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British Journal of Clinical Pharmacology Sep 1998The aim of this study was to investigate whether chloroquine can inhibit drug metabolism in humans, if such inhibition is general or selective for certain enzymes and...
AIMS
The aim of this study was to investigate whether chloroquine can inhibit drug metabolism in humans, if such inhibition is general or selective for certain enzymes and evaluate the potential for and clinical significance of any drug-drug interactions when chloroquine is co-administered with other drugs.
METHODS
The study was conducted in fourteen normal non-smoking healthy male volunteers using a cocktail of five drugs consisting of caffeine, mephenytoin, debrisoquine, chlorzoxazone and dapsone to assess activities of cytochromes P450 (CYP) 1A2, 2C19, 2D6, 2E1 and 3A4 respectively. Dapsone was also used to assess N-acetyltransferase activity. The activities were assessed at baseline, after one and seven daily doses (250 mg daily) of chloroquine and 7 and 14 days after stopping chloroquine dosing.
RESULTS
Chloroquine caused a progressive and significant decrease in CYP2D6 activity as measured by debrisoquine metabolism from first to seventh dose and the activity returned to baseline gradually over 14 days after stopping administration. There was no effect on the metabolism of any of the other probe drugs.
CONCLUSIONS
Chloroquine has been shown to be capable of inhibiting the activity of CYP2D6 in vivo in humans. This effect is selective as activities of other enzymes investigated were not affected. The effect was modest but suggests a potential for drug-drug interactions when co-administered with other drugs that are substrates for this enzyme. The clinical significance of such an interaction will depend on the therapeutic index of any drug involved.
Topics: Adult; Analysis of Variance; Antimalarials; Caffeine; Chloroquine; Chlorzoxazone; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dapsone; Debrisoquin; Drug Administration Schedule; Drug Interactions; Humans; Isoenzymes; Male; Phenotype
PubMed: 9764961
DOI: 10.1046/j.1365-2125.1998.00765.x -
British Journal of Clinical Pharmacology Mar 1998To determine the effect of therapeutic loading doses of halofantrine and chloroquine on CYP2D6 activity in healthy black Zambians. (Clinical Trial)
Clinical Trial
AIMS
To determine the effect of therapeutic loading doses of halofantrine and chloroquine on CYP2D6 activity in healthy black Zambians.
METHODS
Twenty healthy black male Zambians were phenotyped for CYP2D6 activity by measuring the debrisoquine/4-hydroxydebrisoquine ratio in a 0-8 h urine sample after a 10 mg oral dose of debrisoquine hemi-sulphate. The subjects (all 'extensive metabolizer' phenotype with respect to CYP2D6) were randomized into two groups of 10, and 24 h later one group received 1500 mg halofantrine hydrochloride and the other group 1500 mg chloroquine phosphate both orally in divided doses. All subjects were given further 10 mg doses of debrisoquine at 2 h, 1 week and 2 weeks after the last dose of the antimalarial drug, and phenotyped as described above.
RESULTS
The median debrisoquine/4-hydroxydebrisoquine 0-8 h urinary ratio was increased by halofantrine (1.39 to 6.05; P<0.01; 95% confidence intervals 4.00-11.7) and chloroquine (1.96 to 3.91; P<0.01; 95% confidence intervals 1.34-2.66) when debrisoquine was given 2 h after treatment. The decrease in CYP2D6 activity remained statistically significant for 1 week after both drugs. Halofantrine was a significantly more potent inhibitor of CYP2D6 than chloroquine (P=0.037). Phenocopying occurred in two subjects taking halofantrine and one taking chloroquine (i.e. the debrisoquine/4-hydroxydebrisoquine ratios became consistent with the poor metabolizer phenotype).
CONCLUSIONS
Given in therapeutic loading doses, both halofantrine and chloroquine caused significant inhibition of CYP2D6 activity in healthy black Zambians. With respect to halofantrine, this finding reinforces the recommendation that its combination with other drugs known to prolong the QT interval should be avoided, especially those that are metabolized significantly by CYP2D6.
Topics: Administration, Oral; Adult; Antimalarials; Black People; Chloroquine; Chromatography, Gas; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP2D6 Inhibitors; Debrisoquin; Humans; Liver; Male; Phenanthrenes; Zambia
PubMed: 10896408
DOI: 10.1046/j.1365-2125.1998.00671.x -
The Journal of Biological Chemistry Dec 1995Model building studies have intimated a role for aspartic acid 301 in the substrate binding of cytochrome P450 2D6 (CYP2D6). We have tested this hypothesis by generating...
Model building studies have intimated a role for aspartic acid 301 in the substrate binding of cytochrome P450 2D6 (CYP2D6). We have tested this hypothesis by generating a range of CYP2D6 mutants substituting a variety of amino acids at this site. The mutant proteins, which included substitution with a negatively charged glutamic acid residue or neutral asparagine, alanine, or glycine residues, were expressed in Saccharomyces cerevisiae. In addition, a mutant where aspartic acid 301 was deleted was also tested. All the mutants expressed approximately equivalent amounts of recombinant apoprotein and, apart from the alanine 301 and the aspartic acid 301 deletion mutants, gave carbon monoxide difference spectra of similar magnitude to the wild type. In the cases of the alanine and deletion mutants, the amount of holoprotein was significantly reduced or absent relative to the amount of apoprotein, indicating restricted heme incorporation. The glutamic acid mutant was shown to have similar catalytic properties to the wild type enzyme toward the substrates debrisoquine and metoprolol; however, some differences in regioselectivity and ligand binding were observed. The mutants containing neutral amino acids at position 301 exhibited marked reductions in catalytic activity. At low substrate concentrations little, if any, activity toward debrisoquine and metoprolol was measured. However, at a higher substrate concentration (2 mM) some activity was observed (about 10-20% of wild type levels). Consistent with the above findings, the debrisoquine-induced spin changes in the mutant proteins were markedly reduced. These data collectively demonstrate that aspartic acid 301 plays an important role in determining the substrate specificity and activity of CYP2D6 and provide experimental evidence supporting the role of this amino acid in forming an electrostatic interaction between the basic nitrogen atom in CYP2D6 substrates and the carboxylate group of aspartic acid 301.
Topics: Aspartic Acid; Binding Sites; Cytochrome P-450 CYP2D6; Cytochrome P-450 Enzyme System; Humans; Mixed Function Oxygenases; Oxidation-Reduction; Structure-Activity Relationship
PubMed: 7493924
DOI: 10.1074/jbc.270.49.29055 -
The Journal of Biological Chemistry Oct 2001Cytochrome P450 (P450) 2D6 was first identified as the polymorphic human debrisoquine hydroxylase and subsequently shown to catalyze the oxidation of a variety of drugs...
Cytochrome P450 (P450) 2D6 was first identified as the polymorphic human debrisoquine hydroxylase and subsequently shown to catalyze the oxidation of a variety of drugs containing a basic nitrogen. Differences in the regioselectivity of oxidation products formed in systems containing NADPH-P450 reductase/NADPH and the model oxidant cumene hydroperoxide have been proposed by others to be due to an allosteric influence of the reductase on P450 2D6 (Modi, S., Gilham, D. E., Sutcliffe, M. J., Lian, L.-Y., Primrose, W. U., Wolf, C. R., and Roberts, G. C. K. (1997) Biochemistry 36, 4461-4470). We examined the differences in the formation of oxidation products of N-methyl-4-phenyl-1,2,5,6-tetrahydropyridine, metoprolol, and bufuralol between reductase-, cumene hydroperoxide-, and iodosylbenzene-supported systems. Catalytic regioselectivity was not influenced by the presence of the reductase in any of the systems supported by model oxidants, ruling out allosteric influences. The presence of the reductase had little effect on the affinity of P450 2D6 for any of these three substrates. The addition of the reaction remnants of the model oxidants (cumyl alcohol and iodobenzene) to the reductase-supported system did not affect reaction patterns, arguing against steric influences of these products on catalytic regioselectivity. Label from H(2)18O was quantitatively incorporated into 1'-hydroxybufuralol in the iodosylbenzene- but not in the reductase- or cumene hydroperoxide-supported reactions. We conclude that the P450 systems utilizing NADPH-P450 reductase, cumene hydroperoxide, and iodosylbenzene use similar but distinct chemical mechanisms. These differences are the basis for the variable product distributions, not an allosteric influence of the reductase.
Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Allosteric Regulation; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Dopamine Agents; Ethanolamines; Metoprolol; Mixed Function Oxygenases; Models, Chemical; Molecular Conformation; NADPH-Ferrihemoprotein Reductase; Oxidation-Reduction
PubMed: 11509577
DOI: 10.1074/jbc.M106841200 -
British Journal of Clinical Pharmacology Nov 2001The use of multiple probe substrates to evaluate the activity of drug metabolizing enzymes requires that there are no inter-substrate interactions. As part of a series... (Clinical Trial)
Clinical Trial Randomized Controlled Trial
AIMS
The use of multiple probe substrates to evaluate the activity of drug metabolizing enzymes requires that there are no inter-substrate interactions. As part of a series of studies to develop a clinically useful collection of probe substrates that could be given alone or in any combination, we observed an interaction between midazolam (MDZ) and another component of the six-drug cocktail. Published data indicated that the interacting component was likely to be chlorzoxazone. This was investigated as part of a second study. The data relating to the interaction from both studies are reported here.
METHODS
Both studies were performed in 16 healthy subjects. All treatments were given orally after an overnight fast. In study 1, which was performed to a four-period, open, crossover design, subjects received on separate occasions MDZ 5 mg, diclofenac 25 mg, a four drug cocktail (caffeine 100 mg, mephenytoin 100 mg, debrisoquine 10 mg and chlorzoxazone 250 mg) and a six drug cocktail (caffeine 100 mg, mephenytoin 100 mg, debrisoquine 10 mg, chlorzoxazone 250 mg, diclofenac 25 mg and MDZ 5 mg). In study 2, which was performed to a two-period, open, crossover design, subjects received a five drug cocktail (as the six drug cocktail in the first study, but without chlorzoxazone and with diclofenac dose increased to 50 mg) and a six drug cocktail (as five drug cocktail, with chlorzoxazone 250 mg). In both studies, blood samples were taken for measurement of plasma MDZ and 1-hydroxy MDZ (1-OH MDZ) concentrations. In study 1, blood samples were taken up to 12 h post-dose while in study 2 a single sample was taken 2 h after dosing. In study 1, the potential interaction between MDZ and the other components of the six drug cocktail was assessed by comparing AUClast ratios (1-OH MDZ/MDZ) between the two treatments. Additionally, a single sampling timepoint of 2 h post-dose for determination of concentration, rather than AUC, ratios was established. The 2 h plasma concentration ratios from studies 1 and 2 were combined and a pooled analysis performed to compare ratios within each study (to determine the change in ratio when MDZ was dosed with and without chlorzoxazone) and between studies (to determine the consistency of the ratios when MDZ was given either as part of the two six drug cocktails or when given alone and as part of the five drug cocktail).
RESULTS
In study 1, both the AUClast ratio and the 2 h post-dose plasma concentration ratio were reduced when MDZ was given as part of the six drug cocktail in comparison with those for MDZ alone. This was the result of an increase in MDZ, rather than decrease in 1-OH MDZ, concentrations and was considered to result from a reduction in first pass metabolism of MDZ. The geometric mean AUClast values (with 95% CI) for MDZ were 95.6 (79.0, 115.7) and 160.4 (133.6, 192.6) microg l(-1) h when given alone and as part of the six drug cocktail, respectively. The corresponding values for 1-OH MDZ were 789.6 (697.6, 893.6) and 791.4 (701.7, 892.6) microg l(-1) h. The ratio of adjusted geometric mean AUClast ratios for the two treatments was 1.82 (90% CI 1.48, 2.23, P < 0.001). The pooled plasma 1-OH MDZ/MDZ ratio data from both studies showed that the differences in MDZ metabolism observed in study 1 were replicated in study 2. The adjusted geometric mean 1-OH MDZ/MDZ ratios when MDZ was given alone and as part of the six drug cocktail were 7.79 and 4.59, respectively, for study 1 (ratio 1.70, 95% CI 1.36, 2.11, P < 0.001) and 7.64 and 4.60 for study 2 (ratio 1.66, 95% CI 1.34, 2.06, P < 0.001). These data indicate that when given orally chlorzoxazone interacts with MDZ, increasing plasma MDZ concentrations. In contrast, there was no difference between the plasma 1-OH MDZ/MDZ ratios when MDZ was given alone and as part of the five drug cocktail indicating that there were no interactions between MDZ and any of the other components of that cocktail.
CONCLUSIONS
Chlorzoxazone appears to significantly influence the pharmacokinetics of oral MDZ, probably through inhibition of first pass metabolism by CYP3A in the GI tract. Data from these studies and literature evidence showing a further interaction between chlorzoxazone and CYP1A2 substrates and questions concerning the specificity of chlorzoxazone as a probe substrate for CYP2E1, indicate that the use of chlorzoxazone in multisubstrate probe cocktails should be avoided.
Topics: Administration, Oral; Adult; Area Under Curve; Aryl Hydrocarbon Hydroxylases; Caffeine; Chlorzoxazone; Cross-Over Studies; Cytochrome P-450 CYP2E1; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Debrisoquin; Diclofenac; Drug Interactions; Female; Humans; Male; Mephenytoin; Midazolam; Muscle Relaxants, Central; Oxidoreductases, N-Demethylating
PubMed: 11736864
DOI: 10.1046/j.0306-5251.2001.01479.x -
Drugs & Aging 2005Elderly patients are more likely to ingest prescription medications concurrently with botanical supplements, and may therefore be vulnerable to herb-drug interactions.... (Comparative Study)
Comparative Study Randomized Controlled Trial
OBJECTIVES
Elderly patients are more likely to ingest prescription medications concurrently with botanical supplements, and may therefore be vulnerable to herb-drug interactions. Phytochemical-mediated modulation of cytochrome P450 (CYP) activity may underlie many herb-drug interactions. Some evidence suggests that CYP activity may decrease in the elderly. If so, herb-mediated changes in CYP activity may take on greater clinical relevance in this population. In this study, single timepoint, phenotypic metabolic ratios were used to determine whether long-term supplementation of St John's wort, garlic oil, Panax ginseng, and Ginkgo biloba affected CYP1A2, CYP2D6, CYP2E1 or CYP3A4 activity in elderly subjects.
METHODS
Twelve healthy volunteers between the ages of 60 and 76 years (mean age 67 years) were randomly assigned to receive each botanical supplement for 28 days followed by a 30-day washout period. Probe drug cocktails of midazolam, caffeine, chlorzoxazone and debrisoquine were administered before and at the end of supplementation. Pre- and post-supplementation phenotypic ratios were determined for CYP3A4, CYP1A2, CYP2E1 and CYP2D6 using 1-hydroxymidazolam/midazolam serum ratios (1-hour), paraxanthine/caffeine serum ratios (6-hour), 6-hydroxychlorzoxazone/chlorzoxazone serum ratios (2-hour) and debrisoquine urinary recovery ratios (8-hour), respectively. The content of purported 'active' phytochemicals was determined for each supplement.
RESULTS
Comparisons of pre- and post-St John's wort phenotypic ratios revealed significant induction of CYP3A4 (approximately 140%) and CYP2E1 activity (approximately 28%). Garlic oil inhibited CYP2E1 activity by approximately 22%. P. ginseng inhibition of CYP2D6 was statistically significant, but the magnitude of the effect (approximately 7%) did not appear to be clinically relevant. None of the supplements tested in this study appeared to affect CYP1A2 activity.
CONCLUSIONS
Elderly subjects, like their younger counterparts, are susceptible to herb-mediated changes in CYP activity, especially those involving St John's wort. Pharmacokinetic herb-drug interactions stemming from alterations in CYP activity may adversely affect drug efficacy and/or toxicity. When compared with earlier studies that employed young subjects, the data suggest that some age-related changes in CYP responsivity to botanical supplementation may exist. Concomitant ingestion of botanical supplements with prescription medications, therefore, should be strongly discouraged in the elderly.
Topics: Administration, Oral; Aged; Allyl Compounds; Caffeine; Chlorzoxazone; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dietary Supplements; Drug Administration Schedule; Female; Ginkgo biloba; Herb-Drug Interactions; Humans; Hypericum; Isoenzymes; Male; Midazolam; Panax; Phenotype; Plant Preparations; Sulfides
PubMed: 15974642
DOI: 10.2165/00002512-200522060-00006 -
British Journal of Clinical Pharmacology Jul 1986Liver oxidative metabolism, assessed by debrisoquine hydroxylation test, was studied in 107 healthy volunteers and in 71 patients with or without neuroleptic drug...
Liver oxidative metabolism, assessed by debrisoquine hydroxylation test, was studied in 107 healthy volunteers and in 71 patients with or without neuroleptic drug treatment. The mean metabolic ratio (MR = debrisoquine/4-hydroxydebrisoquine excretion in the urine) was 2.8 +/- 0.1 (s.e. mean) in the control group, six persons being poor metabolizers of debrisoquine (MR greater than or equal to 12.6). The mean MR (12.1 +/- 1.5) was significantly higher in those 42 patients taking neuroleptics than in patients without neuroleptics (0.8 +/- 0.1). In the former group, seventeen patients had a MR exceeding 12.6. Oral contraceptives, antiepileptics, benzodiazepines and progestin derivates did not increase MR values, the highest individual ratio being 2.72 in those subjects not receiving neuroleptics. These results suggest a probable competitive inhibition of oxidative metabolism by neuroleptics. This is a phenomenon of potential clinical importance both in patients with an inherited poor metabolic capacity and in patients receiving other drugs like beta-adrenoceptor blocking agents and tricyclic antidepressants oxidized by the same enzyme system.
Topics: Adolescent; Adult; Anticonvulsants; Antipsychotic Agents; Debrisoquin; Female; Humans; Isoquinolines; Liver; Male; Middle Aged; Oxidation-Reduction
PubMed: 2874826
DOI: No ID Found -
Drug Metabolism and Pharmacokinetics 2003The effects of the substitution of glycine at position 42 with various other amino acid residues on the functions of CYP2D6 were studied using debrisoquine (DB) and...
The effects of the substitution of glycine at position 42 with various other amino acid residues on the functions of CYP2D6 were studied using debrisoquine (DB) and bunitrolol (BTL) 4-hydroxylations as indices of drug-metabolizing enzymes. The substitution with hydrophobic amino acid residues such as valine and phenylalanine did not affect the enzymatic properties such as reduced CO-difference spectra, microsomal CYP contents and oxidation activities towards DB and BTL. The substitution of glycine-42 with a polar but noncharged amino acid residue (serine) exhibited a similar reduced CO-different spectrum, but the substitution with a charged basic (lysine and arginine) or acidic (glutamic acid) amino acid residue commonly produced a peak at 420 nm in addition to a Soret peak at 450 nm. Cytochrome P450 contents and microsomal contents of G42S, G42K, G42R and G42E estimated spectrophotometrically and estimated by Western blot analysis, respectively, were lower than those of the wild-type. Kinetic analysis revealed that the substitution of glycine-42 with charged amino acid residues such as lysine, arginine and glutamic acid markedly increased the apparent K(m) values for DB and BTL oxidations without remarkable changes in the V(max) values. The subsitution with noncharged amino acid residues such as serine, valine and phenylalanine did not cause such a marked change in the K(m) values. Efficiencies (V(max)/K(m)) as DB and BTL 4-hydroxylases of CYP2D6 mutant proteins having charged amino acid residues were found to be decreased mainly by increasing their K(m) values. These results indicate that the properties of amino acid residues at position 42 affect the behavior of CYP2D6 proteins such as anchoring into ER membranes, conversion of P450 to P420 and incorporation of heme into apoproteins.
PubMed: 15618721
DOI: 10.2133/dmpk.18.79 -
The Journal of Biological Chemistry Oct 1990The debrisoquine/sparteine-type polymorphism is a clinically important inherited variation of drug metabolism characterized by two phenotypes, the extensive metabolizer...
Multiple mutations of the human cytochrome P450IID6 gene (CYP2D6) in poor metabolizers of debrisoquine. Study of the functional significance of individual mutations by expression of chimeric genes.
The debrisoquine/sparteine-type polymorphism is a clinically important inherited variation of drug metabolism characterized by two phenotypes, the extensive metabolizer and the poor metabolizer (PM). Five to 10 percent of individuals in Caucasian populations are of the PM phenotype and have deficient metabolism of debrisoquine and over 25 other drugs. Our previous studies have revealed absence of cytochrome P450IID6 protein and aberrant splicing of IID6 premRNA in livers of PMs. Moreover, two mutant alleles of the P450IID6 gene locus (CYP2D6) were identified by restriction fragment length analysis to be associated with the PM phenotype. However, the mutations of the CYP2D6 gene causing absent P450IID6 protein have not been defined. Here we report the cloning and sequencing of two types of mutant alleles of CYP2D6 isolated from genomic libraries of three PM individuals. One allele (29-A) was characterized by a single nucleotide deletion in the 5th exon with consequent frameshift and was observed in one individual only. The other type of mutant allele (29-B) was present in all three PM individuals and its sequence contained multiple mutations, notably four base changes causing amino acid changes in exons 1, 2 and 9, and a point mutation at the consensus sequence of the splice site of the 3rd intron. To understand the significance of the individual mutations, chimeric genes were constructed between the wild-type IID6 gene and the mutant 29-B allele or site-specific mutations were introduced into the IID6-cDNA and these DNA constructs were transiently expressed in COS-1 cells. The mutations in exon 1 resulted in a functionally deficient IID6 protein and the mutation at the splice site in absent IID6 protein, whereas the mutations in exons 2 and 9 were of no consequence for IID6 function. Only the mutation at the splice site thus explains the absence of P450IID6 protein in livers of PM individuals and appears to be a common cause of polymorphic drug oxidation.
Topics: Alleles; Base Sequence; Chimera; Cloning, Molecular; Cytochrome P-450 Enzyme System; DNA; Debrisoquin; Exons; Gene Expression; Genes; Humans; Leukocytes; Molecular Sequence Data; Mutation; Oligonucleotide Probes; Phenotype; Polymorphism, Genetic
PubMed: 2211621
DOI: No ID Found -
British Journal of Clinical Pharmacology Dec 19961. The debrisoquine hydroxylase (CYP2D6) is polymorphically distributed. Not only are there differences in the proportions of extensive metabolisers to poor metabolisers...
1. The debrisoquine hydroxylase (CYP2D6) is polymorphically distributed. Not only are there differences in the proportions of extensive metabolisers to poor metabolisers in various ethnic groups, but there are also pronounced variations in the metabolic capacity among those classified as extensive metabolisers. 2. The mean debrisoquine metabolic ratio of Caucasian extensive metabolisers is lower than that for a number of African populations. In the present study, we have searched for novel CYP2D6 mutations to explain the diminished enzyme activity in African populations. 3. Three Zimbabwean Shona subjects with EM phenotypes (metabolic ratios for debrisoquine of 0.4, 1.5 and 10.5 respectively) were selected and the open reading frame of the CYP2D6 gene of each was sequenced. 4. The subject with metabolic ratio of 10.5 was found to be homozygous for an allele with a nucleotide exchange in exon 2, 1111C-->T causing a 107Thr-->Ile amino acid exchange in a conserved region of the enzyme. In addition, he was homozygous for the 2938C-->T and 4268G-->C mutations causing 296Arg-->Ser and 486Ser-->Thr amino acid substitution found in the CYP2D6*2 allele. 5. Seventy-six Zimbabwean Shona subjects were subsequently genotyped for the 1111C-->T mutation and for the intron 1 gene conversion present in the CYP2D6*2 gene. The 1111C-->T mutation was found at an allele frequency of 34% and was only present in alleles carrying the gene conversion in intron 1 indicative for the CYP2D6*2 gene. 6. This allele (CYP2D6*17), containing the 1111C-->T, 2938C-->T and 4268G-->C mutations, was found to be strongly associated with lower capacity for debrisoquine hydroxylation. We therefore postulate that the CYP2D6*17 allele might contribute to the molecular basis of the previously established diminished debrisoquine hydroxylase activity in African Bantu populations.
Topics: Black People; Cytochrome P-450 CYP2D6; DNA; Exons; Genotype; Humans; Mutation; Open Reading Frames; Phenotype; Polymerase Chain Reaction; Polymorphism, Genetic; Zimbabwe
PubMed: 8971426
DOI: 10.1046/j.1365-2125.1996.00489.x