A decade of progress on the genetic basis of coronary artery disease. Practical insights for the internist.

Clinicians are well aware of the importance of a positive family history for coronary artery disease (CAD). Nonetheless, elucidation of the genetic basis of CAD has long proven difficult. The scenario changed in the last decade through the application of modern genomic technologies, like genome-wide association studies (GWAS) and next generation sequencing (NGS). GWAS have discovered over 60 common variants highly associated with CAD. For predictive purposes, such variants have been used to build up Genetic Risk Scores (GRSs), but their incorporation into classical prediction models does not appear substantially outperform the simple addition of family history. To date, the only strong case for the utility of incorporating genetic testing into clinical practice is represented by the diagnosis of Familial Hypercholesterolemia (FH). On the other hand, utilization of genomic techniques has driven formidable advances into the knowledge of CAD pathophysiology, particularly by addressing controversies on the causality of some lipid fractions that had long remained unsolved because of limitations of observational epidemiology. For example, NGS-derived rare variants with strong functional effects on key-genes like ANGPTL4, APOA5, APOC3, LPL, and SCARB1, have proven useful as proxies to demonstrate the causality of triglyceride-rich lipoproteins (TRLs) at variance with HDL-cholesterol concentration, thus contributing to tear down a dogma from classical epidemiology. Moreover, such variants have paved the way for the development of new biologic drugs (i.e. monoclonal antibodies or antisense oligonucleotides) targeting key proteins like PCSK9, Lipoprotein(a), and apolipoprotein C3. Such drugs are currently under active investigation, with first results being extremely promising.

DNA methylation and gene expression profiles show novel regulatory pathways in hepatocellular carcinoma.

BACKGROUND: Alcohol is a well-known risk factor for hepatocellular carcinoma (HCC), but the mechanisms underlying the alcohol-related hepatocarcinogenesis are still poorly understood. Alcohol alters the provision of methyl groups within the hepatic one-carbon metabolism, possibly inducing aberrant DNA methylation. Whether specific pathways are epigenetically regulated in alcohol-associated HCC is, however, unknown. The aim of the present study was to investigate the genome-wide promoter DNA methylation and gene expression profiles in non-viral, alcohol-associated HCC. From eight HCC patients undergoing curative surgery, array-based DNA methylation and gene expression data of all annotated genes were analyzed by comparing HCC tissue and homologous cancer-free liver tissue. RESULTS: After merging the DNA methylation with gene expression data, we identified 159 hypermethylated-repressed, 30 hypomethylated-induced, 49 hypermethylated-induced, and 56 hypomethylated-repressed genes. Notably, promoter DNA methylation emerged as a novel regulatory mechanism for the transcriptional repression of genes controlling the retinol metabolism (ADH1A, ADH1B, ADH6, CYP3A43, CYP4A22, RDH16), iron homeostasis (HAMP), one-carbon metabolism (SHMT1), and genes with a putative, newly identified function as tumor suppressors (FAM107A, IGFALS, MT1G, MT1H, RNF180). CONCLUSIONS: A genome-wide DNA methylation approach merged with array-based gene expression profiles allowed identifying a number of novel, epigenetically regulated candidate tumor-suppressor genes in alcohol-associated hepatocarcinogenesis. Retinol metabolism genes and SHMT1 are also epigenetically regulated through promoter DNA methylation in alcohol-associated HCC. Due to the reversibility of epigenetic mechanisms by environmental/nutritional factors, these findings may open up to novel interventional strategies for hepatocarcinogenesis prevention in HCC related to alcohol, a modifiable dietary component.

Global DNA methylation and hydroxymethylation differ in hepatocellular carcinoma and cholangiocarcinoma and relate to survival rate.

UNLABELLED: In addition to DNA methylation, hydroxymethylation of DNA is recognized as a novel epigenetic mark. Primary liver cancers, i.e., hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC), are highly prevalent but epigenetically poorly characterized, so far. In the present study we measured global methylcytosine (mCyt) and hydroxymethylcytosine (hmCyt) in HCC and CC tissues and in peripheral blood mononuclear cell (PBMC) DNA to define mCyt and hmCyt status and, accordingly, the survival rate. Both mCyt and hmCyt were measured by a liquid chromatography/tandem mass spectrometry method in neoplastic and homologous nonneoplastic tissues, i.e., liver and gallbladder, and in PBMCs of 31 HCC and 16 CC patients. Content of mCyt was notably lower in HCC than in CC tissues (3.97% versus 5.26%, respectively; P < 0.0001). Significantly reduced mCyt was also detected in HCC compared to nonneoplastic tissue (3.97% versus 4.82% mCyt, respectively; P < 0.0001), but no such difference was found for CC versus homologous nonneoplastic tissue. Hydroxymethylation was significantly decreased in HCC versus nonneoplastic liver tissue (0.044 versus 0.128, respectively; P < 0.0001) and in CC versus both liver and gallbladder nonneoplastic tissue (0.030 versus 0.124, P = 0.026, and 0.030 versus 0.123, P = 0.006, respectively). When the survival rate was evaluated according to mCyt PBMC content by Kaplan-Meier analysis, patients with mCyt ≥5.59% had a significantly higher life expectancy than those with mCyt <5.59% (P = 0.034) at a follow-up period up to 48 months. CONCLUSION: A significant DNA hypomethylation distinguishes HCC from CC, while DNA hypo-hydroxymethylation characterizes both HCC and CC, and a PBMC DNA mCyt content ≥5.59% relates to a favorable outcome in primary liver cancers.

Paraoxonases: ancient substrate hunters and their evolving role in ischemic heart disease.

Interest in the role of paraoxonases (PON) in cardiovascular research has increased substantially over the past two decades. These multifaceted and pleiotropic enzymes are encoded by three highly conserved genes (PON1, PON2, and PON3) located on chromosome 7q21.3-22.1. Phylogenetic analysis suggests that PON2 is the ancient gene from which PON1 and PON3 arose via gene duplication. Although PON are primarily lactonases with overlapping, but distinct specificities, their physiologic substrates remain poorly characterized. The most interesting characteristic of PON, however, is their multifunctional roles in various biochemical pathways. These include protection against oxidative damage and lipid peroxidation, contribution to innate immunity, detoxification of reactive molecules, bioactivation of drugs, modulation of endoplasmic reticulum stress, and regulation of cell proliferation/apoptosis. In general, PON appear as "hunters" of old and new substrates often involved in athero- and thrombogenesis. Although reduced PON activity appears associated with increased cardiovascular risk, the correlation between PON genotype and ischemic heart disease remains controversial. In this review, we examine the biochemical pathways impacted by these unique enzymes and investigate the potential use of PON as diagnostic tools and their impact on development of future therapeutic strategies.

In type 1 diabetes a subset of anti-coxsackievirus B4 antibodies recognize autoantigens and induce apoptosis of pancreatic beta cells.

Type 1 diabetes is characterized by autoimmune destruction of pancreatic beta cells. The role played by autoantibodies directed against beta cells antigens in the pathogenesis of the disease is still unclear. Coxsackievirus B infection has been linked to the onset of type 1 diabetes; however its precise role has not been elucidated yet. To clarify these issues, we screened a random peptide library with sera obtained from 58 patients with recent onset type 1 diabetes, before insulin therapy. We identified an immunodominant peptide recognized by the majority of individual patients'sera, that shares homology with Coxsackievirus B4 VP1 protein and with beta-cell specific autoantigens such as phogrin, phosphofructokinase and voltage-gated L-type calcium channels known to regulate beta cell apoptosis. Antibodies against the peptide affinity-purified from patients' sera, recognized the viral protein and autoantigens; moreover, such antibodies induced apoptosis of the beta cells upon binding the L-type calcium channels expressed on the beta cell surface, suggesting a calcium dependent mechanism. Our results provide evidence that in autoimmune diabetes a subset of anti-Coxsackievirus antibodies are able to induce apoptosis of pancreatic beta cells which is considered the most critical and final step in the development of autoimmune diabetes without which clinical manifestations do not occur.

Global DNA hypomethylation in peripheral blood mononuclear cells as a biomarker of cancer risk.

BACKGROUND: Global DNA hypomethylation is an early molecular event in carcinogenesis. Whether methylation measured in peripheral blood mononuclear cells (PBMCs) DNA is a clinically reliable biomarker for early detection or cancer risk assessment is to be established. METHODS: From an original sample-set of 753 male and female adults (ages 64.8 ± 7.3 years), PBMCs DNA methylation was measured in 68 subjects with history of cancer at time of enrollment and 62 who developed cancer during follow-up. Age- and sex-matched controls for prevalent and incident cancer cases (n = 68 and 58, respectively) were also selected. Global DNA methylation was assessed by liquid chromatography/mass spectrometry (LC/MS). Methylenetetrahydrofolate reductase (MTHFR) 677C>T genotype and plasma folate concentrations were also determined for the known gene-nutrient interaction affecting DNA methylation. RESULTS: Cancer subjects had significantly lower PBMCs-DNA methylation than controls [4.39 (95% confidence intervals (CI), 4.25-4.53) vs. 5.13 (95% CI, 5.03-5.21) %mCyt/(mCyt+Cyt); P < 0.0001]. A DNA methylation threshold of 4.74% clearly categorized patients with cancer from controls so that those with DNA methylation less than 4.74% showed an increased prevalence of cancer than those with higher levels (91.5% vs. 19%; P < 0.001). Subjects with cancer at follow-up had, already at enrollment, reduced DNA methylation as compared with controls [4.34 (95% CI, 4.24-4.51) vs. 5.08 (95% CI, 5.05-5.22) %mCyt/(mCyt+Cyt); P < 0.0001]. Moreover, MTHFR677C>T genotype and folate interact for determining DNA methylation, so that MTHFR677TT carriers with low folate had the lowest DNA methylation and concordantly showed a higher prevalence of cancer history (OR, 7.04; 95% CI, 1.52-32.63; P = 0.013). CONCLUSIONS: Genomic PBMCs-DNA methylation may be a useful epigenetic biomarker for early detection and cancer risk estimation. IMPACT: This study identifies a threshold for PBMCs-DNA methylation to detect cancer-affected from cancer-free subjects and an at-risk condition for cancer based on genomic DNA methylation and MTHFR677C>T-folate status.

Promoter methylation in coagulation F7 gene influences plasma FVII concentrations and relates to coronary artery disease.

BACKGROUND: Plasma factor VII concentrations (FVIIa), a marker of coronary artery disease (CAD) risk, are influenced by genetic markers at the promoter site: the A2 allele, due to a 10bp insertion at position -323, is a determinant of lower FVIIa concentrations and reduced CAD risk, while the -402A allele, due to a G>A substitution, confers increased transcriptional activity in vitro resulting in higher FVIIa. Transcriptional regulation of F7 by epigenetic features is, however, still unknown as is the inter-relationship of genetic and epigenetic modifications at the promoter site. OBJECTIVE: To investigate a possible epigenetic regulation of the F7 gene at the promoter region and its link with functional F7 polymorphisms at the same site. METHODS AND RESULTS: F7 promoter methylation and its relation to F7 promoter polymorphisms in modulating FVIIa and CAD risk were evaluated by methyl-specific PCR and bisulfite sequencing techniques in 253 subjects, of whom 168 had CAD and 88 were CAD-free. Plasma FVIIa was inversely related to methylation in A1A1 and -402GG, that is in the absence of the rare A2 and -402A allele. The higher FVIIa paralleled the lower methylation in A1A1 compared to A2A2 (p=0.035), while no variation in methylation was associated with the different -402G>A genotypes. The modulation of methylation-induced FVIIa concentrations was observed only in A1A1 where the higher methylation resulting in lower FVIIa was prevalent within the CAD-free group compared to the CAD group (p=0.011). CONCLUSIONS: Epigenetic regulation through methylation of F7 promoter is associated with CAD by affecting plasma FVIIa concentrations in A1A1 genotypes.

Low levels of serum paraoxonase activities are characteristic of metabolic syndrome and may influence the metabolic-syndrome-related risk of coronary artery disease.

Low concentrations of plasma high-density lipoprotein (HDLs) are characteristic in metabolic syndrome (MS). The antioxidant ability of HDLs is, at least in part, attributable to pleiotropic serum paraoxonase (PON1). Different PON1 activities have been assessed in 293 subjects with (n = 88) or without MS (n = 205) and with (n = 195) or without (n = 98) angiographically proven coronary artery disease (CAD). MS subjects had low PON1 activities, with a progressively decreasing trend by increasing the number of MS abnormalities. The activity versus 7-O-diethyl phosphoryl,3-cyano,4-methyl,7-hydroxycoumarin (DEPCyMC), which is considered a surrogate marker of PON1 concentration, showed the most significant association with MS, independently of both HDL and apolipoprotein A-I levels. Subjects with MS and low DEPCyMCase activity had the highest CAD risk (OR 4.34 with 95% CI 1.44-13.10), while no significant increase of risk was found among those with MS but high DEPCyMCase activity (OR 1.45 with 95% CI 0.47-4.46). Our results suggest that low PON1 concentrations are typical in MS and may modulate the MS-related risk of CAD.

Folic acid effects on s-adenosylmethionine, s-adenosylhomocysteine, and DNA methylation in patients with intermediate hyperhomocysteinemia.

OBJECTIVE: Folic acid (FA) supplementation decreases homocysteine (tHcy) levels. However, little is known about the effects of FA treatment on DNA methylation or plasma S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) concentrations. The purpose of this study was to investigate the effects of FA supplementation on AdoMet, AdoHcy, and genomic DNA methylation in hyperhomocysteinemic subjects without end-stage renal disease. METHODS: To evaluate the effects of 5 mg FA/d for 8 weeks, we recruited 7 hyperhomocysteinemic MTHFR677TT patients (tHcy >30 µmol/L) with normal renal function. RESULTS: FA supplementation induced a decrease in tHcy (from 51.1 ± 21 at baseline to 26.1 ± 27 µmol/L after folate supplementation; p < 0.01). A parallel increase was seen in plasma AdoMet concentrations and the AdoMet/AdoHcy ratio (p < 0.05). However, FA supplementation had no effect on global DNA methylation levels in the present study. CONCLUSIONS: Supraphysiologic FA supplementation can modulate biochemical markers in one-carbon metabolism such as tHcy, AdoMet, and the AdoMet/AdoHcy ratio in hyperhomocysteinemic subjects. However, the reduction in homocysteinemia and the increased availability of methyl compounds provided by vitamin supplementation may not be sufficient to affect genomic DNA methylation.

Polymorphisms of cyclo-oxygenases and 5-lipo-oxygenase-activating protein are associated with chronic spontaneous urticaria and urinary leukotriene E4.

The mechanisms of chronic spontaneous urticaria (CSU) continue to be unknown. Our working hypothesis is that polymorphisms of cyclo-oxygenases and 5-lipo-oxygenase-activating protein may be involved in the pathways leading to CSU. We examined five candidate polymorphisms of cyclo-oxygenases 1 and 2 and of 5-lipo-oxygenase-activating protein in 109 controls and in 94 CSU patients from Northern Italy. We also examined the levels of urinary leukotriene E4 (LTE4) before and after challenge with ASA. A multiple regression model was found to show that COX-2 5'UTR T/G, COX-2 Exon 10 T/C, and FLAP -336 G/A polymorphisms were significantly associated with CSU, with the minor allele more represented in CSU group. Similar results were obtained as regards the specific association with ASA-tolerated CSU and ASA-exacerbated CSU. Evaluating a polygenic model, reflecting the sum of the concomitant alleles associated with CSU (i.e. COX-2 5'UTR G allele, COX-2 Exon 10 C allele, and FLAP -336 G/A allele), the proportion of CSU patients increased progressively with the increasing number of unfavourable alleles. Finally, in a linear regression model after adjustment for disease status COX-1 22 T carriership remained a significant predictor of post-challenge high urinary LTE4 levels. Our results support the hypothesis that polymorphisms of cyclo-oxygenases and 5-lipo-oxygenase-activating protein may be associated with CSU.

Abnormal modulation of cell protective systems in response to ischemic/reperfusion injury is important in the development of mouse sickle cell hepatopathy.

BACKGROUND: Sickle cell disease, a genetic red cell disorder inherited in an autosomal recessive manner, occurs throughout the world. Hepatic dysfunction and liver damage may be present in sickle cell disease, but the pathogenesis of these conditions is only partially understood. DESIGN AND METHODS: Transgenic mice with sickle cell disease (SAD mice) and wild-type mice were exposed to an ischemic/reperfusion stress. The following parameters were evaluated: hematologic profile, transaminase and bilirubin levels, liver histopathology, and mRNA levels of nuclear factor-κB p65, endothelial nitric oxide synthase, inducible nitric oxide synthase, heme oxygenase-1 and phosphodiesterase-1, -2, -3, and -4 genes in hepatocytes obtained by laser-capture microdissection. Immunoblotting was used to analyze the expression of the following proteins: nuclear factor-κB p65 and phospho-nuclear factor-κB p65, heme oxygenase-1, biliverdin reductase, heat shock protein-70, heat shock protein-27 and peroxiredoxin-6. A subgroup of SAD mice was treated with the phosphodiesterase-4 inhibitor rolipram (30 mg/Kg/day by gavage) during the ischemic/reperfusion protocol. RESULTS: In SAD mice the ischemic/reperfusion stress induced liver damage compatible with sickle cell disease hepatopathy, which was associated with: (i) lack of hypoxia-induced nuclear factor-κB p65 activation; (ii) imbalance in the endothelial/inducible nitric oxide synthase response to ischemic/reperfusion stress; (iii) lack of hypoxia-induced increased expression of heme oxygenase-1/biliverdin reductase paralleled by a compensatory increased expression of heat shock proteins 70 and 27 and peroxiredoxin-6; and (iv) up-regulation of the phosphodiesterase-1, -2, -3, and -4 genes. In SAD mice the phosphodiesterase-4 inhibitor rolipram attenuated the ischemic/reperfusion-related microcirculatory dysfunction, reduced the inflammatory cell infiltration and induced the heme oxygenase-1/biliverdin reductase cytoprotective systems. CONCLUSIONS: In SAD mice, sickle cell hepatopathy is associated with perturbed nuclear factor-κB p65 signaling with an imbalance of endothelial/inducible nitric oxide synthase levels, lack of heme oxygenase-1/biliverdin reductase expression and up-regulation of two novel cytoprotective systems: heat shock protein-27 and peroxiredoxin-6.

Effects of shock wave therapy in the skin of patients with progressive systemic sclerosis: a pilot study.

Vasculopathy, immunological abnormalities, and excessive tissue fibrosis are key elements in the pathogenesis of progressive systemic sclerosis (SSc). Extracorporeal shock waves (ESW) have anti-inflammatory and regenerative effects on different tissues. We hypothesized that ESW can reduce endothelial cell damage and skin fibrosis in patients with SSc. We enrolled 30 patients affected by SSc, 29 females and 1 male. Rodnan Skin Score (RSS) and Visuo-Analogical Scale (VAS) for skin wellness were performed before and immediately after ESW therapy (ESWT) and at 7, 30, 60, and 90 days after the treatment. Sonographic examination of the patients' arms was performed before and 7, 30, 60, 90 days after treatment. Blood samples were obtained before and 30 and 60 days after treatment to measure serological levels of von Willebrand factor, vascular endothelial growth factor, intracellular adhesion molecule-1, monocyte chemotactic protein-1. The number of endothelial progenitor cells (EPCs) and circulating endothelial cells (CECs) were determined at the same time points. After ESWT we observed a rapid and persistent reduction of RSS and decrease of VAS. There was no difference in skin thickness before and after ESWT; however, we observed a more regular skin structure and an improvement in skin vascularization 90 days after treatment. EPCs and CECs increased 60 and 90 days after treatment, while serological biomarkers showed no variation before and after therapy. In conclusion, ESWT resulted in an improvement of VAS, RSS, and of skin vascular score, and in an increase of CECs and EPCs.

Polymorphisms at LDLR locus may be associated with coronary artery disease through modulation of coagulation factor VIII activity and independently from lipid profile.

High levels of coagulation factor VIII (FVIII) have been associated with cardiovascular disease. Low-density lipoprotein receptor (LDLR) has been recently demonstrated to contribute to FVIII clearance from plasma. The aim of this study was to evaluate 3 single nucleotide polymorphisms in SMARCA4-LDLR gene locus (rs1122608, rs2228671, and rs688) and FVIII coagulant activity (FVIII:c) in subjects with (n = 692) or without (n = 291) angiographically confirmed coronary artery disease (CAD). High FVIII:c levels were an independent risk factor for CAD. The rs688 and rs2228671 genotypes were predictors of FVIII:c with T alleles associated with higher FVIII:c levels. The rs2228671T allele was associated also with reduced total and LDL-cholesterol levels. With respect to the risk of CAD, no association was found for rs2228671. Consistently with higher FVIII:c levels, the rs688T allele was associated with CAD, whereas, consistently with a favorable lipid profile, the rs1122608T allele was associated with a decreased CAD prevalence. After adjustment for classic cardiovascular risk factors, including plasma lipids, rs688 remained associated with CAD (OR for T carriers: 1.67 with 95% confidence interval, 1.10-2.54). Haplotype analysis confirmed such results. Our data suggest that polymorphisms at LDLR locus modulate FVIII:c levels and may be associated with CAD risk independently from plasma lipids.

Gene expression profiling in circulating endothelial cells from systemic sclerosis patients shows an altered control of apoptosis and angiogenesis that is modified by iloprost infusion.

INTRODUCTION: Circulating endothelial cells are increased in patients affected by systemic sclerosis (SSc) and their number strongly correlates with vascular damage. The effects of iloprost in systemic sclerosis are only partially known. We aimed at studying the gene expression profile of circulating endothelial cells and the effects of iloprost infusion and gene expression in patients with systemic sclerosis. METHODS: We enrolled 50 patients affected by systemic sclerosis, 37 patients without and 13 patients with digital ulcers. Blood samples were collected from all patients before and 72 hours after either a single day or five days eight hours iloprost infusion. Blood samples were also collected from 50 sex- and age-matched healthy controls. Circulating endothelial cells and endothelial progenitors cells were detected in the peripheral blood of patients with systemic sclerosis by flow cytometry with a four-colour panel of antibodies. Statistical analysis was performed with the SPSS 16 statistical package.Circulating endothelial cells were then isolated from peripheral blood by immunomagnetic CD45 negative selection for the gene array study. RESULTS: The number of both circulating endothelial cells and progenitors was significantly higher in patients affected by systemic sclerosis than in controls and among patients in those with digital ulcers than in patients without them. Circulating endothelial cells and progenitors number increased after iloprost infusion. Gene array analysis of endothelial cells showed a different transcriptional profile in patients compared to controls. Indeed, patients displayed an altered expression of genes involved in the control of apoptosis and angiogenesis. Iloprost infusion had a profound impact on endothelial cells gene expression since the treatment was able to modulate a very high number of transcripts. CONCLUSIONS: We report here that circulating endothelial cells in patients with systemic sclerosis show an altered expression of genes involved in the control of apoptosis and angiogenesis. Moreover we describe that iloprost infusion has a strong effect on endothelial cells and progenitors since it is able to modulate both their number and their gene expression profile.

Generation of anti-NAG-2 mAb from patients' memory B cells: implications for a novel therapeutic strategy in systemic sclerosis.

We have previously reported that antibodies directed against the cytomegalovirus-derived protein UL94 cross react with the cell surface tetraspanin transmembrane 4 superfamily member 7 (TM4SF7 or NAG-2) molecule inducing apoptosis of endothelial cells and activation of fibroblasts in patients with systemic sclerosis (SSc). We aimed at generating a non-functional mAb directed against NAG-2 from patients' memory B cells. Direct and competitive ELISA methods have been used to evaluate the binding of antibodies from scleroderma patients' and controls' sera to the NAG-2 peptide. IgG memory B cells were sorted, EBV transformed and cloned to obtain NAG-2-specific mAbs. Endothelial cells and fibroblasts were cultured under standard conditions and used for functional assays. Anti-NAG-2-purified antibodies obtained from patients' Ig induce endothelial cell apoptosis and fibroblast proliferation. Patients' Igs depleted of the anti-NAG-2 fraction do not exert such functional activity. Therefore, the NAG-2 molecule represents a potential novel candidate for therapeutic intervention in SSc. Here, we describe the generation of a human mAb directed against the NAG-2 molecule. Such mAb does not retain any functional property and is able to block the effect of serum pathogenetic anti-NAG-2 antibodies. The majority of SSc patients present antibodies directed against tetraspanin NAG-2 and mediate both endothelial cell apoptosis and fibroblast proliferation, features of the disease. The anti-NAG-2 human mAb we have obtained blocks signal transduction and therefore may be a potential candidate for a new treatment in SSc, a disease where the current biological therapies have little or no efficacy.

Effects of female sex hormones and contraceptive pill on the diagnostic work-up for primary aldosteronism.

OBJECTIVES: Due to the widespread use of the aldosterone to renin ratio (ARR), primary aldosteronism is currently recognized as a frequent cause of secondary hypertension. After a positive screening, primary aldosteronism diagnosis needs confirmation by an inhibitory test such as intravenous saline load (ivSLT). The aim of the present study was to investigate the role of female hormones in primary aldosteronism diagnosis, by evaluating possible differences by sex on ARR screening, on the rate of ivSLT response and analyzing the influence of free and oral contraceptive-induced menstrual cycle on ARR. METHODS: We examined ARR in 103 healthy normotensive volunteers, 81 hypertensive patients who underwent ivSLT, 33 healthy women during free menstrual cycle and after oral contraceptive therapy. RESULTS: A significantly higher proportion of normotensive women than men had an elevated ARR (13.6 versus 2.3%, P < 0.05). In 44 out of 81 hypertensive patients, diagnosis of primary aldosteronism was confirmed by ivSLT. Patients with positive and negative ivSLT differed only for sex distribution: 85.2% of men had the primary aldosteronism diagnosis confirmed, compared with 38.9% of women. In healthy women, renin and aldosterone concentrations increased from the follicular to luteal phase of menstrual period, with unchanged ARR. By contrast, renin nearly halved, aldosterone slightly decreased and ARR doubled after oral contraceptive therapy. CONCLUSION: ARR screening fails to predict positive ivSLT in most (60.2%) hypertensive women as compared with 14.8% of hypertensive men. ARR is more often increased in normotensive women than men. Oral contraceptive may affect ARR contributing to the diagnostic inaccuracy in women.

Identification of a novel antibody associated with autoimmune pancreatitis.

BACKGROUND: Autoimmune pancreatitis is characterized by an inflammatory process that leads to organ dysfunction. The cause of the disease is unknown. Its autoimmune origin has been suggested but never proved, and little is known about the pathogenesis of this condition. METHODS: To identify pathogenetically relevant autoantigen targets, we screened a random peptide library with pooled IgG obtained from 20 patients with autoimmune pancreatitis. Peptide-specific antibodies were detected in serum specimens obtained from the patients. RESULTS: Among the detected peptides, peptide AIP(1-7) was recognized by the serum specimens from 18 of 20 patients with autoimmune pancreatitis and by serum specimens from 4 of 40 patients with pancreatic cancer, but not by serum specimens from healthy controls. The peptide showed homology with an amino acid sequence of plasminogen-binding protein (PBP) of Helicobacter pylori and with ubiquitin-protein ligase E3 component n-recognin 2 (UBR2), an enzyme highly expressed in acinar cells of the pancreas. Antibodies against the PBP peptide were detected in 19 of 20 patients with autoimmune pancreatitis (95%) and in 4 of 40 patients with pancreatic cancer (10%). Such reactivity was not detected in patients with alcohol-induced chronic pancreatitis or intraductal papillary mucinous neoplasm. The results were validated in another series of patients with autoimmune pancreatitis or pancreatic cancer: 14 of 15 patients with autoimmune pancreatitis (93%) and 1 of 70 patients with pancreatic cancer (1%) had a positive test for anti-PBP peptide antibodies. When the training and validation groups were combined, the test was positive in 33 of 35 patients with autoimmune pancreatitis (94%) and in 5 of 110 patients with pancreatic cancer (5%). CONCLUSIONS: The antibody that we identified was detected in most patients with autoimmune pancreatitis but also in some patients with pancreatic cancer, making it an imperfect test to distinguish between these two conditions.

Reduced serum hepcidin levels in patients with chronic hepatitis C.

BACKGROUND/AIMS: Patients with chronic hepatitis C (CHC) often have increased liver iron, a condition associated with reduced sustained response to antiviral therapy, more rapid progression to cirrhosis, and development of hepatocellular carcinoma. The hepatic hormone hepcidin is the major regulator of iron metabolism and inhibits iron absorption and recycling from erythrophagocytosis. Hepcidin decrease is a possible pathophysiological mechanism of iron overload in CHC, but studies in humans have been hampered so far by the lack of reliable quantitative assays for the 25-amino acid bioactive peptide in serum (s-hepcidin). METHODS: Using a recently validated immunoassay, we measured s-hepcidin levels in 81 untreated CHC patients and 57 controls with rigorous definition of normal iron status. All CHC patients underwent liver biopsy with histological iron score. RESULTS: s-hepcidin was significantly lower in CHC patients than in controls (geometric means with 95% confidence intervals: 33.7, 21.5-52.9 versus 90.9, 76.1-108.4 ng/mL, respectively; p<0.001). In CHC patients, s-hepcidin significantly correlated with serum ferritin and histological total iron score, but not with s-interleukin-6. After stratification for ferritin quartiles, s-hepcidin increased significantly across quartiles in both controls and CHC patients (chi for trend, p<0.001). However, in CHC patients, s-hepcidin was significantly lower than in controls for each corresponding quartile (analysis of variance, p<0.001). CONCLUSIONS: These results, together with very recent studies in animal and cellular models, indicate that although hepcidin regulation by iron stores is maintained in CHC, the suppression of this hormone by hepatitis C virus is likely an important factor in liver iron accumulation in this condition.

Additive effect of LRP8/APOER2 R952Q variant to APOE epsilon2/epsilon3/epsilon4 genotype in modulating apolipoprotein E concentration and the risk of myocardial infarction: a case-control study.

BACKGROUND: The R952Q variant in the low density lipoprotein receptor-related protein 8 (LRP8)/apolipoprotein E receptor 2 (ApoER2) gene has been recently associated with familial and premature myocardial infarction (MI) by means of genome-wide linkage scan/association studies. We were interested in the possible interaction of the R952Q variant with another established cardiovascular genetic risk factor belonging to the same pathway, namely apolipoprotein E (APOE) epsilon2/epsilon3/epsilon4 genotype, in modulating apolipoprotein E (ApoE) plasma levels and risk of MI. METHODS: In the Italian cohort used to confirm the association of the R952Q variant with MI, we assessed lipid profile, apolipoprotein concentrations, and APOE epsilon2/epsilon3/epsilon4 genotype. Complete data were available for a total of 681 subjects in a case-control setting (287 controls and 394 patients with MI). RESULTS: Plasma ApoE levels decreased progressively across R952Q genotypes (mean levels +/- SD = RR: 0.045 +/- 0.020, RQ: 0.044 +/- 0.014, QQ: 0.040 +/- 0.008 g/l; P for trend = 0.047). Combination with APOE genotypes revealed an additive effect on ApoE levels, with the highest level observed in RR/non-carriers of the E4 allele (0.046 +/- 0.021 g/l), and the lowest level in QQ/E4 carriers (0.035 +/- 0.009 g/l; P for trend = 0.010). QQ/E4 was also the combined genotype with the most significant association with MI (OR 3.88 with 95%CI 1.08-13.9 as compared with RR/non-carriers E4). CONCLUSION: Our data suggest that LRP8 R952Q variant may have an additive effect to APOE epsilon2/epsilon3/epsilon4 genotype in determining ApoE concentrations and risk of MI in an Italian population.

Novel serum paraoxonase activity assays are associated with coronary artery disease.

BACKGROUND: Serum paraoxonase (PON1) exerts antiatherogenic effects. Novel PON1 enzymatic tests have been recently developed: 5-thiobutyl butyrolactone (TBBL) estimates PON1 lactonase activity, whereas 7-O-diethylphosphoryl-3-cyano-4-methyl-7-hydroxycoumarin (DEPCyMC) is considered a surrogate marker of PON1 concentration. The TBBL to DEPCyMC ratio provides the normalized lactonase activity (NLA), which may reflect the degree of PON1 lactonase catalytic stimulation. The aim of this study was to evaluate for the first time TBBLase and DEPCyMCase activity in patients with coronary artery disease (CAD). METHODS: An angiography-based case-control study was conducted, including 300 sex- and age-matched subjects [100 CAD-free, 100 CAD without myocardial infarction (MI) and 100 CAD with MI]. RESULTS: A low DEPCyMCase activity (lowest vs. highest tertile: OR 2.96, 95% CI 1.18-7.43) and a high NLA (highest vs. lowest tertile: OR 3.25, 95% CI 1.28-8.26) were both associated with CAD, independent of classical atherosclerosis risk factors, lipid-lowering therapy and PON1 genotype. Total TBBLase activity was, however, not different in CAD compared to CAD-free subjects. CONCLUSIONS: Novel PON1 activity assays may be associated with CAD. In this study, CAD patients had low DEPCyMCase activity, a possible marker of low PON1 concentration, but showed a high stimulation of PON1 lactonase activity.