Sebastian Blesa

Evaluation of clinical diagnosis criteria of familial ligand defective apoB 100 and lipoprotein phenotype comparison between LDL receptor gene mutations affecting ligand-binding domain and the R3500Q mutation of the apoB gene in patients from a South European population

Familial hypercholesterolemia (FH) and familial defective apoB 100 (FDB) are characterized by increased plasma low-density lipoprotein cholesterol (LDLc) levels and risk of coronary heart disease (CHD). FDB is clinically indistinguishable from FH. The aims of this study were to evaluate clinical diagnosis criteria for FDB and to compare the lipoprotein phenotype between carriers of LDL receptor (LDLR) gene mutations that affect the ligand-binding domain and subjects with the R3500Q mutation in apoB gene. We studied 213 subjects (113 probands) with FH and 19 heterozygous FDB subjects. Genetic diagnosis was determined by following a protocol based on Southern blot and polymerase chain reaction-single strand conformation polymorphism (SSCP) analysis. Thirty FH carriers of LDLR gene missense mutations that affect ligand-binding domain were matched by age, gender, and body mass index to the 19 FDB subjects (R3500Q mutation). Lipoprotein phenotype comparison was conducted between the 2 groups. FH patients showed plasma total and LDL cholesterol levels significantly higher than those in FDB patients. Three FDB showed plasma total and LDLc values in the normal range. Using the 1999 clinical Med-Ped criteria for diagnosis of genetic hypercholesterolemia, no FDB subjects had a confirmed diagnosis; it was probable in 36% of the subjects, it was possible in 32% of the subjects, and it could be excluded in the remaining 32% of the subjects. We conclude that the FDB lipoprotein phenotype was significantly less severe than that observed in FH carriers of LDLR gene missense ligand-binding domain mutations. Clinical Med-Ped diagnosis criteria tend to under-diagnose FDB.

Different Impacts of Cardiovascular Risk Factors on Oxidative Stress

The objective of the study was to evaluate oxidative stress (OS) status in subjects with different cardiovascular risk factors. With this in mind, we have studied three models of high cardiovascular risk: hypertension (HT) with and without metabolic syndrome, familial hypercholesterolemia (FH) and familial combined hyperlipidemia (FCH) with and without insulin resistance. Oxidative stress markers (oxidized/reduced glutathione ratio, 8-oxo-deoxyguanosine and malondialdehide) together with the activity of antioxidant enzyme triad (superoxide dismutase, catalase, glutathione peroxidase) and activation of both pro-oxidant enzyme (NAPDH oxidase components) and AGTR1 genes, as well as antioxidant enzyme genes (CuZn-SOD, CAT, GPX1, GSR, GSS and TXN) were measured in mononuclear cells of controls (n = 20) and patients (n = 90) by assessing mRNA levels. Activity of some of these antioxidant enzymes was also tested. An increase in OS and pro-oxidant gene mRNA values was observed in patients compared to controls. The hypertensive group showed not only the highest OS values, but also the highest pro-oxidant activation compared to those observed in the other groups. In addition, in HT a significantly reduced antioxidant activity and mRNA induction of antioxidant genes were found when compared to controls and the other groups. In FH and FCH, the activation of pro-oxidant enzymes was also higher and antioxidant ones lower than in the control group, although it did not reach the values obtained in hypertensives. The thioredoxin system was more activated in patients as compared to controls, and the highest levels were in hypertensives. The increased oxidative status in the presence of cardiovascular risk factors is a consequence of both the activation of pro-oxidant mechanisms and the reduction of the antioxidant ones. The altered response of the main cytoplasmic antioxidant systems largely contributes to OS despite the apparent attempt of the thioredoxin system to control it.

Effects of taurine on pulmonary responses to antigen in sensitized Brown-Norway rats.

Oxidative stress appears relevant to asthma. Therefore, the effects of the antioxidant taurine (oral, 1 and 3 mmol x kg(-1) x day(-1) for 7 days before challenge) were examined on antigen-induced responses in sensitized Brown-Norway rats. Taurine did not reduce the bronchospasm produced by aerosol antigen but prevented airway hyperreactivity to 5-hydroxytryptamine (5-HT) at 24 h after antigen exposure, and reduced the eosinophils (from 0.178+/-0.038x10(6) to 0.044+/-0.014x10(6)* and 0.048+/-0.013x10(6)* cells ml(-1) in antigen and antigen+taurine 1 or 3 mmol x kg(-1), respectively; *P<0.05 vs. antigen), lipid hydroperoxides, and Evans blue dye extravasation in bronchoalveolar lavage fluid. Taurine levels in bronchoalveolar lavage fluid from antigen-challenged rats were higher than control values but treatment with taurine failed to further increase these levels. In conclusion, oral taurine showed beneficial effects in an in vivo model of experimental asthma, which confirm and extend the previous positive findings obtained in other models of lung injury.

One-year follow-up of clinical, metabolic and oxidative stress profile of morbid obese patients after laparoscopic sleeve gastrectomy. 8-oxo-dG as a clinical marker

Obesity has grown worldwide over the last few decades. In its different degrees, obesity is accompanied by many clinical and biochemical alterations reflecting the pathological condition of various body tissues. Among the mechanisms underlying the pathogenesis of obesity and associated complications, oxidative stress (OS) may be playing an important role. In the present study, we have characterized at systemic level the degree of OS status in a group of morbid obese patients (BMI>40 kg/m2) at basal sate and its modulation during one year after bariatric surgery using the laparoscopic sleeve gastrectomy (LSG) technique. As compared with normal weight subjects matched in age, peripheral blood mononuclear cells (PBMc) of obese patients present a significant reduction of the antioxidant enzyme activities superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) as well as a significant increase of the oxidized/reduced glutathione ratio (GSSG/GSH) in these cells. Lipid peroxidation is significantly increased in the patient group as shown by the increased levels of malondialdehyde (MDA) in PBMc and the amount of F2-Isoprostanes (F2-IsoPs) released in urine. In addition, the DNA damage product 8-oxo-7,8-2′-deoxyguanosine (8-oxo-dG) was also observed to be increased in serum and urine of morbid obese patients as compared with the control group. After LSG, an improvement of their ponderal and metabolic profile was accompanied by a progressive recovery of antioxidant enzyme activities and the decline of oxidative byproducts both in PBMc and biological fluids. The observed changes of urinary 8-oxo-dG levels correlate positively with its serum concentration, the lipid peroxidation products MDA and F2-IsoPs, triglycerides, glucose, insulin, HOMA index and body weight and negatively with the percentage of weight and BMI loss and antioxidant activities. We conclude that the analysis of urinary 8-oxo-dG could be validated as a useful marker for the monitoring of ponderal and metabolic status of morbid obese patients.

Coexistence of EGFR, KRAS, BRAF, and PIK3CA Mutations and ALK Rearrangement in a Comprehensive Cohort of 326 Consecutive Spanish Nonsquamous NSCLC Patients

Introduction Molecular screening is crucial for the care of nonsquamous non–small‐cell lung cancer (NSCLC) patients. The coexistence of mutations could have important consequences regarding treatment. We described the mutational patterns and coexistence among patients and their outcomes after targeted treatment. Materials and Methods Data from consecutive patients with newly diagnosed nonsquamous NSCLC were prospectively collected. Next‐generation sequencing analysis of mutational hotspots in the EGFR, KRAS, PIK3CA, and BRAF genes and analysis of anaplastic lymphoma kinase (ALK) rearrangement were performed. Results A total of 326 patients with nonsquamous NSCLC were identified. Of the 326 patients, 240 (73.6%) had EGFR, 141 (43.3%) KRAS, 137 (42.0%) BRAF, 130 (39.9%) PIK3CA mutation and 148 (45.4%) ALK rearrangement determined. Of the 240 with EGFR determination, 24.1% harbored EGFR mutations. Of these, 16.3% were activating mutations (43.6%, exon 19 deletion; 46.1%, exon 21; and 10.3%, exon 18) and 7.9% were nonsensitizing EGFR mutations. Furthermore, 39.0% had KRAS mutations, 2.9% BRAF mutations, 10.0% PIK3CA mutations, and 8.8% ALK rearrangements. Of the 154 stage IV patients with ≥ 1 mutations, analysis showed 19 coexisting cases (12.3%). Of 8 patients receiving targeted treatment, 6 had no response. Both responders to targeted treatment had coexistent PIK3CA mutations. Conclusion Driver mutations can coexist in nonsquamous NSCLC. In our cohort, 12.3% of cases with stage IV disease had multiple mutations. Targeted treatment might not be as effective in patients with coexisting mutations; however, coexistence with PIK3CA might not preclude a response. Micro‐Abstract Molecular information from 326 nonsquamous non–small‐cell lung cancer patients is presented with the aim of determining driver mutation coexistence and response to treatment. To the best of our knowledge, limited data have been presented on this subject. We found a 12% rate of mutational coexistence using next‐generation sequencing and anaplastic lymphoma kinase immunohistochemical analysis. Most cases with coexisting mutations did not respond well to targeted treatment.

Determinantes de la lipemia posprandial medida como perfil diurno de triglicéridos en personas no diabéticas con normolipemia

BACKGROUND AND OBJECTIVE We decided to evaluate the clinical and biochemical predictors of postprandial lipemia, measured as daylong capillarly triglycerides (TGc) profiles, in normolipidemic non diabetic subjects. PATIENTS AND METHOD We studied 76 normolipidemic non diabetic subjects (45 premenopausal females). Accutrend was used to measure daylong TGc profiles during 3 days in 6 previously standardized points: fasting, pre and 3 h after dinner and lunch and at bedtime. The area under the curve of TGc (AUC-TGc) was determined as expression of postprandial lipemia. RESULTS Males showed significantly higher AUC-TGc (26.20 [11.00] vs 19.12 [6.57] in females; p < 0.001). Obese showed significantly higher values of AUC-TGc (27.87 [12.47] vs 20.05 [7.04]; p < 0.01). The AUC-TGc correlated with: age (r = 0.242; p < 0.05), body mass index (r = 0.312; p < 0.01), waist circumference (r = 0.394; p < 0.01), fasting plasma triglyceride (r = 0.634; p < 0.001), fasting insulinemia (r = 0.485; p < 0.001) and fasting HOMA (r = 0.484; p < 0.001). The multivariate analysis showed that HOMA (regression coefficient: 0.352; p = 0.02) and waist circumference (regression coefficient: 0.4; p = 0.05) were independent predictors of the AUC-TGc. CONCLUSIONS Independent determinants of postprandial lipemia were waist circumference and HOMA.

Ghrelin Gene Variants Influence on Metabolic Syndrome Components in Aged Spanish Population

Background The role of genetic variations within the ghrelin gene on cardiometabolic profile and nutritional status is still not clear in humans, particularly in elderly people. Objectives We investigated six SNPs of the ghrelin gene and their relationship with metabolic syndrome (MS) components. Subjects and Methods 824 subjects (413 men/411 women, age 77.31±5.04) participating in the Mataró aging study (n = 310) and the Hortega study (n = 514) were analyzed. Anthropometric variables, ghrelin, lipids, glucose and blood pressure levels were measured, and distribution of SNPs -994CT (rs26312), -604GA (rs27647), -501AC (rs26802), R51Q (rs34911341), M72L (rs696217) and L90G (rs4684677) of the ghrelin gene evaluated. Genotypes were determined by multiplex PCR and SNaPshot minisequencing. MS (IDF criteria) was found in 54.9%. Results No association between any of the SNPs and levels of total fasting circulating ghrelin levels was found. C/A-A/A genotype of M72L was associated with increased risk of central obesity according to IDF criteria, while G/A-G/G genotypes of -604GA with reduced risk. A/A genotype of -501AC polymorphism was associated to decreased BMI. In relation to lipid profile, the same genotypes of -604GA were associated with increased total cholesterol and LDL-cholesterol and -501AC with reduced triglycerides. There were no associations with systolic or diastolic blood pressure levels or with hypertension, glucose levels or diabetes and ghrelin polymorphisms. However, G/G genotype of -604GA was associated with glucose >100 mg/dL. Haplotype analysis showed that only one haplotype is associated with increased risk of waist circumference and central obesity. The analysis of subjects by gender showed an important and different association of these polymorphisms regarding MS parameters. Conclusion Ghrelin gene variants -604GA, -501AC and M72L are associated with certain components of MS, in particular to BMI and lipid profile in elderly Spanish subjects.

Polymorphisms in Endothelin System Genes, Arsenic Levels and Obesity Risk

Background/Objectives Obesity has been linked to morbidity and mortality through increased risk for many chronic diseases. Endothelin (EDN) system has been related to endothelial function but it can be involved in lipid metabolism regulation: Receptor type A (EDNRA) activates lipolysis in adipocytes, the two endothelin receptors mediate arsenic-stimulated adipocyte dysfunction, and endothelin system can regulate adiposity by modulating adiponectin activity in different situations and, therefore, influence obesity development. The aim of the present study was to analyze if single nucleotide polymorphisms (SNPs) in the EDN system could be associated with human obesity. Subjects/Methods We analyzed two samples of general-population-based studies from two different regions of Spain: the VALCAR Study, 468 subjects from the area of Valencia, and the Hortega Study, 1502 subjects from the area of Valladolid. Eighteen SNPs throughout five genes were analyzed using SNPlex. Results We found associations for two polymorphisms of the EDNRB gene which codifies for EDN receptor type B. Genotypes AG and AA of the rs5351 were associated with a lower risk for obesity in the VALCAR sample (p=0.048, OR=0.63) and in the Hortega sample (p=0.001, OR=0.62). Moreover, in the rs3759475 polymorphism, genotypes CT and TT were also associated with lower risk for obesity in the Hortega sample (p=0.0037, OR=0.66) and in the VALCAR sample we found the same tendency (p=0.12, OR=0.70). Furthermore, upon studying the pooled population, we found a stronger association with obesity (p=0.0001, OR=0.61 and p=0.0008, OR=0.66 for rs5351 and rs3759475, respectively). Regarding plasma arsenic levels, we have found a positive association for the two SNPs studied with obesity risk in individuals with higher arsenic levels in plasma: rs5351 (p=0.0054, OR=0.51) and rs3759475 (p=0.009, OR=0.53) Conclusions Our results support the hypothesis that polymorphisms of the EDNRB gene may influence the susceptibility to obesity and can interact with plasma arsenic levels.

Estudio del defecto familiar de uniÓn de la apolipoproteína B100 en una poblaciÓn mediterránea

Fundamento y objetivo Comparar las caracteristicas clinicobioquimicas del defecto familiar de union de la apolipoproteina B100 (DFB) con la hipercolesterolemia familiar (HF) heterocigota en una poblacion mediterranea del sur de Europa donde existen pocos datos al respecto, estudiar la prevalencia y el posible efecto fundador del DFB en una zona de la Comunidad Valenciana y conocer las caracteristicas clinicobioquimicas del DFB. Sujetos y metodo Hemos estudiado a 19 heterocigotos con DFB (8 varones) pertenecientes a 12 familias, portadores de la mutacion R3500Q del gen de la apolipoproteina B, y a 57 heterocigotos con HF (24 varones) geneticamente caracterizados, seleccionados de forma aleatoria entre 213 y apareados con los sujetos con DFB por edad y sexo. En todos los sujetos se determinaron de forma estandarizada las concentraciones de lipidos y apolipoproteinas B y A-I. Resultados Encontramos un efecto fundador en un area rural aislada de la Comunidad Valenciana para la mutacion R3500Q del gen de la apolipoproteina B, causante del DFB. La prevalencia del DFB en esta zona es de 4/350. Comparados con los casos de HF, los heterocigotos con DFB presentan menor prevalencia de xantomas tendinosos y de cardiopatia isquemica y concentraciones significativamente menores de colesterol total y unido a lipoproteinas de baja densidad y de alta densidad, asi como de apolipoproteinas B y A-I. Conclusiones Existe una zona geografica en la Comunidad Valenciana con una alta prevalencia del DFB por un efecto fundador. Ademas, en nuestra poblacion, el fenotipo lipoproteinico y la expresion clinica de la hipercolesterolemia en sujetos con DFB son menos graves que los de los sujetos con HF.

8P EGFR, KRAS, BRAF, and PI3K mutations and ALK rearrangement in 327 consecutive Spanish non-squamous NSCLC patients.

P. Martin Martorell1, M. Huerta1, A. Compañ Quilis2, M. Abellán3, S. Blesa3, F. Chaves3, D. Dualde Beltrán4, A. Fernández Do Nascimento1, A. Insa1. 1 Hematology and Medical Oncology, Hospital Clinico Universitario de Valencia, Valencia, Spain , 2 Pathology, Hospital Clinico Universitario de Valencia, Valencia, Spain, 3 Genotyping and Genetic Diagnosis Unit, Hospital Clinico Universitario de Valencia, Valencia, Spain, 4 Radiation Oncology, Hospital Clinico Universitario de Valencia, Valencia, Spain