SLC6 neurotransmitter transporters: structure, function, and regulation

The neurotransmitter transporters (NTTs) belonging to the solute carrier 6 (SLC6) gene family (also referred to as the neurotransmitter-sodium-symporter family or Na(+)/Cl(-)-dependent transporters) comprise a group of nine sodium- and chloride-dependent plasma membrane transporters for the monoamine neurotransmitters serotonin (5-hydroxytryptamine), dopamine, and norepinephrine, and the amino acid neurotransmitters GABA and glycine. The SLC6 NTTs are widely expressed in the mammalian brain and play an essential role in regulating neurotransmitter signaling and homeostasis by mediating uptake of released neurotransmitters from the extracellular space into neurons and glial cells. The transporters are targets for a wide range of therapeutic drugs used in treatment of psychiatric diseases, including major depression, anxiety disorders, attention deficit hyperactivity disorder and epilepsy. Furthermore, psychostimulants such as cocaine and amphetamines have the SLC6 NTTs as primary targets. Beginning with the determination of a high-resolution structure of a prokaryotic homolog of the mammalian SLC6 transporters in 2005, the understanding of the molecular structure, function, and pharmacology of these proteins has advanced rapidly. Furthermore, intensive efforts have been directed toward understanding the molecular and cellular mechanisms involved in regulation of the activity of this important class of transporters, leading to new methodological developments and important insights. This review provides an update of these advances and their implications for the current understanding of the SLC6 NTTs.     

Pharmacogenomics of CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4, CYP3A5 and MDR1 in Vietnam

Aim  The aim of this study was to obtain pharmacogenetic data in a Vietnamese population on genes coding for proteins involved in the elimination of drugs currently used for the treatment of malaria and human immunodeficiency virus/acquired immunodeficiency syndrome. Method  The main polymorphisms on the cytochrome P450 (CYP) genes, CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4 and CYP3A5, and the multi-drug resistance 1 gene (MDR1) were genotyped in 78 healthy Vietnamese subjects. Pharmacokinetic metrics were available for CYP2A6 (coumarin), CYP2C19 (mephenytoin), CYP2D6 (metoprolol) and CYP3As (midazolam), allowing correlations with the determined genotype. Results  In the CYP2 family, we detected alleles CYP2A6*4 (12%) and *5 (15%); CYP2B6*4 (8%), *6 (27%); CYP2C19*2 (31%) and *3 (6%); CYP2D6*4, *5, *10 (1, 8 and 44%, respectively). In the CYP3A family, CYP3A4*1B was detected at a low frequency (2%), whereas CYP3A5 *3 was detected at a frequency of 67%. The MDR1 3435T allele was present with a prevalence of 40%. Allele proportions in our cohort were compared with those reported for other Asian populations. CYP2C19 genotypes were associated to the S-4′-OH-mephenytoin/S-mephenytoin ratio quantified in plasma 4 h after intake of 100 mg mephenytoin. While CYP2D6 genotypes were partially reflected by the α-OH-metroprolol/metoprolol ratio in plasma 4 h after dosing, no correlation existed between midazolam plasma concentrations 4 h post-dose and CYP3A genotypes. Conclusions  The Vietnamese subjects of our study cohort presented allele prevalences in drug-metabolising enzymes that were generally comparable with those reported in other Asian populations. Deviations were found for CYP2A6*4 compared to a Chinese population (12 vs. 5%, respectively; P = 0.023), CYP2A6*5 compared with a Korean population (15 vs. <1%, respectively; P < 0.0001), a Malaysian population (1%; P < 0.0001) and a Chinese population (1%; P < 0.0001); CYP2B6*6 compared with a Korean population (27 vs. 12%; P = 0.002) and a Japanese population (16%; P = 0.021). Pharmacokinetic metrics versus genotype analysis reinforces the view that the predictive value of certain globally common variants (e.g. CYP2D6 single nucleotide polymorphisms) should be evaluated in a population-specific manner.     

Pharmacogenomics of CYP2D6: molecular genetics, interethnic differences and clinical importance

CYP2D6 has received intense attention since the beginning of the pharmacogenetic era in the 1970s. This is because of its involvement in the metabolism of more than 25% of the marketed drugs, the large geographical and inter-ethnic differences in the genetic polymorphism and possible drug-induced toxicity. Many interesting reviews have been published on CYP2D6 and this review aims to reinstate the importance of the genetic polymorphism of CYP2D6 in different populations as well as some clinical implications and important drug interactions.     

Occludin: structure, function and regulation

Epithelial and/or endothelial barriers play a critical role in animal, including human, life forms. The tight junction (TJ) is an essential component of these barriers. Occludin is a major component of the TJ. The structure of occludin, including its gene splice variants and protein essential components have been elucidated. Phosphorylation/dephosphorylation plays a major role in regulation of occludin and TJ. Disruption of occludin regulation is an important aspect of a number of diseases. Strategies to prevent and/or reverse occludin downregulation may be an important therapeutic target.     

CYP2A13-catalysed coumarin metabolism: comparison with CYP2A5 and CYP2A6

1. We investigated the total metabolism of coumarin by baculovirus (BV)-expressed CYP2A13 and compared it with metabolism by BV-expressed CYP2A6. The major coumarin metabolite formed by CYP2A13 was 7-hydroxycoumarin, which accounted for 43% of the total metabolism. The product of 3,4-epoxidation, o-hydroxyphenylacetaldehyde (o-HPA), accounted for 30% of the total metabolites. 2. The K(m) and V(max) for CYP2A13-mediated coumarin 7-hydroxylation were 0.48+/-0.07 micro m and 0.15+/-0.006 nmol min(-1) nmol(-1) CYP, respectively. The V(max) of coumarin 7-hydroxylation by CYP2A13 was about 16-fold lower than that of CYP2A6, whereas the K(m) was 10-fold lower. 3. In the mouse, there were two orthologues for CYP2A6: CYP2A4 and CYP2A5, which differed by only 11 amino acids. However, CYP2A5 is an efficient coumarin 7-hydroxylase, where as CYP2A4 is not. We report here that BV-expressed CYP2A4 metabolizes coumarin by 3,4-epoxidation. Two products of the 3,4-epoxidation pathway, o-HPA and o-hydroxyphenylacetic acid (o-HPAA), were detected by radioflow HPLC. 4. The K(m) and V(max) for the coumarin 3,4-epoxidation by CYP2A4 were 8.7+/-3.6 micro m and 0.20+/-0.04 nmol min(-1) nmol(-1) CYP, respectively. Coumarin 7-hydroxylation by CYP2A5 was more than 200 times more efficient than 3,4 epoxidation by CYP2A4.     

The omega-class glutathione transferases: structure, function, and genetics

The omega class of glutathione transferases (GSTs) is a relatively ancient member of the cytosolic GST superfamily, and the omega-class GSTs are found in plants, animals, and some microbial species. The omega-class GSTs exhibit the canonical GST fold, but, unlike other GSTs, the omega-class GSTs have a cysteine residue in their active site. Consequently, the omega-class GSTs catalyze a range of thiol transferase and reduction reactions that are not catalyzed by members of the other classes. Human GSTO1-1 can catalyze the reduction of monomethylarsonic acid (V), but this does not appear to be physiologically important in cases of high environmental arsenic exposure. GSTO1-1 also plays an important role in the biotransformation of reactive α-haloketones to nontoxic acetophenones. Genetic variation is common in the omega-class GST genes, and variants that result in deficiency of GSTO1-1 have been characterized. Genetic linkage studies have discovered associations between GSTO genes and the age at onset of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The mechanism underlying this association with neurological disease may derive from the capacity of omega-class GSTs to mitigate oxidative stress or their role in activating the proinflammatory cytokine, interleukin-1β.     

Effect of eurycomanone on cytochrome P450 isoforms CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 in vitro

Eurycomanone, an active constituent isolated from Eurycoma longifolia Jack, was examined for modulatory effects on cytochrome P450 (CYP) isoforms CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 using in vitro assays. The IC₅₀ value was determined to assess the potencies of modulation for each CYP isoform. Our results indicated that eurycomanone did not potently inhibit any of the CYP isoforms investigated, with IC₅₀ values greater than 250 μg/ml. Hence there appears to be little likelihood of drug–herb interaction between eurycomanone or herbal products with high content of this compound and CYP drug substrates via CYP inhibition.     

Beta-adrenergic receptors: structure, function and regulation

The diverse effects of the catecholamines (CA) epinephine and norepinephrine are mediated by a family of specific receptors (adrenergic receptors, AR). The beta-AR is a glycoprotein present in the membrane of a number of cell types. This receptor is closely associated with at least two other proteins (Gs and adenylate cyclase enzyme, AC) to form the beta-AR complex. The beta-AR recognizes the CA and is coupled to Gs which stimulates the effector enzyme AC. This enzyme converts ATP to cAMP and is the effector of the beta-AR complex. Thus the beta-AR is a G-coupled receptor which acts by raising intracellular levels of cAMP. The beta-AR is an important site of regulatory modifications through a variety of mechanisms. The best characterized is known as homologous desensitization: when the receptor is exposed to repeated stimulus by the agonist (CA), its responsiveness wanes, probably to compensate this potentially dangerous overstimulation. The gene for mammalian beta2-AR has been recently cloned and the predicted amino-acid sequence now opens the field to identification of the protein structures involved in receptor functions. The beta2-AR protein is characterized by the presence of seven membrane spanning regions. Study of the structure, function and regulation of the beta-AR will extend our knowledge of the role of beta-AR in pathological conditions and suggest new therapeutic approaches.     

A silent mutation (2939G>A, exon 6; CYP2D6*59) leading to impaired expression and function of CYP2D6

We analyzed CYP2D6 in two individuals characterized by impaired sparteine oxidation (intermediate metabolizer phenotype) and genotype 2D62/4 (1661G>C; 2850C>T; 4180G>C) usually associated with normal function. Full genomic sequencing and haplotype analysis confirmed the previously identified silent mutation 2939G>A in exon 6 (former allele variant 2D62J, now termed 2D659), as well as an additional novel 2291G>A change in intron 4. Transient expression in Huh7 hepatoma cells of the entire CYP2D6 gene of constructs carrying either both or only the 2939G>A change resulted in about three-fold reduced levels of mRNA, immunoreactive 2D6 protein and propafenone hydroxylase activity. These data demonstrate profound effects of a silent mutation on expression and function of CYP2D6, resulting in impaired drug oxidation phenotype. The 2939G>A single nucleotide polymorphism in exon 6 was present heterozygously in two individuals out of 308 (0.65%), corresponding to an allele frequency of 0.3%. Genotyping for this mutation thus improves phenotype-genotype correlation for CYP2D6 and may help to predict adverse drug treatment events.     

A novel CYP2A6 allele, CYP2A6*23, impairs enzyme function in vitro and in vivo and decreases smoking in a population of Black-African descent

OBJECTIVES: CYP2A6 is the main enzyme involved in nicotine metabolism in humans. We have identified a novel allele, CYP2A6*23 (2161C>T, R203C), in individuals of Black-African descent and investigated its impact on enzyme activity and association with smoking status. METHODS: Wild-type and variant enzymes containing amino acid changes R203C (CYP2A6*23), R203S (CYP2A6*16) and V365M (CYP2A6*17) were expressed in Escherichia coli. The effect of CYP2A6*23 in vivo was examined in individuals of Black-African descent given 4 mg oral nicotine. RESULTS: CYP2A6*23 occurred at an allele frequency of 2.0% in individuals of Black-African descent (N=560 alleles, 95% confidence interval, 0.8-3.1%) and was not detected in Caucasians (N=334 alleles), Chinese (N=288 alleles) or Japanese (N=104 alleles). In vitro, CYP2A6.23 had greatly reduced activity toward nicotine C-oxidation similar to CYP2A6.17, as well as reduced coumarin 7-hydroxylation. Conversely, CYP2A6.16 did not differ in activity compared with the wild-type enzyme. The trans-3'-hydroxycotinine to cotinine ratio, a phenotypic measure of CYP2A6 activity in vivo, was lower in CYP2A6*1/*23 and CYP2A6*23/*23 individuals (mean adjusted ratio of 0.60, n=5) compared with CYP2A6*1/*1 individuals (mean adjusted ratio of 1.21, n=150) (P<0.04). CYP2A6*23 trended toward a higher allele frequency in nonsmokers (3.1%, N=9/286 alleles) compared with smokers (0.7%, N=2/274 alleles) (P=0.06). CONCLUSION: These results suggest the novel CYP2A6*23 allele impairs enzyme function in vitro and in vivo and trends toward an association with lower risk of smoking.     

Ethnic variation in CYP2A6*7, CYP2A6*8 and CYP2A6*10 as assessed with a novel haplotyping method

Cytochrome P450 2A6 is the main human nicotine metabolizing enzyme coded for by a highly polymorphic gene, CYP2A6. CYP2A6*7, CYP2A6*8 and CYP2A6*10 are variant alleles common to Asian ethnicities. The CYP2A6*7 and CYP2A6*8 alleles each contain a non-synonymous single nucleotide polymorphism (SNP) 6558T>C and 6600G>T, respectively, whereas the CYP2A6*10 haplotype allele contains both. We have developed the first haplotyping assay; it can unambiguously distinguish the CYP2A6*7, CYP2A6*8 and CYP2A6*10 alleles. The allele frequencies of these three variants were assessed using the novel haplotyping assay in Chinese-Canadian (n=112), Chinese-American (n=221), Taiwanese (n=319), Korean-American (n=207) and Japanese-Canadian (n=64) populations, as well as in Caucasian (n=110) and African-Canadian (n=113) populations. Our new method demonstrated higher frequencies of CYP2A6*7 and CYP2A6*10, and a lower frequency of CYP2A6*8 in Asian populations, but no significant change of allele frequencies in Caucasian or African-Canadian populations.     

Gene-gene interactions between CYP2B6 and CYP2A6 in nicotine metabolism

OBJECTIVES: CYP2A6 is the major enzyme involved in nicotine metabolism, yet large interindividual variations in the rate of nicotine metabolism exist within groups of individuals having the same CYP2A6 genotype. We investigated the influence of genetic variation in another potential nicotine-metabolizing enzyme, CYP2B6, and its interaction with CYP2A6, on the metabolism of nicotine. METHODS: Two hundred and twelve healthy Caucasian adult twin volunteers underwent an intravenous infusion of stable isotope-labeled nicotine and its major metabolite, cotinine, for characterization of pharmacokinetic and metabolism phenotypes. Five CYP2B6 genetic polymorphisms causing amino acid substitutions (R22C, Q172 H, S259R, K262R, and R487C) were analyzed. RESULTS: We observed that the CYP2B6*6 haplotype (defined as having both Q172 H and K262R variants) was associated with faster nicotine and cotinine clearance, and that such associations were more prominent among individuals having decreased-activity CYP2A6 genotypes. Statistically significant interactions between CYP2B6 and CYP2A6 genotypes were observed (P<0.003 for nicotine clearance and P<0.002 for cotinine clearance). CONCLUSIONS: Our results indicate that CYP2B6 genetic variation is associated with the metabolism of nicotine and cotinine among individuals with decreased CYP2A6 activity. Further investigation of the roles of CYP2B6 and the interaction between CYP2B6 and CYP2A6 genotypes in mediating nicotine dependence and tobacco-related diseases is merited.     

Growth hormone regulation and developmental expression of rat hepatic CYP3A18, CYP3A9, and CYP3A2

The present study investigated the role of growth hormone (GH) in hepatic CYP3A18 and CYP3A9 expression in prepubertal and adult male rats. For comparison, the effects of GH on CYP3A2 expression were also measured. Initial experiments demonstrated that CYP3A18 mRNA levels were greater during puberty and adulthood than during the prepubertal period, CYP3A9 mRNA was not expressed until puberty and its expression increased in adulthood, and CYP3A2 mRNA levels were relatively constant from prepuberty to adult life. Hypophysectomy, which results in the loss of multiple pituitary factors including GH, increased CYP3A2 and CYP3A18 mRNA expression 3- to 4-fold, but it did not affect CYP3A9 mRNA levels or CYP3A-mediated testosterone 2beta- or 6beta-hydroxylase activity in adult rats. GH administered as twice daily s.c. injections (0.12 microg/g body weight) to hypophysectomized or intact adult rats did not affect CYP3A18 or CYP3A9 mRNA expression. The same treatment decreased CYP3A2 mRNA and protein and testosterone 2beta- and 6beta-hydroxylase activity levels in intact but not hypophysectomized rats. However, in intact prepubertal rats, intermittent GH administration decreased CYP3A18 and CYP3A2 mRNA levels, but a higher dosage (3.6 microg/g) was required to suppress CYP3A2. Overall, the present study demonstrated that: (a) the constitutive expression of CYP3A18, CYP3A9, and CYP3A2 does not require the presence of GH, (b) CYP3A18 is more sensitive than CYP3A9 to GH modulation in adult rats; and (c) CYP3A2 is less sensitive to the suppressive influence of GH during the prepubertal period than during adult life.     

Dose-dependent inhibition of CYP1A2, CYP2C19 and CYP2D6 by citalopram,fluoxetine, fluvoxamine and paroxetine

Objectives: The purpose of this pharmacokinetic study was to investigate the dose-dependent inhibition of model substrates for CYP2D6, CYP2C19 and CYP1A2 by four marketed selective serotonin reuptake inhibitors (SSRIs): citalopram, fluoxetine, fluvoxamine and paroxetine. Methods: The study was carried out as an in vivo single-dose study including 24 young, healthy men. All volunteers had been identified as sparteine- and mephenytoin-extensive metabolisers. The volunteers received in randomised order, at weekly intervals, increasing single oral doses of one of the four SSRIs, followed 3 h later by sparteine (CYP2D6), mephenytoin (CYP2C19) and caffeine (CYP1A2) tests. Fluoxetine was given at 3-week intervals because of the long half-life of fluoxetine and its metabolite norfluoxetine. Citalopram, fluoxetine and paroxetine were given in doses of 10, 20, 40 and 80 mg and fluvoxamine was given in doses of 25, 50, 100 and 200 mg. Results: With increasing doses, there was a statistically significant increase in the sparteine metabolic ratio (MR) (P < 0.01, Page’s test for trend) for all four SSRIs. The increase was modest after intake of citalopram and fluvoxamine, while the increase was more pronounced after fluoxetine intake, although no volunteers changed phenotype from extensive metabolisers to poor metabolisers. Three of the six volunteers changed phenotype from extensive metabolisers to poor metabolisers after intake of 40 or 80 mg paroxetine. There was a statistically significant increase in the mephenytoin S/R ratio (P < 0.01, Page’s test for trend) with increasing doses of fluoxetine and fluvoxamine, but not after citalopram and paroxetine. However, no volunteers changed phenotype from extensive to poor metabolisers of S-mephenytoin. After intake of fluvoxamine, the urinary excretion of the metabolites related to N3 demethylation of caffeine were below the limit of quantification, whereas there were no significant changes in the urinary caffeine metabolic ratios after intake of the other three SSRIs. Conclusion: This investigation confirms that paroxetine and fluoxetine are potent inhibitors of CYP2D6, that fluvoxamine and fluoxetine are moderate inhibitors of CYP2C19 and that fluvoxamine is a potent inhibitor of CYP1A2 in humans in vivo. The clinical prediction of interaction from single-dose experiments may have to take the degree of accumulation during steady-state after multiple doses into account. Received: 11 December 1995/Accepted in revised form: 29 February 1996     

Molecular modelling of CYP2B6, the human CYP2B isoform, by homology with the substrate-bound CYP102 crystal structure: evaluation of CYP2B6 substrate characteristics, the cytochrome b5 binding site and comparisons with CYP2B1 and CYP2B4.

1. Molecular modelling studies of CYP2B isoforms from rat (CYP2B1), rabbit (CYP2B4) and man (CYP2B6) are reported, with particular emphasis on substrate interactions with the human CYP2B isoform, CYP2B6. 2. The findings represent an advance on our previous study that focused primarily on the rat CYP2B isoform, CYP2B1, and involved homology modelling with substrate-free CYP102. 3. The current work utilizes the recently published substrate-bound CYP102 crystal structure as a template for construction of the CYP2B subfamily isoforms and shows, in particular, that known CYP2B6 substrate specificity and regioselectivity can be rationalized by putative active site interactions.     

Multiple Human Cytochromes Contribute to Biotransformation of Dextromethorphan In-vitro: Role of CYP2C9, CYP2C19, CYP2D6, and CYP3A

Cytochromes mediating the biotransformation of dextromethorphan to dextrorphan and 3-methoxymorphinan, its principal metabolites in man, have been studied by use of liver microsomes and microsomes containing individual cytochromes expressed by cDNA-transfected human lymphoblastoid cells. In-vitro formation of dextrorphan from dextromethorphan by liver microsomes was mediated principally by a high-affinity enzyme (K_m (substrate concentration producing maximum reaction velocity) 3–13 μM). Formation of dextrorphan from 25 μM dextromethorphan was strongly inhibited by quinidine (IC50 (concentration resulting in 50% inhibition) = 0.37 μm); inhibition by sulphaphenazole was approximately 18% and omeprazole and ketoconazole had minimal effect. Dextrorphan was formed from dextromethorphan by microsomes from cDNA-transfected lymphoblastoid cells expressing CYP2C9, ?2C19, and ?2D6 but not by those expressing CYP1A2, ?2E1 or ?3A4. Despite the low in-vivo abundance of CYP2D6, this cytochrome was identified as the dominant enzyme mediating dextrorphan formation at substrate concentrations below 10 μM. Formation of 3-methoxy-morphinan from dextromethorphan in liver microsomes proceeded with a mean K_m of 259 μM. For formation of 3-methoxymorphinan from 25 μM dextromethorphan the IC50 for ketoconazole was 1.15 μM; sulphaphenazole, omeprazole and quinidine had little effect. 3-Methoxymorphinan was formed by microsomes from cDNA-transfected lymphoblastoid cells expressing CYP2C9, ?2C19, ?2D6, and ?3A4, but not by those expressing CYP1A2 or ?2E1. CYP2C19 had the highest affinity (K_m = 49 μM) whereas CYP3A4 had the lowest (K_m = 1155 μM). Relative abundances of the four cytochromes were determined in liver microsomes by use of the relative activity factor approach. After adjustment for relative abundance, CYP3A4 was identified as the dominant enzyme mediating 3-methoxymorphinan formation from dextromethorphan, although CYP2C9 and ?2C19 were estimated to contribute to 3-methoxymorphinan formation, particularly at low substrate concentrations. Although formation of dextrorphan from dextromethorphan appears to be sufficiently specific to be used as an in-vitro or in-vivo index reaction for profiling of CYP2D6 activity, the findings raise questions about the specificity of 3-methoxymorphinan formation as an index of CYP3A activity.     

Molecular genetics of CYP2D6: clinical relevance with focus on psychotropic drugs

Cytochrome P450 CYP2D6 is the most extensively characterized polymorphic drug-metabolizing enzyme. A deficiency of the CYP2D6 enzyme is inherited as an autosomal recessive trait; these subjects (7% of Caucasians, about 1% of Orientals) are classified as poor metabolizers. Among the rest (extensive metabolizers), enzyme activity is highly variable, from extremely high in ultrarapid metabolizers, to markedly reduced in intermediate metabolizers. The CYP2D6 gene is highly polymorphic, with more than 70 allelic variants described so far. Of these, more than 15 encode an inactive or no enzyme at all. Others encode enzyme with reduced, "normal" or increased enzyme activity. The CYP2D6 gene shows marked interethnic variability, with interpopulation differences in allele frequency and existence of "population-specific" allelic variants, for instance among Orientals and Black Africans. The CYP2D6 enzyme catalyses the metabolism of a large number of clinically important drugs including antidepressants, neuroleptics, some antiarrhythmics, lipophilic beta-adrenoceptor blockers and opioids. The present-day knowledge on the influence of the genetic variability in CYP2D6 on the clinical pharmacokinetics and therapeutic effects/adverse effects of psychotropic drugs is reviewed.     

Characterization of a genotype previously designated as CYP2A6 D-type: CYP2A6*4B,another entire gene deletion allele of the CYP2A6 gene in Japanese

CYP2A6 is known as an enzyme responsible for the metabolism of several clincally used drugs such as tegafur. Previously, we found two novel genotypes of the CYP2A6 gene, D-type and E-type, and the E-type was clarified to be homozygous for the CYP2A6*4A allele. On the other hand, since the D-type was reported to lack regions from at least intron 5 to a part of exon 9 of the CYP2A6 gene, it caused a misunderstanding that the D-type would be a partial CYP2A6 gene-deleted allele. In this paper, we demonstrate that the D-type is a genotype heterozygous for the CYP2A6*4A and another novel entire CYP2A6 gene-deleted allele, CYP2A6*4B, by analyzing a Japanese family including parents genotyped as the CYP2A6*4A/4A and CYP2A6*1A/*4B, respectively.