGCCTCAACG
7833
C
2587_2590 delGACT
ATCAGCTCAGCCCCCCCGAGACCTGA
F
7846
ATCAGCTCAGCCCCCCCGAGACCTGAC
8093
GACT
ATCAGCTCAGCCCCCCCGAGACCTGAG
8133
del
(5) 15-PLEX
3948T>G
TCTCAGCAGGTGCCTG
F
4873
TCTCAGCAGGTGCCTGG
5160
G
TCTCAGCAGGTGCCTT
5200
T
-1235A>G
GCACCACCCAGCCTAAT
R
5084
GCACCACCCAGCCTAATC
5332
G
GCACCACCCAGCCTAATT
5411
A
137_138insT
GAAGTCCACATGCAGCA
R
5188
GAAGTCCACATGCAGCAA
5460
T
GAAGTCCACATGCAGCAG
5476
—
4155C>T
CAGCCCCGGCCCAGCCAC
F
5376
CAGCCCCGGCCCAGCCACC
5623
C
CAGCCCCGGCCCAGCCACT
5703
T
82C>T
GTGTAGCGTGCAGCCCAGC
R
5830
GTGTAGCGTGCAGCCCAGCA
6101
T
GTGTAGCGTGCAGCCCAGCG
6117
C
-1584C>G
CTGGACAACTTGGAAGAACC
F
6135
CTGGACAACTTGGAAGAACCC
6382
C
CTGGACAACTTGGAAGAACCG
6422
G
2575C>A
GACCTGGGACCCAGCCCAGCC
F
6362
GACCTGGGACCCAGCCCAGCCC
6609
C
GACCTGGGACCCAGCCCAGCCA
6633
A
843T>G
ACTAGGACCTGTAGTCTGGGG
F
6502
ACTAGGACCTGTAGTCTGGGGG
6789
G
ACTAGGACCTGTAGTCTGGGGT
6829
T
-740C>T
ACAGACTCACACTGACACTTAG
R
6672
ACAGACTCACACTGACACTTAGA
6944
T
ACAGACTCACACTGACACTTAGG
6960
C
-678G>A
CTTTGTGTGGGTGATTTTCTGC
F
6769
CTTTGTGTGGGTGATTTTCTGCA
7041
A
CTTTGTGTGGGTGATTTTCTGCG
7057
G
-750_-749 delGA
TGTGACTGGTGTGTGTGAGAGA
F
6902
TGTGACTGGTGTGTGTGAGAGAA
7173
del
TGTGACTGGTGTGTGTGAGAGAG
7189
GA
2291G>A
CACTCGCCAAGTGCCAGCCTCCAC
R
7204
CACTCGCCAAGTGCCAGCCTCCACC
7451
G
CACTCGCCAAGTGCCAGCCTCCACT
7531
A
-1426C>T
GTGTGCCACCACGTCTAGCTTTTT
R
7286
GTGTGCCACCACGTCTAGCTTTTTA
7533
T
GTGTGCCACCACGTCTAGCTTTTTG
7573
C
1758G>T>A
TTGTGCCGCCTTCGCCAACCACTCC
F
7490
TTGTGCCGCCTTCGCCAACCACTCCA
7761
A
TTGTGCCGCCTTCGCCAACCACTCCG
7777
G
TTGTGCCGCCTTCGCCAACCACTCCT
7817
T
-1000G>A
ACATCCTCCCGGGCTGCCTGAGGGT
R
7635
ACATCCTCCCGGGCTGCCTGAGGGTC
7882
G
ACATCCTCCCGGGCTGCCTGAGGGTT
7962
A
UD: Unextended mini–sequencing primer Direction; F: Forward UEP direction; R: Reverse UEP direction; UM: Unextended mini–sequencing primers Mass; ELM: Extended primer Lower Mass; EHM: Extended primer Higher Mass; ELC: Extended primer lower Mass nucleotide Called; EHC: Extended primer higher Mass nucleotide Called; All molecular weight masses are expressed in Dalton (Da).Table 4
List of SNPs excluded in assay design validation and correlation to transcriptional variations.
SNPs excluded
Variations
Reason
-1770G>A
—
Cross-hybridization
-1298G>A
—
Primer dimers
-1253A>G
—
Primer dimers
-1237_-1236insAA
—
Proximal SNP -1235A>G Primer dimers
1973_1974insG
Frameshift
Proximal SNP 1979T>C
1976G>A
—
Proximal SNP 1979T>C
1978C>T
—
Proximal SNP 1979T>C
2097A>G
—
Primer dimers
2470T>C
—
Cross hybridization
4042G>A
—
Primer dimersTable 5
CYP2D6 allele frequencies in 250 healthy Sardinian people. Total chromosomes number = 500. Human cytochrome P450 Allele Nomenclature Committee [7] served as reference for variant allele and correlated enzymatic activity.
Variant allele
Number of chromosomes
Frequency (%)
Correlated enzymatic activity
*
1A
148
29.6
EM
*1B
5
1.0
EM
*1D
4
0.8
EM
*1E
—
—
EM
*2A
75
15.0
EM
*2B
—
—
EM
*2D
—
—
EM
*2E
—
—
EM
*2F
—
—
EM
*2G
—
—
EM
*2K
—
—
EM
*2L
11
2.2
EM
*2M
8
1.6
EM
SH3
7
1.4
Not known
SH4
1
0.2
Not known
*3A
—
—
PM
*3B
11
2.2
PM
*4A
84
16.8
PM
*4B
—
—
PM
*4D
—
—
PM
*4K
—
—
PM
*4L
—
—
PM
*4M
—
—
PM
*4N
—
—
PM
*5
5
1.0
PM
*6A
1
0.2
PM
*6C
—
—
PM
*6D
—
—
PM
*7
—
—
PM
*8
—
—
PM
*9
1
0.2
IM
*10A
—
—
IM
*10B
27
5.4
IM
*11
—
—
PM
*12
—
—
PM
*14A
—
—
PM
*14B
—
—
IM
*15
3
0.6
PM
*17
—
—
IM
*19
—
—
PM
*20
1
0.2
PM
*22
—
—
Not known
*23
—
—
Not known
*24
—
—
Not known
*25
—
—
Not known
*26
—
—
Not known
*27
—
—
EM
*28
4
0.8
Not known
*29
—
—
IM
*30
—
—
Not known
*31
—
—
PM
*32
—
—
Not known
*33
—
—
EM
*35A
5
1.0
EM
*36
—
—
IM
*37
—
—
Not known
*38
—
—
PM
*39
—
—
EM
*41
46
9.2
IM
SH1
41
8.2
Not known
SH2
2
0.4
Not known
*43
—
—
Not known
*58
—
—
Not known
*59
—
—
IM
*64
—
—
Not known
*65
—
—
Not known
*1xN
4
0.8
UM
*2xN
6
1.2
UM
SH1,2,3,4 = Sardinian haplotype 1,2,3,4 [5, 22–25]. CYP2D6*5,*1xN; and *2xN alleles were evaluated by The CYP2D6 Applied Biosystems CNV Assay in [5].


## 2.1.4. iPLEX Reactions

iPLEX reactions were carried out following the Sequenom standard low/medium-plex protocol [6, 26, 27], with minor modifications. Because of the length of the primary PCR fragment and the high GC content, we increased the denaturation time at 94°C from 5 to 30 sec and the annealing temperature from 52 to 56°C. An iPLEX reaction cocktail was added to the amplification products and thermocycled to process the iPLEX reaction, which involved the enzymatic addition of one of the four mass-modified nucleotides, 2′,3′-dideoxynucleoside-5′-triphosphate (ddNTP), into the polymorphic site. During the iPLEX reaction, each primer was extended by one of the ddNTPs which terminated the extension of primers, thus producing allele-specific extension products of different masses. The iPLEX reaction cocktail included 0.222X iPLEX buffer plus, 0.5X iPLEX termination mix, 0.5X iPLEX enzyme, and UEPs, which were divided into four mass groups, according to the position of their respective mass peaks to use the whole spectrum (final concentrations of 0.8–2.0 μM). In the iPLEX termination mix, all four ddNTPs are present at the same concentration. The reactions were performed using a two cycling loop program: initial denaturation at 94°C for 30 s; followed by 40 cycles of 94°C for 30 s, 56°C for 5 s, and 80°C for 5 s. This annealing and extension procedure was repeated four times (to give a total of 200 short cycles), followed by a final extension step of 3 min at 72°C. After desalting by addition of 6 mg clean resin (Sequenom), each 384-well sample was diluted with 16 μL of sterilized H2Odd.Multiplex PCR reactions, SAP dephosphorylation, and iPLEX reactions were performed using Thermo-Fast 384 PCR Plates (ABgene, Epsom, UK) and a DNA Engine Tetrad 2 Peltier Thermal Cycler (Bio-Rad, CA, USA). All pipetting steps were performed using the automatic station Matrix PlateMate 2 × 2 (Sequenom).


## 2.1.5. MALDI-TOF MS Measurement

An aliquot ranging from 15 to 20 nL of each iPLEX reaction product was loaded in a 384-spot SpectroChip (Sequenom) using the MassArray Nanodispenser (Samsung, Seoul, Repubic of Korea). SpectroChip analysis was performed by MassARRAY Compact System (Sequenom). After laser desorption/ionization, automated spectra acquisition analysis was performed and interpreted using Sequenom MassARRAY RT version 3.3 software. Examples of multiplex mass spectrum and cluster plot distributions are shown in Figures1 and 2.Figure 1
An example of 15–plex mass spectrum is shown (Well 5). List of SNPs investigated in this plex is shown in Table3. The figure shows two examples highlighted in different colours. Unextend primer (UEP) peak is marked by an asterisk and dotted arrow, while the solid arrows indicate the presence of the two different alleles. Dotted vertical lines represent UEPs and extended primers (EPs) expected masses. In blue, an AG heterozygous genotype example is shown. *: −1235A>G UEP; expected mass = 5084 Dalton (Da). G: −1235A>G EP; expected mass = 5332 Da. A: −1235A>G EP; expected mass = 5411 Da. In red, a CC wild type homozygous genotype example is shown. *: 82C>T UEP; expected mass = 5830 Da. T: 82C>T not EP; expected mass = 6101 Da. C: 82C>T EP; expected mass = 6117 Da.In (a) a Cluster plot for 1661G>C SNP is shown. Homozygous wild type G/G samples are displayed in (α), homozygous mutate C/C samples in (β) and heterozygous G/C samples in (γ); in (δ) and (ε) are displayed outlier samples resulting in gene copy number variation [5]; (ζ) negative control (H2Odd). In (b) a Mass Spectra for the detection of 1661G>C in a heterozygous G/C sample, displayed in (a) position (γ). In (c) a Mass Spectra for the detection of 1661G>C in the outlier sample, displayed in (a) position (δ), and resulting in CYP2D6*1×N allele [5]. In (d) Mass Spectra for the detection of 1661G > C in an outlier sample, displayed in (a) position (ε), and resulting in CYP2D6*2×N allele [5].
(a)
(b)
(c)
(d)


## 2.2.CYP2D6 Single Allele Genotyping

Following direction of our previous work [5], we decided to apply the single allele protocol creating a single allele genotyping method MALDI-TOF MS based. For each sample, a double long PCR reaction was carried out using P-1584_WT or P-1584_MUT [5] (Table 2) as forward primers. The reverse primer was 2D6-R [19] for both PCR reactions. In this way, we were able to directly determine a direct and correct chromosome phase in samples presenting with a heterozygous status for −1584G>C SNP. PCR reactions were performed in a final volume of 5 μL using 20 ng genomic DNA, 400 μM of each PCR primer (Metabion), 0.2 U QIAGEN LongRange PCR enzyme, 1X QIAGEN LongRange PCR buffer (containing MgCl2 25 mM), and 800 μM Invitrogen dNTP set PCR grade. The PCR conditions were as follows: initial denaturation at 93°C for 3 min; 10 cycles at 93°C for 30 s, 67°C for 30 s, and 68°C for 3 min; 25 cycles at 93°C for 30 s, 65°C for 30 s, and 68°C for 6 min. SAP dephosphorylation, iPLEX Reactions and MALDI-TOF MS measurement were performed without modifications as indicated in paragraph 1 “CYP2D6 Genotyping Platform”. In multiplexed assay 5, −1584G>C UEP (Table 3) was excluded. Examples of cluster distribution for novel 3176C>T and 3948T>G SNPs [5, 17, 18] are shown in Figure 3.MALDI-TOF MS cluster plot distribution for 3176C>T [17] and 3948G>T [18] newly discovered SNPs [5]. In each cluster, there are visualized 32 samples presenting heterozygous status for −1584G>C SNP and submitted to both types of PCR analysis, LongRange PCR (Section 2, paragraph 1) and the two single allele PCR (Section 2, paragraph 2), for a total of 96 PCR analyses. (a) Cluster plot for 3176C>T SNP. (α) homozygous wild type C/C samples; (β) one out of the two heterozygous C/T samples presenting heterozygous status for −1584G>C SNP also; (γ) the same heterozygous sample analysed in single allele PCR; (ζ) negative control (H2Odd). (b) Cluster plot for 3948T>G SNP. (α) homozygous wild type T/T samples; (β) the only heterozygous T/G sample; (γ) the only heterozygous sample analysed in single allele PCR; (ζ) negative control (H2Odd).
(a)
(b)


## 3. Results and Discussion

ACYP2D6 screening assay was developed using the Sequenom MassARRAY platform to simultaneously identify the most frequent and some rare CYP2D6 Caucasian alleles. We have modified the basic Sequenom iPLEX assay and used a new primary PCR strategy based on the amplification of the entire gene [5] coupled with multiplex primer extension reactions. This strategy avoids false genotyping, which would result in nonspecific coamplification of the homologous pseudogenes CYP2D7P and CYP2D8P, and, secondly, it reduces the number of PCR primers used to select regions containing the targeted polymorphisms. Multiplexing was performed for 69 SNPs, which represents 66 of the most frequent and some rarer variants and subvariants reported to date in the Caucasian population [5, 7, 9–16] and known to be responsible for absent, reduced or extensive metabolic activity (Tables 1 and 5). Due to the high possibility of recombination, it was possible to insert African, African-American (CYP2D6*2L, 2*M, 4*N, 6*4, and 6*5) and Asian (CYP2D6*3A,4B*, 4L*, 14*,36*, and 39*) variants in the study. A sample of 250 unrelated healthy Sardinian individuals analyzed in [5] was submitted to MALDI-TOF MS genotyping for these 69 CYP2D6 SNPs. An example of multiplex mass spectrum is shown in Figure 1. In Figure 2(a), an example of the cluster plot distribution for the 1661G>C SNP is shown. Spectrum peak intensities were not correctly balanced in some heterozygous samples (Figures 2(c) and 2(d)), which appeared as outliers in the cluster distribution (Figure 2(a) point δ and  ε). The CYP2D6 Applied Biosystems Copy Number Variation (CNV) Assay used to analyze these DNAs in our previous work [5] detected the presence of duplications or multiplications in 100% of the analyzed samples presenting this kind of distribution. Furthermore, in samples presenting a heterozygous status for −1584G>C SNP, we applied long PCR single allele analysis [5] to MALDI-TOF MS screening assays and inferred a direct haplotype phase. To verify if our CYP2D6 platform works correctly, we compared genotyping results and haplotype phase elaborated in the two screening platforms with our results found in [5]. A consensus of 100% was found for all samples (Table 5). Moreover, for all samples presenting the 214G>C SNP in homo- or heterozygous status in MALDI-TOF MS analysis, it was possible to verify in our previous sequencing analysis [5] the presence of all SNPs correlated to gene conversion to CYP2D7P in Intron 1 (Figure 4), which indicated the reliability of MALDI-TOF analysis for this variation.(a) Electropherograms of a homozygouswild type and (b) a homozygous mutate sample for CYP2D6/CYP2D7P gene conversion in Intron 1 analyzed in [5]. SNPs are circled in purple; reference positions are indicated under each SNP. For all samples presenting 214G>C SNP in homo- or heterozygous status in MALDI-TOF MS analysis, it was confirmed the presence of all SNPs correlated to gene conversion in Intron 1 by sequencing analysis.
(a)
(b)


## 4. Conclusions

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. Polymorphisms inCYP2D6 gene have been thoroughly investigated, including their associations with the incidence of adverse reactions. In this study, we have developed a reliable medium-throughput genotyping platform using the Sequenom MassARRAY system to provide a simultaneous screening of the most relevant CYP2D6 gene variants in several hundreds of subjects. MALDI-TOF MS-based analyses have the potential to become a useful approach in clinical diagnoses, as they are very flexible and applicable to individualized genetic therapies. The screening platform developed in this study, coupled with The CYP2D6 Applied Biosystems CNV Assay, provides a robust alternative to many currently available CYP450-genotyping approaches and aims to increase the number of responders and to limit the incidences of adverse events. Moreover, by modifying long PCR Forward primer, we have implemented a rapid strategy to infer the phase by direct analysis using MALDI-TOS MS multiplex assays. Concordance between data found in this work and our direct genomic DNA sequencing analysis done in [5] reliably validated our MALDI-TOF MS-based platform.



---

*Source: 609797-2013-05-12.xml*