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MALDI-TOF mass spectrometry for multiplex genotyping of CYP2B6 single-nucleotide polymorphisms.

Pharmacogenetic diagnostics is a promising approach to predict the pharmacokinetics, as well as the effects and adverse drug reactions, of a drug in an individual patient (1, 2). The majority of all clinically used drugs are oxidatively metabolized by isoenzymes of the cytochrome P450 (CYP) [4] superfamily, several of which are functionally highly variable, leading to different drug oxidation phenotypes within the population. Of established clinical importance are the genetic variations of CYP2D6, [5] CYP2C19, and CYP2C9 (3-5). Compared with these well-studied genes, CYP2B6 has only recently been recognized as a gene that codes for a highly variable enzyme of potential clinical importance (6). Clinically relevant drug substrates include the antidepressant bupropion (7-9), the antineoplastic agent cyclophosphamide (10, 11), the anaesthetic propofol (12), the antimalarial artemisinine (13), the anti-Parkinsonian selegiline (14), and the non-nucleosidic reverse transcriptase inhibitor efavirenz (15).

The CYP2B6 gene is located, together with a closely related pseudogene CYP2B7P1, in a CYP2 gene cluster on chromosome 19 (16). In contrast to the above-mentioned CYP genes, variations in CYP2B6 have been discovered through reverse genetics studies, i.e., by initial sequencing and variation scanning studies and subsequent analysis of the functional consequences of the identified variations (17-25). More than 100 DNA variations, including numerous nonsynonymous variations, as well as silent, promoter, and intronic changes, were found within the CYP2B6 gene, many of them showing extensive linkage disequilibrium giving rise to distinct haplotypes (26). The spectrum of functional consequences of these variations is wide and includes null alleles with no detectable function and/or expression (alleles CYP2B6 * 8, * 12, * 15, * 18, * 21), alleles with partially reduced function/ expression (CYP2B6 * 5, * 6, * 7, * 11, * 14, * 19, * 20, * 21) (17,19, 21, 23), and alleles with increased expression [CYP2B6 * 22; (24)]. Some of these variations are rare, but many are common, with allele frequencies between 10% and almost 50%, depending on the population studied (22,23).

The clinical relevance of CYP2B6 variations has recently been demonstrated for the anti-HIV drug efavirenz. The common clinical practice of administering the same dose to all patients leads to profound differences in drug plasma concentrations, which are correlated with patient genotype (27-29). Patients with high drug concentrations are at risk of developing concentration-related central nervous system toxicity, including insomnia, fatigue, and headache and often leading to discontinuation of the therapy. Thus, for a drug such as efavirenz, dose adjustment based on CYP2B6 genotype could prevent administration of too-high doses, thus increasing the safety and efficacy of the therapy.

In recent years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) proved to be a superior technology for the detection of single-nucleotide variations (SNPs). This method has several advantages over other methods, including high accuracy because of the direct measurement of molecular masses, high sensitivity in the detection of both homozygous and heterozygous base changes, and high-throughput and cost-effective multiplex capability (30-38). We developed a MALDI-TOF MS genotyping method to determine 15 SNPs in the CYP2B6 gene.

Materials and Methods

DNA SAMPLES

Anonymized blood samples were obtained from individuals of white European origin. All individuals gave written informed consent to genetic testing. The study was approved by the ethics committees of the Medical Faculties of the Charit6, Humboldt-University Berlin, and of the University of Tubingen, and written informed consent was obtained from each patient. Genomic DNA was prepared by standard methods from whole blood samples as described previously (17, 21).

MALDI-TOF MASS SPECTROMETRIC CYP2B6 GENOTYPING ASSAY

The CYP2B6 promoter sequence (AC 24497632), gene sequence (NG_000008.5 and AC023172), and the CYP2B7 pseudogene sequence (A0008537) were used for specific CYP2B6 primer design with the AlignX program of the VectorNTI Suite 9 package (InforMax, Inc.). All reactions, including PCR amplification, shrimp alkaline phosphatase treatment, and base extension, were performed in 384 microtiter plates (ABgene, Epsom) with a Puredisk pipette robot (Cybio). PCR amplification and primer extension reaction were carried out in a DYADTM PCR thermal cycler (MJ Research), and no-template controls were carried along in every plate to exclude contaminations.

Multiplex PCR. PCBs (final volume, 8 [micro]L) contained 50 ng of DNA, 0.2 units of HotStarTaq polymerase, 1 mmol/L Mg[Cl.sub.2] (Qiagen GmbH), and the desired primers (MWG Biotech AG) at their optimized concentrations (see Table 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol53/issue1). A tag (5'-ACGTTGGATG) was included in the primer sequence to equalize extreme relative percentage-GC contents (39). PCR conditions were 95 [degrees]C for 15 min followed by 45 cycles at 95 [degrees]C for 30 s, 60 [degrees]C or 61 [degrees]C (assay 5) for 1 min, and 72 [degrees]C for 1 min; and finally 72 [degrees]C for 10 min. After dephosphorylation of excess dNTPs with 0.3 units of shrimp alkaline phosphatase in 1 x Rx-Buffer (Amersham) at 37 [degrees]C for 20 min, 85 [degrees]C for 10 min, and 20 [degrees]C for 1 s, the PCR products were used as templates for the primer extension reactions. CYP2B6-specific amplification was verified for all primer pairs by sequence analysis of products obtained in single reactions.

Primer extension reactions. Extension reactions (final volume, 16 [micro]L) contained 1 [micro]L of buffer C (500 mmol/L Tris-HCL, pH 9.5;100 mmol/L Mg[Cl.sub.2]), 6 mmol/L Mg[Cl.sub.2], 1 U of Termipol DNA polymerase I (all from Solis Biodyne), and extension primers at optimized concentrations (see Table 2 in the online Data Supplement). Assay-specific deoxynucleoside triphosphates (dATP, dGTP, dTTP; Amersham) and dideoxynucleoside triphosphates (ddA, ddC, ddG, ddT; BioLog Life Science Institute) were added at 0.12 mmol/L. In assays 1, 2, 3, and 5, dCTP (Amersham) and dGTP were added at 0.25 mmol/L. Extension reactions were performed at 94 [degrees]C for 4 min followed by 55 cycles at 94 [degrees]C for 30 s and 52 [degrees]C (assays 1 and 3), 56 [degrees]C (assay 2), or 60 [degrees]C (assay 4 and 5) for 30 s, and 72 [degrees]C for 30 s; and finally 72 [degrees]C for 2 min. The final nucleotide extension products were treated with a cationic exchange resin (AG[R] 50W-X8 Resin; Bio-Rad Laboratories, Inc.) for 30 min to remove salts.

MALDI-TOF MS measurement. We spotted and air dried 1 [micro]L of 3-hydroxypicolinic acid matrix (45 mmol/L and 3 mmol/L diammonium hydrogen citrate solution) onto a 384-format MTP AnchorChip[TM] 400/384TF target plate (Bruker Daltonik, spot size 400 Am). The extension reaction products (0.5 [micro]L) were dispensed on the matrix and air dried. The target plate was then inserted into the Ultraflex MALDI-TOF mass spectrometer (Bruker Daltonik), and analysis was performed with 180 nitrogen laser shots for each sample. The mass range of the MS instrument was set at 4000-9000 Da. Genotyping calls were made with the assistance of the GENOTOOLS software (Bruker Daltonik). DNA samples not automatically assigned were determined manually.

GENERATION OF CONTROL SAMPLES

Control samples are needed for each SNP in heterozygous and homozygous form to exclude artifacts such as pausing peaks or other DNA fragments. To generate a homozygous variant control sample from an available heterozygous sample, a PCR product was generated with the appropriate PCR primers (see Table 1 in the online Data Supplement). The 2 alleles were separated by subcloning the fragments in TOP10 cells by positive selection with a TOPO TA Cloning[R] Kit vector (Invitrogen) containing the F plasmid ccdB killer gene (40). DNA was isolated from bacteria and sequenced to verify the presence of the SNP. If no heterozygous sample was available, we used a previously developed touchdown PCR method to introduce the variation in a wild-type (wt) sample. A primer located within the amplified region and containing the variation of interest was designed with a melting temperature ~8 [degrees]C above that of the amplification primers (see Table 1 in the online Data Supplement), as described previously (41). The primer sequences are available on request. Sequence verification was performed with AB Big Dye[R] Terminator Mix v1.1. (Applied Biosystems) and the ABI Prism 310 Genetic Analyzer (Applied Biosystems).

ALLELE NOMENCLATURE AND STATISTICS

Base numbering and allele definitions were according to the published recommendations of the CYPallele Nomenclature Committee (http://www.imm.ki.se/CYPalleles/criteria.htm). Numbering was based on the cDNA with the full-length cDNA sequence published by Yamano et al. (42) defined as the wt (CYP2B6 * 1). We used the DeFinetti program (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl) to test genotype frequencies for conformance with Hardy-Weinberg equilibrium. Data were compiled according to the genotype and allele frequencies estimated from the observed numbers of each specific allele and with the assistance of the PHASE program v.2.0.2 (43, 44). The frequency of each SNP in our study participants is given with the 95% confidence interval (CI).

Results

SELECTION OF SNPS FOR GENOTYPING

To choose relevant SNPs for CYP2B6 genotyping we considered more than 100 known and rare variations, most of which have been published and are publicly available in databases (CYPallele nomenclature: http://www.cypalleles.ki.se; dbSNP: http://www.ncbi.nhn.nih.gov/SNP/). Relevant data for the 15 SNPs finally selected are summarized in Table 1. They include the 2 most frequent amino acid variations, Q172H and K262R in exons 4 and 5, respectively, and the common R487C variation in exon 9, all of which are associated with lower expression in human liver (17). We also selected the more rare SNPs M46V, G99E, K139E, and I391N, described as phenotypic null alleles in Caucasians, and the R140Q SNP that strongly affects enzyme activity (21). Furthermore, the following amino acid variations reported in Ghanaian or African-American individuals were included: R29T, D257N, I328T, R336C, and P428T, some of which were found to result in small or undetectable amounts of residual protein (23). A novel SNP, V183I, found by sequence analysis in a Ghanaian individual (data not shown) was also included. Finally, we enclosed the -82T [right arrow] C promoter SNP, which was recently shown to cause enhanced transcription and to result in increased expression (24).

The c.64C [right arrow] T (R22T) variation, defining the CYP2B6 * 2 allele, was thus far not included because it was shown to be comparable to the wt in expression and function (17, 20).

DEVELOPMENT OF MALDI-TOF MS ASSAYS

For the specific amplification of CYP2B6 gene fragments, it was important to (a) effectively discriminate between CYP2B6 and the pseudogene CYP2B7, and (b) minimize the possibility that nucleotide variations within the PCR-and extension-primer sequences interfered with amplification. To meet these requirements, the PCR primer pairs (see Table 1 in the online Data Supplement) were designed to bind to CYP2B6 in regions of low homology to CYP2B7, mostly found in intronic or exon/intron boundary regions. The only exon not included in the multiplex PCR assays is exon 6, for which no relevant variations had been described to date. All primer pairs were shown to be functional in various combinations up to a single-tube multiplex amplification reaction for all 8 amplicons. However, a lower degree of multiplexing was finally applied because not all extension reactions could be carried out simultaneously. The compatibility of the extension primers is strongly restricted by their sequence, molecular masses, and annealing temperatures. Individual extension primers were optimized with respect to primer length and concentration and tested with positive controls for all 3 genotypes (homozygous wt, homozygous variant, and heterozygous). The control DNAs were selected from previously genotyped genomic DNA samples or generated recombinantly as described in Materials and Methods. By testing various combinations, we arrived at a final design comprising 5 multiplex assays (assays 1-5; see Table 2 in the online Data Supplement). Representative MALDI-TOF MS spectra for all 5 assays are shown in Fig. 1.

EVALUATION OF THE MALDI-TOF MS GENOTYPING ASSAY

To evaluate the established CYP2B6 genotyping assay, we analyzed 287 genomic DNA samples from individuals of Caucasian origin who had previously been genotyped for some of the SNPs by various other methods. In total, this amounted to 4305 individual genotype determinations. Fig. 2 shows the relative intensities of the mass signals corresponding to the 2 primer extension products of a given CYP2B6 SNP, plotted against each other, for the 4 most frequent variations. As evident from this analysis, the mass spectral data were clearly structured into 3 groups, i.e., homozygous wt/wt and mutant (mut)/mut genotypes found along the axes, and wt/mut heterozygotes scattered in-between (no-template controls with relative signal intensities of 0 are indicated with white points and black from the middle). In total, 2300 genotypes have been previously determined by other methods, including sequencing, allele-specific 5'-nuclease assay (TagMan), denaturing high-performance liquid chromatography, and/or PCR-restriction fragment length polymorphism (RFLP). In 9 cases (0.4%), MALDI-TOF MS analysis resulted in a genotype discrepant from that of the earlier studies. Retrospective inspection revealed that PCR-RFLP assays had been used for the earlier analyses of these samples and that the fragment profiles were ambiguous and difficult to interpret. After resequencing these samples, the MALDI-TOF MS outcomes were confirmed in all 9 cases.

FREQUENCY ANALYSIS OF CYP2B6 VARIATIONS IN CAUCASIANS

The allele and genotype frequencies observed in the analyzed population of 287 Caucasians are summarized in Table 2. All genotypes were shown to be in Hardy--Weinberg equilibrium. Five variations previously observed in persons of African or African-American origin were not detected in any Caucasian sample. The most frequent SNPs were the amino acid substitutions K262R (27.4%), Q172H (25.2%), and R487C (9.7%), followed by the promoter SNP -82C (2.1%). The variations M46V, G99E, K139E, R140Q, V183I, and I391N were present at a frequency of <1% (Table 2). Results of haplotype analysis with the PHASE program indicated that the most frequent alleles were the wt allele (* 1; 59%) and * 6 (24.6%). The other CYP2B6 alleles and their frequencies were * 4 (2.3%), * 5(7.3%), * 11 (0.35%), * 12 (0.17%), * 13 (0.52), * 14 (0.52%), * 15 (0.7%), and * 22 (2.1%). The alleles * 17, * 18, * 19, and * 21 were not found in this population.

[FIGURE 1 OMITTED]

Discussion

We developed a comprehensive SNP analysis method based on MALDI-TOF MS for the human drug metabolizing CYP2B6 gene and the application of this genotyping method to a study population of European Caucasian origin. The design of the assay consists of 5 multiplex subassays with carefully optimized protocols for 15 selected SNPs, thus allowing us to analyze SNP subsets and to incorporate additional SNPs for analysis if, for example, novel predictive SNPs are discovered. MALDI-TOF MS is a sensitive, accurate and reproducible method for the detection of oligonucleotides. In contrast to other genotyping technologies that rely on hybridization, MALDI-TOF MS directly measures the molecular weight of the oligonucleotide and therefore has several principal and methodologic advantages, which have been summarized in previous articles (31-33, 45). Further advantages of MALDI-TOF MS over some other methods are the option for highly automated processes and the relative ease of set up for multiplex assays, thus effectively reducing time and cost while increasing sample throughput.

Similar to other cytochrome P450 genes, CYP2B6 represents a particular challenge with respect to developing gene-specific assays because of its high homology to the pseudogene CYP2B7P1 (46). Because no variation analysis has been carried out for CYP2B7, the influence of contaminating CYP2B6 amplicons with CYP2B7 is unpredictable. We designed intronic primers such that each of them harbored at least 1 nucleotide difference from the pseudogene sequence, and we controlled each amplicon for its specificity by direct sequencing. Occasionally contamination was observed and PCR conditions were then modified to improve specificity. It is thus important that such controls are performed when setting up this or similar assays. The use of a common tagging sequence at the 5'-end of the amplification primers to level out differences in GC content proved to be advantageous for multiplexing (39). Under optimized conditions, the entire set of primer pairs gave satisfactory results in a single multiplex amplification reaction.

The combination of the various primer extension reactions into multiplex assays is another critical aspect, and support by suitable software is highly recommended to allow the optimization of several different parameters, i.e., GC content, molecular mass range, annealing temperatures, dimers, and hairpin loop formation. We tested the accuracy of conversion of the unextended primer into allele-specific analyte by use of either control DNAs from previously sequenced/ genotyped samples or recombinantly generated controls. To achieve highest multiplexing levels, we tested many primer combinations, leading us to the final assay design consisting of 5 multiplex assays. More than 4 extension primers could not be combined into a single-multiplex extension reaction, mainly because of cross-binding problems and different extension mixes necessary for the 15 SNPs (see Table 2 in the online Data Supplement).

[FIGURE 2 OMITTED]

For evaluation, we analyzed all 15 SNPs in 287 genomic DNA samples of white European origin. As shown in Fig. 2 for the most frequent CYP2B6 variations, the MALDI-TOF MS data obtained for the 3 possible genotypes (wt, mut, and heterozygous) were clearly structured into 3 clusters. On average, ~5% (equivalent to 14 samples) were not automatically identified and had to be assigned manually, resulting in an overall automated call rate of >95% for all 15 SNPs. Thus, with the assays presented, a reliable high-throughput genotyping of CYP2B6 variations is achievable. The comparison of the MALDI-TOF MS results with those obtained by other methods revealed a few discrepant results, which could be assigned to 2 distinct RFLP assays. These divergent results were retrospectively shown to be caused by erroneous data interpretation of the primary RFLP data, and the MALDI-TOF MS outcomes could be verified in all cases by sequencing, emphasizing the high accuracy of this method.

After completion of the mass spectrometric analysis, 9 genotypes (0.2%) of the 4305 total SNP analyses remained undetermined even after 3 repeated analyses. The reason for this is currently unclear. However, because only 4 different SNPs were affected (see Table 2) and because PCR amplification was successful in each case, we speculate that unknown sequence variations within primer binding sites may have prevented the binding and/or extension of the respective primers. The overall genotyping success rate for the 287 samples was thus higher than 99% in 3 of the 5 assays, and even 100% in 2 assays, which confirms the high technical reliability of MALDI-TOF MS (47).

The SNP frequencies observed in this study confirm and extend current knowledge on CYP2B6 genetic variation in the middle European population. CYP2B6 variations have been discovered relatively recently, and thus their value for predicting drug response is largely unexplored. However, clinical applications of CYP2B6 genotyping so far include bupropion, used in smoking cessation (48); cyclophosphamide, in lupus nephritis (49) and in hematologic malignancies (50); and efavirenz, an anti-HIV agent (27-29). In particular, the consistent results of multiple studies on efavirenz pharmacogenetics emphasize the potential of CYP2B6 variations in predicting patient variability in drug side effects, response, and toxicity. To date, only the more common variations of CYP2B6 have been included in these studies, in part because comprehensive genotyping assays have not been available (51-53). In our assay, we have included numerous recently discovered, less frequent SNPs, which have pronounced effects on expression/ function and which together account for 5% to 10% of functionally variant alleles, depending on the ethnic origin of the population studied (Table 1) (19, 21, 23, 25). Their determination in the course of clinical studies should thus help to improve our understanding of the phenotype-genotype relationship and enhance the predictive value of CYP2B6 genotyping.

We are grateful to Igor Liebermann for technical assistance and to Dr. Werner Schroth (Stuttgart, Germany) for helpful discussions. This work was supported in part by the H.W. & J. Hector Foundation (Weinheim, Germany), the Robert Bosch Foundation (Stuttgart, Germany), and the German Federal Ministry of Education and Research (grant 0313080D).

Received June 13, 2006; accepted October 12, 2006. Previously published online at DOI: 10.1373/clinchem.2006.074856

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(53.) Futatsugawa Y, Kubota T, Ishiguro A, Suzuki H, Ishikawa H, Iga T. PCR-based haplotype determination to distinguish CYP2B6 * 1/ * 7 and * 5/ * 6. Clin Chem 2004;50:1472-3.

[4] Nonstandard abbreviations: CYP, cytochrome P450; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; SNP, single-nucleotide polymorphism; wt, wild-type; mut, mutant; RFLP, restriction fragment length polymorphism.

[5] Human genes: CYP2B6, cytochrome P450, family 2, subfamily B, polypeptide 6; CYP2B7P1, cytochrome P450, family 2, subfamily B, polypeptide 7 pseudogene 1; CYP2D6, cytochrome P450, family 2, subfamily D, polypeptide 6; CYP2C19, cytochrome P450, family 2, subfamily C, polypeptide 19; CYP2C9, cytochrome P450, family 2, subfamily C, polypeptide 9.

JULIA K. BLIEVERNICHT, [1] ELKE SCHAEFFELER, [1] KATHRIN KLEIN, [1] MICHEL EICHELBAUM, [1,2] MATTHIAS SCHWAB, [1] and ULRICH M. ZANGER [1] *

[1] Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.

[2] Division of Clinical Pharmacology, University Hospital Tuebingen, Tuebingen, Germany.

* Address correspondence to this author at: Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstr. 112, 70376 Stuttgart, Germany. Fax 49-(0)711-85-92-95; e-mail uli.zanger@ikp-stuttgart.de.
Table 1. Selected SNPs and their functional consequences.

Position (a) Reference dbSNP Allele

g.-82T[right arrow]C (p) (24) * 22
c.86G[right arrow]3C (23) * 17
g.86 (e1) rs34284776
c.136A[right arrow]G (21) * 11
g.136 (e1) rs35303484
c.296G[right arrow]A (21) * 12
g.12820 (e2) rs36060847
c.415A[right arrow]G (19, 23) * 8, * 13
g.13072 (e3) rs12721655
c.419G[right arrow]A (21) * 14
g.13076 (e3) rs35773040
c.516G[right arrow]T (17, 19) * 6, * 7, * 9, *13,
 * 19, * 20
g.15631 (e4) rs3745274
c.547G[right arrow]A (e4) Novel SNP to be assigned
g.15662G[right arrow]A
c.769G[right arrow]A (e5) (23) to be assigned
g.18038 (e5) rs34646544
c.785A[right arrow]G (17) * 4, * 6, * 7, * 13,
 * 16, * 19, * 20
g.18053 (e5) rs2279343
c.983T[right arrow]C (23) * 16, * 18
g.21011 (e7) rs28399499
c.1006C[right arrow]T (23) * 19
g.21034 (e7) rs34826503
c.1172T[right arrow]A (21) * 15
g.21388 (e8) rs35979566
c.1282C[right arrow]A (23) * 21
g.21498 (e8) rs35010098
c.1459C[right arrow]T (17) * 5, * 7
g.25505 (e9) rs3211371

Position (a) Protein Relevance

g.-82T[right arrow]C (p) 2B6.1 Increased expression and activity
c.86G[right arrow]3C R29P Increased frequency in
 African-Americans
g.86 (e1)
c.136A[right arrow]G M46V Phenotypic null allele
g.136 (e1)
c.296G[right arrow]A G99E Phenotypic null allele
g.12820 (e2)
c.415A[right arrow]G K139E Phenotypic null allele
g.13072 (e3)
c.419G[right arrow]A R140Q Reduced activity
g.13076 (e3)
c.516G[right arrow]T Q172H Reduced expression and activity
g.15631 (e4)
c.547G[right arrow]A (e4) V183I Undetermined
g.15662G[right arrow]A
c.769G[right arrow]A (e5) D257N Undetermined
g.18038 (e5)
c.785A[right arrow]G K262R Reduced expression and activity
g.18053 (e5)
c.983T[right arrow]C I328T Phenotypic null allele
g.21011 (e7)
c.1006C[right arrow]T R336C Strongly reduced protein and
 activity
g.21034 (e7)
c.1172T[right arrow]A I391N Phenotypic null allele
g.21388 (e8)
c.1282C[right arrow]A P428T Phenotypic null allele
g.21498 (e8)
c.1459C[right arrow]T R487C Reduced expression and activity
g.25505 (e9)

(a) Position based on cDNA numbering [cDNA (c.)] (33) or genomic DNA
(g.), with 1 corresponding to A of ATG (CYP allele nomenclature
website at http://www.imm.ki.se/cypalleles); e, exon; p, promoter.

Table 2. CYP2B6 SNP and genotype frequencies in 287 Caucasians.

c. SNP (a) g. SNP (a) SNP frequency,
 % (95% CI) (b)

Promoter -82T[right arrow]C 2.1 (1.1-3.)
86G[right arrow]C 86G[right arrow]C 0.0
136A[right arrow]G 136A[right arrow]G 0.3 (0.0-1.3)
296G[right arrow]A 12.820G[right arrow]A 0.2 (0.0-1.0)
415A[right arrow]G 13.072A[right arrow]G 0.5 (0.1-1.5)
419G[right arrow]A 13.076G[right arrow]A 0.5 (0.1-1.5)
516G[right arrow]T 15.631G[right arrow]T 25.2 (21.6-28.9)
547G[right arrow]A 15.662G[right arrow]A 0.5 (0.1-1.5)
769G[right arrow]A 18.037G[right arrow]A 0.0
785A[right arrow]G 18.053A[right arrow]G 27.4 (23.7-31.2)
983T[right arrow]C 21.011T[right arrow]C 0.0
1006C[right arrow]T 21.034C[right arrow]T 0.0
1172T[right arrow]A 21.388T[right arrow]A 0.7 (0.1-1.8)
1282C[right arrow]A 21.498C[right arrow]A 0.0
1459C[right arrow]T 25.505C[right arrow]T 9.7 (7.5-12.5)

c. SNP (a) Undetermined, % (c) WT/WT, %

Promoter 0.0 95.8 (n=275)
86G[right arrow]C 0.7 (n=2) 99.3 (n=285)
136A[right arrow]G 0.0 99.3 (n=285)
296G[right arrow]A 1.4 (n=4) 98.3 (n=282)
415A[right arrow]G 0.7 (n=2) 98.3 (n=282)
419G[right arrow]A 0.0 99.0 (n=284)
516G[right arrow]T 0.35 (n=1) 55.4 (n=159)
547G[right arrow]A 0.0 99.0 (n=284)
769G[right arrow]A 0.0 100 (n=287)
785A[right arrow]G 0.0 51.9 (n=149)
983T[right arrow]C 0.0 100 (n=287)
1006C[right arrow]T 0.0 100 (n=287)
1172T[right arrow]A 0.0 98.6 (n=283)
1282C[right arrow]A 0.0 100 (n=287)
1459C[right arrow]T 0.0 81.2 (n=233)

c. SNP (a) WT/MT, % (b) MT/MT, % (b) HWE (b) [x.sup.2]
 (P value)

Promoter 4.2 (n=12) 0.0 0.13 (0.72)
86G[right arrow]C 0.0 0.0 0.0
136A[right arrow]G 0.7 (n=2) 0.0 0.003 (0.95)
296G[right arrow]A 0.3 (n=1) 0.0 0.0009 (0.97)
415A[right arrow]G 1.0 (n=3) 0.0 0.008 (0.93)
419G[right arrow]A 1.0 (n=3) 0.0 0.008 (0.93)
516G[right arrow]T 38.3 (n=110) 5.9 (n=17) 0.12 (0.72)
547G[right arrow]A 1.0 (n=3) 0.0 0.008 (0.93)
769G[right arrow]A 0.0 0.0 0.0
785A[right arrow]G 41.5 (n=119) 6.6 (n=19) 0.54 (0.46)
983T[right arrow]C 0.0 0.0 0.0
1006C[right arrow]T 0.0 0.0 0.0
1172T[right arrow]A 1.4 (n=4) 0.0 0.01 (0.90)
1282C[right arrow]A 0.0 0.0 0.0
1459C[right arrow]T 18.1 (n=52) 0.7 (n=2) 0.24 (0.62)

(a) See Table 1 for explanation of SNP numbering.

(b) CI, confidence interval; WT, wild type; MT, mutant;
HWE, Hardy--Weinberg equilibrium.

(c) Number of samples lacking the specific signal in at least 3
determinations.
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Title Annotation:Molecular Diagnostics and Genetics
Author:Blievernicht, Julia K.; Schaeffeler, Elke; Klein, Kathrin; Eichelbaum, Michel; Schwab, Matthias; Zan
Publication:Clinical Chemistry
Date:Jan 1, 2007
Words:6164
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