Copy number variations (CNVs) are a type of genetic variation involving from 50 base pairs (bps) to millions of bps and, in a general point of view, can include alterations of complete chromosomes. As CNVs mean the gain or loss of DNA sequences, their detection requires specific techniques and analysis. We have developed Easy One-Step Amplification and Labeling for CNV Detection (EOSAL-CNV) by fragment analysis in a DNA sequencer. The procedure is based on a single PCR reaction for amplification and labeling of all fragments included. The protocol includes specific primers for the amplification of the regions of interest with a tail in each of the primers (one for forward and another for the reverse primers) together with primers for tail amplification. One of the primers for tail amplification is labeled with a fluorophore, allowing the amplification and labeling in the same reaction. Combination of several tail pairs and labels allows the detection of DNA fragment by different fluorophores and increases the number of fragments that can be analyzed in one reaction. PCR products can be analyzed without any purification on a DNA sequencer for fragment detection and quantification. Finally, simple and easy calculations allow the detection of fragments with deletions or extra copies. The use of EOSAL-CNV allows simplifying and reducing costs in sample analysis for CNV detection.

Copy number variations (CNVs) are a type of genetic variation involving from 50 base pairs (bps) to millions of bps and, in a general point of view, can include alterations of complete chromosomes. As CNVs mean the gain or loss of DNA sequences, their detection requires specific techniques and analysis. We have developed Easy One-Step Amplification and Labeling for CNV Detection (EOSAL-CNV) by fragment analysis in a DNA sequencer. The procedure is based on a single PCR reaction for amplification and labeling of all fragments included. The protocol includes specific primers for the amplification of the regions of interest with a tail in each of the primers (one for forward and another for the reverse primers) together with primers for tail amplification. One of the primers for tail amplification is labeled with a fluorophore, allowing the amplification and labeling in the same reaction. Combination of several tail pairs and labels allows the detection of DNA fragment by different fluorophores and increases the number of fragments that can be analyzed in one reaction. PCR products can be analyzed without any purification on a DNA sequencer for fragment detection and quantification. Finally, simple and easy calculations allow the detection of fragments with deletions or extra copies. The use of EOSAL-CNV allows simplifying and reducing costs in sample analysis for CNV detection.

INTRODUCTION:Molecular-matched therapies have revolutionized cancer treatment. We evaluated the improvement in clinical outcomes of applying an in-house customized Next Generation Sequencing panel in a single institution.METHODS:Patients with advanced solid tumors were molecularly selected to receive a molecular-matched treatment into early phase clinical trials versus best investigators choice, according to the evaluation of a multidisciplinary molecular tumor board. The primary endpoint was progression-free survival (PFS) assessed by the ratio of patients presenting 1.3-fold longer PFS on matched therapy (PFS2) than with prior therapy (PFS1).RESULTS:Of a total of 231 molecularly screened patients, 87 were eligible for analysis. Patients who received matched therapy had a higher median PFS2 (6.47 months; 95% CI, 2.24-14.43) compared to those who received standard therapy (2.76 months; 95% CI, 2.14-3.91, Log-rank p = 0.022). The proportion of patients with a PFS2/PFS1 ratio over 1.3 was significantly higher in the experimental arm (0.33 vs 0.08; p = 0.008).DISCUSSION:We demonstrate the pivotal role of the institutional molecular tumor board in evaluating the results of a customized NGS panel. This process optimizes the selection of available therapies, improving disease control. Prospective randomized trials are needed to confirm this approach and open the door to expanded drug access.

Assessment of TMB for response stratification of cancer patients is emerging as a new biomarker for immunotherapy. TMB is defined as the total number of non-synonymous somatic mutations and it approximates the amount of neoantigens. Whole exome sequencing is recognized as the best approach to quantify TMB, nevertheless the cost and access represent relevant limitations. The aim of this investigation is to evaluate the capacity of out small panel to predict response to immunotherapy based on the calculation of a min-TMB score. Patients with advanced tumors were screened for r targetable alterations. A customized hotspots NGS panel containing 87 genes, including 4 full CDS genes was used. Somatic mutations were selected as follow: 1) present in our somatic variant database; 2) mutation within cancer hotspot regions, in non-intronic regions with a population allele frequency < 0.1; 3) mutations with a known population frequency < 0.05%, with an unknown population frequency and not intronic; 4) Mutations in samples with a VAF over the selected thresholds (3%, 5% and 10%). A descriptive observational analysis was performed. Multivariable Cox regression analysis was carried out evaluating age, PS and TMB with R v3.6.2. Among 255 patients analyzed with our customized panel, only 46 received immunotherapy after the molecular screening as I or II line and were included. 25 received treatment with standard checkpoint inhibitors, while 21 received experimental immunotherapy. Median age was 61 (54-65). Tumor types represented were NSCLC, Melanoma HNSCC, GI and gynecological tumors. Median PFS was 5.4 (3.7-8.2) months. TMB was calculated and over patients treated with standard immunotherapy, the 3% non-synonymous mutations remained the only significant prognostic factor of PFS in the multivariable model (p=0.045). The feasibility of the TMB score calculation depending on the panel size remains controversial. Our customized small panel shows that it could help in the identification of a predictive TMB score for those patients candidate to receive checkpoint inhibitors. Although these finding are promising, further investigation is needed.

Macronutrients represent risk factors for hyperlipidemia or diabetes. Lipid alterations and type 2 diabetes mellitus are global health problems. Overexpression of sterol regulatory element-binding factor (Srebf2) in transgenic animals is linked to elevated cholesterol levels and diabetes development. We investigated the impact of increased Srebf2 locus expression and the effects of control and high-fat, high-sucrose (HFHS) diets on body weight, glucose and lipid metabolisms in transgenic mice (S-mice). Wild type (WT) and S-mice were fed with both diets for 16 weeks. Plasma glucose, insulin and lipids were assessed (n = 25). Immunostainings were performed in liver, pancreas and fat (N = 10). Expression of Ldlr and Hmgcr in liver was performed by RT-PCR (N = 8). Control diet: S-mice showed reduced weight, insulin, total and HDL cholesterol and triglycerides (TG). HFHS diet widened differences in weight, total and HDL cholesterol, insulin and HOMA index but increased TG in S-mice. In S-mice, adipocyte size was lower while HFHS diet produced lower increase, pancreatic β-cell mass was lower with both diets and Srebf2, Ldlr and Hmgcr mRNA levels were higher while HFHS diet produced a rise in Srebf2 and Hmgcr levels. Srebf2 complete gene overexpression seems to have beneficial effects on metabolic parameters and to protect against HFHS diet effects.

BACKGROUND The specific characteristics of copy number variations (CNVs) require specific methods of detection and characterization. We developed the Easy One-Step Amplification and Labeling procedure for CNV detection (EOSAL-CNV), a new method based on proportional amplification and labeling of amplicons in 1 PCR. METHODS We used tailed primers for specific amplification and a pair of labeling probes (only 1 labeled) for amplification and labeling of all amplicons in just 1 reaction. Products were loaded directly onto a capillary DNA sequencer for fragment sizing and quantification. Data obtained could be analyzed by Microsoft Excel spreadsheet or EOSAL-CNV analysis software. We developed the protocol using the LDLR (low density lipoprotein receptor) gene including 23 samples with 8 different CNVs. After optimizing the protocol, it was used for genes in the following multiplexes: BRCA1 (BRCA1 DNA repair associated), BRCA2 (BRCA2 DNA repair associated), CHEK2 (checkpoint kinase 2), MLH1 (mutL homolog 1) plus MSH6 (mutS homolog 6), MSH2 (mutS homolog 2) plus EPCAM (epithelial cell adhesion molecule) and chromosome 17 (especially the TP53 [tumor protein 53] gene). We compared our procedure with multiplex ligation-dependent probe amplification (MLPA). RESULTS The simple procedure for CNV detection required 150 min, with <10 min of handwork. After analyzing >240 samples, EOSAL-CNV excluded the presence of CNVs in all controls, and in all cases, results were identical using MLPA and EOSAL-CNV. Analysis of the 17p region in tumor samples showed 100% similarity between fluorescent in situ hybridization and EOSAL-CNV. CONCLUSIONS EOSAL-CNV allowed reliable, fast, easy detection and characterization of CNVs. It provides an alternative to targeted analysis methods such as MLPA.

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/m(2)) 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-oxod-G 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.

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.

Although it was initially thought many driver alterations were mutually exclusive, it might not be so. These pathways may interact in a dynamic way. Whether these mutations are present in different cellular clones that emerge as a result of treatment pressure or they co-exist in the same cell is not yet fully clear. Between Nov. 2011Aug. 2014 clinical data from consecutive newly diagnosed lung cancer patients in our center were prospectively collected in a database. Mutational analysis of EGFR, KRAS, BRAF and PI3K as well as ALK rearrangement was determined in all metastatic Nsq-NSCLC. EGFR mutational analysis was carried out using next generation sequencing using the Cobas and Junior diagnostic method. KRAS, BRAF and Pi3Kanalysis was determined by next generation sequencing using Sequencing Multiplex and Junior System. ALK rearrangement was carried out by FISH and confirmed by IHQ (D5F3, Ventana diagnostics). 327 patients were diagnosed with Nsq-NSCLC. 216 (66%) of the 327 patients had EGFR analysed, 132 (40.3%) KRAS, 128 (39.1%) BRAF, 122 (37.3%) PI3K and 126 (38.5%) ALK. Of the 216 with EGFR determination, 58 (26.8%) harbored EGFR mutations. Exon 19 deletions (44.4%), exon 21 mutations (L858R and L861Q – 47.2%), exon 18 mutations G719A/C/S (13.8%). Fifty four (40.9%) KRAS mutations, 4 (3.1%) BRAF mutations, 12 (9.8%) PI3K mutations and 8 (6.3%) ALK re-arranged.14 had coexistent mutations: 2 EGFR/ALK re-arrangements, 9 EGFR/KRAS, 4 EGFR/PI3K, and 2 EGFR/BRAF. 3 patients had triple mutations in EGFR/BRAF/ALK, and 2 in EGFR/KRAS/PI3K. We show that these mutations may co-exist previous to receiving targeted therapy. Whether they represent a primary or acquired resistance to targeted therapy is important to determine (data to be presented on the tissue origin – primary tumor vs metastasis). In our series, 78.5% of the patients were heavy smokers. Most patients were not exposed to targeted therapy, but of those who were, most did not respond.

Background/objectivesObesity 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/methodsWe 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.ResultsWe 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).ConclusionsOur results support the hypothesis that polymorphisms of the EDNRB gene may influence the susceptibility to obesity and can interact with plasma arsenic levels.

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.

Background: Autosomal dominant hypercholesterolemias (ADHs) are characterised by increased plasma levels of total and LDL cholesterol, predisposing to premature atherosclerosis. ADHs comprise several diseases with undistinguishable phenotype, caused by mutations in different genes: LDLR, APOB and PCSK9. Genetic studies are usually performed in patients with altered cholesterol levels. However, some persons carrying pathogenic mutations are normocholesterolemic and there are no further studies about this subject. We have studied the frequency of families and individuals carrying ADH mutations who do not present the disease in Spanish population.Methods: We have analysed genes known to cause ADH by direct sequencing in 24 ADH families (215 members). Functional effect of some LDLR gene mutations was assessed by transfecting cultured cells with plasmids.Results: Six families with mutations presented 7 mutation carriers who did not show ADH phenotype: 30% of ADH families presented normocholesterolemic individuals, and 7% of carriers of pathogenic mutations did not show ADH phenotype. We have analysed the effect of some of these mutations and they are responsible for impaired LDL receptor function. We have excluded mutations in APOB and PCSK9 genes that could reduce LDLc levels.Conclusions: An important percentage of ADH families presented individuals who do not show an ADH phenotype, but who are able to transmit the pathogenic mutation to their offspring. Genetic study of all subjects in ADH families should be performed in order to identify normocholesterolemic carriers that allow the detection of mutations in their descendants and the prevention of the disease consequences. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

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.

CONTEXT Autosomal dominant hypercholesterolemia (ADH) is a genetic disorder characterized by increased low-density lipoprotein (LDL)-cholesterol levels, leading to high risk of premature cardiovascular disease. More than 900 mutations in LDL receptor, six in APOB and 10 in PCSK9 have been identified as a cause of the disease in different populations. All known mutations in PCSK9 causing hypercholesterolemia produce an increase in the enzymatic activity of this protease. Up to now, there are data about the implication of PCSK9 in ADH in a low number of populations, not including a Spanish population. OBJECTIVE The objective of the study was to study the prevalence of PCSK9 mutations in ADH Spanish population. PARTICIPANTS We screened PCSK9 gene in 42 independent ADH patients in whom mutations in LDL receptor and APOB genes had been excluded. RESULTS None of the known mutations causing ADH was detected in our sample, but we found two variations in the promoter region that could cause ADH, c.-288G>A and c.-332C>A (each in one proband). The analysis of the effect of these two variations on the transcription activity of the PCSK9 promoter showed that c.-288G>A did not modify the transcription, whereas c.-332C>A variant caused a 2.5-fold increase when compared with the wild-type sequence, either with or without lovastatin. CONCLUSIONS PCSK9 is a rare cause of ADH in Spanish population and, up to what we know, none of the previously described mutations has been detected. We have identified a new mutation that could cause ADH by increasing the transcription of PCSK9.

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.