In the Balkan and Taiwan, the relationship between exposure to aristolochic acid and risk of urothelial neoplasms was inferred from the A>T genetic hallmark in TP53 gene from malignant cells. This study aimed to characterize the TP53 mutational spectrum in urothelial cancers consecutive to Aristolochic Acid Nephropathy in Belgium. Serial frozen tumor sections from female patients (n = 5) exposed to aristolochic acid during weight-loss regimen were alternatively used either for p53 immunostaining or laser microdissection. Tissue areas with at least 60% p53-positive nuclei were selected for microdissecting sections according to p53-positive matching areas. All areas appeared to be carcinoma in situ. After DNA extraction, mutations in the TP53 hot spot region (exons 5-8) were identified using nested-PCR and sequencing. False-negative controls consisted in microdissecting fresh-frozen tumor tissues both from a patient with a Li-Fraumeni syndrome who carried a p53 constitutional mutation, and from KRas mutated adenocarcinomas. To rule out false-positive results potentially generated by microdissection and nested-PCR, a phenacetin-associated urothelial carcinoma and normal fresh ureteral tissues (n = 4) were processed with high laser power. No unexpected results being identified, molecular analysis was pursued on malignant tissues, showing at least one mutation in all (six different mutations in two) patients, with 13/16 exonic (nonsense, 2; missense, 11) and 3/16 intronic (one splice site) mutations. They were distributed as transitions (n = 7) or transversions (n = 9), with an equal prevalence of A>T and G>T (3/16 each). While current results are in line with A>T prevalence previously reported in Balkan and Taiwan studies, they also demonstrate that multiple mutations in the TP53 hot spot region and a high frequency of G>T transversion appear as a complementary signature reflecting the toxicity of a cumulative dose of aristolochic acid ingested over a short period of time.

Background and Aim: Previous data indicate that the urinary losartan/E-3174 ratio is a marker for cytochrome P450 (CYP) 2C9 activity in vivo. The functional impact of CYP2C9(star)5, (star)6, (star)8, and (star)11 polymorphisms in vivo has not been investigated previously in humans.Methods: A single oral dose of losartan (25 mg) was given to 19 Beninese subjects with CYP2C9(star)1/(star)1 (n = 9), (star)1/(star)5 (n = 1), (star)1/(star)6 (n = 1), (star)1/(star)8 (n = 2), (star)1/(star)11 (n = 3), (star)5/(star)6 (n = 1), (star)5/(star)8 (n = 1), and (star)8/(star)11 (n = 1) genotypes. Concentrations of losartan and its active metabolite E-3174 were determined in urine from 0 to 8 hours by HPLC. The losartan/E-3174 metabolic ratio was used as a measure of losartan oxidation in vivo.Results: The urinary losartan/E-3174 ratio in the various genotypes was as follows: 1.85 +/- 2.4 (mean +/- SD) for CYP2C9(star)1/(star)1, 14.6 for CYP2C9(star)1/star5, 4.2 for CYP2C9(star)1/(star)6, 188 for CYP2C9(star)5/(star)6, 11.6 for CYP2C9(star)5/(star)8, 0.44 +/- 0.13 (mean +/- SD) for CYP2C9(star)1/(star)8, 2.2 for CYP2C9(star)8/(star)11, and 5.72 +/- 4.5 (mean +/- SD) for CYP2C9(star)1/(star)11. Compared with the CYP2C9(star)1/(star)1 genotypes, the losartan/E-3174 ratio was significantly different in the CYP2C9(star)5 allele carriers (CYP2C9(star)1/(star)5, CYP2C9(star)5/(star)8, and CYP2C9(star)5/(star)6 genotypes) (P = .01, Mann-Whitney) but was not different in CYP2C9(star)1/(star)8 (P = .16) and CYP2C9(star)1/(star)11 (P = .11) carriers. The urinary losartan/E-3174 ratio of the single CYP2C9(star)1/(star)6 subject was higher than the 95% confidence interval of the mean of the CYP2C9(star)1/(star)1 group (0.0-3.7), whereas the metabolic ratio of the CYP2C9(star)8/(star)11 carrier was inside the 95% confidence interval of the means of the CYP2C9(star)1/(star)1 and CYP2C9(star)1/(star)11 groups (0.0-18).Conclusions: The CYP2C9(star)5 and (star)6 alleles are associated with decreased enzyme activity in vivo compared with the wild-type variant, whereas the CYP2C9(star)8 and (star)11 variants did not appear to have large in vivo effects.

UNLABELLED:Background and aim Previous data indicate that the urinary losartan/E-3174 ratio is a marker for cytochrome P450 (CYP) 2C9 activity in vivo. The functional impact of CYP2C9*5, *6, *8, and *11 polymorphisms in vivo has not been investigated previously in humans.METHODS:A single oral dose of losartan (25 mg) was given to 19 Beninese subjects with CYP2C9*1/*1 (n = 9), *1/*5 (n = 1), *1/*6 (n = 1), *1/*8 (n = 2), *1/*11 (n = 3), *5/*6 (n = 1), *5/*8 (n = 1), and *8/*11 (n = 1) genotypes. Concentrations of losartan and its active metabolite E-3174 were determined in urine from 0 to 8 hours by HPLC. The losartan/E-3174 metabolic ratio was used as a measure of losartan oxidation in vivo.RESULTS:The urinary losartan/E-3174 ratio in the various genotypes was as follows: 1.85 +/- 2.4 (mean +/- SD) for CYP2C9*1/*1, 14.6 for CYP2C9*1/*5, 4.2 for CYP2C9*1/*6, 188 for CYP2C9*5/*6, 11.6 for CYP2C9*5/*8, 0.44 +/- 0.13 (mean +/- SD) for CYP2C9*1/*8, 2.2 for CYP2C9*8/*11, and 5.72 +/- 4.5 (mean +/- SD) for CYP2C9*1/*11. Compared with the CYP2C9*1/*1 genotypes, the losartan/E-3174 ratio was significantly different in the CYP2C9*5 allele carriers (CYP2C9*1/*5, CYP2C9*5/*8, and CYP2C9*5/*6 genotypes) (P =.01, Mann-Whitney) but was not different in CYP2C9*1/*8 (P =.16) and CYP2C9*1/*11 (P =.11) carriers. The urinary losartan/E-3174 ratio of the single CYP2C9*1/*6 subject was higher than the 95% confidence interval of the mean of the CYP2C9*1/*1 group (0.0-3.7), whereas the metabolic ratio of the CYP2C9*8/*11 carrier was inside the 95% confidence interval of the means of the CYP2C9*1/*1 and CYP2C9*1/*11 groups (0.0-18).CONCLUSIONS:The CYP2C9*5 and *6 alleles are associated with decreased enzyme activity in vivo compared with the wild-type variant, whereas the CYP2C9*8 and *11 variants did not appear to have large in vivo effects.

β-Thalassemia is characterized by the reduced production of β-globin chains as a result of mutations in the β-globin gene (1). This reduction is predictable when mutations occur in the coding sequence, but not when they occur in the 5′- and 3′-untranslated regions (UTRs), the locus control region (LCR), the promoter, or the introns. Whether such mutations are involved in the reduction of the β-globin chain production or are simple polymorphisms cannot always be inferred from clinical data. Transient transfection studies with a β-globin promoter and an heterologous reporter gene have shown that promoter mutations can decrease transcription (2) and are then associated with the β-thalassemia phenotype, as illustrated by the −30T→A mutation (3). However, such studies have often failed to provide clear-cut data regarding the transcriptional effect of a mutation or a deletion occurring in a noncoding sequence (4), and quantitative data are lacking. To bypass these limitations and to mimic as closely as possible the regulatory mechanisms of β-human globin gene expression in vivo, we created a construct (pBLG), in which the entire human β-globin gene was cloned behind the β-μLCR. Whereas previous assays used constructs bearing HS2 as a single LCR enhancer element (5)(6), we used the entire β-μLCR because it has been shown that the other three HS elements play also a key role in β-globin transcription (7)(8)(9)(10)(11). Nucleotides changes in various untranscribed or untranslated parts of the β-globin gene representing thalassemic mutations or deletions were introduced in the construct. All the mutations assessed in our study were found in members of proband families presenting with β-thalassemia or were created by directed mutagenesis. In addition to the wild type, variant pBLG constructs carrying the following mutations were generated: −101C→T, +20C→T, IVS-I-108T→C, and IVS-I-110G→A mutations (12) …

The molecular basis of beta-thalassemia was investigated at the DNA level in 28 Belgians from 14 unrelated families. All the patients were heterozygous for beta-thalassaemia. Seven different mutations were identified using a combination of dot-blot hybridization with allele-specific oligonucleotide probes and direct automated fluorescence-based DNA sequencing. Among these mutations, four are commonly found in the Mediterraneans - codon 8 (-AA), IVS-I-1 (G --> A), IVS-I-6 (T --> C) and codon 39 (C --> T)-and two have occasionally been reported-initiation codon (T --> C) and codon 35 (C --> A). The last mutation, a -CC deletion at codons 38/39, appears to be a novel mutation and can routinely be investigated by AvaII restriction on amplified DNA. We report our findings, discuss the diversity of the mutations found in Belgium and show the usefulness of direct DNA sequencing in a population in which the molecular defects of beta-thalassaemia have yet to be characterized and in which screening is hampered by the wide range of potential mutations.

The construction of a new wide-host-range, restriction-site bank, cosmid-cloning vehicle (pJRD215) is described. The wide-host-range properties and the ability to be transferred by conjugation, extend genetic engineering to those Gram-negative species that cannot be transformed. The vector permits the cloning of genes from Gram-negative bacteria using a complementation screening procedure in a mutant host. This procedure is simplified by the possibility of construction of a cosmid gene bank so that only a few hundred clones need to be screened. Subsequent subcloning of the gene of interest is facilitated by the presence of at least 23 unique cloning sites.

An improved restriction site bank vector has been constructed from plasmid pJRD158. The new version is smaller and contains 43 unique restriction sites. It should greatly facilitate cloning versatility by providing unique sites for most commercially available restriction enzymes.

An improved restriction site bank vector has been constructed from plasmid pJRD158. The new version is smaller and contains 43 unique restriction sites. It should greatly facilitate cloning versatility by providing unique sites for most commercially available restriction enzymes.