The rs3771863 single nucleotide polymorphism of the TACR1 gene is associated to a lower risk of sicca syndrome in fibromyalgia patients.

OBJECTIVES: Fibromyalgia (FM) has been associated with affective spectrum disorders and other chronic pain disorders, which tend to co-occur in individuals and co-aggregate among families. The objective of our study was to investigate the genetic risk factors associated with the presence of related symptoms and with disease severity in subjects affected with FM. METHODS: Two independent cohorts of subjects diagnosed with FM according to the 1990 ACR criteria were studied. A genetic array composed of 320 single nucleotide polymorphisms (SNPs) was analysed in a discovery cohort comprised by 564 patients, and the most suggestive variants were genotyped in a replication cohort, comprised by 397 subjects. The associated conditions and related symptoms analysed were: the presence of depression, sleep disorders, headache, myofascial syndrome, irritable bowel syndrome, chronic fatigue syndrome, vertiginous syndrome, chronic cystitis, and sicca syndrome. FM severity was assessed by the Fibromyalgia Impact Questionnaire and the Hospital Anxiety and Depression Scale. Analyses were adjusted by elapsed time from pain onset, and a meta-analysis was performed to pool the results. RESULTS: Minor allele of the rs3771863 SNP from the TACR1 gene showed a significant association with a lower risk of sicca syndrome (pooled and adjusted OR 0.56, [95%CI 0.42-0.76], p=0.00022). CONCLUSIONS: Our findings indicate a role of the TACR1 gene in the development of sicca syndrome in subjects affected with FM.

Lutein, zeaxanthin, macular pigment, and visual function in adult cystic fibrosis patients.

BACKGROUND: Pancreatic insufficiency in cystic fibrosis (CF), even with replacement pancreatic enzyme therapy, is often associated with decreased carotenoid absorption. Because the macular pigment of the retina is largely derived from 2 carotenoids, lutein and zeaxanthin, the decreased serum concentrations seen in CF may have consequences for ocular and retinal health OBJECTIVES: Our aims were to determine plasma carotenoid concentrations, determine absorption and distribution of macular pigment, and assess retinal health and visual function in CF patients. DESIGN: In 10 adult CF patients (ages 21-47 y) and 10 age- and sex-matched healthy control subjects, we measured macular pigment density in vivo, measured serum lutein and zeaxanthin concentrations, and comprehensively assessed visual performance (including contrast sensitivity, color discrimination, and retinal function) under conditions of daylight illumination. RESULTS: Serum lutein and zeaxanthin were significantly reduced (P < 0.005) in CF patients ( +/- SD: 87 +/- 36.1 and 27 +/- 15.8 nmol/L, respectively) compared with control subjects (190 +/- 72.1 and 75 +/- 23.6 nmol/L, respectively). Although macular pigment optical density was significantly lower (P < 0.0001) in the CF group (0.24 +/- 0.11) than in the control group (0.53 +/- 0.12), no significant differences in visual function were observed. CONCLUSIONS: Adults with CF have dramatically low serum and macular concentrations of carotenoids (lutein and zeaxanthin), but their ocular status and visual function are surprisingly good. The clinical implications of low plasma concentrations of carotenoids in CF are yet to be clarified.

Pectins and pectic-oligosaccharides inhibit Escherichia coli O157:H7 Shiga toxin as directed towards the human colonic cell line HT29.

Pectins and pectic-oligosaccharides, as derived by controlled enzymatic hydrolysis, were evaluated for their ability to interfere with the toxicity of Shiga-like toxins from Escherichia coli O157:H7. Both types of material resulted in some degree of protection but this was significantly higher (P>0.01) with the oligosaccharide fractions (giving 90-100% cell survival, compared to 70-80% with the polymer). An effect of methylation on the protective effect was detected with lower degrees being more active. The pectic-oligosaccharides and galabiose, the minimum toxin receptor analogue, were shown to inhibit toxicity and were both protective at 10 mg x ml(-1), but not at lower concentrations.

Pectin and pectic-oligosaccharides induce apoptosis in in vitro human colonic adenocarcinoma cells.

BACKGROUND: Dietary fibres have been associated with decreased risk of various cancers, although the mechanisms are unclear. Induction of apoptosis in tumour cells is thought to be an important protective mechanism against colorectal cancer. This work investigates the effects of pectins and pectic-oligosaccharides (POS) on the human colonic adenocarcinoma cell line HT29. MATERIALS AND METHODS: The anti-proliferative effects of pectin and POS were studied by testing the HT29 cells for cytotoxicity, differentiation and/or apoptosis by lactate dehydrogenase, alkaline phosphatase and caspase-3 activity assays. DNA agarose gel electrophoresis was also carried out. RESULTS: A significant reduction in attached cell numbers was observed after three days incubation. This decrease was neither due to cells undergoing necrosis nor differentiation. Increased apoptosis frequency, after incubation with 1% (w/v) pectin and/or POS, was demonstrated by caspase-3 activity and DNA laddering on agarose gel electrophoresis. CONCLUSION: Dietary pectins and their degradation products may contribute to the reported protective effects of fruits against colon cancer.

A DNA microarray for the detection of point mutations and copy number variation causing familial hypercholesterolemia in Europe.

To facilitate genetic cascade screening for familial hypercholesterolemia (FH) in Europe, two versions (7 and 9) of a DNA microarray were developed to detect the most frequent point mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin 9 (PCSK9) genes. The design of these microarrays is based on LIPOchip, version 4, which detects 191 LDLR and APOB mutations identified in Spanish patients with FH. A major improvement of LIPOchip, versions 7 and 9, is the ability to detect copy number variation (deletions or duplications of entire exons) in LDLR, thus abolishing the need to perform multiplex ligase-dependent probe amplification in patients with FH. The aim of this study was to validate a tool capable of detecting point mutations and copy number variations simultaneously and to evaluate its use and the newly developed software for analysis in clinical practice by reanalysis of several patients with known mutations causing FH. With the help of these validations, several aspects were analyzed, improved, and implemented in a newer version, which was evaluated through an internal validation.

Comparative study of polymorphism frequencies of the CYP2D6, CYP3A5, CYP2C8 and IL-10 genes in Mexican and Spanish women with breast cancer.

AIM: Pharmacogenetic studies in breast cancer (BC) may predict the efficacy of tamoxifen and the toxicity of paclitaxel and capecitabine. We determined the frequency of polymorphisms in the CYP2D6 gene associated with activation of tamoxifen, and those of the genes CYP2C8, CYP3A5 and DPYD associated with toxicity of paclitaxel and capecitabine. We also included a IL-10 gene polymorphism associated with advanced tumor stage at diagnosis. PATIENTS & METHODS: Genomic DNAs from 241 BC patients from northeast Mexico were genotyped using DNA microarray technology. RESULTS: For tamoxifen processing, CYP2D6 genotyping predicted that 90.8% of patients were normal metabolizers, 4.2% ultrarapid, 2.1% intermediate and 2.9% poor metabolizers. For paclitaxel and the CYP2C8 gene, 75.3% were normal, 23.4% intermediate and 1.3% poor metabolizers. Regarding the DPYD gene, only one patient was a poor metabolizer. For the IL-10 gene, 47.1% were poor metabolizers. CONCLUSION: These results contribute valuable information towards personalizing BC chemotherapy in Mexican women.

Molecular characterization of familial hypercholesterolemia in Spain.

Familial hypercholesterolemia (FH), characterized by isolated elevation of plasmatic low-density lipoprotein (LDL) cholesterol and premature coronary heart disease (CHD), is associated with mutations in three major genes: LDL receptor (LDLR), apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin 9 (PCSK9). We have analyzed 5430 Spanish index cases and 2223 relatives since 2004 with LIPOchip(®) genetic diagnostic platform, a microarray for the detection of Spanish common mutations in these three genes, including copy number variation (CNV) in LDLR, followed by sequencing analysis of the coding regions of LDLR and exon 26 of APOB, when the result is negative. Samples were received from hospitals of all around Spain. The preferred clinical criterion to diagnose FH was Dutch Lipid Clinic Network (DLCN) score. Our results show that there is a broad spectrum of mutations in the LDLR gene in Spain since about 400 different mutations were detected, distributed along almost the whole LDLR gene. Mutations in APOB (mainly p.Arg3527Gln) covered 6.5% of positive cases and only one PCSK9 mutation was detected. We found correlation between more severe mutations and the clinical diagnosis but also that 28% of FH patients harboring mutations do not have a definite clinical diagnosis. This study analyzes the mutation spectrum in Spain, remarks the importance of genetic diagnosis of FH patients, as well as the cascade screening, and shows how it is being carried out in Spain.

Interactions between age and apoE genotype on fasting and postprandial triglycerides levels.

OBJECTIVE: The influences of genetic determinants on the magnitude of postprandial lipaemia are presently unclear. Here the impact of the common apolipoprotein (apo)E epsilon mutation on the postprandial triglyceride (TG) response is determined, along with an assessment of genotype penetrance according to age, body mass index and gender. METHODS AND RESULTS: Healthy adults (n=251) underwent a postprandial investigation, in which blood samples were taken at regular intervals after a test breakfast (0 min, 49 g fat) and lunch (330 min, 29 g fat) until 480 min after the test breakfast. There was a significant impact of apoE genotype on fasting total cholesterol (TC), (P=0.027), LDL-cholesterol (LDL-C), (P=0.008), and %LDL(3) (P=0.001), with higher and lower levels in the E4 and E2 carriers respectively relative to the E3/E3 genotype. Reflective of a higher fasting TG (P=0.001), a significantly higher area under the curve for the postprandial TG response (TG AUC) was evident in the E4 carriers relative to the E3/E3 group (P=0.038). In the group as a whole, a significant age×genotype interaction was observed for fasting TC (P=0.021). In the participants>50 years there was a significant impact of genotype on TC (P=0.005), LDL-C (P=0.001) and TAG AUC (P=0.028). CONCLUSIONS: It is possible that an exaggerated postprandial lipaemia contributes to the increased coronary heart disease risk associated with carriers of the E4 allele; an effect which is more evident in older adults.

Contribution of apolipoprotein E genotype and docosahexaenoic acid to the LDL-cholesterol response to fish oil.

OBJECTIVES: To investigate the impact of apolipoprotein E (apoE) genotype on the response of the plasma lipoprotein profile to eicosapentaenoic acid (EPA) versus docosahexaenoic acid (DHA) intervention in humans. METHODS AND RESULTS: 38 healthy normolipidaemic males, prospectively recruited on the basis of apoE genotype (n=20 E3/E3 and n=18 E3/E4), completed a double-blind placebo-controlled cross-over trial, consisting of 3 x 4 week intervention arms of either control oil, EPA-rich oil (ERO, 3.3g EPA/day) or DHA-rich oil (DRO, 3.7g DHA/day) in random order, separated by 10 week wash-out periods. A significant genotype-independent 28% and 19% reduction in plasma triglycerides in response to ERO and DRO was observed. For total cholesterol (TC), no significant treatment effects were evident; however a significant genotype by treatment interaction emerged (P=0.045), with a differential response to ERO and DRO in E4 carriers. Although the genotype x treatment interaction for LDL-cholesterol (P=0.089) did not reach significance, within DRO treatment analysis indicated a 10% increase in LDL (P=0.029) in E4 carriers with a non-significant 4% reduction in E3/E3 individuals. A genotype-independent increase in LDL mass was observed following DRO intervention (P=0.018). Competitive uptake studies in HepG2 cells using plasma very low density lipoproteins (VLDL) from the human trial, indicated that following DRO treatment, VLDL(2) fractions obtained from E3/E4 individuals resulted in a significant 32% (P=0.002) reduction in LDL uptake relative to the control. CONCLUSIONS: High dose DHA supplementation is associated with increases in total cholesterol in E4 carriers, which appears to be due to an increase in LDL-C and may in part negate the cardioprotective action of DHA in this population subgroup.

Errors and reproducibility of DNA array-based detection of allelic variants in ADME genes: PHARMAchip.

AIMS: Differences in adverse drug reactions can be explained by genetic variations, especially if they determine the expression of certain protein effectors and/or drug-metabolizing enzymes. Over the last decade, several tests screening for the most frequent polymorphisms in drug-metabolizing enzymes have been marketed for research and diagnostic purposes. The aim of this study was to assess the suitability of PHARMAchip for the genotyping of polymorphisms of genes associated with drug metabolism and response as an alternative to Jurilab Ltd's DrugMEt Test. MATERIALS & METHODS: In this observational study, performed using 100 previously genotyped DNA samples, we report on common genes included in the two different tests examined: the former DrugMEt test and the recently introduced PHARMAchip test. RESULTS & CONCLUSION: Although these tests are based on different methodological approaches, we have found a high concordance of results between both methods. Some of the discrepancies between tests were related to allelic variants not monitored in a particular microarray and the quality of the genomic DNA used.

Influence of apoA-V gene variants on postprandial triglyceride metabolism: impact of gender.

Although apolipoprotein A-V (apoA-V) polymorphisms have been consistently associated with fasting triglyceride (TG) levels, their impact on postprandial lipemia remains relatively unknown. In this study, we investigate the impact of two common apoA-V polymorphisms (-1131 T>C and S19W) and apoA-V haplotypes on fasting and postprandial lipid metabolism in adults in the United Kingdom (n = 259). Compared with the wild-type TT, apoA-V -1131 TC heterozygotes had 15% (P = 0.057) and 21% (P = 0.002) higher fasting TG and postprandial TG area under the curve (AUC), respectively. Significant (P = 0.038) and nearly significant (P = 0.057) gender x genotype interactions were observed for fasting TG and TG AUC, with a greater impact of genotype in males. Lower HDL-cholesterol was associated with the rare TC genotype (P = 0.047). Significant linkage disequilibrium was found between the apoA-V -1131 T>C and the apoC-III 3238 C>G variants, with univariate analysis indicating an impact of this apoC-III single nucleotide polymorphism (SNP) on TG AUC (P = 0.015). However, in linear regression analysis, a significant independent association with TG AUC (P = 0.007) was only evident for the apoA-V -1131 T>C SNP, indicating a greater relative importance of the apoA-V genotype.

Polymorphisms in the Apolipoprotein L1 gene and their effects on blood lipid and glucose levels in middle age males.

Apolipoprotein L1 in plasma is associated with high-density lipoprotein. Novel APOL1 polymorphisms are investigated along with the association of two common haplotypes (Lysl66Glu, Ile244Met, Lys271Arg) with circulating lipid and glucose levels. Although the amino acid substitutions occur in the amphipathic alpha helices region involved in lipid binding, these substitutions were found not to independently account for variability in circulating lipid and glucose levels in 149 middle age males.

Egb 761 in the postgenomic era: new tools from molecular biology for the study of complex products such as Ginkgo biloba extract.

The decoding of the human genome has completely changed our views on medicine. Beyond sequencing, tools of the postgenomic era may lead to a better understanding of various therapies, especially those with a complex effect on numerous cellular components and functions. The development of high-density oligonucleotide microarrays led to pioneer studies on the multiple gene expression effects exhibited by Ginkgo biloba extract EGb 761, changing traditional pharmacology and medicine concepts. Instead of studying a simple gene or protein, a global investigation of all genes or proteins at once can give insights of the complexity of biological systems.

Lycopene inhibits the growth of normal human prostate epithelial cells in vitro.

Lycopene has repeatedly been shown to inhibit the growth of human prostate cells in vitro. However, previous studies with lycopene have focused on cancer specimens, and it is still unclear whether this carotenoid affects the growth of normal human prostate cells as well. Therefore, we investigated the effects of lycopene on normal human prostate epithelial cells (PrEC) by treating them with synthetic all-E-lycopene (up to 5 micromol/L) and assessing proliferation via [3H]thymidine incorporation. The effects of lycopene on cell cycle progression were investigated via flow cytometry. To elucidate whether lycopene modulates cyclins involved in cell cycle progression, protein expressions of cyclins D1 and E were analyzed. The results show that lycopene significantly inhibited the growth of PrEC in a dose-dependent fashion. Flow cytometry revealed a significant cell cycle arrest in the G0/G1 phase. This effect was confirmed by inhibition of cyclin D1 protein expression, whereas cyclin E levels remained unchanged. The results demonstrate that lycopene inhibits growth of nonneoplastic PrEC in vitro. We hypothesize that lycopene might likewise inhibit the growth of prostatic epithelial cells in vivo. This might have an effect on prostate development and/or on enlargement of prostate tissue as found in benign prostate hyperplasia, a potential precursor of prostate cancer.

Tissue-specific gene expression of prolactin receptor in the acute-phase response induced by lipopolysaccharides.

Acute inflammation can elicit a defense reaction known as the acute-phase response (APR) that is crucial for reestablishing homeostasis in the host. The role for prolactin (PRL) as an immunomodulatory factor maintaining homeostasis under conditions of stress has been proposed; however, its function during the APR remains unclear. Previously, it was shown that proinflammatory cytokines characteristic of the APR (TNF-alpha, IL-1beta, and IFNgamma) induced the expression of the PRL receptor (PRLR) by pulmonary fibroblasts in vitro. Here, we investigated the in vivo expression of PRLR during lipopolysaccharide (LPS)-induced APR in various tissues of the mouse. We show that PRLR mRNA and protein levels were downregulated in hepatic tissues after intraperitoneal LPS injection. Downregulation of PRLR in the liver was confirmed by immunohistochemistry. A suppressive effect on mRNA expression was also observed in prostate, seminal vesicle, kidney, heart, and lung tissues. However, PRLR mRNA levels were increased in the thymus, and no changes were observed in the spleen. The proportion of transcripts for the different receptor isoforms (long, S1, S2, and S3) in liver and thymus was not altered by LPS injection. These findings suggest a complex tissue-specific regulation of PRLR expression in the context of the APR.