Integrated CYP2D6 interrogation for multiethnic copy number and tandem allele detection.

AIM: To comprehensively interrogate CYP2D6 by integrating genotyping, copy number analysis and novel strategies to identify CYP2D6*36 and characterize CYP2D6 duplications. METHODS: Genotyping of 16 CYP2D6 alleles, multiplex ligation-dependent probe amplification (MLPA) and CYP2D6*36 and duplication allele-specific genotyping were performed on 427 African-American, Asian, Caucasian, Hispanic, and Ashkenazi Jewish individuals. RESULTS: A novel PCR strategy determined that almost half of all CYP2D6*10 (100C>T) alleles are actually *36 (isolated or in tandem with *10) and all identified duplication alleles were characterized. Integrated results from all testing platforms enabled the refinement of genotype frequencies across all studied populations. CONCLUSION: The polymorphic CYP2D6 gene requires comprehensive interrogation to characterize allelic variation across ethnicities, which was enabled in this study by integrating multiplexed genotyping, MLPA copy number analysis, novel PCR strategies and duplication allele-specific genotyping.

Multi-ethnic SULT1A1 copy number profiling with multiplex ligation-dependent probe amplification.

AIM: To develop a SULT1A1 multiplex ligation-dependent probe amplification assay and to investigate multi-ethnic copy number variant frequencies. METHODS: A novel multiplex ligation-dependent probe amplification assay was developed and tested on 472 African-American, Asian, Caucasian, Hispanic and Ashkenazi Jewish individuals. RESULTS: The frequencies of atypical total copy number (i.e., greater or less than two) were 38.7% for Hispanics, 38.9% for Ashkenazi Jewish, 43.2% for Caucasians, 53.6% for Asians and 64.1% for African-Americans. Heterozygous SULT1A1 deletion carriers (slow sulfators) were most common among Caucasians (8.4%), whereas African-Americans had the highest frequencies of three or more copies (rapid sulfators; 60.9%). CONCLUSION: Different ethnic and racial populations have varying degrees of SULT1A1-mediated sulfation activity, which warrants further research and that may have utility for drug response prediction among SULT1A1-metabolized medications.

Warfarin pharmacogenetics: a controlled dose-response study in healthy subjects.

The aim of this study was to determine how genetic variants contribute to warfarin dosing variability when non-genetic factors are controlled. Thirty healthy subjects were subjected to a warfarin dosing algorithm with daily international normalized ratio (INR) measurements to INR ≥ 2.0, then off warfarin to INR ≤ 1.2. The primary outcome was the cumulative dose required to achieve INR ≥ 2.0 for 2 consecutive days. CYP2C9 (p=0.004) and VKORC1 (p=0.02) variant carriers required lower cumulative doses, and CYP4F2 carriers required higher doses (p=0.04). Subjects with variants in both CYP2C9 and VKORC1 required fewer days to reach INR ≥ 2.0 than wild-type subjects or those with variants in CYP2C9 or VKORC1 (p=0.01). Genetic contribution to dose variability (~62%) was greater than previously reported, suggesting that uncontrolled clinical variables influence the effect of these variants. In conclusion, genotype-guided warfarin-dosing algorithms may rely more on genetic variables in healthier individuals than in patients with clinical confounders.

An allele-specific PCR system for rapid detection and discrimination of the CYP2C19∗4A, ∗4B, and ∗17 alleles: implications for clopidogrel response testing.

CYP2C19 is involved in the metabolism of clinically relevant drugs, including the antiplatelet prodrug clopidogrel, which has prompted interest in clinical CYP2C19 genotyping. The CYP2C19∗4B allele is defined by both gain-of-function [c.-806C>T (∗17)] and loss-of-function [c.1A>G (∗4)] variants on the same haplotype; however, current genotyping and sequencing assays are unable to determine the phase of these variants. Thus, the aim of this study was to develop an assay that could rapidly detect and discriminate the related ∗4A, ∗4B, and ∗17 alleles. An allele-specific PCR assay, composed of four unique primer mixes that specifically interrogate the defining ∗17 and ∗4 variants, was developed by using samples (n = 20) with known genotypes, including the ∗4A, ∗4B, and/or ∗17 alleles. The assay was validated by testing 135 blinded samples, and the results were correlated with CYP2C19 genotyping and allele-specific cloning/sequencing. Importantly, among the six ∗4 carriers in the validation cohort, after allele-specific PCR testing both samples with a ∗1/∗4 genotype were reclassified to ∗1/∗4A, all three samples with a ∗4/∗17 genotype were reclassified to ∗1/∗4B, and a sample with a ∗4/∗17/∗17 genotype was reclassified to ∗4B/∗17. In conclusion, this rapid and robust allele-specific PCR assay can refine CYP2C19 genotyping and metabolizer phenotype classification by determining the phase of the defining ∗17 and ∗4 variants, which may have utility when testing CYP2C19 for clopidogrel response.

Frequency of the cholesteryl ester storage disease common LIPA E8SJM mutation (c.894G>A) in various racial and ethnic groups.

UNLABELLED: Cholesteryl ester storage disease (CESD) and Wolman disease are autosomal recessive later-onset and severe infantile disorders, respectively, which result from the deficient activity of lysosomal acid lipase (LAL). LAL is encoded by LIPA (10q23.31) and the most common mutation associated with CESD is an exon 8 splice junction mutation (c.894G>A; E8SJM), which expresses only ∼3%-5% of normally spliced LAL. However, the frequency of c.894G>A is unknown in most populations. To estimate the prevalence of CESD in different populations, the frequencies of the c.894G>A mutation were determined in 10,000 LIPA alleles from healthy African-American, Asian, Caucasian, Hispanic, and Ashkenazi Jewish individuals from the greater New York metropolitan area and 6,578 LIPA alleles from African-American, Caucasian, and Hispanic subjects enrolled in the Dallas Heart Study. The combined c.894G>A allele frequencies from the two cohorts ranged from 0.0005 (Asian) to 0.0017 (Caucasian and Hispanic), which translated to carrier frequencies of 1 in 1,000 to ∼1 in 300, respectively. No African-American heterozygotes were detected. Additionally, by surveying the available literature, c.894G>A was estimated to account for 60% (95% confidence interval [CI]: 51%-69%) of reported mutations among multiethnic CESD patients. Using this estimate, the predicted prevalence of CESD in the Caucasian and Hispanic populations is ∼0.8 per 100,000 (∼1 in 130,000; 95% CI: ∼1 in 90,000 to 1 in 170,000). CONCLUSION: These data indicate that CESD may be underdiagnosed in the general Caucasian and Hispanic populations, which is important since clinical trials of enzyme replacement therapy for LAL deficiency are currently being developed. Moreover, future studies on CESD prevalence in African and Asian populations may require full-gene LIPA sequencing to determine heterozygote frequencies, since c.894G>A is not common in these racial groups.

Copy number variation and warfarin dosing: evaluation of CYP2C9, VKORC1, CYP4F2, GGCX and CALU.

AIM: To determine if copy number variants contribute to warfarin dose requirements, we investigated CYP2C9, VKORC1, CYP4F2, GGCX and CALU for deletions and duplications in a multiethnic patient population treated with therapeutic doses of warfarin. PATIENTS & METHODS: DNA samples from 178 patients were subjected to copy number analyses by multiplex ligation-dependent probe amplification or quantitative PCR assays. Additionally, the CYP2C9 exon 8 insertion/deletion polymorphism (rs71668942) was examined among the patient cohort and 1750 additional multiethnic healthy individuals. RESULTS: All patients carried two copies of CYP2C9 by multiplex ligation-dependent probe amplification and no exon 8 deletion carriers were detected. Similarly, quantitative PCR assays for VKORC1, CYP4F2, GGCX and CALU identified two copies in all populations. CONCLUSION: These data indicate that copy number variants in the principal genes involved in warfarin dose variability (CYP2C9, VKORC1), including genes with lesser effect (CYP4F2, GGCX), and those that may be more relevant among certain racial groups (CALU), are rare in multiethnic populations, including African-Americans.