A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) has a prevalence of over 20% in Western societies. Affected individuals are at risk of developing both cirrhosis and hepatocellular cancer (HCC). Presently there is no cost effective population based means of identifying cirrhotic individuals and even if there were, our ability to perform HCC surveillance in the at risk group is inadequate. We have performed a pilot proteomic study to assess this as a strategy for serum biomarker detection. METHODS: 2D Gel electrophoresis was performed on immune depleted sera from 3 groups of patients, namely those with (1) pre-cirrhotic NAFLD (2) cirrhotic NAFLD and (3) cirrhotic NAFLD with co-existing HCC. Five spots differentiating at least one of these three groups were characterised by mass spectroscopy. An ELISA assay was optimised and a cross sectional study assessing one of these serum spots was performed on serum from 45 patients with steatohepatitis related cirrhosis and HCC and compared to 77 patients with histologically staged steatohepatitis. RESULTS: Four of the spots identified were apolipoprotein isoforms, the pattern of which was able to differentiate the three groups. The 5th spot, seen in the serum of cirrhotic individuals and more markedly in those with HCC, was identified as CD5 antigen like (CD5L). By ELISA assay, although CD5L was markedly elevated in a number of cirrhotic individuals with HCC, its overall ability to distinguish non-cancer from cancer individuals as determined by AUC ROC analysis was poor. However, serum CD5L was dramatically increased, independently of age, sex, and the presence of necroinflammation, in the serum of individuals with NAFLD cirrhosis relative to those with pre-cirrhotic disease. CONCLUSION: This novel proteomic strategy has identified a number of candidate biomarkers which may have benefit in the surveillance and diagnosis of individuals with chronic liver disease and/or HCC.

Contribution of CYP2C9 to variability in vitamin K antagonist metabolism.

CYP2C9 is the third most important cytochrome P450 (CYP) in terms of number of drugs metabolised. A considerable amount of information on this isoform is now available with respect to its structural biology, the mechanisms by which it can be induced and the existence of a range of variant alleles, which are often functionally significant. CYP2C9 makes a very important contribution to metabolism of vitamin K antagonist anticoagulants, and is the main oxidising enzyme for S-warfarin and S-acenocoumarol as well as contributing to phenprocoumon metabolism. A large number of studies have now shown that CYP2C9 genotype predicts dose requirement for both warfarin and acenocoumarol, with a possible contribution for phenprocoumon. Patients with variant alleles are likely to require a lower dose and may be at risk of overcoagulation and resultant bleeding, especially during the induction phase of therapy. Although CYP2C9 genotype is clearly a predictor of vitamin K antagonist dose requirement, especially in Caucasian populations in whom variant alleles are common, a number of recent studies have shown that age, genotype for the gene encoding the target gene vitamin K epoxide reductase and concomitant drugs are equally important factors in determining dose. There is a need for prospective studies to assess the value of predicting dose requirement on the basis of all these factors, including the CYP2C9 genotype.

Genotyping for cytochrome P450 polymorphisms.

Protocols for the extraction of DNA from human blood and for genotyping for a number of common cytochrome P450 polymorphisms using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR-single-strand conformational polymorphism (SSCP) analysis are described. Rapid high-throughput techniques are also available for analyses of this type, but they require access to specialized equipment and are not considered here. General guidelines for performing amplification using PCR are described together with electrophoresis protocols for analysis of restriction digests of PCR products with agarose and polyacrylamide gels including the use of polyacrylamide-based gels for SSCP analysis. Protocols for the following specific isoforms and alleles are also provided: CYP1A1 (*2B and *4 alleles), CYP2C8 (*3 and *4 alleles), CYP2C9 (*2, *3, and *11 alleles), CYP2C19 (*2 and *3 alleles), CYP2D6 (*3, *4, *5, and *6 alleles), CYP2E1 (*5A, *5B, and *6 alleles), and CYP3A5 (*3 allele).

The impact of CYP2C9 and VKORC1 genetic polymorphism and patient characteristics upon warfarin dose requirements: proposal for a new dosing regimen.

Current dosing algorithms do not account for genetic and environmental factors for warfarin dose determinations. This study investigated the contribution of age, CYP2C9 and VKORC1 genotype, and body size to warfarin-dose requirements. Studied were 297 patients with stable anticoagulation with a target international normalized ratio (INR) of 2.0 to 3.0. Genetic analyses for CYP2C9 (*2 and *3 alleles) and VKORC1 (-1639 polymorphism) were performed and venous INR and plasma R- and S-warfarin concentrations determined. The mean warfarin daily dose requirement was highest in CYP2C9 homozygous wild-type patients, compared with those with the variant *2 and *3 alleles (P < .001) and highest in patients with the VKORC1 (position -1639) GG genotype compared with those with the GA genotype and the AA genotype (P < .001). Mean warfarin daily dose requirements fell by 0.5 to 0.7 mg per decade between the ages of 20 to 90 years. Age, height, and CYP2C9 genotype significantly contributed to S-warfarin and total warfarin clearance, whereas only age and body size significantly contributed to R-warfarin clearance. The multivariate regression model including the variables of age, CYP2C9 and VKORC1 genotype, and height produced the best model for estimating warfarin dose (R2 = 55%). Based upon the data, a new warfarin dosing regimen has been developed. The validity of the dosing regimen was confirmed in a second cohort of patients on warfarin therapy.

Upstream and coding region CYP2C9 polymorphisms: correlation with warfarin dose and metabolism.

OBJECTIVES: To assess whether CYP2C9 alleles other than CYP2C9*2 and *3 are associated with a low-warfarin dose requirement and the relevance of upstream CYP2C9 polymorphisms to dose requirement and metabolism. METHODS: CYP2C9 exons, intron-exon boundaries and 3 kb of upstream sequence in 20 patients requiring or= 0.01, was obtained. In individuals negative for coding region polymorphisms, neither individual genotypes for T-1188C or DeltaG-2664DeltaT-2665 or particular combinations of haplotype pairs were predictive of dose requirement or S-warfarin total clearance, suggesting neither upstream polymorphism was functionally significant. Dose requirements in CYP2C9*11 heterozygotes were not statistically significantly different from homozygous wild-type individuals. CONCLUSIONS: The coding region non-synonymous polymorphisms associated with the CYP2C9*2 and CYP2C9*3 alleles are the major CYP2C9-related factor affecting warfarin dose in UK Caucasians. Upstream CYP2C9 polymorphisms do not appear to be important independent determinants of dose requirement.

Contribution of age, body size, and CYP2C9 genotype to anticoagulant response to warfarin.

OBJECTIVE: Our objective was to assess the contribution of CYP2C9 genotype, age, body size, and vitamin K and lipid status to warfarin dose requirements. METHODS: Patients with stable warfarin dose requirements and an international normalized ratio (INR) of the prothrombin time within the target range of 2.0 to 3.0 were recruited to the study. On arrival at the clinic in the morning, after an overnight fast, a blood sample was taken from each patient for CYP2C9 genotyping and for determination of venous INR and plasma vitamin K, R- and S-warfarin, and triglyceride concentrations. RESULTS: A total of 121 patients were recruited to the study. CYP2C9 genotyping showed that 74 patients were homozygous wild-type (*1/*1), 30 were heterozygous *1/*2, and 15 were heterozygous *1/*3 genotype. One patient was found to have the genotype *2/*3, and another was found to have the genotype *3/*3. The mean warfarin daily dose requirement in milligrams fell from 4.06 +/- 1.72 mg in homozygous wild-type patients to 3.63 +/- 1.78 mg for *1/*2-positive patients and 2.70 +/- 1.36 mg for *1/*3-positive patients. The multiple linear regression model for warfarin dose indicated significant contributions from age (r = 0.41, P <.001), genotype (r = 0.24, P <.005), and age and genotype together (r = 0.45, P <.005). Although there were significant linear correlations between warfarin dose and body surface area (r = 0.21, P =.02), body weight (r = 0.25, P =.005), and plasma vitamin K concentration (r = 0.18, P <.05), none of these variables made a significant contribution to the regression model for warfarin dose. CYP2C9 genotype had a significant effect on S-warfarin clearance (r = 0.34, P <.0001) but none on R-warfarin clearance. CONCLUSION: This study showed that age and CYP2C9 polymorphism affect warfarin dose requirements in patients receiving long-term therapy and having stable control of anticoagulation. It is anticipated that using dosing regimens modified to take into account the contribution of age and CYP2C9 genotype has the potential to improve the safety of warfarin therapy.

CYP3A5 phenotype-genotype correlations in a British population.

AIMS: To develop a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP)-based assay to genotype for hepatic CYP3A5 expression and to use this assay to study a British population. METHODS: CYP3A5-specific primers were designed with one including a base-pair mismatch to create a RsaI site in samples positive for G6986 (CYP3A5*3 allele) [correction]. Following PCR and RsaI digestion, different band patterns on electrophoresis were predicted for individuals positive for CYP3A5 (CYP3A5*1 allele) compared with those who do not express the gene (CYP3A5*3 homozygotes). The assay was validated by DNA sequencing. DNA samples from a human liver bank consisting of 22 livers whose CYP3A5 expression had been determined by immunoblotting and a group of random individuals (n = 100) from the North-east of England were genotyped by the new assay. RESULTS: In the liver bank, five out of 22 samples expressed CYP3A5 at significant levels (>20 pmol mg-1 protein) and were found to have the genotype CYP3A5*1/CYP3A5*3 by the PCR-RFLP assay. All other liver DNA samples were CYP3A5*3 homozygotes. In the group of 100 random individuals, 13 had the genotype CYP3A5*1/CYP3A5*3 and all others were CYP3A5*3 homozygotes, predicting that 13% (95% confidence interval (CI) 6%, 20%) would show significant hepatic CYP3A5 expression. The frequency for the CYP3A5*1 allele was 0.065 (95% CI 0.032, 0.097). CONCLUSIONS: We have developed a simple assay for the detection of the CYP3A5*1/CYP3A5*3 alleles and shown that in a British population their frequency is similar to that reported previously. We have also shown a good correlation between hepatic CYP3A5 expression and genotype for a British Caucasian liver bank.

Pharmacogenetics of oral anticoagulants.

There is wide interindividual variation in oral anticoagulant dose requirement, which is partly genetically determined. Several cytochrome P450s contribute to oxidative metabolism of oral anticoagulants. The most important of these is CYP2C9, which hydroxylates the S-enantiomers of warfarin, acenocoumarol and phenprocoumon with high catalytic activity. In at least eight separate clinical studies, possession of the CYP2C9*2 or CYP2C9*3 variant alleles, which result in decreased enzyme activity, has been associated with a significant decrease in a mean warfarin dose requirement. Several studies also suggest that possession of a CYP2C9 variant allele is associated with an increased risk of adverse events, such as bleeding. Possession of the CYP2C9*3 variant also appears to be associated with a low acenocoumarol dose requirement. Other genetic factors, such as polymorphisms in the cytochromes P450 that metabolize the R-enantiomers of warfarin and acenocoumarol, may also be relevant to anticoagulant dose. The molecular basis of anticoagulant resistance where a higher than normal dose of anticoagulant is required remains unclear, but could be due to unusually high CYP2C9 activity (pharmacokinetic resistance) or to an abnormality in the target enzyme vitamin K epoxide reductase (pharmacodynamic resistance).