Jose Antonio Gómez-Capilla

Gene expression profiling reveals novel biomarkers in nonsmall cell lung cancer

The development of reliable gene expression profiling technology is having an increasing impact on our understanding of lung cancer biology. Our study aimed to determine any correlation between the phenotypic heterogeneity and genetic diversity of lung cancer. Microarray analysis was performed on a set of 46 tumor samples and 45 paired nontumor samples of nonsmall cell lung cancer (NSCLC) samples to establish gene signatures in primary adenocarcinomas and squamous‐cell carcinomas, determine differentially expressed gene sequences at different stages of the disease and identify sequences with biological significance for tumor progression. After the microarray analysis, the expression level of 92 selected genes was validated by qPCR and the robust Bonferroni test in an independent set of 70 samples composed of 48 tumor samples and 22 nontumor samples. Gene sequences were differentially expressed as a function of tumor type, stage and differentiation grade. High upregulation was observed for KRT15 and PKP1, which may be good markers to distinguish squamous‐cell carcinoma samples. High downregulation was observed for DSG3 in stage IA adenocarcinomas.

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

The development of reliable gene expression profiling technology is having an increasing impact on the understanding of breast cancer biology.

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 5α-Reductase (5α-R) (EC 1.3.99.5), which occurs as two isozymes, type 1 and type 2. Type-1 5α-R is widely distributed in the body, and type-2 5α-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 5α-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 capillary electrophoresis (LIF-CE). We demonstrated that T control the expression of 5α-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.

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.

Multiplex analysis of the most common mutations related to hereditary haemochromatosis: two methods combining specific amplification with capillary electrophoresis

We present the first application of a multiplex multicolour assay for the simultaneous detection of three of the most frequent mutations related to hereditary haemochromatosis (C282Y, H63D and S65C), using fluorescent detection and capillary electrophoresis. We describe two methods: the first is based on a single base extension assay, resulting in a single base difference of the extended products; and the second is a competitive allele‐specific polymerase chain reaction (PCR), based on competition between allele‐specific primers. Specificity of the latter primers is enhanced with a mismatch at the antepenultimate nucleotide. Primers are designed to amplify products of different sizes and with different fluorescent dyes in order to accurately distinguish all possible combinations of genotypes (homozygous and heterozygous for each mutation) in a multiplex PCR analysis. An advantage of the present approach is that capillary electrophoresis analysis of the amplified products enables easy, rapid, unambiguous and high resolution discrimination between wild‐type and mutant alleles, although different mutations may be present in the multiplex analysis. This will facilitate automated genotyping for routine molecular diagnostics and large‐scale genetic studies.

Differential immunohistochemical localization of desmosomal plaque‐related proteins in non‐small‐cell lung cancer

Immunohistochemistry is a highly valuable and widely used tool in the subtyping of lung carcinomas. The aim of this study was to identify markers for the differential diagnosis of non‐small‐cell carcinomas.

Cerebral cortex and amino acid neurotransmitters: Higher levels of aspartic acid but not GABA in the frontal cortex of the rat

Endogenous levels of Aspartic acid, GABA and Glutamic acid plus Glutamine were measured in the frontal, occipital, temporal and parietal cortex. Aspartic acid levels were found higher in the frontal cortex than in the rest of the cortical areas studied. GABA, however, had a homogenous distribution among all cortical areas.

Use of capillary electrophoresis for accurate determination of CAG repeats causing Huntington disease. An oligonucleotide design avoiding shadow bands

Huntington disease (HD) is a neurodegenerative disorder associated with the expansion of a polymorphic trinucleotide CAG repeat in the HD gene. We have developed an assay to accurately determine CAG repeats that combines a novel oligonucleotide design and the resolution of capillary electrophoresis. A mismatch in the second nucleotide from the 3′ end enhanced specificity by avoiding mispriming and diminishing shadow bands and artifactual PCR products. The coupling of capillary electrophoresis analysis with the assay added the advantages of accuracy, high resolution, semi‐automation, rapid analysis and low sample consumption. Analysis of 200 chromosomes in the Spanish population sample studied (control group) gave a peak frequency for 16 CAG repeats and of 7 triplets for CCG repeats. Diagnosis of HD was confirmed in 22 of 34 individuals with a range of CAG repeats from 39 to 52. Predictive testing was also carried out for 19 relatives of the HD families diagnosed at our laboratory. The method proposed in this article provides an accurate sizing of DNA repeats that can be applied to the analysis of DNA size‐related disorders.

Multi-mutational analysis of fifteen common mutations of the glucose 6-phosphate dehydrogenase gene in the Mediterrranean population

BACKGROUND Glucose-6-phosphate dehydrogenase (G6PD) is a cytosolic enzyme encoded by a housekeeping X-linked gene whose main function is to produce NADPH, a key electron donor in the defence against oxidizing agents and in reductive biosynthetic reactions. Many variants of G6PD have been described, mostly produced from missense mutations, with wide ranging levels of enzyme activity and associated clinical symptoms. METHOD A single base extension assay is used, yielding a single base difference of the extended products. Primers are designed to amplify products of different sizes with distinct fluorescent dyes in order to accurately distinguish all possible combinations of genotypes (homozygous and heterozygous for each mutation) in a multiplex PCR analysis. RESULTS We present the first application of a multiplex multicolour assay to detect 15 of the most frequent G6PD-related mutations in Spain, which are studied in three multiplex reactions. Capillary electrophoresis analysis of the amplified products enables easy, rapid, unambiguous and high-resolution discrimination between wild-type and mutant alleles, even though various mutations may be present in the multiplex analysis. CONCLUSION The analytical method described herein offers greater diagnostic power in Spanish and Mediterranean populations and would facilitate automated genotyping in routine molecular diagnostics and large-scale genetic studies (e.g., newborn screening programs).

Quantitative reverse-transcriptase polymerase chain reaction assay for mRNA levels of steroid 5α-reductase isozymes

Many genes that play a major physiologic role present low levels of expression, whose characterization requires accurate quantitation methods of adequate sensitivity. The use of the reverse-transcriptase polymerase chain reaction (RT-PCR) coupled to capillary electrophoresis (CE) for the analysis of steady-state messenger RNA levels has become increasingly widespread in recent years [1–5]. The present paper describes a rapid and accurate RTPCR method coupled to CE to quantitate the mRNA expression of the two 5a-reductase (5a-R) (EC 1.3.99.5) isozymes, 5a-reductase type I (5a-RI), and 5a-reductase type II (5a-RII), which play an important role in virilization in mammals [6]. This method combines the high degree of specificity of competitive PCR with the sensitivity of capillary electrophoresis. Total RNA isolated from the tissue was reverse transcribed (problem cDNA) and mixed with a known amount (number of molecules) of a synthetic internal standard (mimic DNA). Both standard and unknown problem cDNA were then subjected to PCR amplification using the same sets of primers. The amount of the mRNA in question was quantitated by comparing the fluorescence intensity of the problem cDNA with that of a known amount of the internal standard after separation by capillary electrophoresis.