Warfarin is an anticoagulant drug with narrow therapeutic index and high interindividual variability in dose requirement. S-warfarin is metabolized mainly by polymorphic cytochrome P450 (CYP) 2C9. We systematically quantified the influence of CYP2C9 genotype, demographic factors and concomitant drug treatment on warfarin metabolism and maintenance dose. The mean warfarin doses were lower in carriers of one (2.71 mg/day, 59 patients) and two polymorphic alleles (1.64 mg/day, 11 patients) than in carriers of two wild-type alleles (4.88 mg/day, 118 patients). Multiple regression analysis demonstrated that CYP2C9 genotype, age, concomitant treatment with warfarin metabolism inducers and lean body weight contributed significantly to interindividual variability in warfarin dose requirement (adjusted R 2 ¼ 0.37). The same factors, except for age, significantly influenced S-warfarin clearance (adjusted R 2 ¼ 0.42). These results can serve as a starting point for designing prospective studies in patients in the initiation phase of genotype-based warfarin therapy.

The degree of interpatient variability in the warfarin dose required to achieve the desired anticoagulant response can only partly be explained by polymorphisms in the CYP2C9 gene, suggesting that additional genetic factors such as polymorphisms in genes involved in blood coagulation may influence warfarin dose requirement. In total, 165 Caucasian outpatients on stable maintenance warfarin treatment previously genotyped for CYP2C9 were analysed for common polymorphisms in FVII, GGCX and VKORC1 genes. The -402G > A polymorphism and a variable number of repeats in intron 7 of FVII gene did not significantly influence warfarin dose. The mean warfarin doses increased with the number of (CAA) repeats in the GGCX gene, but the differences were significant only in the CYP2C9*1/*1 subgroup of patients (p = 0.032). Common polymorphism (6484C > T) in intron 1 of the VKORC1 gene led to lower warfarin dose requirement; the means were 5.70 (95% C.I. 4.95-6.45), 3.49 (3.07-3.90) and 2.11 (1.80-2.42) mg/day for 6484 CC, CT and TT genotypes, respectively (p < 0.001). In contrast, 9041G > A polymorphism in 3'UTR of theVKORC1 gene led to higher warfarin dose requirement; the means were 3.09 (2.58- 3.60), 4.26 (3.69-4.82) and 5.86 (4.53-7.19) mg/day for 9041 GG, GA and AA genotypes, respectively (p < 0.001). With a regression model we explained 60.0% of variability in warfarin dose, which was due to gene polymorphisms (CYP2C9, VKORC1), age and body-surface-area. When aiming for individualised warfarin therapy, at least VKORC1 polymorphisms should be included in predictive genotyping besides CYP2C9.

We confirmed important interaction between carbamazepine and warfarin metabolism which can be of major clinical importance. If treatment with carbamazepine cannot be avoided, patients taking warfarin should be frequently monitored, especially when initiating or stopping carbamazepine therapy.

Objective: To analyse the mutational spectrum, the associated haplotypes and the genotype -phenotype correlation, and to design a reliable and rational approach for CYP21 mutation detection in Slovenian congenital adrenal hyperplasia (CAH) patients. Design: Molecular analysis of the CYP21 gene was performed in 36 CAH patients and 79 family members. Methods: Southern blotting, sequence-specific PCR amplification (PCR-SSP), sequence-specific oligonucleotide hybridisation (PCR-SSO) and sequencing were used to detect CYP21 gene deletions, conversions and point mutations. Results: CYP21 gene deletion was the most frequent mutation (36.4%). Large gene conversions detectable only by Southern blotting represented 12.1%, and gene conversions involving the promoter region represented 7.6% of the mutated alleles. The most frequent point mutations were: intron 2 splice mutation 16.7%, Ile172Asn mutation 7.6%, Gln318Stop 7.5% and Pro30Leu 12.2% of alleles. A correlation between the genotype and the clinical phenotype similar to those described for large populations was observed. The finding of Pro30Leu mutation linked to a gene conversion could explain the simple virilising (SV) phenotype in compound heterozygotes for the Pro30Leu and a severe mutation. In two siblings with a salt wasting form of CAH (SW-CAH), a novel mutation Ala15Thr was found on the allele characterised by Pro30Leu mutation and gene conversion involving the promoter region. Conclusions: Our genotyping approach allowed reliable diagnosis of CAH in the Slovenian population. The high frequency of CYP21 gene aberrations on Pro30Leu positive alleles justified systematic searching for a gene conversion in the promoter region using the PCR-SSP reaction.

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