debrisoquine oxidation in a white British population. J Med. Genet 1980;17:102-5. 5. Evans DAP, Harmer D, Downham. DY, Whibley EJ, Idle JR,. Ritchie J, et al.
CLIN. CHEM. 41/3, 413-418 (1995)
#{149} Melecular
Pathology
Robust Nonradioactive Oligonucleotide Ligation Assay to Detect a Common Point Mutation in the CYP2D6 Gene Causing Abnormal Drug Metabolism Torben
S. Hansen,”3 Niels E. Petersen,’
Antti Iitifl,2 Ole Blaabjerg,’
A new nonradioactive oligonucleotide ligation assay for the detection of a common point mutation in the CYP2D6 gene is presented. The assay takes advantage of simul-
taneous time-resolved fluorescence measurements of lanthanide-labeled probes and the specificity of T4-DNA ligase in combination with the polymerase chain reaction. This strategy makes it possible to perform the assay using both the wild-type-specific and mutant-specific
probes simultaneously, securing an internal control in each reaction. We show that the allele-specific ligation part of the assay can be performed with great accuracy over a wide range of temperatures, salt concentrations, and T4-DNA ligase concentrations. This eliminates the risk of false-positive
or false-negative
reactions
due to
variations in these factors. Because the assay is simple to perform, is very reliable, and can be partly automated, we conclude that it is well-suited for analysis in a routine laboratory. Indexing Terms: heritable disorders/debrisoquine-.sparteine polymorphism /aIlele-speciflc ligation/poiymerase chain reaction/timeresolved fluorescence assay/biotin-streptavidin
The debrisoquine/sparteine polymorphism is one of the most studied genetic defects of oxidative drug metabolism in humans. The defect is caused by a deficiency in the hepatic cytochrome CYP2D6 (1, 2) and leads to poor metabolism of >25 drugs, including widely used tricyclic antidepressants, -adrenergic blocking agents, and antiarrhythmics (3). The poor metabolizer (PM) phenotype is inherited as an autosomal recessive trait (4-7) and occurs in 5-10% of European Caucasian populations (8 )4#{149} The majority of the populations can be divided into extensive metabolizers (EM) and a small group of so-called ultraextensive metabolizers (UEM) (9). The clinical importance of this polymorphism is well established. if patients are given the recommended doses of some of the above-mentioned drugs, PM individuals will develop toxic plasma concentrations while UEM individuals will suffer therapeutic failurebecause the attained plasma concentration will be far too low (3). The standard procedure to evaluate the meta‘Department of Clinical Chemistry, tal, DK-5000 Odense C, Denmark.
Odense University
Hospi-
2Depment of Biotechnology, University of Turku, Turku, Finland. 3Author for correspondence. Fax +45 6613 2854. 4Nonstandard abbreviations: PCR, polymerase chain reaction; OLA, oligonucleotide ligation assay; PM, poor metabolizer; EM, extensive metabolizer; UEM, ultraextensive metabolizer; and RFLP, restriction fragment length polymorphism. Received July 13, 1994; accepted December 1, 1994.
Per Hyltoft-Petersen,’
and Mogens H#{248}rder’
bolic capacity involves the administration of a probe drug [e.g., sparteine (10)] and measuring the ratio between the parent drug and its hydroxy metabolite in urine. Alternatively, the metabolism of the given drug is measured directly in plasma. In the first case, however, it is generally required that the patient be untreated
while
the
metabolism
of sparteine
is fol-
lowed, and in both cases a conclusive answer is not obtained before a week or more. These problems are circumvented by genotyping the patient. The human CYP2D6 gene has been mapped to chromosome 22 in conjunction with two unprocessed pseudogenes, CYP2D7 and CYP2D8 (11, 12). In the Caucasian wild-type configuration, CYP2D6 and the two pseudogenes reside together on a 29-kb XbaI fragment (12). So far, three mutations have been associated with the PM subgroup. The A mutation is a 1-bp deletion in exon 5 leading to a fraxneshift in the translation of CYP2D6 mRNA (13). The B mutation is a G-to-A transition in the 3’-end splice-site consensus sequence of intron 3, leading to missplicing of the premature transcript (13, 14). The D mutation is a deletion of the entire gene (15). All three mutations lead to either no or a nonfunctional enzyme. In the Caucasian population the B mutation constitutes -75% of all mutant alleles, the D mutation 14%, and the A mutation 5%. Together the three mutations account for 90 -95% of all PM individuals (16-18). Restriction fragment length polymorphism (RFLP) analysis has shown that the A mutation is associated with the 29-kb XbaI fragment (13), the B mutation with both this and a 44-kb XbaI fragment (14, 19), and the D mutation with a 11.5-kb XbaI fragment (15). This heterogeneity makes RFLP informative only in the absence of the 29-kb XbaI fragment, which only occurs in 10% to 25% of Caucasian PM individuals (17, 18, 20). The use of the specificity of T4-DNA ligase to detect point mutations, the so-called oligonucleotide ligation assay (OLA), has previously been reported (21, 22). Here we report a new, simplified, and very safe OLA for detection of the B point mutation in the CYP2D6 gene. The assay relies on time-resolved fluorescence measurements of the lanthanides europium (Eu) and samarium (Sm) (23). Two allele-specific probes differently labeled with Eu and Sm are simultaneously annealed together with a common biotinylated capture probe to a polymerase chain reaction (PCR) product harboring the site of the mutation. Hereafter T4-DNA ligase is added and the homologous probe is captured on streptavidin when ligated to the biotinylated capture probe. The biological variation of a mutation is binary. The analytical variations caused by differences CLINICAL CHEMISTRY, Vol. 41, No. 3, 1995
413
in PCR yield or by pipetting errors can be reduced by determining the ratio of Eu counts to Sm counts. Thus, this ratio will separate the following genotypes: (a) homozygotes B/B or compound heterozygotes B/D; (b) heterozygotes W/B; and (c) homozygotes W/W or heterozygotes W/D. The D/D genotype is indicated indirectly.
of NaCl were as indicated in Fig. 3. The reaction were then transferred to high-capacity streptavithn-coated microtiter wells (Wallac Oy, Turku, Finland) containing 180 L of Delfia assay buffer (Wallac Oy) and incubated for 1 h on a plate shaker. The contents of the wells were then aspirated, denaturated in 200 L of 100 mmo)/L NaOH for 10 mm, and washed once in 200 of 100 mmol/L NaOH Materials and Methods and six times with 200 L of Deffia washing solution. DNA sources. DNA was extracted from 5 mL of Delfia enhancement solution (200 p1) was then added peripheral blood anticoagulated with K2EDTA by a and the plates were incubated for 25 mm on a plate high-salt precipitating-out method (24). shaker before Eu and Sm fluorescence were counted on PCR amplifications. PCR amplifications were cara 1234 Delfia fluorometer. ried out on a Perkin-Elmer (Norwalk, CT) 9600 therEach time an assay was performed, a mock reaction mocycler. The standard procedure to detect the B point of each individual sample was included. The mock mutation (25) was modified as follows: PCR I was in 50 reactions were identical with the test samples except tL of Taq polymerase buffer (60 mmol/L Tris-HC1, pH that no T4-DNA ligase was added. Unless otherwise 8.3, 50 mmol/L KC1, 0.1 g/L gelatin, 2.5 mmol/L of each stated, the mean signal obtained from the mock reacdNTP), 250 pmol/L of each primer 1 and 2, 100-500 ng tions was used for background subtraction. 2) Method 2: As above but with the following modiof genomic DNA, and 2.5 U of Taq polymerase (Boehringer Mannheim, Mannheim, Germany). The reaction fications: Instead of using the thermocycler, the samwas carried out as follows: one cycle at 94#{176}C for 3 mm; ples were placed in a 94#{176}C water bath for 3 mm, 10 cycles at 94#{176}C for 1 mm, 58#{176}C for 1 mm, 72#{176}C for 3 transferred to a water bath for annealing for 10 mm at indicated, and transferred to the labmm; 25 cycles at 94#{176}C for 1 mm, 56.5#{176}C for 1 mm, 72#{176}C the temperature for 3 mm; one cycle at 72#{176}C for 7 mm. PCR II was in 25 oratory bench at room temperature for 10 mm before adding ATP and T4-DNA ligase in 10 p1 of 2 x ligase L of Taq polymerase buffer as above, plus 250 pmoIJL of each primer 1 and 7 or 1 and 8, respectively, 2.5 buffer. The reactions were briefly centrifuged and inmmoL’L of each dNTP, 1.5 L of PCR I product, and 0.5 cubated for 1 h at room temperature before the assay U of Taq polymerase. The following reaction profile was was continued as described above. The amount of used: one cycle at 94#{176}C for 3 mm; 15 cycles at 94#{176}C for T4-DNA ligase was 0.5 U and the final concentration of NaCl was 200 mmoIJL. 1 mm, 56.5#{176}C for 1 mm, 72#{176}C for 2 mm; one cycle at 72#{176}C for 7 mm. (For sequence and numbering of primStatistics. The statistical variance model of balanced ers see ref. 23.) From each PCR II reaction 10 L was repeated subsampling (nested design) was used to estimate of the variance components and calculate CV analyzed on a 1% agarose gel together with molecular mass standards. for within- and between-day analytical variation (27). Synthesis and lanthanide labeling of allele-specific Principle ligation probes. The allele-specific ligation probes were synthesized and labeled as in ref. 26. The number of The principle of the allele-specific ligation assay is 5’-diaminohexane-modified deoxycytidines was 20. illustrated in Fig. 1. Three oligomers are simultaLigation reactions. Five microliters of PCR I reaction product was transferred to MicroAmp reaction A tubes (Perkin-Elmer), and 5 L of 2 X ligation buffer 31 (100 mmol/L Tris-HC1, pH 7.5, 20 mmol/L MgC12, 10 mmoL/L dithiothreitol, 0.2 g/L bovine serum albumin, and NaCl) including 600 fmol of biotinylated capture oligomer (DNA Technology ApS, Aarhus, Denmark) and 1200 finol of each allele-specific ligation probe was B 5 added. The sequences of the allele-specific ligation POH probes and the biotinylated capture probe are given in the legend to Fig. 1. The reaction mixtures were then treated in either of two ways: 1) Method 1: The tubes were placed at 94#{176}C in the Fig. 1. Principle of allele-specific ligation. Perkin-Elmer thermocycler for 3 mm. The temperature 0, target DNA; #{149}, biotinylated common capture probe; , allele-specific was then lowered over a period of 15 min to the ligation probe. Position of the point mutation is at the 3’-end of the allele-specific reaction temperature. Ten microliters of 2 x ligation probe. (4) Capture probe and the Eu-labeled B point mutation-specific probe are annealed to a wild-type target. (B) Capture probe and the Sm-labeled buffer including 1 mmol/L ATP and T4-DNA ligase wild-type-specific probe are annealedto a wild-type target. In the presence of (Pharmacia Biotech, Uppsala, Sweden) was added and T4-DNA ligase only the Sm-labeled wild-type-specific probe would be ligated to the capture probe. Sequence of probes: biotinylated capture probe, the reaction mixtures were incubated at the ligation 5’P-TGGGGGTGGGAGATGC-3’; Sm-labeled wild-type-specifIc probe, temperature for 1 h. The amount of T4-DNA ligase 5’Sm-CGA&AGGGGCGTCC-3; Eu-labeled B mutation-specific probe, 5’Euused, the ligation temperature, and the final concenCGAMGGGGCGTCT-3’. 414
CLINICAL CHEMISTRY, Vol. 41, No. 3, 1995
tration
mixtures
probes differing from each other at the 3’-end. Thus, the two allele-specific probes compete with each other in annealing to the target DNA. The wild-type-specific probe is labeled with Sm at the 5’-end. The B mutationspecific probe is labeled with Eu at the 5’-end. After a period of incubation, T4-DNA ligase is added and the incubation is continued for another period before the reaction mixture is transferred to a streptavidin-coated microtiter well, where the biotmnylated oligomer is captured. The DNA is then denatured and washed in 0.1 mmol/L NaOH, leaving the labeled allele-specific probes only if ligated to the biotinylated common oligomer. After a final wash the products in the wells are quantified by time-resolved fluorescence measurements. The whole procedure is outlined in Fig. 2.
PCR amplification
.1 Heat-denature PCR product and ligation probes
Renature and add T4-DNA
ligase and ATP
Incubate 1 Bind to streptavidin
Denature in 0.1 mmol/L NaOH and wash 4. Count sample
Results
Fig. 2. Flowchart showing the simplicity of the ligation assay.
Method 1. To test the reliability of the method, several factors were varied by using three subjects (previously described) with genotypes W/W, W/B, and B/D as controls (18). Fig. 3 shows percent Eu counts, percent Sm counts, and the ratio Eu/Sm from the respective genotypes as the amount of T4-DNA ligase, the ligation temperature, and the NaCl concentration is varied as indicated. Increasing the amount of T4DNA ligase from 0.25 to 4 U (Fig. 3A-C) does not lead to a corresponding increase in Eu counts, Sm counts or,
neously incubated with the target DNA, which is the 739-bp B fragment obtained in the PCR I step as described in Materials and Methods. One oligomer is common to both alleles and is biotinylated at the 3’-end and phosphorylated at the 5’-end. This oligomer is homologous to the sense strand in a position immediately on the 5’-site of the position of the B point mutation. The other two oligomers are allele-specific A
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Fig. 3. Ligation with increasing amounts of T4-DNA bgase 4-C) increasing ligation temperature (D-F), and increasing NaCI concentration (G-1). AS tests were done in duplicate. Final NaCI concentration (4-F), 100 mmoVL G-I as indicated. Ugation temperature (4-C and G-4), 22’C; D-F as indicated. Amount of T4-DNA In A-C as indicated; D-I, 0.5 U. In A. 0, and G, 100% Eu counts correspond to the mean Eu counts obtainedfrom the B/D genotype at, respectively, all T4-DNA ligase concentrations, all ligation temperatures, and all NaCIconcentrations. In B, E, and H, 100% Sm counts correspond to the mean Sm counts obtalned from the W/W genotype at, respectively, all T4-DNA ligase concentrations, all ligation temperatures, and all NaCI concentrabons. #{149}, B/D genotype; W/B genotype; #{149}, W/W genotype. CLINICAL CHEMISTRY. Vol. 41, No. 3, 1995 415
10000, most important, any dramatic change in the Eu/Sm ratio. When the ligation temperature is changed from 1000 22#{176}C to 40#{176}C (Fig. 3D-F), although the number of total 100 counts might increase slightly with increasing temper10 ature, no effect is observed on the Eu/Sm ratio. ConE t 1 cerning the effect of NaCl (Fig. 3G-I), there is a modest S . $ : 0.1 20 25 30 35 40 45 50 increase in the difference between the Eu/Sm ratios from the respective genotypes with increasing NaCl 0.01 concentration (Fig. 31). This increase is accompanied 0.001 #{149} by a corresponding decrease in the false-positive Eu 0.0001 and Sm counts from the W/W and B/D genotypes, Annealing temperature, ‘C respectively (Fig. 3G and H). Fig. 4. Eu/Sm ratios vs annealing temperature with method 2. T4-DNA ligase, 0.5 U; NaCI,200 mmoVl. Experiments were done in duplicate. Table 1 shows the mean of Eu counts, Sm counts, and Symbols as in FIg. 3. Eu/Sm ratios when all the results from the experiment above are treated as a whole. Despite the fact that the 10000 assay conditions have been changed with respect to T4-DNA ligase concentration, ligation temperature, 1000 and NaCl concentration, there is a clear separation 100 between the W/W, the WIB, and the B/D genotypes, E especially considering the Eu/Sm ratios. Moreover, 10 Ui there is no overlap between the three genotypes, conir sidering the total number of Eu counts or Sm counts. 40 80 120 However, care should be taken against relating the 0.1 genotype to the total number of Eu counts or Sm counts 0.01 alone. % Eu counts Method 2. The Perkin-Elmer 9600 thermocycler Fig. 5. Results of 51 subjects tested by using method 2 with an used in the experiments above has the capability to annealing temperature of 50#{176}C where 100% (X-axis)corresponds to adjust the temperature gradually and is equipped with the value obtained from the B/D control genotype. a lid, giving top heat to the reaction tubes and thus Symbols as in Fig. 3. preventing condensation of water on the inside walls and caps of the tubes during the heat denaturation. To were tested in duplicate. A single reaction of the W/W, test whether such condensation would affect the assay, W/B, and B/D genotypes used to optimize the assay we performed an experiment in which the reactions conditions above were included each day to standardize were heat-denatured at 94#{176}C in a water bath, transthe assay. Fig. 5 shows the results for all samples in ferred to a water bath for annealing at a fixed temperterms of the Eu/Sm ratio against percent Eu counts; ature, and then transferred to the laboratory bench 100% corresponds to the absolute number of Eu counts before adding T4-DNA ligase and ATP. The results obtained with the B/D genotype. Note that this assay (Fig. 4) surprisingly were in general greatly improved does not distinguish between a BID or B/B genotype or in terms of the differences in the Eu/Sm ratios obtained between a W/D or W/W genotype, since the PCR amfrom the respective genotypes. Moreover, this improveplification is not quantitative in itself when performed ment was independent of annealing temperatures beas in this work. Table 2 summarizes the results in tween 22#{176}C and 50#{176}C, although the number of total terms of Eu counts, Sm counts, and Eu/Sm ratios and counts decreased at lower temperatures (not shown). shows about a 20-fold difference between the Eu/Sm Thus, we decided to continue testing the assay by using ratio for the respective genotypes. Moreover, even if no a 94#{176}C water bath for heat denaturation and a 50#{176}C background is subtracted, the three genotypes are water bath for annealing. Healthy randomly chosen subjects (51) were tested. Table 2. Eu counts, Sm counts, and Eu/Sm VS For day-to-day variation, the samples were divided and genotype. assayed on six different days. In all cases the samples A
A
Count, mean Genotype
Table 1. Eu counts, Sm counts, and Eu/Sm VS
B/B or B/D (n = 2 x 3)
genotype. CV,%
Counts, mean
W/B Genotype B/D W/B
W/W
Eu
212 130 105 444 16329
Sm 542 1999 3928
Eu/Sm 403 53 5.0
Eu
Sm
Eu/Sm
8.7
19.7
16.4
14.3 12.7
18.4 10.3
6.6 16.0
Assay conditions are as described in legend to Fig. 3. n = 26 for each genotype.
416 CLINICAL CHEMISTRY, Vol. 41, No. 3,
1995
(n
70629 =
=
Sm 197 1339
2 x 9)
W/W or W/D
(n
Eu 239 179
2 x 39)
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clearly separated from each other with regard to the Eu/Sm ratio. Although the number of total counts varied from day to day, no such variation could be demonstrated with respect to the Eu/Sm ratios (Table 3). Among the 51 subjects, 3 were B/B or BID, 9 were WIB, and 39 were W/W or W/D. The results were all confirmed by the standard procedure (see Materials and Methods and ref. 25), by means of which one person was also found to be A/A or A/D when tested for this mutation. This corresponds well to reports that have found that 7-10% of the Caucasian population are PMs because of the A, B, and D mutations and the relative allele frequencies (18). Discussion We have presented a T4-DNA ligase-based OLA to identify a common point mutation in the CYP2D6 gene. The assay is easy to perform and is extremely reliable over a broad range of annealing temperatures, ligation temperatures, salt concentrations, and T4-DNA ligase concentrations. The assay takes advantages of competitive hybridization and ligation when two differently labeled allele-specific probes are added to a target DNA amplificate together with a biotinylated capture probe common to the two different alleles (21). In this work the labels of choice have been the lanthanides Sm (wild-type-specific probe) and Eu (mutant-type specific probe). The allele-specific probes ligated to the capture probe are then detected by time-resolved fluorescence measurement in a Delfia fluorometer when bound to streptavidin-coated microtiter wells. The strategy to include differently labeled allele-specific probes simultaneously makes it possible to calculate a ratio between the signals from the probes. This efficiently eliminates variations in the assay caused by differences in yield of PCR product or by pipetting errors. In principle the B/B or BID genotype should give rise to an indefinitely high Eu/Sm ratio, a W/W or W/D genotype should give rise to an indefinitely low Eu/Sm ratio, and the Eu/Sm ratio from a W/B genotype should reflect the relative fluorescence sensitivity between Eu and Sm. Though it is not possible to achieve these indefinitely high and low values in practice, the assay makes it possible to discriminate sharply between homozygotes B/B (or compound heterozygotes BID) and heterozygotes W/B on one hand, and between heterozygotes W/B and homozygotes W/W (or compound heterozygotes W/D) on the other. This capability to differentiate between
Table 3. Variation
Eu counts
Eu/Sm
of absolute Eu counts all W/B genotypes. CV,% Total (df=23) 29.5
and Eu/Sm for
Within-day
Between-day
(df=18) 13.2
(df=5) 28.1
10.6
0.0
10.1
Assay conditions as in legend to Fig. 5. df, degrees of freedom.
homozygotes and heterozygotes is a must when dealing with autosomal recessive inherited traits, as is the case with the debrisoquine/sparteine polymorphism in this work. Furthermore, except in rare cases of homozygotes for gene deletions (as for instance the D/D genotype in the debrisoquine/sparteine polymorphism), the assay secures an internal control, as at least one of the allele-specific probes should give rise to a signal. Table 3 indicates that the day-to-day variation in absolute number of counts is reduced considerably by calculating the Eu/Sm ratio. Because of the statistical model of balanced repeated subsampling (27), the between variance component is estimated to be negative and the within component consequently to be larger than the total (due to the lower number of degrees of freedom). This means that the between-day variation is negligible. There seems to be both a significant day-to-day and a within-experiment variation in background signal (typically in the range of 2000 to 5000 counts for Eu and 290 to 370 counts for Sm) that is not correlated to the day-to-day variation in the absolute signal or to a certain analytical preparation (not shown). Since the biological variation for a mutation is binary, the most important reason for the within variation in the Eu/Sm ratio is probably this variation in background signal. However, although a background subtraction improves the results, Table 2 shows that this step is not a necessity. Thus, if background counts are to be subtracted, it is necessary to include only a few T4-DNA ligase-free background samples spread over the microtiter plates and use the mean counts obtained from these for background subtraction. The reason for the improvement of the ligation assay using method 2 compared with method 1 is not very clear. By comparing Tables 1 and 2 one can see that there is no increase in the true-positive signals. Rather, there is a strong reduction in false-positive signals. The reason might be that, with method 2, water from the sample is evaporated and condensed on the inside walls and caps of the tubes. This is not the case when the thermocycler is used because of top heat from the lid. Thus, in method 2 the reaction volume is reduced during heat denaturation and annealing, which leads to a higher concentration of salt, target, and probes. This might change the annealing kinetics such that the homologous probe more efficiently displaces the nonhomologous probe from the target. This assumption is supported by the fact that, regarding the Eu/Sm ratio, method 2 works equally well with annealing temperatures from 22#{176}C to 50#{176}C (Fig. 4). The most important advantages of the OLA approach as performed here are its simplicity and its extreme reliability over a broad range of annealing temperatures, ligation temperatures, salt concentrations, and ligase concentrations on separating genotypes homozygous or heterozygous for a point mutation from each other. In contrast to traditional assays, which often depend on inspection of ethidiu.m bromide-stained gels, the results are presented as pure numbers. This makes CLINICAL CHEMISTRY, Vol. 41, No. 3.
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it possible to automate interpretations. Thus, if the Eu/Sm ratio vs the total signal of Eu counts is not outside well-defined limits, a number can be automatically translated to a genotype or phenotype and answers can be sent directly to the clinician without the need to be inspected by a laboratory specialist. This makes possible the fast and reliable detection of inherited disorders, which might influence diagnosis and treatment of a patient. There is no reason to believe that the reliability of the assay is confined to a G-to-A transition, which is the characteristic of the B point mutation in CYP2D6, and we are currently testing other types of mutations with the assay. By a careful design of primers most PCR amplifications can be performed with the same temperature cycle proffle. Thus, on one microtiter plate either 96 different individuals can be tested for one single mutation or one single individual can be tested for 96 different mutations. This perspective makes the assay interesting as a tool for large-scale analysis at the DNA or RNA level in a routine laboratory. We thank Nina Brogger for expert laboratory technical work and Kim Br#{248}sen, Institute of Medical Biology, Department of Clinical Pharmacology, Odense University Hospital, for kindly providing the control genotypes used. This work was supported by the Institute of Clinical Research, Odense University, Denmark. References 1. Gonzales FJ, Vilbois F, Hardwick JP, McBride OW, Nebert DW, Gelboin IIV, et al. Human debrisoquine 4-hydroxylase (P45011D1): cDNA and deduced amino acid sequence and assignment of the CYP2D locus to chromosome 22. Genomics 1988;2: 174-9. 2. Zanger UM, Vilbois F, Hardwick JP, Meyer UA. Absence of hepatic cytochrome P45Obufl causes genetically deficient debrisoquine oxidation in man. Biochemistry 198827:5447-54. 3. Br#{248}senK, Gram LF. Clinical significance of the sparteine/ debrisoquine oxidation polymorphism. Eur J Clin Pharmacol 1989;36:537-47. 4. Evans DAP, Mahgoub A, Sloan TP, Idle JR. Smith RL A family and population study of the genetic polymorphism of debrisoquine oxidation in a white British population. J Med Genet 1980;17:102-5. 5. Evans DAP, Harmer D, Downham DY, Whibley EJ, Idle JR, Ritchie J, et al. The genetic control of sparteine and debrisoquine metabolism in man with new methods of analyzing bimodal distributions. J Med Genet 1983;20:321-9. 6. Stainer E, Iselius L, Alvan G, Lindsten J, Sjoqvist F. A family study of genetic and environmental factors determining polymorphic hydroxylation of debrisoquine. Clin Pharmacol Ther 1985;
38:394-401. 7. Br#{248}sen K, Otton SV, Gram LF. Sparteine oxidation polymorphism: a family study. Br J Clin Pharmacol 1986;21:661-7. 8. Alvan G, Bechtel P, Iselius L, Gundert-Remy U. Hydroxylation polymorphisms of debrisoquine and mephenytoin in European populations. Eur J Cliii Pharmacol 1990;39:533-7. 9. Johanson I, Lundquist E, Bertilsson L, Dahi ML, Sjoquist F, Ingelrnan-Sundberg M. Inherited amplifications of an active gene in the cytochrome P450 2D locus as a cause of ultrarapid metabolism of debrisoquine. Proc Nati Acad Sci USA 1993;90: 11825-9.
418 CLINICAL CHEMISTRY, Vol. 41. No. 3, 1995
10. Eichelbaum
M, Spannbrucker
N, Steincke B, Dengler JJ.
Defective N-oxidation of sparteine in man: a new pharmacogenetic defect. Eur J Chin Pharmacol 1979;16:183-7. 11. Gonzales FJ, Skoda RC, Kimura S, Umeno M, Zanger UM, Nebert DW, et al. Characterization of the common genetic defect in humans deficient in debrisoquune metabolism. Nature 1988; 31:442-6. 12. Kimura S, Umeno M, Skoda RC, Meyer UA. The human debrisoquine 4-hydroxylase (CYP2D) locus: sequence and identification of the polymorphic CYP2D6 gene, a related gene, and a pseudogene. Am J Hum Genet 1989;45:889-904. 13. Kagimoto M, Heim M, Kagimoto K, Zeugin T, Meyer UA. Multiple mutations of the human cytochrome P45011D6 gene (CYP2D6) in poor metamolizers of debrisoquune. J Biol Chem 1990;265:17209-14.
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