L. DiAnne Bradford

CYP2D6 allele frequency in European Caucasians, Asians, Africans and their descendants

Over 40 cytochrome P450 (CYP) 2D6 allelic variants have been discovered thus far. The alleles may be classified on the basis of the level of activity for which they encode CYP2D6 enzymes, into functional, non-functional and reduced function groups. CYP2D6 allele frequency is known to vary amongst racial/ethnic groups. Generally, for European Caucasians and their descendants, the functional group of alleles are predominant, with a frequency of 71%. Non-functional alleles represent 26% of the variability, mainly CYP2D6*4. In Asians and their close descendants, functional alleles represent only ~ 50% of the frequency of CYP2D6 alleles. Asians and Pacific Islanders have a high frequency (median = 41%) of a reduced function allele, CYP2D6*10, contributing to the population shift to the right of metabolic rates indicating slower metabolism. Information concerning Amerindians from North (Canada), Central and South America indicate comparatively low frequencies of CYP2D6*10, perhaps a ”founders“effect. The freque...

Unique CYP2D6 activity distribution and genotype‐phenotype discordance in black Americans

Although CYP2D6 has been studied extensively in differentpopulation groups, relatively little is known for black Americans.

Cytochrome P4502D6 (CYP2D6) Gene Locus Heterogeneity: Characterization of Gene Duplication Events

Duplications and multiplications of active CYP2D6 genes can cause ultrarapid drug metabolism and lead to therapeutic failure. Multiple functional and non‐functional duplication alleles have been further characterized. Duplications were detected by long‐range polymerase chain reaction (PCR), PCR‐restriction fragment length polymorphism, and sequence analysis. A PCR fragment encompassing the entire duplicated gene was utilized for detailed characterization. Duplications occurred at 1.3, 5.75, and 2.0% in Caucasian, African American, and racially mixed populations, respectively (n=887 total). Of those 28, 47, and 17% were non‐functional CYP2D6*4 × N. Twelve unique duplication alleles were detected: *1 × N, *2 × N, *4 × N, *6 × N, *10 × N, *17 × N, *17 × N[spacer], *29 × N, *35 × N, *43 × N, *45 × N, and a novel non‐functional tandem arrangement of a chimeric 2D7/2D6 and *1 gene. All novel duplications except *35 × N were found in African Americans. Accurate identification of gene duplication events is essential to avoid false‐positive ultrarapid metabolism assignments and thus, overestimation of predicted activity and increased risk for unwanted adverse events.

CYP2D6*36 gene arrangements within the CYP2D6 locus: association of CYP2D6*36 with poor metabolizer status

Unexplained cases of CYP2D6 genotype/phenotype discordance continue to be discovered. In previous studies, several African Americans with a poor metabolizer phenotype carried the reduced function CYP2D6*10 allele in combination with a nonfunctional allele. We pursued the possibility that these alleles harbor either a known sequence variation (i.e., CYP2D6*36 carrying a gene conversion in exon 9 along the CYP2D6*10-defining 100C>T single-nucleotide polymorphism) or novel sequences variation(s). Discordant cases were evaluated by long-range polymerase chain reaction (PCR) to test for gene rearrangement events, and a 6.6-kilobase pair PCR product encompassing the CYP2D6 gene was cloned and entirely sequenced. Thereafter, allele frequencies were determined in different study populations comprising whites, African Americans, and Asians. Analyses covering the CYP2D7 to 2D6 gene region established that CYP2D6*36 did not only exist as a gene duplication (CYP2D6*36x2) or in tandem with *10 (CYP2D6*36+*10), as previously reported, but also by itself. This “single” CYP2D6*36 allele was found in nine African Americans and one Asian, but was absent in the whites tested. Ultimately, the presence of CYP2D6*36 resolved genotype/phenotype discordance in three cases. We also discovered an exon 9 conversion-positive CYP2D6*4 gene in a duplication arrangement (CYP2D6*4Nx2) and a CYP2D6*4 allele lacking 100C>T (CYP2D6*4M) in two white subjects. The discovery of an allele that carries only one CYP2D6*36 gene copy provides unequivocal evidence that both CYP2D6*36 and *36x2 are associated with a poor metabolizer phenotype. Given a combined frequency of between 0.5 and 3% in African Americans and Asians, genotyping for CYP2D6*36 should improve the accuracy of genotype-based phenotype prediction in these populations.

Identification and characterization of novel sequence variations in the cytochrome P4502D6 (CYP2D6) gene in African Americans.

Cytochrome P4502D6 (CYP2D6) genotyping reliably predicts poor metabolizer phenotype in Caucasians, but is less accurate in African Americans. To evaluate discordance we have observed in phenotype to genotype correlation studies, select African American subjects were chosen for complete resequencing of the CYP2D6 gene including 4.2 kb of the CYP2D7-2D6 intergenic region. Comparisons were made to a CYP2D6*1 reference sequence revealing novel SNPs in the upstream, coding and intervening sequences. These sequence variations, defining four functional alleles (CYP2D6*41B, *45A and B and *46), were characterized for their ability to influence splice site strength, transcription level or catalytic protein activity. Furthermore, their frequency was determined in a population of 251 African Americans. A −692TGTG deletion (CYP2D6*45B) did not significantly decrease gene expression, nor could any other upstream SNP explain a genotype-discordant case. CYP2D6*45 and *46 have a combined frequency of 4% and can be identified by a common SNP. Carriers are predicted to exhibit an extensive or intermediate CYP2D6 phenotype.Cytochrome P4502D6 (CYP2D6) genotyping reliably predicts poor metabolizer phenotype in Caucasians, but is less accurate in African Americans. To evaluate discordance we have observed in phenotype to genotype correlation studies, select African American subjects were chosen for complete resequencing of the CYP2D6 gene including 4.2 kb of the CYP2D7-2D6 intergenic region. Comparisons were made to a CYP2D6*1 reference sequence revealing novel SNPs in the upstream, coding and intervening sequences. These sequence variations, defining four functional alleles (CYP2D6*41B, *45A and B and *46), were characterized for their ability to influence splice site strength, transcription level or catalytic protein activity. Furthermore, their frequency was determined in a population of 251 African Americans. A −692TGTG deletion (CYP2D6*45B) did not significantly decrease gene expression, nor could any other upstream SNP explain a genotype-discordant case. CYP2D6*45 and *46 have a combined frequency of 4% and can be identified by a common SNP. Carriers are predicted to exhibit an extensive or intermediate CYP2D6 phenotype.

CYP2D6 Poor Metabolizer Status Can Be Ruled Out by a Single Genotyping Assay for the −1584G Promoter Polymorphism

Accurate prediction of CYP2D6 phenotype from genotype data is important for many clinically relevant drugs. Genotyping strategies targeting allelic variants with diminished or no activity to identify poor metabolizers generally works well in Caucasian (1)(2)(3) and Asian (4)(5) populations, but we have had more limited success (i.e., poor concordance) in African Americans, even after extensive testing (6). For example, genotype analysis predicted poor metabolizer status (by Caucasian-derived antimode definition) in only 4 of 14 individuals, and many individuals predicted to be extensive metabolizers presented as “intermediate” metabolizers (6). A single-nucleotide polymorphism in the CYP2D6 promoter region (−1584G) has been reported to confer higher CYP2D6 activity in vivo than −1584C (7), possibly as a consequence of higher expression of CYP2D6 protein (8). This polymorphism was first associated with CYP2D6*2 alleles, and the CYP nomenclature committee subsequently assigned * 2[−1584G] as CYP2D6 * 2A (and noting that −1584G is probably found on all CYP2D6 * 2 alleles) and * 2[−1584C] as CYP2D6 * 41 , respectively (9). However, −1584G also appears to be linked with the functional CYP2D6 * 35 allele, which has been found in many duplication-negative “ultrarapid” metabolizers (10). For simplicity, we refer to the * 2[−1584G] allele cumulatively as CYP2D6 * 2 because our genotyping procedure does not differentiate among variants CYP2D6 * 2A through K . For reference, −1584G corresponds to −1496G in Ref. (7). The goals of this investigation were ( a ) to explore whether −1584G is exclusively linked to functional allelic variants and hence would allow rapid “positive” identification of extensive metabolizers and reliably rule out poor metabolizer status, and ( b ) to reevaluate the genotype-to-phenotype correlation data in our Caucasian and African-American populations. The use of patients’ DNA samples was approved by the Pediatric Institutional Review Board of Children’s Mercy …

Project Among African-Americans to Explore Risks for Schizophrenia (PAARTNERS): Evidence for Impairment and Heritability of Neurocognitive Functioning in Families of Schizophrenia Patients

OBJECTIVE Neurocognitive impairments in schizophrenia are well replicated and widely regarded as candidate endophenotypes that may facilitate understanding of schizophrenia genetics and pathophysiology. The Project Among African-Americans to Explore Risks for Schizophrenia (PAARTNERS) aims to identify genes underlying liability to schizophrenia. The unprecedented size of its study group (N=1,872), made possible through use of a computerized neurocognitive battery, can help further investigation of the genetics of neurocognition. The current analysis evaluated two characteristics not fully addressed in prior research: 1) heritability of neurocognition in African American families and 2) relationship between neurocognition and psychopathology in families of African American probands with schizophrenia or schizoaffective disorder. METHOD Across eight data collection sites, patients with schizophrenia or schizoaffective disorder (N=610), their biological relatives (N=928), and community comparison subjects (N=334) completed a standardized diagnostic evaluation and the computerized neurocognitive battery. Performance accuracy and response time (speed) were measured separately for 10 neurocognitive domains. RESULTS The patients with schizophrenia or schizoaffective disorder exhibited less accuracy and speed in most neurocognitive domains than their relatives both with and without other psychiatric disorders, who in turn were more impaired than comparison subjects in most domains. Estimated trait heritability after inclusion of the mean effect of diagnostic status, age, and sex revealed significant heritabilities for most neurocognitive domains, with the highest for accuracy of abstraction/flexibility, verbal memory, face memory, spatial processing, and emotion processing and for speed of attention. CONCLUSION Neurocognitive functions in African American families are heritable and associated with schizophrenia. They show potential for gene-mapping studies.

In Silico and In Vitro Identification of MicroRNAs That Regulate Hepatic Nuclear Factor 4α Expression

Hepatic nuclear factor 4α (HNF4A) is a nuclear transcription factor that regulates the expression of many genes involved in drug disposition. To identify additional molecular mechanisms that regulate HNF4A, we identified microRNAs (miRNAs) that target HNF4A expression. In silico analyses suggested that HNF4A is targeted by many miRNAs. We conducted in vitro studies to validate several of these predictions. With use of an HNF4A 3′-untranslated region (UTR) luciferase reporter assay, five of six miRNAs tested significantly down-regulated (∼20–40%) the luciferase activity. In HepG2 cells, miR-34a and miR-449a also down-regulated the expression of both the HNF4A protein and an HNF4A target gene, PXR (∼30–40%). This regulation appeared without reduction in HNF4A mRNA expression, suggesting that they must be blocking HNF4A translation. Using additional bioinformatic algorithms, we identified polymorphisms that are predicted to alter the miRNA targeting of HNF4A. Luciferase assays indicated that miR-34a and miR-449a were less effective in regulating a variant (rs11574744) than the wild-type HNF4A 3′-UTR. In vivo, subjects with the variant HNF4A had lower CYP2D6 enzyme activity, although this result was not statistically significant (p = 0.16). In conclusion, our findings demonstrate strong evidence for a role of miRNAs in the regulation of HNF4A.

CYP2D7-2D6 hybrid tandems: identification of novel CYP2D6 duplication arrangements and implications for phenotype prediction

AIMS Allelic variants of cytochrome P450 CYP2D6 (CYP2D6), such as gene deletion, duplication, multiplication and conversion, contribute to the wide range of CYP2D6 activity. Novel gene arrangements were discovered and characterized. MATERIALS & METHODS DNA from 32 Caucasian and 59 African-American duplication-positive subjects were analyzed by long-range PCR and genotyping to detect CYP2D7-2D6 hybrid tandem alleles. Novel allelic variants were sequenced and a strategy for the detection and analysis of hybrid genes was refined. RESULTS CYP2D7-2D6 hybrid tandem alleles were identified in one African-American and four Caucasian subjects. Three novel hybrid genes were found on CYP2D6*1 and CYP2D6*2 duplication backgrounds and designated CYP2D6*76, *77 and *78. CYP2D7 to 2D6 conversion occurred in introns 1 and 4, and exon 9. All carried a T-insertion in exon 1 abolishing activity. In Caucasians, four out of 33 (12%) of the duplication-positive alleles were hybrid tandems, three CYP2D6*77 + *2 and one CYP2D6*78 + *2. By contrast, in African-Americans only one of 60 duplication-positive alleles was identified as a hybrid tandem. This allele was designated CYP2D6*76 + *1. CONCLUSION Hybrid tandem alleles occur infrequently (<0.25%) in Caucasians, but may explain why not every subject with a CYP2D6 duplication presents with an ultrarapid metabolizer phenotype.

Discovery of a novel nonfunctional cytochrome P450 2D6 allele, CYP2D6*42, in African American subjects

Discovery of a novel nonfunctional cytochrome P450 2D6 allele, CYP2D6*42, in African American subjects To the Editor: Investigating the acquisition of cytochrome P450 (CYP) 2D6 activity during the first year of life, we identified an African American infant who consistently had a CYP2D6 poor metabolizer phenotype at age 2 weeks to 4 months (Table I). His genotype was initially determined as CYP2D6*2/*17 and revised to CYP2D6*41/*40 on testing for the single-nucleotide polymorphism at position 1584G C and the TTT CGC CCC insertion at position 1863. Because