Markus A. Schirmer

The Polymorphisms Asn130Asp and Val174Ala in OATP1B1 and the CYP2C9 Allele *3 Independently Affect Torsemide Pharmacokinetics and Pharmacodynamics

To the editor: We investigated two organic anion transporting polypeptide 1B1 (OATP1B1) polymorphisms in relation to torsemide pharmacokinetics and pharmacodynamics. The major conclusions are as follows: (1) OATP1B1 transports torsemide; (2) OATP1B1 supposedly also transports other CYP2C9 substrates; (3) the polymorphisms Asn130Asp and Val174Ala affected kinetics independently; (4) haplotype analysis was necessary to detect their independent impact; (5) together, CYP2C9, OATP1B1, OAT1, and OAT4 polymorphisms explained nearly 50% pharmacokinetic variation; and (6) this example of significant gene–gene interactions may stimulate future research to conjointly consider enzyme and transporter polymorphisms. We hypothesized genetic variability in hepatocellular torsemide uptake because polymorphisms affecting torsemide’s biotransformation and renal excretion only incompletely explained the variation in its pharmacokinetics. Polymorphisms affecting the hepatocellular uptake of other anions have been reported in OATP1B1 (synonyms SLCO1B1, OATP-C, OATP2, LST1). We explored the impact of the OATP1B1 polymorphisms Asn130Asp and Val174Ala in our study with 10mg torsemide in 99 healthy Caucasians. Asn130Asp (rs2306283) and Val174Ala (rs4149056) had minor allele frequencies of 0.393 and 0.173. They were in moderate linkage disequilibrium (D0 1⁄4 0.62, 95% CI: 0.34–0.8, LOD (logarithm of odds) score: 2.85). Four haplotypes resulted: Asn130Val174 (synonym *1a), Asp130Val174 (*1b), Asn130Ala174 (*5), and Asp130Ala174 observed at allele frequencies of 0.577, 0.246, 0.027, and 0.150. Total oral torsemide clearance (Cl/F) was associated with the Val174Ala polymorphism with 62+3, 46+3, and 41+5ml/min (mean+SE) in Val/Val, Val/Ala, and Ala/Ala carriers (Po0.001) it was not statistically significantly associated with Asn130Asp (55+3, 54+3, 64+3ml/min in Asn/Asn, Asp/Asn, Asp/Asp). It was the haplotype–phenotype analysis that revealed a consistently higher clearance with aspartate130: total oral clearance was 56+3, 73+7, 27+16, and 41+9ml/min in homozygous Asn130Val174, Asp130Val174, Asn130Ala174, and Asp130Ala174 carriers (Po0.01, multiple linear regression). The corresponding extrarenal clearance was 40+3, 56+5, 13+12, and 26+7 (Po0.001). Especially, the Asp130Val174 haplotype was clearly associated with the torsemide clearance (Figure 1). We previously reported that the CYP2C9*3 allele is associated with reduced torsemide biotransformation and that polymorphisms in the organic anion transporters OAT1 and OAT4 alter renal torsemide clearance. The associations with the OATP1B1 alleles and those in CYP2 C9 (Figure 2), OAT1, and OAT4 were independent of each other. A fraction of 15.5% of variation was explained by the OATP1B1 haplotypes, roughly additive to approximately 20% explained by CYP2C9 and the 10% explained by OAT1 and OAT4, resulting in 46.7% variation now explained in the total oral torsemide clearance. This is a prime example showing how pharmacokinetic variation in a substrate of a polymorphic enzyme can be substantially better explained by including genetic variation in a transporter that mediates variable access to this enzyme (Fig u re 2 ). Renal torsemide excretion was higher with lower clearing OATP1B1 alleles and vice versa (Figure 1). Mean renal torsemide excretion was 2.8+0.1, 3.2+0.1, and 4.5+0.1mg with no, one, and two alanine174 alleles (Po0.001). It was not associated with the Asn130Asp po lymorphism (3.1 + 0.1, 3.0 + 0.1, and 2.7+0.2mg with no, one, and two aspartate130 alleles). Again as for clearance, the haplotype–phenotype analysis clarified this: renal torsemide excretion with all three less frequent haplotypes was significantly different from that in carriers of the most common Asn130Val174 haplotype with 3.0+0.1, 2.5+0.2, 4.9+0.5, and 3.6+0.3mg in homozygous carriers of PERSPECTIVES

Genetic polymorphisms of NAD(P)H oxidase: variation in subunit expression and enzyme activity

Genetic polymorphisms in superoxide-producing NAD(P)H oxidase have been linked to cardiovascular diseases including anthracycline-induced cardiotoxicity. We quantified NAD(P)H oxidase activity in granulocytes of 81 healthy Caucasian volunteers (in addition, 51 in an independent confirmatory study) by chemiluminescence using the luminol analogue L-012. Expression of CYBA, NCF4 and RAC2 coding for NAD(P)H oxidase subunits was measured in whole blood cells in 59 study participants by real-time PCR. Of the five variants investigated (−930A>G, 242C>T, 640A>G in CYBA and the recently reported −368G>A in NCF4 and 7508T>A in RAC2), only CYBA 640A>G was consistently associated with superoxide production (640GG carriers 28% less than AA individuals, P=0.05 in each cohort, P=0.005 in combined analysis). RAC2 7508T>A was related to higher expression of RAC2 (P=0.02) and NCF4 (P=0.04). In summary, CYBA 640A>G rather than 242C>T was associated with reduced activity. The quantitatively moderate effect and the high intra-individual variability should be considered for further study design.

Genetic variation in the renal sodium transporters NKCC2, NCC, and ENaC in relation to the effects of loop diuretic drugs.

There is little data on genetic predictors of loop diuretic efficacy in humans. Therefore, we investigated the diuretic effects of single oral doses of bumetanide, frusemide, and torsemide in a crossover study in 97 healthy Caucasians in relation to genetic variation in the renal sodium transporters NKCC2 (coded by SLC12A1), NCC (SLC12A3), and ENaC (three subunits coded by SCNN1A, SCNN1B, and SCNN1G). The NCC alanine 264 allele (Gly264Ala) and the most frequent SCNN1B haplotype were associated with stronger diuresis, indicating lower reabsorbing function of these alleles. The variant alleles of the tightly coupled polymorphisms rs5723 (Leu649Leu) and rs5729 in SCNN1G were associated with weaker diuresis, indicating higher activity. Extended haplotype homozygosity implied evolutionary selection of the NCC alanine 264 allele. In conclusion, acute diuretic effects of loop diuretics were affected by genetic variation in sodium transporters that, in the nephron, are located distally from NKCC2.

A Functional Polymorphism in the NAD(P)H Oxidase Subunit CYBA Is Related to Gene Expression, Enzyme Activity, and Outcome in Non–Hodgkin Lymphoma

NAD(P)H oxidase is a major endogenous source of reactive oxygen species (ROS). ROS may not only be involved in carcinogenesis but also in efficacy of chemotherapeutic agents like doxorubicin. By a comprehensive genotyping approach covering 48 genetic polymorphisms (single-nucleotide polymorphisms) in five subunits of phagocytic NAD(P)H oxidase, we asked whether they affect gene expression, enzymatic activity, and outcome of CHO(E)P chemotherapy. A highly consistent effect was observed for the CYBA 640A>G variant. In peripheral blood granulocytes of 125 healthy volunteers, the G allele of 640A>G was associated with lower NAD(P)H oxidase activity (P = 0.006). Moreover, the G allele was associated with lower mRNA and protein expression (both P = 0.02). Of clinical importance, the outcome of patients suffering from non-Hodgkin lymphoma and treated with CHO(E)P regimen was dependent on the CYBA 640A>G polymorphism. In an exploratory study (n = 401), carriers of 640GG had an event-free survival (EFS) risk ratio of 1.95 [95% confidence interval (95% CI), 1.31-2.90; P = 0.001] compared with 640AA. In a confirmatory set (n = 477), the risk ratios were 1.53 (1.04-2.25, P = 0.03). The complete set of 878 patients showed a relative risk of 1.72 (1.30-2.26) and 1.59 (1.14-2.21) for EFS and overall survival, respectively. Further molecular-biological experiments showed lower expression and reduced stability of transcripts with the G allele in lymphoblastoid cell lines. Transfection of allele-specific plasmids into HEK293 cells elicited lower activity for the G allele in a luciferase reporter gene construct. Thus, CYBA 640A>G was shown to be a functional polymorphism with possible consequences for patients receiving CHO(E)P chemotherapy and might have further implications for other ROS-mediated modalities.

Genetic variation at the CYP2C locus and its association with torsemide biotransformation

In 97 unselected volunteers and two additional homozygous carriers of CYP2C9*3, we investigated the oral clearance of torsemide in relation to 37 polymorphisms at the CYP2C gene locus. Torsemide total oral clearance was linearly associated with the number of CYP2C9*3 alleles (geometric mean: 59, 40 and 20 ml/min in carriers of no, one and two alleles) and so were the methyl- and ring-hydroxylation but not the carboxylation clearance. Haplotypes including the CYP2C9*3 allele were similarly associated with the clearances but no other variant and no haplotype not including the CYP2C9*3 variant. The extended haplotype length (EHL) of the CYP2C9 haplotypes was positively associated with higher activity of the gene product. Torsemide total oral clearance was predictable with r2=82.1% using plasma concentrations at 0.5, 1, 2 and 24 h. In conclusion, torsemides biotransformation strongly depended on the CYP2C9*3 variant but no other. Higher clearance CYP2C9 haplotypes appear to be evolutionarily selected.

Acute Toxicity of Radiochemotherapy in Rectal Cancer Patients: A Risk Particularly for Carriers of the TGFB1 Pro25 variant

PURPOSE Transforming growth factor-beta1 is related to adverse events in radiochemotherapy. We investigated TGFB1 genetic variability in relation to quality of life-impairing acute organ toxicity (QAOT) of neoadjuvant radiochemotherapy under clinical trial conditions. METHODS AND MATERIALS Two independent patient cohorts (n = 88 and n = 75) diagnosed with International Union Against Cancer stage II/III rectal cancer received neoadjuvant radiation doses of 50.4 Gy combined with 5-fluorouracil-based chemotherapy. Toxicity was monitored according to Common Terminology Criteria for Adverse Events. QAOT was defined as a CTCAE grade ≥2 for at least one case of enteritis, proctitis, cystitis, or dermatitis. Nine germline polymorphisms covering the common genetic diversity in the TGFB1 gene were genotyped. RESULTS In both cohorts, all patients carrying the TGFB1 Pro25 variant experienced QAOT (positive predictive value of 100%, adjusted p = 0.0006). In a multivariate logistic regression model, gender, age, body mass index, type of chemotherapy, or disease state had no significant impact on QAOT. CONCLUSION The TGFB1 Pro25 variant could be a relevant marker for individual treatment stratification and carriers may benefit from adaptive clinical care or specific radiation techniques.

Relevance of Sp Binding Site Polymorphism in WWOX for Treatment Outcome in Pancreatic Cancer

Background: A genome-wide association study (GWAS) suggested inherited genetic single-nucleotide polymorphisms (SNPs) affecting overall survival (OS) in advanced pancreatic cancer. To identify robust clinical biomarkers, we tested the strongest reported candidate loci in an independent patient cohort, assessed cellular drug sensitivity, and evaluated molecular effects. Methods: This study comprised 381 patients with histologically verified pancreatic ductal adenocarcinoma treated with gemcitabine-based chemotherapy. The primary outcome was the relationship between germline polymorphisms and OS. Functional assays addressed pharmacological dose-response effects in lymphoblastoid cell lines (LCLs) and pancreatic cancer cell lines (including upon RNAi), gene expression analyses, and allele-specific transcription factor binding. All statistical tests were two-sided. Results: The A allele (26% in Caucasians) at SNP rs11644322 in the putative tumor suppressor gene WWOX conferred worse prognosis. Median OS was 14 months (95% confidence interval [CI] = 12 to 15 months), 13 months (95% CI = 11 to 15 months), and nine months (95% CI = 7 to 12 months) for the GG, GA, and AA genotypes, respectively (P trend < .001 for trend in univariate log-rank assuming a codominant mode of inheritance; advanced disease subgroup P trend < .001). Mean OS was 25 months (95% CI = 21 to 29 months), 19 months (95% CI = 15 to 22 months), and 13 months (95% CI = 10 to 16 months), respectively. This effect held true after adjustment for age, performance status according to Eastern Cooperative Oncology Group classification, TNM, grading, and resection status and was comparable with the strongest established prognostic factors in multivariable analysis. Consistently, reduced responsiveness to gemcitabine, but not 5-fluorouracil, along with lower WWOX expression was demonstrated in LCLs harboring the AA genotype. Likewise, RNAi-mediated WWOX knockdown in pancreatic cancer cells confirmed differential cytostatic drug sensitivity. In electrophoretic mobility shift assays, the A allele exhibited weaker binding of Sp family members Sp1/Sp3. Conclusions: WWOX rs11644322 represents a major predictive factor in gemcitabine-treated pancreatic cancer. Decreased WWOX expression may interfere with gemcitabine sensitivity, and allele-specific binding at rs11644332 might be a causative molecular mechanism behind the observed clinical associations.

Bioinformatic and functional analysis of TGFBR1 polymorphisms.

Objectives The transforming growth factor-&bgr; (TGFB) pathway has substantial impact on cellular functions, cell proliferation, and apoptosis. We used bioinformatics, gene expression, and cell biological assays to evaluate the functionality of frequent inherited germline polymorphisms in the TGFB receptor 1 (TGFBR1). Methods In an exploratory (n=55) and confirmatory (n=106) study, we analyzed the TGFB1 pathway after incubation with TGF&bgr;1 ligand and after exposure to X-rays in peripheral blood human mononuclear cells. Expression of TGFB pathway genes was assessed by real-time PCR, and cellular viability was analyzed by flow cytometry. A total of six polymorphisms including the deletion variant (*6A) were identified to tag currently known common genetic variations in TGFBR1 and were analyzed in relation to the phenotypes. Results In accordance with a negative feedback mechanism, incubations with the ligand TGF&bgr;1 was followed by up-regulation of the intracellular SMAD7 and down-regulation of the SMAD3 mRNA molecules. The TGFBR1*6A deletion variant attenuated the suppression of SMAD3 in response to TGF&bgr;1 (P=0.02, in both studies). Moreover, cells harboring *6A were more sensitive toward cytotoxic effects of irradiation (P=0.001 after adjustment for age and sex). Cells were particularly prone toward radiation toxicity when carrying, in addition to *6A, the variant allele of rs11568785, which exhibits a strong genetic selection signature. Conclusion The *6A deletion and the linked rs11568785 polymorphisms seem to attenuate TGFB signaling. This should be considered not only for clinical–epidemiological studies on cancer susceptibility but may also be relevant for side effects from drugs or radiotherapy.

Genetic polymorphisms in the glucuronosyltransferases 1A1, 1A8 and 1A9 in relation to pharmacokinetics of furosemide

About 30% of the loop diuretic furosemide is eliminated via glucuronidation. According to in vitro data, uridine diphosphate glucuronosyltransferase (UGT) 1A8 may catalyze in this reaction but the quantitatively relevant isoenzyme in humans has not been unambiguously identified. We analyzed whether polymorphisms in UGT1A1, 1A8 and 1A9 are associated with the pharmacokinetics of furosemide.

A novel missense variant in the SDR domain of the WWOX gene leads to complete loss of WWOX protein with early-onset epileptic encephalopathy and severe developmental delay

The human WWOX (WW domain-containing oxidoreductase) gene, originally known as a tumor suppressor gene, has been shown to be important for brain function and development. In recent years, mutations in WWOX have been associated with a wide phenotypic spectrum of autosomal recessively inherited neurodevelopmental disorders. Whole exome sequencing was completed followed by Sanger sequencing to verify segregation of the identified variants. Functional WWOX analysis was performed in fibroblasts of one patient. Transcription and translation were assessed by quantitative real-time PCR and Western blotting. We report two related patients who presented with early epilepsy refractory to treatment, progressive microcephaly, profound developmental delay, and brain MRI abnormalities. Additionally, one of the patients showed bilateral optic atrophy. Whole exome sequencing revealed homozygosity for a novel missense variant affecting the evolutionary conserved amino acid Gln230 in the catalytic short-chain dehydrogenase/reductase (SDR) domain of WWOX in both girls. Functional studies showed normal levels of WWOX transcripts but absence of WWOX protein. To our knowledge, our patients are the first individuals presenting the more severe end of the phenotypic spectrum of WWOX deficiency, although they were only affected by a single missense variant of WWOX. This could be explained by the functional data indicating an impaired translation or premature degradation of the WWOX protein.