UGT1A1 genetic analysis as a diagnostic aid for individuals with unconjugated hyperbilirubinemia.

OBJECTIVE: To assess the clinical utility of UGT1A1 genetic testing and describe the spectrum and prevalence of UGT1A1 variations identified in pediatric unconjugated hyperbilirubinemia (UCH), and to characterize specific genotype-phenotype relationships in suspected Gilbert and Crigler-Najjar syndromes. STUDY DESIGN: A retrospective study was conducted to review clinical information and UGT1A1 genotyping data from 181 pediatric patients referred for UCH. In silico analyses were performed to aid in the assessment of novel UGT1A1 variants. RESULTS: Overall, 146/181 pediatric patients had at least one heterozygous UGT1A1 functional variant. Identified UGT1A1 variants included 17 novel variants, 7 rare star alleles, and 1 rare variant. There were 129 individuals who possessed the TA7 (*28) promoter repeat and 15 individuals who possessed the *6 (c.211G > A) variation. Out of the 104 individuals with accompanying bilirubin levels, 41 individuals did not have identifiable UGT1A1 variants that explained their UCH, although glucose-6-phosphate dehydrogenase deficiency and other causes of UCH could not be ruled out. CONCLUSION: Much of the observed UCH could be attributed to variation at the UGT1A1 locus, and UGT1A1 testing helped to substantiate a genetic diagnosis, thereby aiding in individual and family disease management. Although UGT1A1 variation plays a large role in UCH, genetic assessment of UGT1A1 alone may not be comprehensive. Assessment of additional genes may also be useful to evaluate genetic causes for UCH.

CYP2D6*11 and challenges in clinical genotyping of the highly polymorphic CYP2D6 gene.

CYP2D6 is genotyped clinically for prediction of response to tamoxifen, psychotropic drugs and other medications. Phenotype prediction is dependent upon accurate genotyping. The CYP Allele Nomenclature Committee maintains the allelic nomenclature for CYP2D6; however, in some cases, the list of polymorphisms associated with a given allele is incomplete. Clinical laboratories and in vitro diagnostic manufacturers rely upon this nomenclature, in addition to the literature, to infer allelic function and haplotypes and when they design CYP2D6-testing platforms. This article provides more complete sequencing data for the CYP2D6*11 allele and describes the difficulties encountered in genotyping CYP2D6 when incomplete data are available. The CYP Allele Nomenclature Committee should provide clear information about the completeness of the original data used to define each allele.

Correlation of CYP2B6, CYP2C19, ABCC4 and SOD2 genotype with outcomes in allogeneic blood and marrow transplant patients.

CYP2B6, CYP2C19, ABCC4, and SOD2 have been implicated in adverse drug reactions and survival after cyclophosphamide (CPA) treatment. 110 BMT patients who received high dose CPA treatment were genotyped for variants in these genes and the results were correlated with toxicity and relapse. CYP2B6 genotype significantly influenced overall toxicity suggesting active CYP2B6 alleles led to higher rates of overall toxicity. The p.R487C deficiency allele was significantly associated with a lower rate of overall toxicity and a higher rate of relapse. SOD2 rs4880 V16A polymorphism was associated with significantly less CPA-related overall toxicity and significantly lower relapse rates by Kaplan-Meier analysis although the SOD2 finding regarding relapse was not significant when evaluated by the cumulative incidence function.

Frequency of undetected CYP2D6 hybrid genes in clinical samples: impact on phenotype prediction.

Cytochrome P450 2D6 (CYP2D6) is highly polymorphic. CYP2D6-2D7 hybrid genes can be present in samples containing CYP2D6*4 and CYP2D6*10 alleles. CYP2D7-2D6 hybrid genes can be present in samples with duplication signals and in samples with homozygous genotyping results. The frequency of hybrid genes in clinical samples is unknown. We evaluated 1390 samples for undetected hybrid genes by polymerase chain reaction (PCR) amplification, PCR fragment analysis, TaqMan copy number assays, DNA sequencing, and allele-specific primer extension assay. Of 508 CYP2D6*4-containing samples, 109 (21.5%) harbored CYP2D6*68 + *4-like, whereas 9 (1.8%) harbored CYP2D6*4N + *4-like. Of 209 CYP2D6*10-containing samples, 44 (21.1%) were found to have CYP2D6*36 + *10. Of 332 homozygous samples, 4 (1.2%) harbored a single CYP2D7-2D6 hybrid, and of 341 samples with duplication signals, 25 (7.3%) harbored an undetected CYP2D7-2D6 hybrid. Phenotype before and after accurate genotyping was predicted using a method in clinical use. The presence of hybrid genes had no effect on the phenotype prediction of CYP2D6*4- and CYP2D6*10-containing samples. Four of four (100%) homozygous samples containing a CYP2D7-2D6 gene had a change in predicted phenotype, and 23 of 25 (92%) samples with a duplication signal and a CYP2D7-2D6 gene had a change in predicted phenotype. Four novel genes were identified (CYP2D6*13A1 variants 1 and 2, CYP2D6*13G1, and CYP2D6*13G2), and two novel hybrid tandem structures consisting of CYP2D6*13B + *68×2 + *4-like and CYP2D6*13A1 variant 2 + *1×N were observed.

UGT1A1 sequence variants and bilirubin levels in early postnatal life: a quantitative approach.

BACKGROUND: Fundamental to definitively identifying neonates at risk of developing significant hyperbilirubinemia is a better understanding of the genetic factors associated with early bilirubin rise. Previous genetic studies have focused on the UGT1A1 gene, associating common variation in the coding or promoter regions with qualitative assessments of bilirubin (i.e. significantly elevated or not). These studies have had conflicting results and limited success. We chose to approach the problem by focusing on the quantitative (absolute) change in bilirubin levels early in post-natal life. We apply this approach to the UGT1A1 gene--exploring the contribution of both rare and common variants to early bilirubin changes. METHODS: We sequenced the exons, PBREM, 5'-, and 3'- regions of the UGT1A1 gene in 80 otherwise healthy term neonates who had repeat bilirubin levels measured within the first five days of life. RESULTS: Three novel coding variants were observed, but there was no clear relationship between rare coding variants and bilirubin rise. Adjusted linear regression models fit to evaluate the relationship between changing bilirubin levels and common UGT1A1variants found that among 39 neonates whose bilirubin was resampled within 33 hours, individuals homozygous for the mutant allele of a 3'UTR SNP had significantly smaller changes in bilirubin (P=0.003) than individuals carrying the wild-type allele. CONCLUSIONS: Collectively, rare UGT1A1 coding variants do not appear to play a prominent role in determining early bilirubin levels; however common variants in the 3' UTR of UGT1A1 may modulate the early bilirubin rise. A quantitative approach to evaluating early bilirubin kinetics provides a more robust framework in which to better understand the genetics of neonatal hyperbilirubinemia.

Stimulation of retinoic acid-induced functional sodium iodide symporter (NIS) expression and cytotoxicity of ¹³¹I by carbamazepine in breast cancer cells.

The sodium iodide symporter (NIS) mediates the active iodide uptake in the thyroid gland as well as lactating breast tissue. Recently, we reported significant stimulation of all-trans retinoic acid (atRA)-induced NIS expression in the estrogen-receptor positive human breast cancer cell line MCF-7 by dexamethasone (Dex) in vitro and in vivo, which might offer the potential to image and treat breast cancer with radioiodine. In this study, based on its known interaction with the pregnane-X-receptor (PXR) forming a heterodimer with the retinoid-X-receptor (RXR), we examined the effect of carbamazepine (CBZ), a potent activator of PXR, on atRA-induced NIS expression and therapeutic efficacy of (131)I in MCF-7 cells. For this purpose, functional NIS expression in MCF-7 cells was examined by iodide uptake assay, quantitative real-time PCR as well as Western blot analysis, followed by investigation of (131)I cytotoxicity in vitro after incubation with CBZ (4, 25, 100 µM) in the presence of atRA (1 µM) with or without Dex (100 nM). Incubation with CBZ stimulated atRA-induced iodide accumulation up to twofold in a concentration-dependent manner, while atRA/Dex-stimulated iodide uptake was further stimulated up to 1.5-fold by additional CBZ treatment based on significantly increased NIS mRNA and protein levels. This stimulatory effect of CBZ was shown to be dependent on the PI3K-Akt pathway without involvement of mTOR. In contrast, treatment with CBZ alone had no effect on functional NIS expression. Moreover, selective cytotoxicity of (131)I was significantly increased from approximately 20% in MCF-7 cells treated with atRA alone to 50% after treatment with CBZ in the presence of atRA, which was further enhanced to 90% after combined treatment with atRA/Dex/CBZ. In conclusion, CBZ represents another potent stimulator of atRA-induced functional NIS expression resulting in an enhanced selective killing effect of (131)I in MCF-7 breast cancer cells.

CYP2C19 variation and citalopram response.

OBJECTIVE: Variations in cytochrome P450 (CYP) genes have been shown to be associated with both accelerated and delayed pharmacokinetic clearance of many psychotropic medications. Citalopram is metabolized by three CYP enzymes. CYP2C19 and CYP3A4 play a primary role in citalopram metabolism, whereas CYP2D6 plays a secondary role. METHODS: The Sequenced Treatment Alternatives to Relieve Depression sample was used to examine the relationship between variations in the CYP2C19 and CYP2D6 genes and remission of depressive symptoms and tolerance to treatment with citalopram. The primary analyses were of the White non-Hispanic patients adherent to the study protocol (n= 1074). RESULTS: Generally, patients who had CYP2C19 genotypes associated with decreased metabolism were less likely to tolerate citalopram than those with increased metabolism, although this difference was not statistically significant (P = 0.06). However, patients with the inactive 2C19*2 allele had significantly lower odds of tolerance (P = 0.02). Patients with the poor metabolism CYP2C19 genotype-based category who were classified as citalopram tolerant were more likely to experience remission (P = 0.03). No relationship between CYP2D6 genotype-based categories and either remission or tolerance was identified, although exploratory analyses identified a potential interaction between CYP2C19 and CYP2D6 effects. CONCLUSION: Despite several limitations including the lack of serum drug levels, this study showed that variations in CYP2C19 were associated with tolerance and remission in a large sample of White non-Hispanic patients treated with citalopram.

Effect of cytochrome P450 enzyme polymorphisms on pharmacokinetics of venlafaxine.

This study examines the relationship between blood concentrations of venlafaxine and its active metabolite, O-desmethyl venlafaxine (ODV), and genetic variants of the cytochrome P450 enzymes CYP2D6 and CYP2C19 in human subjects. Trough blood concentrations were measured at steady state in patients treated with venlafaxine extended release in a clinical practice setting. CYP2D6 and CYP2C19 genotypes were converted to activity scores based on known activity levels of the two alleles comprising a genotype. After adjusting for drug dose and gender effects, higher CYP2D6 and CYP2C19 activity scores were significantly associated with lower venlafaxine concentrations (P < 0.001 for each). Only CYP2D6 was associated with the concentration of ODV (P < 0.001), in which genotypes with more active alleles were associated with higher ODV concentrations. The sum of venlafaxine plus ODV concentration showed the same pattern as venlafaxine concentrations with CYP2D6 and CYP2C19 genotypes with higher activity scores being associated with a lower venlafaxine plus ODV concentration (2D6 P = 0.01; 2C19 P < 0.001). Because allelic variants in both CYP2D6 and CYP2C19 influence the total concentration of the active compounds venlafaxine and ODV, both CYP2D6 and CYP2C19 genotypes should be considered when using pharmacogenomic information for venlafaxine dose alterations.

Warfarin genotyping reduces hospitalization rates results from the MM-WES (Medco-Mayo Warfarin Effectiveness study).

OBJECTIVES: This study was designed to determine whether genotype testing for patients initiating warfarin treatment will reduce the incidence of hospitalizations, including those due to bleeding or thromboembolism. BACKGROUND: Genotypic variations in CYP2C9 and VKORC1 have been shown to predict warfarin dosing, but no large-scale studies have prospectively evaluated the clinical effectiveness of genotyping in naturalistic settings across the U.S. METHODS: This national, prospective, comparative effectiveness study compared the 6-month incidence of hospitalization in patients receiving warfarin genotyping (n = 896) versus a matched historical control group (n = 2,688). To evaluate for temporal changes in the outcomes of warfarin treatment, a secondary analysis compared outcomes for 2 external control groups drawn from the same 2 time periods. RESULTS: Compared with the historical control group, the genotyped cohort had 31% fewer hospitalizations overall (adjusted hazard ratio [HR]: 0.69, 95% confidence interval [CI]: 0.58 to 0.82, p < 0.001) and 28% fewer hospitalizations for bleeding or thromboembolism (HR: 0.72, 95% CI: 0.53 to 0.97, p = 0.029) during the 6-month follow-up period. Findings from a per-protocol analysis were even stronger: 33% lower risk of all-cause hospitalization (HR: 0.67, 95% CI: 0.55 to 0.81, p < 0.001) and 43% lower risk of hospitalization for bleeding or thromboembolism (HR: 0.57, 95% CI: 0.39 to 0.83, p = 0.003) in patients who were genotyped. During the same period, there was no difference in outcomes between the 2 external control groups. CONCLUSIONS: Warfarin genotyping reduced the risk of hospitalization in outpatients initiating warfarin. (The Clinical and Economic Impact of Pharmacogenomic Testing of Warfarin Therapy in Typical Community Practice Settings [MHSMayoWarf1]; NCT00830570).

Warfarin sensitivity genotyping: a review of the literature and summary of patient experience.

The antithrombotic benefits of warfarin are countered by a narrow therapeutic index that contributes to excessive bleeding or cerebrovascular clotting and stroke in some patients. This article reviews the current literature describing warfarin sensitivity genotyping and compares the results of that review to the findings of our study in 189 patients at Mayo Clinic conducted between June 2001 and April 2003. For the review of the literature, we identified relevant peer-reviewed articles by searching the Web of Knowledge using key word warfarin-related adverse event. For the 189 Mayo Clinic patients initiating warfarin therapy to achieve a target international normalized ratio (INR) in the range of 2.0 to 3.5, we analyzed the CYP2C9 (cytochrome P450 2C9) and VKORC1 (vitamin K epoxide reductase complex, subunit 1) genetic loci to study the relationship among the initial warfarin dose, steady-state dose, time to achieve steady-state dose, variations in INR, and allelic variance. Results were compared with those previously reported in the literature for 637 patients. The relationships between allelic variants and warfarin sensitivity found in our study of Mayo Clinic patients are fundamentally the same as in those reported by others. The Mayo Clinic population is predominantly white and shows considerable allelic variability in CYP2C9 and VKORC1. Certain of these alleles are associated with increased sensitivity to warfarin. Polymorphisms in CYP2C9 and VKORC1 have a considerable effect on warfarin dose in white people. A correlation between steady-state warfarin dose and allelic variants of CYP2C9 and VKORC1 has been demonstrated by many previous reports and is reconfirmed in this report. The allelic variants found to most affect warfarin sensitivity are CYP2C9*1*1-VKORC1BB (less warfarin sensitivity than typical); CYP2C9*1*1-VKORC1AA (considerable variance in INR throughout initiation); CYP2C9*1*2-VKORC1AB (more sensitivity to warfarin than typical); CYP2C9*1*3-VKORC1AB (much more sensitivity to warfarin than typical); CYP2C9*1*2-VKORC1AB (much more sensitivity to warfarin than typical); CYP2C9*1*3-VKORC1AA (much more sensitivity to warfarin than typical); and CYP2C9*2*2-VKORC1AB (much more sensitivity to warfarin than typical). Although we were unable to show an association between allelic variants and initial warfarin dose or dose escalation, an association was seen between allelic variant and steady-state warfarin dose. White people show considerable variance in CYP2C9 allele types, whereas people of Asian or African descent infrequently carry CYP2C9 allelic variants. The VKORC1AA allele associated with high warfarin sensitivity predominates in those of Asian descent, whereas white people and those of African descent show diversity, carrying either the VKORC1BB, an allele associated with low warfarin sensitivity, or VKORC1AB or VKORC1AA, alleles associated with moderate and high warfarin sensitivity, respectively.

CYP2D6: novel genomic structures and alleles.

OBJECTIVE: CYP2D6 is a polymorphic gene. It has been observed to be deleted, to be duplicated and to undergo recombination events involving the CYP2D7 pseudogene and surrounding sequences. The objective of this study was to discover the genomic structure of CYP2D6 recombinants that interfere with clinical genotyping platforms that are available today. METHODS: Clinical samples containing rare homozygous CYP2D6 alleles, ambiguous readouts, and those with duplication signals and two different alleles were analyzed by long-range PCR amplification of individual genes, PCR fragment analysis, allele-specific primer extension assay, and DNA sequencing to characterize alleles and genomic structure. RESULTS: Novel alleles, genomic structures, and the DNA sequence of these structures are described. Interestingly, in 49 of 50 DNA samples that had CYP2D6 gene duplications or multiplications where two alleles were detected, the chromosome containing the duplication or multiplication had identical tandem alleles. CONCLUSION: Several new CYP2D6 alleles and genomic structures are described which will be useful for CYP2D6 genotyping. The findings suggest that the recombination events responsible for CYP2D6 duplications and multiplications are because of mechanisms other than interchromosomal crossover during meiosis.

Pharmacogenetics of solid tumors: directed therapy in breast, lung, and colorectal cancer: a paper from the 2008 william beaumont hospital symposium on molecular pathology.

Genetic variability in drug-metabolizing enzymes and signaling pathways affects chemotherapy-related toxicity and treatment outcome in cancer. In breast and colorectal cancer, polymorphisms in metabolic enzymes involved in tamoxifen and irinotecan therapies has led the U.S. Food and Drug Administration to address genetic factors relevant to patient consideration of treatment with these compounds. Tamoxifen therapeutic failure in breast cancer has been associated with reduced CYP2D6 activity due to inefficient activation of tamoxifen. Irinotecan toxicity in colorectal cancer is more common in patients with reduced-activity UGT1A alleles, resulting in excessive exposure to the potent SN-38 metabolite. In colorectal and lung cancers, somatic mutations in the epidermal growth factor receptor and downstream signaling molecules have been associated with the therapeutic outcome of epidermal growth factor receptor-directed therapies. This review discusses the current knowledge regarding the utility of single gene-UGT1A1, CYP2D6, EGFR, and KRAS-or multigene analysis, for optimizing breast, colorectal, and lung cancer therapy. Current advances in these areas highlight how pharmacogenetics help personalized decision-making for patient management.

Use of cyst fluid CEA, CA19-9, and amylase for evaluation of pancreatic lesions.

OBJECTIVES: The study goals were development of reference intervals and an interpretive algorithm for pancreatic cyst fluid tumor markers. DESIGN AND METHODS: 442 pancreatic cyst fluids were tested for CEA, CA19-9, and amylase. RESULTS: CEA>30 ng/mL discriminates mucinous from non-mucinous cysts. After CEA analysis, amylase and CA19-9 segregate non-mucinous and mucinous subtypes, respectively. CONCLUSIONS: Pancreatic cyst fluid tumor markers supplement other diagnostic measures. This study provides estimated reference intervals and an algorithm for interpretation.

Pharmacogenomics of tamoxifen and irinotecan therapies.

Genetic variability in drugmetabolizing enzymes affects the toxicity and efficacy of many compounds, including the chemotherapeutic agents irinotecan and tamoxifen. The correlation of clinical response to polymorphisms in enzymes associated with metabolism of these two drugs has led to the recommendation that patients who receive them undergo genotyping analysis. Irinotecan toxicity in patients who have colorectal cancer has been linked to reduced activity of uridine diphosphate-glucuronyltransferase 1A1 (UGT1A1). Reduced cytochrome P450 (CYP) 2D6 activity leads to therapeutic failure of tamoxifen in the prevention and treatment of breast cancer, as a result of absence of conversion of the prodrug to its active forms. This article discusses current knowledge of the usefulness of UGT1A1 and CYP2D6 genotyping in the context of cancer chemotherapy and highlights the need for additional studies to clarify the many issues remaining.

Cytochrome P450 2D6 genotype variation and venlafaxine dosage.

OBJECTIVE: To determine whether the presence or absence of a fully functioning cytochrome P450 2D6 allele was associated with the dosage of the antidepressant drug venlafaxine in patients who had either adverse effects or absence of a therapeutic response to treatment with the immediate release or extended release form of venlafaxine. PATIENTS AND METHODS: We reviewed the electronic medical records of 199 patients enrolled in a previous pharmacogenomic study (June 1, 2002 through April 30, 2004) who had either adverse effects or the absence of a therapeutic response to treatment with psychotropic medications. This review identified 38 patients previously treated with venlafaxine immediate release or extended release and subsequently genotyped for the 2D6 gene with a commercial genotyping assay. Their dosage was examined along with their 2D6 genotype to determine whether the presence or absence of a fully functioning 2D6 allele was associated with their venlafaxine dosage. RESULTS: Of the 38 patients, 5 had a 2D6 genotype that consisted of 1 inactive allele and 1 allele associated with decreased activity. None of these 5 patients were able to tolerate treatment with more than 75 mg/d of venlafaxine. The remaining 33 patients had at least 1 fully active 2D6 allele, 26 of whom had been able to tolerate treatment with 150 mg/d or more of venlafaxine (P less than .002). CONCLUSION: Genetic variations of the P450 2D6 gene may contribute to patient-specific variation in response to treatment with venlafaxine. Physicians should be alert to the possibility that an adverse reaction may indicate a slow metabolizer and consider genotyping such patients.

Comparison of three methods for genotyping the UGT1A1 (TA)n repeat polymorphism.

OBJECTIVES: The UGT1A1 promoter contains a (TA)n repeat polymorphism. The 7 repeat allele is associated with decreased enzyme activity and patients homozygous for this allele treated with irinotecan may experience life-threatening toxicity. Here, we have compared three methods [DNA sequencing, fragment analysis, and the Invader assay (Third Wave Technologies)] for genotyping this polymorphism. RESULTS: All of the DNA samples (n=119) had concordant genotype calls between the sequencing and size-based methods. The Invader method was also concordant if the genotypes were 6/6, 6/7, or 7/7. Both the size-based method and the Invader method had straightforward data analysis, while interpretation of the sequencing results was occasionally more challenging. The Invader method required more concentrated DNA for analysis, was more expensive, and had a limited genotyping spectrum. CONCLUSION: All three methods were valuable for genotyping the UGT1A1 (TA)n repeat, with the sequencing and size-based assays having the fewest drawbacks.

The impact of CYP allelic variation on antidepressant metabolism: a review.

Psychiatric diseases that are treated with antidepressants are the leading causes of morbidity and mortality in humankind. Although antidepressants are generally well tolerated and widely available, they are not equally effective in all patients and only 35 - 45% of patients treated for depression with these drugs recover to premorbid levels of functioning. There is a need for an effective, individualized approach to antidepressant selection. One promising lead in the development of personalized medicine is the emerging field of pharmacogenomics, whereby pharmacologic agents are selected on the basis of the genotype of patients, with particular attention to drug targets and phase I- and phase II-metabolizing enzymes. This review article focuses on phase I antidepressant-metabolizing enzymes (e.g., relevant CYP enzymes). The authors first briefly review CYP nomenclature, the relevant members of the CYP superfamily and their alleles, the metabolic categories and CYP antidepressant substrates, inhibitors and inducers. The literature on the impact of CYP polymorphisms on antidepressant metabolism are also reviewed.