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Genetic analysis of the CYP2D6 locus in a Hong Kong Chinese population.

Debrisoquine-4-hydroxylase, a cytochrome cytochrome (sī`təkrōm'), protein containing heme (see coenzyme) that participates in the phase of biochemical respiration called oxidative phosphorylation.  P450 enzyme known as CYP CYP

In currencies, this is the abbreviation for the Cyprus Pound.

Notes:
The currency market, also known as the Foreign Exchange market, is the largest financial market in the world, with a daily average volume of over US $1 trillion.
2D6 enzyme, metabolizes different types of pharmacotherapeutic drugs, such as tricyclic antidepressants and neuroleptics Neuroleptics
Any of a class of drugs used to treat psychotic conditions.

Mentioned in: Stuttering, Tardive Dyskinesia
. The gene encoding for CYP2D6 enzyme, CYP2D6, is part of a cluster on chromosome 22 that includes two to three related pseudogenes (1). Starting at the 5' end of the cluster, there are two nonfunctional pseudogenes, CYP2D8P and CYP2D7AP (an additional pseudogene CYP2D7BP can also be found), followed by the active gene, CYP2D6. Several mutated alleles of the CYP2D6 locus have been identified and associated with alterations in the metabolism of debrisoquine and related drugs. Consequently, the activity of the CYP2D6 enzyme ranges from ultrarapid (ultrarapid me tabolizer) to a complete absence [poor metabolizer poor metabolizer Pharmacology A person who metabolizes a probe drug–the rate of which is related to the metabolizing cytochrome P-450 enzyme–slower than others; a person can be a PM of one probe drug, and an extensive metabolizer of another.  (PM)]. [3] Individuals homozygous ho·mo·zy·gous
adj.
Having the same alleles at one or more gene loci on homologous chromosome segments.


Homozygous
Identical genes controlling a specified inherited trait.
 or heterozygous het·er·o·zy·gous
adj.
1. Having different alleles at one or more corresponding chromosomal loci.

2. Of or relating to a heterozygote.
 for deficient CYP2D6 alleles metabolize drugs at lower rates, which leads to an increased risk of side effects Side effects

Effects of a proposed project on other parts of the firm.
 and drug toxicity. On the other hand, individuals with duplication of either the active gene CYP2D6 or the CYP2D6*2 allele allele (əlēl`): see genetics.
allele

Any one of two or more alternative forms of a gene that may occur alternatively at a given site on a chromosome.
 metabolize drugs at ultrarapid rate, which may lead to therapeutic failure.

In addition to interindividual variability in CYP2D6 enzyme activity Enzyme activity
A measure of the ability of an enzyme to catalyze a specific reaction.

Mentioned in: Glucose-6-Phosphate Dehydrogenase Deficiency
, the incidence of PMs of debrisoquine has been shown to vary between different populations. The prevalence of PMs ranges from <1% in some Oriental populations to as high as 19% in some black populations (2). In particular, differences between Caucasian and Oriental populations have been studied exhaustively (3-8). Interracial in·ter·ra·cial  
adj.
Relating to, involving, or representing different races: interracial fellowship; an interracial neighborhood.
 differences are explained by an unequal distribution of the alleles of the CYP2D6 gene among different populations. The defective alleles CYP2D6*3 and CYP2D6*4 that give rise to the PM phenotype in Caucasians are rarely found in Chinese, explaining the low frequency of PMs in this population. However, these two races differ not only in the incidence of PMs but also in the activity of debrisoquine hydroxylase within the extensive metabolizer (EM) phenotype. The distribution of the debrisoquine metabolic ratios is shifted toward higher values among Chinese EMs, indicating lower CYP2D6 enzyme activity. These individuals are classified as intermediate metabolizers. The lower enzyme activity in Oriental EMs is associated with the frequent presence of the allele CYP2D*10 and its variants, CYP2D*10A and CYP2D*10B. These alleles contain a [C.sub.188] [right arrow] T mutation that causes a [Pro.sub.34] [right arrow] Ser amino acid amino acid (əmē`nō), any one of a class of simple organic compounds containing carbon, hydrogen, oxygen, nitrogen, and in certain cases sulfur. These compounds are the building blocks of proteins.  substitution, leading to the formation of an unstable enzyme with lower metabolic activity (3).

Earlier studies have indicated that there may be differences in CYP2D6 enzyme activity not only between the major races but also among different Oriental populations (4-8). Consequently, further studies on different Oriental ethnic groups were urgently needed to provide important genotypic data for facilitating rational therapeutics. In this study, we analyzed the molecular basis of the CYP2D6 polymorphism polymorphism, of minerals, property of crystallizing in two or more distinct forms. Calcium carbonate is dimorphous (two forms), crystallizing as calcite or aragonite. Titanium dioxide is trimorphous; its three forms are brookite, anatase (or octahedrite), and rutile.  in a Hong Kong Hong Kong (hŏng kŏng), Mandarin Xianggang, special administrative region of China, formerly a British crown colony (2005 est. pop. 6,899,000), land area 422 sq mi (1,092 sq km), adjacent to Guangdong prov.  Chinese population, aiming at characterizing the distribution of known alleles of the CYP2D6 locus. The genotyping analysis presented here comprises the identification of 10 CYP2D6 alleles (including the wild type) by PCR-based methods (Table 1). CYP2D6 alleles are designated according to according to
prep.
1. As stated or indicated by; on the authority of: according to historians.

2. In keeping with: according to instructions.

3.
 the current nomenclature (9).

Materials and Methods

INDIVIDUALS

One hundred nineteen unrelated Chinese individuals participated in the genotyping tests after giving their informed consent. The subjects include 70 healthy volunteers and 49 patients diagnosed with depressive illness according to DSM 1. DSM - Data Structure Manager.

An object-oriented language by J.E. Rumbaugh and M.E. Loomis of GE, similar to C++. It is used in implementation of CAD/CAE software. DSM is written in DSM and C and produces C as output.
 N (10). This research project was approved by the local ethics committee ethics committee A multidisciplinary hospital body composed of a broad spectrum of personnel–eg, physicians, nurses, social workers, priests, and others, which addresses the moral and ethical issues within the hospital. See DNR, Institutional review board. .

STATISTICS

The frequency of a determined CYP2D6*X allele in a sample of N individuals was estimated by ([2n.sub.x/x] + [n.sub.x/-])/2N, where [n.sub.x/x] is the number of individuals homozygous for X, and [n.sub.x/-] is the number of individuals heterozygous for the X allele. Allele frequencies in different populations were compared using Z-test for proportions.

GENOTYPE DETERMINATION

Blood samples (12 mL) were collected in Vacutte[R] (Greiner) tubes containing EDTA EDTA: see chelating agents.  as an anticoagulant. DNA DNA: see nucleic acid.
DNA
 or deoxyribonucleic acid

One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes.
 was extracted using a QIAamp blood kit (Qiagen). PCR PCR polymerase chain reaction.

PCR
abbr.
polymerase chain reaction


Polymerase chain reaction (PCR) 
 followed by either restriction-enzyme digestion of the amplified product or allele-specific PCR (ASPCR ASPCR American Society for the Prevention of Cruelty to Robots ) tests was used to identify some of the CYP2D6 alleles. The mutations analyzed for each of the alleles and primers and/or enzymes used for the corresponding tests are summarized in Table 1. Sequences and locations of the primers for each PCR/ASPCR reaction are specified in Table 2.

The first step was the amplification of a 5093-bp genomic DNA genomic DNA
n.
The full complement of DNA contained in the genome of a cell or organism.
 fragment, containing all nine CYP2D6 exons, to be used as an amplicon for the subsequent PCR-based tests. Genomic DNA (400 ng) was used as template and the Expand Long Template PCR System[TM] (Boehringer) was applied. The reaction was carried out in a 25-[micro]L mixture containing buffer 3 (supplied with the kit), 2.5 mmol/L Mg[Cl.sub.2], 500 [micro]mol/L deoxynucleotide triphosphates, 0.5 [micro]mol/L of each of the CYP2D6-specific primers (2D6F and 2D6R; see Table 2) and 2 U of the enzyme mixture (Taq and Pwo polymerases). The cycle profile was as follows: predenaturation at 94[degrees]C for 2 min, followed by 10 cycles of denaturation denaturation, term used to describe the loss of native, higher-order structure of protein molecules in solution. Most globular proteins exhibit complicated three-dimensional folding described as secondary, tertiary, and quarternary structures.  at 94[degrees]C for 1 min, annealing at 60[degrees]C for 30 s, and synthesis at 68[degrees]C for 4 min. These 10 cycles were followed by 30 additional cycles in which the elongation time was increased by 20 s in each cycle. The final elongation step was 7 min at 68[degrees]C. Approximately 10 ng of the 5093-bp PCR product was used as a template in the subsequent nested PCR reactions. Detection of the mutations was performed as per the corresponding references listed in Table 1. Only the modifications and/or new assays are described in this report. All PCR reactions were carried out in a PTC-200 DNA Engine thermocycler (MJ Research) or in a Master-cycler Gradient thermocycler (Eppendorf). PCR products were detected in ethidium bromide-agarose gels.

FREQUENCY OF THE [C.sub.188] [right arrow] T MUTATION

Additional precautions to increase specificity were taken when checking for mutations in exon Exon

In split genes, a portion that is included in the ribonucleic acid (RNA) transcript of a gene and survives processing of the RNA in the cell nucleus to become part of a spliced messenger RNA (mRNA) or structural RNA in the cell cytoplasm.
 1 and 2, especially for the mutation [C.sub.188] [right arrow] T, which is responsible for decreased CYP2D6 enzyme activity in Orientals (3, 6). To exclude any possible artifact attributable to the coamplification of CYP2D7P, primer M, whose sequence is unique to CYP2D6, was used in combination with primer A to amplify exons 1 and 2 of the active gene. PCR was carried out using the same cycling conditions as those to amplify the whole gene. The concentrations of Mg[Cl.sub.2] and the primers were 2.5 mmol/L and 0.4 [micro]mol/L, respectively. The 1193-bp PCR product (amplicon AM) was used as a template for the subsequent rounds of PCR reactions to allow detection of mutations in introns 1 and 2. For [C.sub.188] [right arrow] T, primers A and B were used. A parallel PCR reaction using primers A and 10B was carried out to detect the CYP2D7P-derived sequence in the region 302-333, characteristic of CYP2D6*2, and to rule out any possible misinterpretation of [C.sub.188] [right arrow] T (3). The final concentrations of the PCR reagents for a 25-[micro]L reaction were as follows: 0.8 mmol/L Mg[Cl.sub.2], 40 [micro]mol/L dNTPs, 0.250 [micro]mol/L of each primer, and 0.5 U of the Taq polymerase Taq polymerase ("Taq Pol," or simply "Taq") is a thermostable polymerase used in polymerase chain reaction to check for the presence or absence of a gene by amplifying a DNA fragment. It replaced E.coli DNA polymerase in PCR because of the temperature conditions of PCR.  enzyme (Boehringer Mannheim). The cycle profile was as follows: predenaturation at 94[degrees]C for 2 min, followed by 25 cycles of denaturation at 94[degrees]C for 1 min, annealing at 66[degrees]C for 30 s, and synthesis at 72[degrees]C for 1.5 min. The final elongation step was 7 min at 72[degrees]C. The PCR products obtained, which were 270 by when A and B were used for amplification and 230 by when A and 10B were used, were digested by HphI as described previously (6).

FREQUENCY OF THE [T.sub.226] INSERTION MUTATION

In CYP2D8P and CYP2D7P pseudogenes, a T exists at the 226 site corresponding to exon 1 of the wild-type CYP2D6 gene (11,12). Hence, to exclude artifacts caused by coamplification of pseudogenes, amplicon AM was used as a template for subsequent reactions. PCR products generated by the primer pairs A and B (270 bp) and A and 10B (230 bp) were digested by BspMI (11).

FREQUENCY OF THE [C.sub.1127] [right arrow] T MUTATION

The mutation [C.sub.1127] [right arrow] T was checked by amplifying 10 ng of amplicon AM with allele-specific primers 1127W and 1127A, and the reverse primer M. The final concentrations of the PCR reagents were as follows: 0.8 mmol/L Mg[Cl.sub.2], 40 [micro]mol/L dNTPs, 0.200 [micro]mol/L of each primer, and 0.25 U of Taq polymerase (Boehringer Mannheim). Cycling conditions were as follows: predenaturation at 97[degrees]C for 2 min, followed by 25 cycles of denaturation at 94[degrees]C for 1 min, annealing at 62[degrees]C for 30 s, and synthesis at 72[degrees]C for 1 min. The final elongation step was 7 min at 72[degrees]C.

FREQUENCY OF THE [G.sub.1749] [right arrow] C MUTATION

ASPCR by competitive oligonucleotide priming (13) was used for the detection of this mutation. The previously amplified CYP2D6 gene was used as a template in a PCR reaction buffer similar to that described above for [C.sub.188] [right arrow] T. Primers E, F, and 1749W or 1749A (for the parallel reaction) were used. Cycling conditions were as follows: predenaturation at 97[degrees]C for 2 min, followed by 20 cycles of denaturation at 94[degrees]C for 1 min, annealing at 66[degrees]C for 30 s, and synthesis at 72[degrees]C for 1 min. The final elongation step was 7 min at 72[degrees]C.

FREQUENCY OF THE CYP2D6*5 ALLELE

The deletion allele CYP2D6*5 was identified using primers Z and Y, which are located in CYP2D7 and in the 3'-flanking region of CYP2D6, respectively (Table 2). We applied the method described by Griese et al. (14) with some modifications. PCR was performed on a PTC-200 DNA Engine thermocycler using the Expand Long Template PCR System. The reaction was carried out in a 25-[micro]L mixture containing buffer 3 (supplied with the kit), Mg[Cl.sub.2] at 2.25 mmol/L, deoxynucleotide triphosphates at 500 [micro]mol/L each, each of the primers at 0.3 [micro]mol/L, and 500 ng of genomic DNA as template. The cycle profile was as follows: predenaturation at 93[degrees]C for 1 min, followed by 39 cycles of denaturation at 93[degrees]C for 1 min, annealing at 63[degrees]C for 30 s, and synthesis at 68[degrees]C for 6 min. The final elongation step was 7 min at 72[degrees]C. Carriers of allele *5 revealed a 6.2-kb PCR fragment amplification. This assay was validated using as a positive control a DNA sample from an individual showing (by Southern blot) a XbaI 11.5-kb restriction fragment Noun 1. restriction fragment - the fragment of DNA that is produced by cleaving DNA with a restriction enzyme
fragment - a piece broken off or cut off of something else; "a fragment of rock"
 indicative of CYP2D*5 (gift from Dr. J Noun 1. Dr. J - United States basketball forward (born in 1950)
Erving, Julius Erving, Julius Winfield Erving
. M. Agundez, Department of Pharmacology, Medical School, University of Extremadura, Badajoz, Spain). Detection of the CYP2D6*5 allele was submitted to different PCR tests to confirm the results. To this end, primer Y was used in combination with primer CYP13 under the same PCR conditions. This assay yielded a 3.5-kb fragment (15).

Results and Discussion

CYP2D6 ALLELES

DNA from all individuals was analyzed for most key mutations as described by Daly et al. (9) (see Table 1). The frequencies of the CYP2D6 alleles investigated in this study are listed in Table 3, and a comparison with the CYP2D6 frequencies found in other studies conducted in Chinese populations is presented in Table 4. We found 5 known alleles out of 10 CYP2D6 alleles analyzed. Our genotype results are generally in agreement with the published sequences at the investigated target sites. Each of the CYP2D6*2 alleles contained the CYP2D7-derived sequence in the region 302-333 in intron Intron

In split genes, a portion that is included in ribonucleic acid (RNA) transcripts but is removed from within a transcript during RNA processing and is rapidly degraded.
 1. However, the converted intronic sequence was also found in two CYP2D6*1 alleles (wild type). In addition, one individual presented an *2 allele with an additional [C.sub.1127] [right arrow] T mutation, a variant described previously (16), and two other individuals presented *2 alleles without mutation [C.sub.2938] [right arrow] T. On the other hand, two individuals homozygous for the CYP2D6*10 alleles were found to have mutation [C.sub.2938] [right arrow] T, which is characteristic of allele *2. The presence of this particular mutation, which causes an [Arg.sub.296] [right arrow] Cys amino acid substitution, can be considered of high functional importance as exemplified by its significant role in the CYP2D6*17 allele (17). Further studies on alterations of the enzymatic activity caused by the combinations of these mutations are on going. The frequency of allele *2 was somewhat lower than that found in previous studies conducted in Chinese populations (Table 4) (7). As for CYP2D6*10 alleles, the frequencies were 10.51% for the variant *10A and 54.20% for the variant *10B. The CYP2D6*10C allele was not analyzed because it is present together with the 10*B allele in Oriental subjects with a 44-kb XbaI haplotype haplotype /hap·lo·type/ (-tip) the group of alleles of linked genes, e.g., the HLA complex, contributed by either parent; the haploid genetic constitution contributed by either parent.

hap·lo·type
n.
 (3, 8). The prevalence of mutation [C.sub.188] [right arrow] T, which is characteristic of the *10 alleles, was 64.71%. As shown in Table 4, the frequency of [C.sub.188] [right arrow] T was higher than that found in Chinese living in Sweden (51%) (3, 7) and similar to that found in Taiwanese Chinese (70%) and Chinese living in Singapore (62%) (6,18). These differences in the frequency of [C.sub.188] [right arrow] T were not statistically significant and could be attributed to the different methods applied for its detection (3).

Alleles CYP2D6*15 (11), CYP2D6*16 (12), and CYP2D6*8/*14 (19, 20) seem to be very rare mutant alleles in Caucasians as well as in Orientals. To date, no studies on their prevalence have been done in Oriental populations, and our results show that alleles *15 and *16 are rare in these population. Recently, three *14 alleles were characterized in a sample of 124 (1.2%) Taiwanese Chinese subjects by Wang et al. (20).

As expected, CYP2D6*4D, which is the main allele responsible for the PM phenotype in Caucasians (21), was not detected in our study.

CYP2D*J9 was first detected in Japanese (22, 23) with a very low frequency (0.7%). Because of the similarity between the Japanese and Chinese populations, we thought it would be of interest to find the distribution of CYP2D*J9 in Chinese because it has not been studied previously. However, this allele was not found in the Hong Kong population studied.

CYP2D6 GENOTYPES

We found eight different genotypes, which are listed together with their respective frequencies in Table 5. As expected, the most frequent genotype was *10/*10, followed by *1/*10. Individuals homozygous for allele *10 have impaired metabolism of debrisoquine-4-hydroxylase, which explains the right shift in the metabolic ratio of debrisoquine among Chinese when compared with Caucasian EMs.

No significant differences in allele distribution or genotype were observed between psychiatric patients and healthy individuals. However, it is worth mentioning that psychiatric patients showed a higher frequency overall of allele *2 when compared with the controls (data not shown). This observation requires further study because discrepancies in the of catalytic activity of allele *2 have been reported (24). In addition, allele *2 is the main CYP2D6 variant behind the genetic mechanism that originates duplications of the active CYP2D6 gene that gives rise to the ultrarapid metabolizer phenotype (25, 26). Thus, there may be clinical relevance for the drug treatment of psychiatric patients harboring this allele.

In Orientals, PCR-based detection of the *10/*10 genotype should be recommended when CYP2D6-metabolized drugs are applied.

Received June 29, 1999; accepted October 15, 1999.

References

(1.) Kimura S, Umeno M, Skoda RC, Meyer UA, Gonzalez F. The human debrisoquine 4-hydroxylase (CYP2D6) locus: sequence and identification of the polymorphic polymorphic - polymorphism  CYP2D6 gene, a related gene and a pseudogene. Am J Hum Genet genet: see civet.  1989;45:889-904.

(2.) Bertilsson L. Geographical/interracial differences in polymorphic drug oxidation. Clin Pharmacokinet 1995;29:192-209.

(3.) Johansson I, Oscarsson M, Yue Q-Y, Bertilsson L, Sjovist F, Ingelman-Sundberg M. Genetic analysis of the Chinese cytochrome P450 locus: characterization of variant CYP2D6 genes present in subjects with diminished capacity This doctrine recognizes that although, at the time the offense was committed, an accused was not suffering from a mental disease or defect sufficient to exonerate him or her from all criminal responsibility, the accused's mental capacity may have been diminished by intoxication,  for debrisoquine hydroxylation. Mol Pharmacol 1994;46:452-9.

(4.) Horai Y, Nakano M, Ishizaki T, Ishikawa K, Zhou H-H, Zhou B-J, et al. Metoprolol metoprolol /met·o·pro·lol/ (met?ah-pro´lol) a cardioselective ß used in the form of the succinate and tartrate salts in the treatment of hypertension, chronic angina pectoris, and myocardial infarction.  and mephenytoin oxidation polymorphisms in Far Eastern Oriental subjects: Japanese versus mainland Chinese. Clin Pharmacol Ther 1989;46:198-207.

(5.) Sohn D-R, Shin S-G, Park C-W, Kusaka M, Chiba K, Ishizaki T. Metoprolol oxidation polymorphism in a Korean population: comparison with native Japanese and Chinese populations. Br J Clin Pharmacol 1991;32:504-7.

(6.) Wang SL, Huang JD, Lai MD, Liu BH, Lai ML. Molecular basis of genetic variation in debrisoquin hydroxylation in Chinese subjects: polymorphism in RFLP RFLP
abbr.
restriction fragment length polymorphism



RFLP

restriction fragment length polymorphism.

RFLP 
 and DNA sequence DNA sequence Genetics The precise order of bases–A,T,G,C–in a segment of DNA, gene, chromosome, or an entire genome. See Base pair, Base sequence analysis, Chromosome, Gene, Genome.  of CYP2D6. Clin Pharmacol Ther 1993;53:410-8.

(7.) Dahl M-L, Yue Q-Y, Roh H-K, Johansson I, Sawe J, Sjwist F, Bertilsson L. Genetic analysis of the CYP2D6 locus in relation to debrisoquine hydroxylation capacity in Korean, Japanese and Chinese subjects. Pharmacogenetics Pharmacogenetics Definition

Pharmacogenetics is the study of how the actions of and reactions to drugs vary with the patient's genes.
Description
 1995;5:159-64.

(8.) Roh H-K, Dahl M-L, Johansson I, Ingelman-Sundberg M, Cha Y-M, Bertilsson L. Debrisoquine and S-mephenytoin hydroxylation phenotypes and genotypes in a Korean population. Pharmacogenetics 1996;6:441-7.

(9.) Daly AK, Brockmoller J, Broly F, Eichelbaum M, Evans WE, Gonzalez FJ, et al. Nomenclature for human CYP2D6 alleles. Pharmacogenetics 1996;6:193-201.

(10.) American Psychiatric Association. Diagnostic and statistical manual of mental disorders Diagnostic and Statistical Manual of Mental Disorders /Di·ag·nos·tic and Sta·tis·ti·cal Man·u·al of Men·tal Dis·or·ders/ (DSM) a categorical system of classification of mental disorders, published by the American Psychiatric Association, that delineates objective , 4th ed. Washington, DC: American Psychiatric Association, 1994:317-92.

(11.) Sachse C, Brockmoller J, Bauer S, Reum T, Roots I. A rare insertion of T226 in exon 1 of CYP2D6 causes a frameshift and is associated with the poor metabolizer phenotype: CYP2D6*15. Pharmacogenetics 1996;6:269-72.

(12.) Daly AK, Fairbrother KS, Andreassen OA, London SJ, Idle JR, Steen VM. Characterization and PCR based detection oftwo different hybrid CYP2D7P/CYP2D6 alleles associated with the poor metabolizer phenotype. Pharmacogenetics 1996;6:319-28.

(13.) Bottema CDK Cdk

cyclin-dependent protein kinase.
, Sommer SS. PCR amplification of specific alleles: Rapid detection of known mutations and polymorphism. Mutat Res 1993;288:93-102.

(14.) Griese EU, Zanger UM, Brudermanns U, Gaedigk A, Mikus G, Morike K, et al. Assessment of the predictive power of genotypes for the in-vivo catalytic function of CYP2D6 in a German population. Pharmacogenetics 1998;8:15-26.

(15.) Daly AK, Steen VM, Fairbrother KS, Idle JR. CYP2D6 multiallelism. Methods Enzymol 1996;272:199-210.

(16.) Marez D, Legrand M, Sabbaagh N, Lo Guidice JM, Spire C, Lafitte JJ, et al. Polymorphism of the cytochrome P450 CYP2D6 gene in a European population: characterization of 48 mutations and 53 alleles, their frequencies and evolution. Pharmacogenetics 1997; 7:193-202.

(17.) Oscarson M, Hidestrand M, Johansson I, Ingelman-Sundberg M. A combination of mutations in the CYP2D*17 (CYP2D6Z) allele causes alterations in enzyme function. Mol Pharmacol 1997;52: 1034-40.

(18.) Lee EJD EJD European Journal of Dermatology
EJD Executive Juris Doctor (degree) 
, Jeyaseelan K. Frequency of human CYP2D6 mutant alleles in a normal Chinese population. Br J Clin Pharmacol 1994;37:605-7.

(19.) Broly F, Marez D, Sabbaagh N, Legrand M, Millecamps S, Lo Guidice JM, et al. An efficient strategy for detection of known and new mutations of the CYP2D6 gene using single strand conformation polymorphism Definition
Single strand conformation polymorphism (SSCP) is defined as conformational difference of single stranded nucleotide sequences of identical length as induced by differences in the sequences under certain experimental conditions.
 analysis. Pharmacogenetics 1995;5:373-84.

(20.) Wang SL, Lai MD, Huang JD. G169R mutation diminishes the metabolic activity of CYP2D6 in Chinese. Drug Metab Dispos 1999; 27:385-8.

(21.) Heim HH, Meyer UA. Genotyping of poor metabolizers of debrisoquine by allele-specific PCR amplification. Lancet 1990;336: 529-32.

(22.) Yokoi T, Kosaka Y, Chida M, Chiba K, Nakamura H, Ishizaki T, et al. A new CYP2D6 allele with a nine base insertion in exon 9 in a Japanese population associated with poor metabolizer phenotype. Pharmacogenetics 1996;5:395-441.

(23.) Yokoi T, Kamataki T. Genetic polymorphism of drug metabolizing enzymes: new mutations in CYP2D6 and CYP2A6 genes in Japanese. Pharmacol Res 1998;15:517-24.

(24.) Sachse C, Brockmoller J, Bauer S, Roots I. Cytochrome P450 2D6 variants in a Caucasian population: allele frequencies and phenotypic consequences. Am J Hum Genet 1997;60:284-95.

(25.) Johansson I, Lundqvist E, Bertilsson L, Dahl M-L, Sjovist F, Ingelman-Sundberg M. Inherited amplification of an active gene in the cytochrome P450 CYP2D6 locus as a cause of ultrarapid metabolism of debrisoquine. Proc Natl Acad Sci U S A 1993;90: 11825-9.

(26.) Lundqvist E, Johansson J, Ingelman-Sundberg M. Genetic mechanisms for duplication and multiplication of the human CYP2D6 gene and methods for detection of duplicated CYP2D6 genes. Gene 1999;226:327-38.

(27.) Wang SL, Lai MD, Lai ML, Huang JD. R296C and other CYP2D6 mutations in Chinese. Pharmacogenetics 1995;5:385-8.

(28.) Chen S, Chou W-H, Blouin RA, Mao Z, Humphries LL, Meek QC, et al. The cytochrome P450 2D6 (CYP2D6) enzyme polymorphism: screening costs and influence in clinical outcomes in psychiatry. Clin Pharm Ther 1996;5:522-34.

[3] Nonstandard non·stan·dard  
adj.
1. Varying from or not adhering to the standard: nonstandard lengths of board.

2.
 abbreviations: PM, poor metabolizer; EM, extensive metabolizer; and ASPCR, allele-specific PCR.

Merce Garcia-Barcelo, [1] * Lok Yee Chow, [1] Helen Fung Kum Chiu, [1] Yun Kowk Wing, [1] Dominic Tak Shing Lee, [1] Kwok Lim Lam, [1] and Mary Miu Yee Waye [2]

[1] Department of Psychiatry, 11/F Room 134046, Prince of Wales Hospital
This article is about a hospital in Hong Kong. For the hospital in Sydney, Australia, see Prince of Wales Hospital, Sydney. There also exists another Prince of Wales Hospital in the United Kingdom.
, The Chinese University of Hong Kong The motto of the university is "博文約禮" in Chinese, meaning "to broaden one's intellectual horizon and keep within the bounds of propriety". , Shatin, New Territories, Hong Kong.

[2] Department of Biochemistry, Room 608, 6/F Mong Man Wai For the actor, see .
Mong Man Wai (蒙民偉) is the chairman of the Shun Hing Group, the distributor of Matsushita products (National, Panasonic, Technics) in Hong Kong.

He used to study in La Salle College Hong Kong.
 Building, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.

* Author for correspondence. Fax 852-2646-2284; e-mail b025744@ mailserv.cuhk.edu.hk.
Table 1. Analysis of the mutations and identification of CYP2D6
alleles by PCR-based methods.

Allele (a)      Mutations analyzed (b)               Primers used (c)

CYP2D6*1        Wild type
CYP2D6*2        Conversion 2D6-2D7 at 302-333       A, M, 10B
                [G.sub.1749] [right arrow] C        E, F, 1749W, 1749A
                [G.sub.2938] [right arrow] T        G, H
                [G.sub.4268] [right arrow] C        J, N

CYP2D6*4D       [G.sub.188] [right arrow] T         A, B, M
                [G.sub.1127] [right arrow] T        A, M, 1127W, 1127A
                [G.sub.1749] [right arrow] C        E, F, 1749W, 1749A
                [G.sub.1934] [right arrow] A        E, F
                [G.sub.4268] [right arrow] C        J, N

CYP2D6*5        Deletion of CYP2D6 gene             Z, Y; CYP13
CYP2D6*8/*14    [G.sub.188] [right arrow] T         A, B, M
                [G.sub.1846] [right arrow] T/A      E, F
                [G.sub.2938] [right arrow] T        G, H
                [G.sub.4268] [right arrow] C        J, N

CYP2D6*10A      [G.sub.188] [right arrow] T         A, B, M
                [G.sub.1749] [right arrow] C        E, F, 1749W, 1749A
                [G.sub.4268] [right arrow] C        J, N

CYP2D6*10B      [C.sub.1127] [right arrow] T        A, M, 1127W, 1127A
                  and all mutations of
                  CYP2D6*10A
CYP2D6*J9       9-bp insertion in exon 9            9S, 9AS
CYP2D6*15       [T.sub.226] insertion               A, M, B/10B
CYP2D6*16       Deletion between CYP2D6/2D7         Y, Z

Mutations analyzed (b)         Enzyme (d)       References

Wild type
Conversion 2D6-2D7 at 302-333               (24)
[G.sub.1749] [right arrow] C                See Materials and Methods
[G.sub.2938] [right arrow] T   HhaI         (27)
[G.sub.4268] [right arrow] C   BstEII       (6)

[G.sub.188] [right arrow] T    HphI         (6) and see Materials and
                                               Methods
[G.sub.1127] [right arrow] T                See Materials and Methods
[G.sub.1749] [right arrow] C                See Materials and Methods
[G.sub.1934] [right arrow] A   BstNI        (6)
[G.sub.4268] [right arrow] C   BstEII       (6)

Deletion of CYP2D6 gene                     (14)(15)
[G.sub.188] [right arrow] T    HphI         (6) and see Materials and
                                            Methods
[G.sub.1846] [right arrow]
  T/A                          MspI         (20)(27)(28)
[G.sub.2938] [right arrow] T   HhaI         (27)
[G.sub.4268] [right arrow] C   BstEII       (6)

[G.sub.188] [right arrow] T    HphI         (6) and Materials and
                                            Methods
[G.sub.1749] [right arrow] C                See Materials and Methods
[G.sub.4268] [right arrow] C   BstEII       (6)

[C.sub.1127] [right arrow] T                See Materials and Methods
  and all mutations of
  CYP2D6*10A
9-bp insertion in exon 9                    (22)
[T.sub.226] insertion          BspMI        (11) and see Materials
                                              and Methods
Deletion between CYP2D6/2D7                 (14)

(a) Alleles designated according to the current nomenclature (9).

(b) Sites numbered according to Kimura et al. (1).

(c) PCR was applied to the active CYP2D6 gene, which was amplified
previously with primers 2D6F and 2D6R (see Table 2 and text).

(d) Restriction enzyme used to detect the mutation.

Table 2. Sequences and locations of primers used in PCR reactions.

Name                   Sequence                    Location (a)

2D6F      5'-CCA GAA GGC TTT GCA GGC TTC A-3'    -253 to-232
2D6R      5'-ACT GAG CCC TGG GAG GTA GGT A-3'    4840 to 4819
A         5'-CCA TTT GGT AGT GAG GCA GGT AT-3'   68 to 90
B         5'-CAC CAT CCA TGT TTG CTT CTG GT-3'   338 to 316
10B       5'-GTG GTG GGG CAT CCT CAG G-3'        320 to 302 (7P)
1127W     5'-TCA CCC AGA TCC TGG GTT TC-3'       1105 to 1127
1127A     5'-TCA CCC AGA TCC TGG GTT TT-3'       1105 to 1127
E         5'-GTG GAT GGT GGG GCT AAT GCC TT-3'   1637 to 1659
F         5'-CAG AGA CTC CTC GGT CTC TCG CT-3'   2124 to 2102
1749W     5'-GGAG CAG AGG CGC TTC TCC GTG-3'     1729 to 1749
1749A     5'-GGAG CAG AGG CGC TTC TCC GTC-3'     1729 to 1749
G         5'-CCG TTC TGT CCC GAG TAT GCT CT-3'   2859 to 2882
H         5'-GGC TAT CAC CAG GTG CTG GTG CT-3'   3485 to 3463
J         5'-GAG ACA AAC CAG GAC CTG CCA-3'      3632 to 3652
M         5'-CCC CAC TCG CTG GCC TGT TTC A-3'    1261 to 1237
N         5'-GCC TCA ACG TAC CCC TGT CTC-3'      +118 to +98
9S        5'-AGC TCT TCC TCT TCT TCA CC-3'       4167 to 4186
9AS       5'-CAG GAA AGC AAA GAC ACC ATG-3'      4264 to 4244
Y         5'-TGA GCT AAG GCA CCC ACA CT-3'       +3462 to +3443
Z         5'-GCT ACC CCG TTC TAT CCC C-3'        2823 to 2841 (b)
CYP-13    5'-ACC GGG CAC CTG GGT ACT CCT CA-3'   7020 to 7040 (b,c)

(a) Location of primers numbered according to Kimura et al. (1).
Negative and positive numbers indicate locations in the 5'- and
3'-flanking regions, respectively.

(b) Positions corresponding to CYP2D7 pseudogene.

(c) Position in sequence GenBank accession no. X58467.

Table 3. Frequencies of the CYP2D6 alleles identified in the
present study.

Alleles               Number of          Frequency, %
                       variants

CYP2D6*1                  54            22.69 (54/238)
CYP2D6*2                  19             7.98 (19/238)
CYP2D6*4D                  0             0
CYP2D6*5                  11             4.62 (11/238)
CYP2D6*/*14                0             0
CYP2D6*10A                25            10.51 (25/238)
CYP2D6*10B               129            54.20 (129/238)
CYP2D6*15                  0             0
CYP2D6*16                  0             0
CYP2D6*J9                  0             0
Total                    238           100

Table 4. Frequencies of CYP2D6 alleles found in Chinese populations.

                                          Frequency, %

                                n (a)      CYP2D6*1     CYP2D6*2

This study
  Hong Kong Chinese             119         22.69         7.98
Wang and co-workers (6)(20)
  Chinese Taiwanese             124         NA (b)         NA
Lee and Jeyaseelan (18)
  Chinese Singaporian            93           NA           NA
Dahl et al. (7)
  Mainland Chinese               21          21.0         26.0
Johansson et al. (3)
  Chinese living in Sweden      113          29.6         13.4

                                          Frequency, %

                              CYP2D6*4D    CYP2D6*5    CYP2D6*8/14

This study
  Hong Kong Chinese               0          4.62           0
Wang and co-workers (6)(20)
  Chinese Taiwanese              0.8          NA           1.2
Lee and Jeyaseelan (18)
  Chinese Singaporian             0           NA           NA
Dahl et al. (7)
  Mainland Chinese               NA          2.0           NA
Johansson et al. (3)
  Chinese living in Sweden       NA          5.73          NA

                                    Frequency, %

                              CYP2D6*10    CYP2D6*J9

This study
  Hong Kong Chinese             64.71         0
Wang and co-workers (6)(20)
  Chinese Taiwanese             70.0          NA
Lee and Jeyaseelan (18)
  Chinese Singaporian           62.0          NA
Dahl et al. (7)
  Mainland Chinese              48.0          NA
Johansson et al. (3)
  Chinese living in Sweden      50.7          NA

(a) Number of individuals per study.

(b) NA, data not available.

(c) 115 subjects (3).

Table 5. Frequency of CYP2D6 genotypes.

Genotype     Number of subjects        Frequency, %

*1/*1                 5                    4.20
*1/*2                 4                    3.36
*1/*5                 4                    3.36
*1/*10               36                   30.25
*10/*5                6                    5.04
*10/*10              49                   41.17
*2/*5                 1                    0.84
*2/*10               14                   11.78
Total               119                  100
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Title Annotation:Molecular Diagnostics and Genetics
Author:Garcia-Barcelo, Merce; Chow, Lok Yee; Chiu, Helen Fung Kum; Wing, Yun Kowk; Lee, Dominic Tak Shing;
Publication:Clinical Chemistry
Date:Jan 1, 2000
Words:4701
Previous Article:Pitfalls in the diagnosis of patients with a partial dihydropyrimidine dehydrogenase deficiency.
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