Quantitative-fluorescent-PCR versus full karyotyping in prenatal diagnosis of common chromosome aneuploidies in southern Spain.

BACKGROUND: Quantitative-fluorescent polymerase chain reaction (QF-PCR) is a reliable, rapid, and economic technique for prenatal diagnosis of the most common abnormalities. However, conventional karyotyping is expensive and requires a much longer time to yield results. It is currently under debate whether the replacement or restriction of karyotyping reduces the quality of prenatal test results. This study was undertaken to determine the percentage of clinically significant chromosomal abnormalities that would not be detected if QF-PCR was the main analysis method and karyotyping reserved for cases with increased nuchal translucency (NT) and/or abnormal ultrasound findings and to estimate the difference in cost between QF-PCR and full karyotyping. METHODS: Nine hundred twenty-eight pregnant women underwent an invasive procedure at our center between May 2009 and December 2012, yielding 580 (62.5%) chorionic villous samples and 348 (37.5%) amniotic fluid samples. Samples were studied by both QF-PCR and full karyotyping. Karyotyping and detailed ultrasound findings were retrospectively analyzed. RESULTS: If QF-PCR was the main analytic method and full karyotyping reserved for cases with elevated NT (≥4.5) and/or abnormal ultrasound findings, 12.7% of the patients would have required full karyotyping, 99% of the clinically significant chromosomal abnormalities would have been detected, and the cost would have been 54% lower than a policy of full karyotyping for all. CONCLUSIONS: Detailed prenatal ultrasound scan can reduce the need for conventional karyotyping as a complement to QF-PCR in most prenatal samples, offering rapid results and reducing parental anxiety and healthcare costs.

Identification of de novo mutations of Duchénnè/Becker muscular dystrophies in southern Spain.

BACKGROUND: Duchénnè/Becker muscular dystrophies (DMD/BMD) are X-linked diseases, which are caused by a de novo gene mutation in one-third of affected males. The study objectives were to determine the incidence of DMD/BMD in Andalusia (Spain) and to establish the percentage of affected males in whom a de novo gene mutation was responsible. METHODS: Multiplex ligation-dependent probe amplification (MLPA) technology was applied to determine the incidence of DMD/BMD in 84 males with suspicion of the disease and 106 female relatives. RESULTS: Dystrophin gene exon deletion (89.5%) or duplication (10.5%) was detected in 38 of the 84 males by MLPA technology; de novo mutations account for 4 (16.7%) of the 24 mother-son pairs studied. CONCLUSIONS: MLPA technology is adequate for the molecular diagnosis of DMD/BMD and establishes whether the mother carries the molecular alteration responsible for the disease, a highly relevant issue for genetic counseling.

Adjusting the dose of tamoxifen in patients with early breast cancer and CYP2D6 poor metabolizer phenotype.

BACKGROUND: CYP2D6 is a key enzyme in tamoxifen metabolism, transforming it into its main active metabolite, endoxifen. Poor CYP2D6 metabolizers (PM) have lower endoxifen plasma concentrations and possibly benefit less from treatment with tamoxifen. We evaluated tamoxifen dose adjustment in CYP2D6 PM patients in order to obtain plasma concentrations of endoxifen comparable to patients with extensive CYP2D6 metabolism (EM). PATIENTS AND METHODS: Comprehensive CYP2D6 genotyping and plasma tamoxifen metabolite concentrations were performed among 249 breast cancer patients in adjuvant treatment with tamoxifen. Tamoxifen dose was increased in PM patients to 40 mg and to 60 mg daily for a 4-month period each, repeating tamoxifen metabolite measurements on completion of each dose increase. We compared the endoxifen levels between EM and PM patients, and among the PM patients at each dose level of tamoxifen (20, 40 and 60 mg). RESULTS: Eleven PM patients (4.7%) were identified. The mean baseline endoxifen concentration in EM patients (11.30 ng/ml) was higher compared to the PM patients (2.33 ng/ml; p < 0.001). In relation to the 20 mg dose, increasing the tamoxifen dose to 40 and 60 mg in PM patients significantly raised the endoxifen concentration to 8.38 ng/ml (OR 3.59; p = 0.013) and to 9.30 ng/ml (OR 3.99; p = 0.007), respectively. These concentrations were comparable to those observed in EM patients receiving 20 mg of tamoxifen (p = 0.13 and p = 0.64, respectively). CONCLUSION: In CYP2D6 PM patients, increasing the standard tamoxifen dose two-fold or three-fold raises endoxifen concentrations to levels similar to those of patients with EM phenotype.

Influence of CYP2D6 polymorphisms on serum levels of tamoxifen metabolites in Spanish women with breast cancer.

BACKGROUND: Estrogen receptor-positive breast cancer tumors depend on estrogen signaling for their growth and replication and can be treated by anti-estrogen therapy with tamoxifen. Polymorphisms of the CYP2D6 and CYP2C19 genes are associated with an impaired response to tamoxifen. The study objective was to investigate the impact of genetic polymorphisms in CYP2D6 and CYP2C19 on the pharmacokinetics of tamoxifen and its metabolites in Spanish women with estrogen receptor-positive breast cancer who were candidates for tamoxifen therapy. METHODS: We studied 90 women with estrogen receptor-positive breast cancer, using the AmpliChip CYP450 test to determine CYP2D6 and CYP2C19 gene variants. Plasma levels of tamoxifen and its metabolites were quantified by high-performance liquid chromatography. RESULTS: The CYP2D6 phenotype was extensive metabolizer in 80%, intermediate metabolizer in 12.2%, ultra-rapid metabolizer in 2.2%, and poor metabolizer in 5.6% of patients, and the allele frequency was 35.0% for allele (*)1, 21.0% for *2, and 18.9% for *4. All poor metabolizers in this series were *4/*4, and their endoxifen and 4-hydroxy tamoxifen levels were 25% lower than those of extensive metabolizers. CYP2C19*2 allele, which has been related to breast cancer outcomes, was detected in 15.6% of the studied alleles. CONCLUSION: CYP2D6*4/*4 genotype was inversely associated with 4-hydroxy tamoxifen and endoxifen levels. According to these results, CYP2D6 and CYP2C19 genotyping appears advisable before the prescription of tamoxifen therapy.

Gene expression signatures in breast cancer distinguish phenotype characteristics, histologic subtypes, and tumor invasiveness.

BACKGROUND: The development of reliable gene expression profiling technology is having an increasing impact on the understanding of breast cancer biology. METHODS: In this study, microarray analysis was performed to establish gene signatures for different breast cancer phenotypes, to determine differentially expressed gene sequences at different stages of the disease, and to identify sequences with biologic significance for tumor progression. Samples were taken from patients before their treatment. After microarray analysis, the expression level of 153 selected genes was studied by real-time quantitative polymerase chain reaction analysis. RESULTS: Several gene sequences were expressed differentially in tumor samples versus control samples and also were associated with different breast cancer phenotypes, estrogen receptor status, tumor histology, and grade of tumor differentiation. In lymph node-negative tumors were identified a set of genes related to tumor differentiation grade. CONCLUSIONS: Several differentially expressed gene sequences were identified at different stages of breast cancer.

Semiquantitative RT-PCR method coupled to capillary electrophoresis to study 5alpha-reductase mRNA isozymes in rat ventral prostate in different androgen status.

The development and growth of the prostate gland depends on androgen stimulation. Dihydrotestosterone (DHT) is the primary androgen responsible for prostate development and also for the pathogenesis of benign prostatic hyperplasia (BPH). The incidence of prostate cancer (PCa) and benign prostatic hypertrophy (BPH) continues to rise in the Western world. DHT is synthesized in prostate from circulating testosterone (T) through the action of 5alpha-Reductase (5alpha-R) (EC 1.3.99.5), which occurs as two isozymes, type 1 and type 2. Type-1 5alpha-R is widely distributed in the body, and type-2 5alpha-R is confined to androgen-dependent structures. Both types are expressed in the prostate: type-2 isozyme is implicated in BPH and PCa; type-1 isozyme is also increased in some prostatic adenocarcinomas. In recent years, various inhibitors of type-2 isozyme or of both type-1 and type-2 isozyme have been used in prostatic diseases. In this work we present measurements of mRNA levels of steroid 5alpha-R isozymes in the ventral prostate of rats of different androgen status. We used a novel method that combines the high specificity of semiquantitive PCR with the sensitivity of laser-induced fluorescence capillary electrophoresis (LIF-CE). We demonstrated that T control the expression of 5alpha-R2 isozyme in rat prostrate. This approach could be of great value for the study of prostate diseases in humans and would allow study at the transcriptional level the effects of drugs that inhibit either or both of these isozymes.