Multiplex ligation-dependent probe amplification for detection of genomic alterations in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is the commonest form of leukaemia in adults in Western countries. We performed multiplex ligation-dependent probe amplification (MLPA) analysis in 50 CLL patients to identify multiple genomic CLL-specific targets, including genes located at 13q14, 17p13 (TP53), 11q23 (ATM) and chromosome 12, and compared the results with those obtained with fluorescence in situ hybridization (FISH). There was a good correlation between MLPA and FISH results, as most alterations (89%) were detected by both techniques. Only three cases with a low percentage (<25%) of cells carrying the alterations were not detected by MLPA. On the other hand, as MLPA uses multiple probes it identified intragenic or small alterations undetected by FISH in three cases. MLPA also detected alterations in 8q24 (MYC) and 6q25-26. In summary, unlike interphase FISH, MLPA enabled the simultaneous analysis of many samples with automated data processing at a low cost. Therefore, the combination of robust multiplexing and high throughput makes MLPA a useful technique for the analysis of genomic alterations in CLL.

Association of CYP2D6 ultrarapid metabolizer genotype with deficient patient satisfaction regarding methadone maintenance treatment.

OBJECTIVE: The activity of cytochrome P-450 enzyme 2D6 (CYP2D6) could be related to heroin-dependent patient satisfaction with methadone maintenance treatment. We sought to compare satisfaction with the usual methadone treatment in patients who are ultrarapid, extensive or poor metabolizers, according to CYP2D6 genotyping. METHODS: Two hundred and five heroin-dependent patients filled out the Verona Service Satisfaction Scale for methadone maintenance treatment (VSSS-MT), before CYP2D6 genotyping. RESULTS: VSSS-MT overall scores were comparable in the poor metabolizer (N=9) and extensive metabolizer (N=185) groups, although they were higher in poor metabolizers and extensive metabolizers taken together than in the ultrarapid metabolizers (N=11) (p<0.003). Likewise, ultrarapid metabolizers scored higher than the rest of the sample on the VSSS-MT Basic Interventions subscale (p<001). Regarding this subscale, no poor metabolizers felt dissatisfied, and ultrarapid metabolizer males (N=7) reported lower satisfaction than ultrarapid metabolizer females (N=4) (p<0.022). Ultrarapid metabolizer genotype accounted for 4.2% of the variance on the VSSS-MT total scores, and 5.0% on the Basic Intervention scores. CONCLUSION: Heroin-dependent patients who are CYP2D6 ultrarapid metabolizers according to genotyping present deficient satisfaction with methadone maintenance treatment.

Characterization of variant alleles of cytochrome CYP2D6 in a Spanish population.

The CYP2D6 gene codes for a P450 monooxygenase which is involved in the biotransformation of a large number of commonly prescribed drugs. Adverse drug effects and therapeutic failure can be related to abnormal CYP2D6 activity. We investigated the allele and genotype frequencies of cytochrome P4502D6 in a Spanish population to predict the prevalence of ultra-rapid and poor metabolizer phenotypes in our population and to design a feasible CYP2D6 genotyping protocol. The study included 105 healthy unrelated Spanish Caucasian volunteers. CYP2D6 genotyping was performed by a combination of long-PCR, direct sequencing and allele-specific real-time PCR. The frequency of the wild-type CYP2D6*1 allele was 31%. The alleles coding for slightly (CYP2D6*2) or moderately (*9 and *10) reduced activity showed frequencies of 40.47, 2.38 and 1.90%, respectively. Frequencies of defective alleles *3, *4, *5 and *6 were 0.95, 13.8, 3.33 and 0.95%, respectively. The defective CYP2D6 alleles *7, *8, *12, *14, *15 and *21 were not found. Duplicated CYP2D6 alleles were detected at a frequency of 4.27%. Our protocol allows the identification of the four inactive CYP2D6 alleles (*3, *4, *5 and *6) and the detection of alleles with CYP2D6 *1, CYP2D6 *2 and CYP2D6*4 gene duplications. Testing for this reduced CYP2D6 allele set would facilitate its use in clinical practice by assisting in the development of individualized pharmacotherapy.

Single nucleotide polymorphism in the 5' tandem repeat sequences of thymidylate synthase gene predicts for response to fluorouracil-based chemotherapy in advanced colorectal cancer patients.

Thymidylate synthase (TS) is the primary target of 5-fluorouracil (5-FU). A VNTR polymorphism in the TS promoter region is associated with the efficacy of 5-FU-based chemotherapy in colorectal cancer. A common G>C SNP at the 12th nucleotide of the second repeat in the TS*3 alleles has been recently described. The combination of SNP and VNTR allows the definition of 3 TS alleles: *2, *3G and *3C. The aim of our study was to evaluate the predictive value of clinical response and survival of these new defined TS alleles. TS genotypes of 89 patients diagnosed with metastatic colorectal cancer and undergoing 5-FU-based chemotherapy were carried out. The clinical outcome was evaluated according to the genotype (high expression genotype: *2R/*3G; *3C/*3G; *3G/*3G; and low expression genotype: *2R/*2R; *2R/*3C; *3C/*3C. A higher overall response was observed in the group of patients with a low expression genotype (p = 0.035). The probability of achieving a clinical response of patients with a low expression-related genotype was 2.9 higher than that of the other group (95% CI = 1.03-5.6, p = 0.04). The median time to progression was 12 months and 9 months in the low and high expression groups, respectively (p = 0.07, log rank test). Overall survival was significantly longer in the low expression group. In this group the median OS was not achieved at 50 months of follow-up in contrast to the 20 months observed in the high expression group (p = 0.03). TS genotype was an independent predictor of progression-free and overall survival in the Cox regression models after adjustment to the other clinical variables. The selection of patients who are likely to respond to 5-FU therapy may be considerably improved if the TS genotype were to include both the VNTR and the SNP located within the promoter region of the gene.

Hormone-specific regulation of the kidney androgen-regulated gene promoter in cultured mouse renal proximal-tubule cells.

The kidney androgen-regulated protein (KAP) is specifically expressed and differentially regulated by androgens and tri-iodothyronine (T(3)) in intact mouse early (PCT) and late (PR) proximal-tubule cells. Until now, detailed characterization of the molecular elements mediating androgen-responsive gene expression in the kidney has been hampered by the lack of appropriate cultured cell systems suitable for DNA transfection studies. In the present study we have analysed the hormone-dependent transactivation of the KAP gene promoter in immortalized differentiated PCT and PR proximal-tubule cells derived from L-PK/Tag1 transgenic mice. Transient transfection studies with different KAP promoter constructs indicated that a 224 bp-truncated fragment was sufficient to mediate cell-specific expression of the KAP promoter. Dihydrotestosterone (DHT) stimulated in an androgen-dependent manner the transactivation of KAP in PCT and PR cells, while mutation of a putative androgen-response element (ARE) sequence located at -39 bp from the transcription initiation site abolished the transactivation induced by DHT. Furthermore, insulin-like growth factor 1 (IGF-1), but not T(3), enhanced the androgen-dependent transactivation of KAP in cultured PCT cells. These results demonstrate that the short 224 bp fragment of the KAP promoter is sufficient to drive the proximal-tubule androgen-specific regulated expression of KAP and reveal synergistic interactions between IGF-1 and androgens for KAP regulation in PCT cells.