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Background: Studies on childhood obesity mainly focus on the genetic component and on the lifestyle that may be associated with the development of obesity. However, the study of perinatal factors in their programming effect toward future obesity in children or adults is somewhat more recent, and there are still mechanisms to be disentangled. Summary: In this narrative review, a comprehensive route based on the influence of some early factors in life in the contribution to later obesity development is presented. Maternal pre-pregnancy BMI and gestational weight gain have been pointed out as independent determinants of infant later adiposity. Lifestyle interventions could have an impact on pregnant mothers through epigenetic mechanisms capable of redirecting the genetic expression of their children toward a future healthy weight and body composition and dietary-related microbiome modifications in mothers and newborns might also be related. After birth, infant feeding during the first months of life is directly associated with its body composition and nutritional status. From this point of view, all the expert committees in the world are committed to promote exclusive breastfeeding up to 6 months of age and to continue at least until the first year of life together with complementary feeding based on healthy dietary patterns such as Mediterranean Diet. Key Messages: To develop future effective programs to tackle early obesity, it is necessary not only by controlling lifestyle behaviors like infant feeding but also understanding the role of other mechanisms like the effect of perinatal factors such as maternal diet during pregnancy, epigenetics, or microbiome.

In general terms, fetal growth restriction (FGR) is considered the impossibility of achieving the genetically determined potential size. In the vast majority of cases, it is related to uteroplacental insufficiency. Although its origin remains unknown and causes are only known in 30% of cases, it is believed to be related to an interaction of environmental and genetic factors with either a fetal or maternal origin. One hypothesis is that alterations in the gastrointestinal microbiota composition, and thus alteration in the immune response, could play a role in FGR development. We performed an observational, prospective study in a subpopulation affected with FGR to elucidate the implications of this microbiota on the FGR condition. A total of 63 fetuses with FGR diagnosed in the third trimester as defined by the Delphi consensus, and 63 fetuses with fetal growth appropriate for gestational age will be recruited. Obstetric and nutritional information will be registered by means of specific questionnaires. We will collect maternal fecal samples between 30 to 36 weeks, intrapartum samples (maternal feces, maternal and cord blood) and postpartum samples (meconium and new-born feces at 6 weeks of life). Samples will be analyzed in the Department of Biochemistry and Molecular Biology II, Nutrition and Food Technology Institute of the University of Granada (UGR), for the determination of the gastrointestinal microbiota composition and its relationship with inflammatory biomarkers. This study will contribute to a better understanding of the influence of gastrointestinal microbiota and related inflammatory biomarkers in the development of FGR. Trial registration: NCT04047966. Registered August 7, 2019, during the recruitment stage. Retrospectively registered. Ongoing research., Instituto de Salud Calos III, Fondos de Desarrollo Regional Europeos (FEDER) PI17/01215

Asthma is a multifactorial condition where patients with identical clinical diagnoses do not have the same clinical history or respond to treatment. This clinical heterogeneity is reflected in the definition of two main endotypes. We aimed to explore the metabolic and microbiota signatures that characterize the clinical allergic asthma phenotype in obese children. Methods: We used a multi-omics approach combining clinical data, plasma and fecal inflammatory biomarkers, metagenomics, and metabolomics data in a cohort of allergic asthmatic children. Results: We observed that the obese allergic asthmatic phenotype was markedly associated with higher levels of leptin and lower relative proportions of plasma acetate and a member from the Clostridiales order. Moreover, allergic children with a worse asthma outcome showed higher levels of large unstained cells, fecal D lactate and D/L lactate ratio, and with a higher relative proportion of plasma creatinine and an unclassified family member from the RF39 order belonging to the Mollicutes class. Otherwise, children with persistent asthma presented lower levels of plasma citrate and dimethylsulfone. Conclusion: Our integrative approach shows the molecular heterogeneity of the allergic asthma phenotype while highlighting the use of omics technologies to examine the clinical phenotype at a more holistic level., Fundación Progreso y Salud Project PI-0373-2014, Redes temáticas de investigación cooperativa RETIC Red SAMID RD12/0026/0015

The aim of this systematic review is to evaluate whether the use of probiotics has any effect on the components of metabolic syndrome (MetS) before patients develop type 2 diabetes. A qualitative systematic review, following the Cochrane methodology, and a comprehensive literature search of randomized controlled trials (RCTs) were conducted in PubMed and Scopus from inception until 4 July 2019. According to our inclusion criteria, nine clinical studies were finally analyzed, corresponding to six RCTs. Probiotics intake in patients with MetS resulted in improvements in body mass index, blood pressure, glucose metabolism, and lipid profile in some studies. Regarding inflammatory biomarkers, probiotics also positively affected the soluble vascular cell adhesion molecule 1 (sVCAM-1), interleukine-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), vascular endothelial growth factor (VEGF), and thrombomodulin. Despite the diversity of the published studies, the intake of probiotics for patients with MetS may offer a discrete improvement in some of the clinical characteristics of the MetS and a decrease in inflammatory biomarkers. Nevertheless, these beneficial effects seem to be marginal compared to drug therapy and a healthy lifestyle and clinically non-relevant.

Background: S100A4 is a metastasis-associated protein also reported as a promising marker for dysfunctional white adipose tissue (WAT) and insulin resistance (IR) in adult and adolescent populations. Objective: We aimed to evaluate the association between the protein S100A4 and obesity and IR in children and during pubertal development. Design and methods: The study design consisted of three cross-sectional populations of 249, 11 and 19 prepubertal children respectively (named study population 1, 2 and 3), and a longitudinal population of 53 girls undergoing sexual maturation (study population 4). All subjects were classified into experimental groups according to their sex, obesity and IR status. All study populations counted on anthropometry, glucose, and lipid metabolism, inflammation and cardiovascular biomarkers as well as S100A4 plasma levels measured. The study population 1 was intended as the discovery population in which to elucidate the relationship between Obesity-IR and S100A4 plasma levels in prepubertal children. The cross-sectional populations 2 and 3 further counted on WAT gene expression data for investigating the molecular basis of this association. Instead, the longitudinal study population 4 presented blood whole-genome DNA methylation data at each temporal record, allowing deepening into the Obesity-IR-S1004 relationship during puberty as well as deciphering plausible epigenetic mechanisms altering S100A4 plasma levels. Results: S100A4 plasma levels were strongly associated with several metabolic and anthropometric outcomes, namely IR, in prepubertal non-diabetic obese children. We also found highly significant positive associations during the course of puberty between the increase in S100A4 levels and the increase in HOMA-IR (P = 0.0003, FDR = 0.005) and insulin levels (P = 0.0003, FDR = 0.005). Methylation in two-enhancer related CpG sites of the S100A4 region (cg07245635 and cg10447638) was associated with IR biomarkers at the prepubertal stage and with longitudinal changes in these measurements. We further reported an association between visceral WAT (vWAT) S100A4 expression and HOMA-IR, insulin levels and BMI Z-Score, but not with circulating S100A4. Conclusions: We report for the first time the association of S100A4 with IR and WAT dysfunction in prepubertal populations as well as how the change in plasma S100A4 levels accompanies longitudinal trajectories of IR in children during pubertal development. Moreover, we propose epigenetic changes in two methylation sites and an altered S100A4 vWAT expression as plausible molecular mechanisms underlying this disturbance in obesity.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This paper will be part of Augusto Anguita-Ruiz’s doctorate, which is being performed under the “Nutrition and Food Sciences Program” at the University of Granada., Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-40482-0., Tenomodulin (TNMD) is a type II transmembrane glycoprotein that has been recently linked to obesity, and it is highly expressed in obese adipose tissue. Several sex-dependent associations have been observed between single-nucleotide polymorphisms (SNPs) of the TNMD gene, which is located in the X-chromosome, and obesity, type 2 diabetes mellitus (T2DM), and metabolic syndrome in adults. On the other hand, results are lacking for children. We aimed (i) to study the association between TNMD genetic variants and metabolic complications related to childhood obesity and (ii) to investigate the function of TNMD in human adipocytes. We conducted a case-control, multicenter study in 915 Spanish children and demonstrated significant positive associations between TNMD genetic variants and BMI z-score, waist circumference, fasting glucose, and insulin resistance in boys, highlighting the SNP rs4828038. Additionally, we showed a BMI-adjusted inverse association with waist circumference in girls. Second, in vitro experiments revealed that TNMD is involved in adipogenesis, along with glucose and lipid metabolism in differentiated adipocytes, and these effects may be mediated through AMPK activation. Hence, these results suggest that TNMD genetic variants could be potentially useful as early life risk indicators for obesity and T2DM. In addition, we support the fact that TNMD exhibits significant metabolic functions in adipocytes., This work was supported by Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I + D + I), Instituto de Salud Carlos III-Fondo de Investigación Sanitaria (Projects numbers PI020826, PI051968, PI1102042, PI1600871), RETIC (Red SAMID RD12/0026/0015), Fondo Europeo De Desarrollo Regional (FEDER) and the Junta de Andalucía (project number CTS-6770); Secretaría General de Universidades, Investigación y Tecnología. Consejería de Economía, Innovación y Ciencia).

Background Obesity is characterized by increased fat mass and is associated with the development of insulin resistance syndrome (IRS), usually known as metabolic syndrome. The alteration of the intestinal microbiota composition has a role in the development of IRS associated with obesity, and probiotics, which are live microorganisms that confer a health benefit to the host, contribute to restore intestinal microbiota homeostasis and lower peripheral tissue insulin resistance. We aim to evaluate the effects of the probiotic strain Lactobacillus reuteri (L. reuteri) V3401 on the composition of intestinal microbiota, markers of insulin resistance and biomarkers of inflammation, cardiovascular risk, and hepatic steatosis in patients with overweight and obesity exhibiting IRS. Methods/design We describe a randomized, double-blind, crossover, placebo-controlled, and single-centre trial. Sixty participants (aged 18 to 65 years) diagnosed with IRS will be randomized in a 1:1 ratio to receive either a daily dose of placebo or 5 × 109 colony-forming units of L. reuteri V3401. The study will consist of two intervention periods of 12 weeks separated by a washout period of 6 weeks and preceded by another washout period of 2 weeks. The primary outcome will be the change in plasma lipopolysaccharide (LPS) levels at 12 weeks. Secondary outcomes will include anthropometric parameters, lipid profile, glucose metabolism, microbiota composition, hepatic steatosis, and inflammatory and cardiovascular biomarkers. Blood and stool samples will be collected at baseline, at the midpoint (only stool samples) and immediately after each intervention period. Luminex technology will be used to measure interleukins. For statistical analysis, a mixed ANOVA model will be employed to calculate changes in the outcome variables. Discussion This is the first time that L. reuteri V3401 will be evaluated in patients with IRS. Therefore, this study will provide valuable scientific information about the effects of this strain in metabolic syndrome patients. Trial registration The trial has been retrospectively registered in ClinicalTrials.gov on the 23rd November 2016 (ID: NCT02972567), during the recruitment phase. Electronic supplementary material The online version of this article (10.1186/s12906-018-2371-x) contains supplementary material, which is available to authorized users.

Inflammatory bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic inflammation of the small intestine and colon caused by a dysregulated immune response to host intestinal microbiota in genetically susceptible subjects. A number of fermented dairy products contain lactic acid bacteria (LAB) and bifidobacteria, some of which have been characterized as probiotics that can modify the gut microbiota and may be beneficial for the treatment and the prevention of IBD. The objective of this review was to carry out a systematic search of LAB and bifidobacteria probiotics and IBD, using the PubMed and Scopus databases, defined by a specific equation using MeSH terms and limited to human clinical trials. The use of probiotics and/or synbiotics has positive effects in the treatment and maintenance of UC, whereas in CD clear effectiveness has only been shown for synbiotics. Furthermore, in other associated IBD pathologies, such as pouchitis and cholangitis, LAB and bifidobacteria probiotics can provide a benefit through the improvement of clinical symptoms. However, more studies are needed to understand their mechanisms of action and in this way to understand the effect of probiotics prior to their use as coadjuvants in the therapy and prevention of IBD conditions.

Obesity and asthma are two chronic conditions that affect millions of people. Genetic and lifestyle factors such as diet, physical activity, and early exposure to micro-organisms are important factors that may contribute to the escalating prevalence of both conditions. The prevalence of asthma is higher in obese individuals. Recently, two major phenotypes of asthma with obesity have been described: one phenotype of early-onset asthma that is aggravated by obesity, and a second phenotype of later-onset asthma that predominantly affects women. Systemic inflammation and mechanical effect, both due to the expansion of the adipose tissue, have been proposed as the main reasons for the association between obesity and asthma. However, the mechanisms involved are not yet fully understood. Moreover, it has also been suggested that insulin resistance syndrome can have a role in the association between these conditions. The intestinal microbiota is an important factor in the development of the immune system, and can be considered a link between obesity and asthma. In the obese state, higher lipopolysaccharide (LPS) serum levels as a consequence of a microbiota dysbiosis have been found. In addition, changes in microbiota composition result in a modification of carbohydrate fermentation capacity, therefore modifying short chain fatty acid (SCFA) levels. The main objective of this review is to summarize the principal findings that link obesity and asthma.

The following are available online at https://www.mdpi.com/2072-6643/11/8/1761/s1: Figure S1, Rarefaction curves; Figure S2, Alpha diversity measured by means of the Shannon index (H); Figure S3, Bacterial beta diversity; Table S1, Gastrointestinal microbiome normalized data, This paper will be part of Carmen Tenorio Jimenez’s doctorate, which is being completed as part of Programa de Doctorado en Seguridad de los Alimentos at the University of Jaén, Spain. We also wish to thank all the participants who generously contributed to the study., Previous studies have reported that probiotics may improve clinical and inflammatory parameters in patients with obesity and metabolic syndrome (MetS). Lactobacillus (L.) reuteri V3401 has shown promising results on the components of MetS in animal studies. We aimed to evaluate the effects of L. reuteri V3401 together with healthy lifestyle recommendations on adult patients with MetS. Methods: We carried out a randomized, crossover, placebo-controlled, single-center trial in which we included 53 adult patients newly diagnosed with MetS. Patients were block randomly allocated by body mass index (BMI) and sex to receive a capsule containing either the probiotic L. reuteri V3401 (5 × 109 colony-forming units) or a placebo once daily for 12 weeks. Anthropometric variables, biochemical and inflammatory biomarkers, as well as the gastrointestinal microbiome composition were determined. Results: There were no differences between groups in the clinical characteristics of MetS. However, we found that interleukin-6 (IL-6) and soluble vascular cell adhesion molecule 1 (sVCAM-1) diminished by effect of the treatment with L. reuteri V3401. Analysis of the gastrointestinal microbiome revealed a rise in the proportion of Verrucomicrobia. Conclusions: Consumption of L. reuteri V3401 improved selected inflammatory parameters and modified the gastrointestinal microbiome. Further studies are needed to ascertain additional beneficial effects of other probiotic strains in MetS as well as the mechanisms by which such effects are exerted., This study is part of the grant entitled “Guía para la sustanciación de declaraciones de salud en alimentos: funciones inmune, cognitiva y síndrome metabólico”, funded by the company Biosearch life (Granada, Spain), reference 3006, managed by Fundación General Universidad de Granada, Spain.

Background Thrombophilia is defined as an inherited or acquired abnormality of hemostasis predisposing to thrombosis. While the most common thrombophilia has a genetic origin and is manifested by elevated circulating antiphospholipid antibodies, about 40% of cases presenting with thrombosis are acquired. Factor V Leiden G1691A, prothrombin G20210A, MTHFR C677T, and Factor XII C46T mutations are associated with the risk of developing thrombophilia. Methods In this study, a method using single base extension assay coupled with fluorescent detection and capillary electrophoresis was applied to simultaneously detect G1691A, G20210A, C677T and C46T mutations in 1499 patients from Spain with suspicion of thrombotic disease. Results Out of these individuals, 5.4% were heterozygous for G20210A mutation, 9.21% were heterozygous and 0.20% homozygous for G1691A mutation, 46.36% were heterozygous and 20.71% homozygous for MTHFR mutation, and 30.41% were heterozygous and 3.4% homozygous for C46T mutation. Conclusion We applied an accurate, simple, semi-automatic, and cost-effective method to simultaneously detect the main thrombophilia-related mutations, allowing us to determine the frequency of these mutations in a Spanish population.

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.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.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.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.

Exogenous nucleotides (NT) have been reported to exert a reparative role in animal models of intestinal and hepatic damage. Thus, the administration of NT in the diet of rats with thioacetamide-induced liver cirrhosis normalized many of the histological and biochemical alterations produced by this hepatotoxin. We are currently studying the mechanism by which NT exert this effect using cell culture models. The aim of this work was to investigate whether exogenous nucleosides (NS) modulate the proliferation of hepatocytes. We used fetal rat hepatocytes, which, unlike adult hepatocytes, are proliferative cells. Fetal rat primary hepatocytes were incubated with mixtures of NS, and cell proliferation was studied. NS added to the medium of fetal hepatocytes were taken up in a selective fashion by the cells. Cell proliferation was enhanced, as demonstrated by the induction of c-myc and h-ras gene expression as well as by the higher percentage of cells in S phase, and exogenous NS increased the expression of alpha-fetoprotein. These results suggest that exogenous NS may in fact stimulate proliferation of hepatic cells and help preserve the undifferentiated state of fetal rat hepatocytes.

The potential for the positive manipulation of the gut microbiome through the introduction of beneficial microbes, as also known as probiotics, is currently an active area of investigation. The FAO/WHO define probiotics as live microorganisms that confer a health benefit to the host when administered in adequate amounts. However, dead bacteria and bacterial molecular components may also exhibit probiotic properties. The results of clinical studies have demonstrated the clinical potential of probiotics in many pathologies, such as allergic diseases, diarrhea, inflammatory bowel disease and viral infection. Several mechanisms have been proposed to explain the beneficial effects of probiotics, most of which involve gene expression regulation in specific tissues, particularly the intestine and liver. Therefore, the modulation of gene expression mediated by probiotics is an important issue that warrants further investigation. In the present paper, we performed a systematic review of the probiotic-mediated modulation of gene expression that is associated with the immune system and inflammation. Between January 1990 to February 2014, PubMed was searched for articles that were published in English using the MeSH terms “probiotics" and "gene expression" combined with “intestines", "liver", "enterocytes", "antigen-presenting cells", "dendritic cells", "immune system", and "inflammation". Two hundred and five original articles matching these criteria were initially selected, although only those articles that included specific gene expression results (77) were later considered for this review and separated into three major topics: the regulation of immunity and inflammatory gene expression in the gut, in inflammatory diseases of the gut and in the liver. Particular strains of Bifidobacteria , Lactobacilli , Escherichia coli , Propionibacterium , Bacillus and Saccharomyces influence the gene expression of mucins, Toll-like receptors, caspases, nuclear factor-κB, and interleukins and lead mainly to an anti-inflammatory response in cultured enterocytes. In addition, the interaction of commensal bacteria and probiotics with the surface of antigenpresenting cells in vitro results in the downregulation of pro-inflammatory genes that are linked to inflammatory signaling pathways, whereas other anti-inflammatory genes are upregulated. The effects of probiotics have been extensively investigated in animal models ranging from fish to mice, rats and piglets. These bacteria induce a tolerogenic and hyporesponsive immune response in which many genes that are related to the immune system, in particular those genes expressing anti-inflammatory cytokines, are upregulated. By contrast, information related to gene expression in human intestinal cells mediated by the action of probiotics is scarce. There is a need for further clinical studies that evaluate the mechanism of action of probiotics both in healthy humans and in patients with chronic diseases. These types of clinical studies are necessary for addressing the influence of these microorganisms in gene expression for different pathways, particularly those that are associated with the immune response, and to better understand the role that probiotics might have in the prevention and treatment of disease.

We have previously described the safety and immunomodulatory effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 in healthy volunteers. The scope of this work was to evaluate the effects of these probiotic strains on the hepatic steatosis of obese rats. We used the Zucker rat as a genetic model of obesity. Zucker-Leprfa/fa rats received one of three probiotic strains, a mixture of L. paracasei CNCM I-4034 and B. breve CNCM I-4035, or a placebo for 30 days. An additional group of Zucker-lean+/fa rats received a placebo for 30 days. No alterations in intestinal histology, in the epithelial, lamina propria, muscular layers of the ileal or colonic mucosa, or the submucosae, were observed in any of the experimental groups. Triacylglycerol content decreased in the liver of Zucker-Leprfa/fa rats that were fed L. rhamnosus, B. breve, or the mixture of B. breve and L. paracasei. Likewise, the area corresponding to neutral lipids was significantly smaller in the liver of all four groups of Zucker-Leprfa/fa rats that received probiotics than in rats fed the placebo. Zucker-Leprfa/fa rats exhibited significantly greater serum LPS levels than Zucker-lean+/fa rats upon administration of placebo for 30 days. In contrast, all four groups of obese Zucker-Leprfa/fa rats that received LAB strains exhibited serum LPS concentrations similar to those of Zucker-lean+/fa rats. Serum TNF-α levels decreased in the Zucker-Leprfa/fa rats that received B. breve, L. rhamnosus, or the mixture, whereas L. paracasei feeding decreased IL-6 levels in the serum of Zucker-Leprfa/fa rats. In conclusion, the probiotic strains reduced hepatic steatosis in part by lowering serum LPS, and had an anti-inflammatory effect in obese Zucker rats., Part of the research currently in progress in the authors' laboratory is funded by the company Hero Spain, S. A. through the grant #3545 managed by the Fundacion General Empresa-Universidad de Granada.

We carried out a molecular analysis of 350 chromosomes from 55 families originating from the South of Spain (Andalucia) who were diagnosed with cystic fibrosis (CF). We used polymerase chain reaction, followed by an oligonucleotide ligation assay (OLA) and sequence-coded separation using capillary electrophoresis. A frequency of 43.5% for DF508 was found, making it the most common CF mutation in our sample. Seven more mutations (G542X, R334W, R1162X, 2789+5G !A, R117H, DI507 and W1282X) were detected and accounted for 24.7% of the total. The remaining mutations (31.8%) were undetectable with the methodology used in this study. D 2001 Elsevier

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.

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.

Clinical manifestations of cystic fibrosis (CF) are variable. Genetic and environmental factors that determine whether an individual will develop associated complications are still under investigation. The present study reports the genetic analysis of a family with different clinical forms of CF and addresses the difficulty of CF diagnosis in an individual with mutant alleles G542X and R117H because of the variable phenotype associated with R117H mutation. Both children in this family were heterozygous for G542X/R117H with the same thymine sequence (7T/9T) in intron 8 of CF transmembrane conductance regulator. The girl was diagnosed with CF, whereas the boy was diagnosed with azoospermia as the sole clinical manifestation. The possible implication of the hemochromatosis gene as a CF modifier locus was analyzed because the 2 children had the same genotype. No genetic differences were detected between brother and sister that explained the different clinical manifestations of CF.

Since the initial characterization of the predominant mutation (ΔF508) in cystic fibrosis (CF), more than 1000 pathogenic mutations and numerous polymorphisms have been identified in the CFTR gene (1). The frequencies and types of CFTR mutations vary according to the geographic and ethnic origins of the population under study. A recent worldwide survey revealed great mutational heterogeneity for CF in the Mediterranean region (2). Spain may have the highest heterogeneity of CF mutations among Mediterranean countries, with more than 75 different mutations detected, representing 90.2% of the CF chromosomes (3). On the other hand, only 10 mutations had a frequency higher than 1%, and these accounted for 74.2% of the CF chromosomes studied. Among the 75 different CFTR mutations detected to date in Spain, 56 are not included in the commercial Applied Biosystems assay, which may account for the numerous uncharacterized CFTR alleles in a previous study in Southern Spain (4). We attempted to solve this analytical problem by developing a PCR method that uses fluorescent detection and capillary electrophoresis to detect 11 of the most frequent CFTR mutations not included in the Applied Biosystems assay. If used in conjunction with the Applied Biosystems assay, our novel PCR technique could increase the rate of CF allele detection among European populations. Mutations in the CFTR gene were studied in 140 samples from patients diagnosed with CF at the 12 de Octubre Hospital (Madrid, Spain) or the IRO, Hospital Duran I Reynals (Barcelona, Spain), and from patients with suspicion of CF referred to the Hospital Universitario “San Cecilio” (Granada, Spain). Human genomic DNA was extracted from whole-blood samples by use of a QIAamp® Blood Mini Kit (Qiagen). The DNA concentrations in samples were determined spectrophotometrically by use of a UV-1603 Shimadzu spectrophotometer. Oligonucleotide primers were designed to avoid the formation of primer-dimers, …