Elisabetta Trabetti

C-reactive protein (CRP) is a sensitive biomarker of chronic low-grade inflammation and is associated with multiple complex diseases. The genetic determinants of chronic inflammation remain largely unknown, and the causal role of CRP in several clinical outcomes is debated. We performed two genome-wide association studies (GWASs), on HapMap and 1000 Genomes imputed data, of circulating amounts of CRP by using data from 88 studies comprising 204,402 European individuals. Additionally, we performed in silico functional analyses and Mendelian randomization analyses with several clinical outcomes. The GWAS meta-analyses of CRP revealed 58 distinct genetic loci (p < 5 × 10-8). After adjustment for body mass index in the regression analysis, the associations at all except three loci remained. The lead variants at the distinct loci explained up to 7.0% of the variance in circulating amounts of CRP. We identified 66 gene sets that were organized in two substantially correlated clusters, one mainly composed of immune pathways and the other characterized by metabolic pathways in the liver. Mendelian randomization analyses revealed a causal protective effect of CRP on schizophrenia and a risk-increasing effect on bipolar disorder. Our findings provide further insights into the biology of inflammation and could lead to interventions for treating inflammation and its clinical consequences.

Imputing genotypes from reference panels created by whole-genome sequencing (WGS) provides a cost-effective strategy for augmenting the single-nucleotide polymorphism (SNP) content of genome-wide arrays. The UK10K Cohorts project has generated a data set of 3,781 whole genomes sequenced at low depth (average 7x), aiming to exhaustively characterize genetic variation down to 0.1% minor allele frequency in the British population. Here we demonstrate the value of this resource for improving imputation accuracy at rare and low-frequency variants in both a UK and an Italian population. We show that large increases in imputation accuracy can be achieved by re-phasing WGS reference panels after initial genotype calling. We also present a method for combining WGS panels to improve variant coverage and downstream imputation accuracy, which we illustrate by integrating 7,562 WGS haplotypes from the UK10K project with 2,184 haplotypes from the 1000 Genomes Project. Finally, we introduce a novel approximation that maintains speed without sacrificing imputation accuracy for rare variants.

The delta-5 and delta-6 desaturases, encoded by FADS1 and FADS2 genes, are key enzymes in polyunsaturated fatty acid (PUFA) metabolism that catalyze the conversion of linoleic acid (LA) into arachidonic acid (AA) and that of alpha-linolenic acid (ALA) into eicosapentaenoic acid (EPA). Single-nucleotide polymorphisms (SNPs) in FADS1 and FADS2 have been associated with different concentrations of AA and LA, and those associations have possible functional consequences for desaturase activity.We aimed to evaluate the possible association among FADS genotypes, desaturase activity, inflammation, and coronary artery disease (CAD).Thirteen FADS SNPs and the ratio of AA to LA (AA/LA) on red blood cell (RBC) membranes, a marker of desaturase activity, were evaluated in 876 subjects with (n = 610) or without (n = 266) angiographically documented CAD.Both AA/LA and the ratio of EPA to ALA (EPA/ALA) were higher in patients with CAD than in those without CAD, but, in a multiple logistic regression model, only a higher AA/LA resulted an independent risk factor for CAD (odds ratio: 2.55; 95% CI: 1.61, 4.05 for higher compared with lower ratio tertile; P for trend < 0.001). Furthermore, concentrations of high-sensitivity C-reactive protein increased progressively across tertiles of AA/LA. Graded increases in high-sensitivity C-reactive protein concentrations and CAD risk were related to the carriership of FADS haplotypes, including the alleles associated with a higher ratio.In populations following a Western diet, subjects carrying FADS haplotypes that are associated with higher desaturase activity may be prone to a proinflammatory response favoring atherosclerotic vascular damage.

High blood pressure is a major risk factor for cardiovascular disease and premature death. However, there is limited knowledge on specific causal genes and pathways. To better understand the genetics of blood pressure, we genotyped 242,296 rare, low-frequency and common genetic variants in up to 192,763 individuals and used ∼155,063 samples for independent replication. We identified 30 new blood pressure- or hypertension-associated genetic regions in the general population, including 3 rare missense variants in RBM47, COL21A1 and RRAS with larger effects (>1.5 mm Hg/allele) than common variants. Multiple rare nonsense and missense variant associations were found in A2ML1, and a low-frequency nonsense variant in ENPEP was identified. Our data extend the spectrum of allelic variation underlying blood pressure traits and hypertension, provide new insights into the pathophysiology of hypertension and indicate new targets for clinical intervention.

Some individuals with autism spectrum disorder (ASD) carry functional mutations rarely observed in the general population. We explored the genes disrupted by these variants from joint analysis of protein-truncating variants (PTVs), missense variants and copy number variants (CNVs) in a cohort of 63,237 individuals. We discovered 72 genes associated with ASD at false discovery rate (FDR) ≤ 0.001 (185 at FDR ≤ 0.05). De novo PTVs, damaging missense variants and CNVs represented 57.5%, 21.1% and 8.44% of association evidence, while CNVs conferred greatest relative risk. Meta-analysis with cohorts ascertained for developmental delay (DD) (n = 91,605) yielded 373 genes associated with ASD/DD at FDR ≤ 0.001 (664 at FDR ≤ 0.05), some of which differed in relative frequency of mutation between ASD and DD cohorts. The DD-associated genes were enriched in transcriptomes of progenitor and immature neuronal cells, whereas genes showing stronger evidence in ASD were more enriched in maturing neurons and overlapped with schizophrenia-associated genes, emphasizing that these neuropsychiatric disorders may share common pathways to risk.

Abstract Most genes associated with neurodevelopmental disorders (NDDs) were identified with an excess of de novo mutations (DNMs) but the significance in case–control mutation burden analysis is unestablished. Here, we sequence 63 genes in 16,294 NDD cases and an additional 62 genes in 6,211 NDD cases. By combining these with published data, we assess a total of 125 genes in over 16,000 NDD cases and compare the mutation burden to nonpsychiatric controls from ExAC. We identify 48 genes (25 newly reported) showing significant burden of ultra-rare (MAF &lt; 0.01%) gene-disruptive mutations (FDR 5%), six of which reach family-wise error rate (FWER) significance ( p &lt; 1.25E−06). Among these 125 targeted genes, we also reevaluate DNM excess in 17,426 NDD trios with 6,499 new autism trios. We identify 90 genes enriched for DNMs (FDR 5%; e.g., GABRG2 and UIMC1 ); of which, 61 reach FWER significance ( p &lt; 3.64E−07; e.g., CASZ1 ). In addition to doubling the number of patients for many NDD risk genes, we present phenotype–genotype correlations for seven risk genes ( CTCF , HNRNPU , KCNQ3 , ZBTB18 , TCF12 , SPEN , and LEO1 ) based on this large-scale targeted sequencing effort.

Abstract Polymorphisms of the human Δ‐5 ( FADS1 ) and Δ‐6 ( FADS2 ) desaturase genes have been recently described to be associated with the level of several long‐chain n‐3 and n‐6 polyunsaturated fatty acids (PUFAs) in serum phospholipids. We have genotyped 13 single nucleotide polymorphisms (SNPs) located on the FADS1 – FADS2 – FADS3 gene cluster (chromosome 11q12–13.1) in 658 Italian adults (78% males; mean age 59.7 ± 11.1 years) participating in the Verona Heart Project. Polymorphisms and statistically inferred haplotypes showed a strong association with arachidonic acid (C20:4n‐6) levels in serum phospholipids and in erythrocyte cell membranes (rs174545 adjusted P value for multiple tests, P &lt; 0.0001 and P &lt; 0.0001, respectively). Other significant associations were observed for linoleic (C18:2n‐6), alpha‐linolenic (C18:3n‐3) and eicosadienoic (C20:2n‐6) acids. Minor allele homozygotes and heterozygotes were associated to higher levels of linoleic, alpha‐linolenic, eicosadienoic and lower levels of arachidonic acid. No significant association was observed for stearidonic (C18:4n‐3), eicosapentaenoic (C20:5n‐3) and docosahexaenoic (C22:6n‐3) acids levels. The observed strong association of FADS gene polymorphisms with the levels of arachidonic acid, which is a precursor of molecules involved in inflammation and immunity processes, suggests that SNPs of the FADS 1 and FADS 2 gene region are worth studying in diseases related to inflammatory conditions or alterations in the concentration of PUFAs.

Metabolic activity of cytochrome P450 (CYP) 3A4 has been associated with clopidogrel response variability. Because metabolic activity of CYP3A4 is genetically regulated, we hypothesized that genetic variations of this enzyme may contribute to clopidogrel response variability.The CYP3A4*1B, CYP3A4*3, IVS7+258A>G, IVS7+894C>T, and IVS10+12G>A polymorphisms of the CYP3A4 gene were assessed in 82 patients in a steady phase of clopidogrel therapy. Glycoprotein (platelet glycoprotein (GP) IIb/IIIa receptor activation and platelet aggregation were assessed. A cohort of 45 clopidogrel-naïve patients was studied to determine the modulating effects of these polymorphisms after loading dose (300 mg) administration. Only the IVS7+258A>G, IVS7+894C>T, and IVS10+12G>A polymorphisms were sufficiently polymorphic. During the steady phase of clopidogrel treatment, IVS10+12A allele carriers had reduced GP IIb/IIIa activation (P=0.025) and better responsiveness (P=0.02); similarly, clopidogrel-naïve patients carriers of the IVS10+12A allele had reduced GP IIb/IIIa activation during the first 24 hours after a loading dose (P=0.025), increased platelet inhibition (P=0.006), and a more optimal drug response (P=0.003). This polymorphism did not influence platelet aggregation profiles. No association was observed between the other polymorphisms and clopidogrel responsiveness.The IVS10+12G>A polymorphism of the CYP3A4 gene modulates platelet activation in patients treated with clopidogrel and may therefore contribute to clopidogrel response variability.

Abstract Moderate elevation of plasma total homocysteine (tHcy) is a strong and independent risk factor for coronary artery disease (CAD). It can result from genetic or nutrient-related disturbances in the transsulfuration or remethylation pathways for Hcy metabolism. A point mutation (C677T; Ala-to-Val) in the gene encoding the 5,10-methylenetetrahydrofolate reductase (MTHFR) has been recently reported to render the enzyme thermolabile and less active. Studies on the role of this mutation as a risk factor for CAD have given conflicting results. We studied a total of 415 subjects, 278 with angiographically documented multivessel CAD and 137 with angiographically documented normal coronary arteries. The overall frequency of the MTHFR V/V homozygous genotype was 15.7% (with 52.5% heterozygous and 31.8% normal). Subgroup analysis showed no significant differences between CAD and CAD-free subjects. A genotype/phenotype correlation study showed a marked effect of folate on the association between MTHFR genotypes and tHcy. Among individuals with folate levels below the median (11.5 nmol/L), fasting tHcy was significantly increased not only in V/V homozygotes (by 59%) but also, at intermediate values, in A/V heterozygotes (by 21% on average). Conversely, the mutation resulted neutral with respect to tHcy levels in subjects with adequate folate levels. We conclude that, in our population, the MTHFR C677T mutation is rather common, but it does not appear to be associated per se to CAD. A genetic-environmental interaction may contribute to the vascular risk by elevating tHcy when folate status is low.

We performed a genome-wide association study on 1,292 individuals with abdominal aortic aneurysms (AAAs) and 30,503 controls from Iceland and The Netherlands, with a follow-up of top markers in up to 3,267 individuals with AAAs and 7,451 controls. The A allele of rs7025486 on 9q33 was found to associate with AAA, with an odds ratio (OR) of 1.21 and P = 4.6 x 10(-10). In tests for association with other vascular diseases, we found that rs7025486[A] is associated with early onset myocardial infarction (OR = 1.18, P = 3.1 x 10(-5)), peripheral arterial disease (OR = 1.14, P = 3.9 x 10(-5)) and pulmonary embolism (OR = 1.20, P = 0.00030), but not with intracranial aneurysm or ischemic stroke. No association was observed between rs7025486[A] and common risk factors for arterial and venous diseases-that is, smoking, lipid levels, obesity, type 2 diabetes and hypertension. Rs7025486 is located within DAB2IP, which encodes an inhibitor of cell growth and survival.

Asthma is a multifactorial disease influenced by genetic and environmental factors. In the past decade, several loci and >100 genes have been found to be associated with the disease in at least one population. Among these loci, region 12q13-24 has been implicated in asthma etiology in multiple populations, suggesting that it harbors one or more asthma susceptibility genes. We performed linkage and association analyses by transmission/disequilibrium test and case-control analysis in the candidate region 12q13-24, using the Sardinian founder population, in which limited heterogeneity of pathogenetic alleles for monogenic and complex disorders as well as of environmental conditions should facilitate the study of multifactorial traits. We analyzed our cohort, using a cutoff age of 13 years at asthma onset, and detected significant linkage to a portion of 12q13-24. We identified IRAK-M as the gene contributing to the linkage and showed that it is associated with early-onset persistent asthma. We defined protective and predisposing SNP haplotypes and replicated associations in an outbred Italian population. Sequence analysis in patients found mutations, including inactivating lesions, in the IRAK-M coding region. Immunohistochemistry of lung biopsies showed that IRAK-M is highly expressed in epithelial cells. We report that IRAK-M is involved in the pathogenesis of early-onset persistent asthma. IRAK-M, a negative regulator of the Toll-like receptor/IL-1R pathways, is a master regulator of NF- kappa B and inflammation. Our data suggest a mechanistic link between hyperactivation of the innate immune system and chronic airway inflammation and indicate IRAK-M as a potential target for therapeutic intervention against asthma.

Clopidogrel inhibits the ADP subtype P2Y(12) receptor. Recently, polymorphisms of this receptor have been associated with different degrees of platelet aggregation in healthy volunteers and have been suggested to modulate clopidogrel response. However, the role of gene sequence variations of the P2Y(12) receptor in patients treated with clopidogrel has not yet been assessed.The T744C polymorphism of the P2Y(12) receptor gene was assessed in 119 patients: 36 undergoing coronary stenting receiving a 300 mg loading dose (Group A) and 83 on long-term clopidogrel (75 mg/day) treatment (Group B). Patients were divided into 2 subgroups according to the presence or absence of the C allele: carriers (CT heterozygotes and CC homozygotes) and non-carriers (TT homozygotes). Platelet aggregation, assessed by light transmittance aggregometry following ADP, collagen, TRAP and epinephrine stimuli, and platelet activation (GP IIb/IIIa activation and P-selectin expression), assessed by whole blood flow cytometry in ADP and TRAP-stimulated platelets, were performed. Platelet function was assessed at baseline and 4 and 24 h following clopidogrel loading dose in Group A and when patients where on clopidogrel treatment for at least 1 month in Group B.The genotype distribution of Group A was: 22/36 (61.1%) non-carriers and 14/36 (38.9%) carriers of the C allele; Group B: 57/83 (68.7%) non-carriers and 26/83 (31.3%) carriers of the C allele. There were no differences between groups for all the assessed platelet function assays.The T744C polymorphism of the P2Y(12) receptor gene does not modulate platelet response to clopidogrel either in the early or long-term phases of treatment. This specific gene polymorphism alone is therefore unlikely to be the cause of variability in individual response to antiplatelet therapy.

Apolipoprotein C-III (apoC-III) is a marker of triglyceride (TG)-rich lipoproteins, which are often increased in metabolic syndrome (MS). The T−455C polymorphism in the insulin-responsive element of the APOC3 gene influences TG and apoC-III levels. To evaluate the contribution of apoC-III levels and T−455C polymorphisms in the coronary artery disease (CAD) risk of MS patients, we studied 873 patients, 549 with CAD and 251 with normal coronary arteries. Patients were classified also as having or not having MS (MS, n=270; MS-free, n=603). Lipids, insulin, apolipoprotein levels, and APOC3 T−455C genotypes were evaluated. ApoC-III levels were significantly increased in MS patients, and the probability of having MS was correlated with increasing quartiles of apoC-III levels. MS patients with CAD had significantly higher apoC-III levels than did CAD-free MS patients. The carriership for the −455C variant multiplied the probability of CAD in MS in an allele-specific way and was associated with increased apoC-III and TG levels. Obesity was less frequent in MS carriers of the −455C allele than in MS noncarriers (21.6% vs. 34.8%, <i>P</i> < 0.05). In conclusion, apoC-III-rich lipoprotein metabolism and the APOC3 polymorphism have relevant impacts on the CAD risk of MS patents.

We sought to replicate the association between the kinesin-like protein 6 (KIF6) Trp719Arg polymorphism (rs20455), and clinical coronary artery disease (CAD). Recent prospective studies suggest that carriers of the 719Arg allele in KIF6are at increased risk of clinical CAD compared with noncarriers. The KIF6Trp719Arg polymorphism (rs20455) was genotyped in 19 case-control studies of nonfatal CAD either as part of a genome-wide association study or in a formal attempt to replicate the initial positive reports. A total of 17,000 cases and 39,369 controls of European descent as well as a modest number of South Asians, African Americans, Hispanics, East Asians, and admixed cases and controls were successfully genotyped. None of the 19 studies demonstrated an increased risk of CAD in carriers of the 719Arg allele compared with noncarriers. Regression analyses and fixed-effects meta-analyses ruled out with high degree of confidence an increase of ≥2% in the risk of CAD among European 719Arg carriers. We also observed no increase in the risk of CAD among 719Arg carriers in the subset of Europeans with early-onset disease (younger than 50 years of age for men and younger than 60 years of age for women) compared with similarly aged controls as well as all non-European subgroups. The KIF6Trp719Arg polymorphism was not associated with the risk of clinical CAD in this large replication study.

Atherosclerosis affects aorta, coronary, carotid, and iliac arteries most frequently than any other body vessel. There may be common molecular pathways sustaining this process. Plaque presence and diffusion is revealed by circulating factors that can mediate systemic reaction leading to plaque rupture and thrombosis.We used DNA microarrays and meta-analysis to study how the presence of calcified plaque modifies human coronary and carotid gene expression. We identified a series of potential human atherogenic genes that are integrated in functional networks involved in atherosclerosis. Caveolae and JAK/STAT pathways, and S100A9/S100A8 interacting proteins are certainly involved in the development of vascular disease. We found that the system of caveolae is directly connected with genes that respond to hormone receptors, and indirectly with the apoptosis pathway. Cytokines, chemokines and growth factors released in the blood flux were investigated in parallel. High levels of RANTES, IL-1ra, MIP-1 alpha, MIP-1 beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-17, PDGF-BB, VEGF and IFN-gamma were found in plasma of atherosclerotic patients and might also be integrated in the molecular networks underlying atherosclerotic modifications of these vessels.The pattern of cytokine and S100A9/S100A8 up-regulation characterizes atherosclerosis as a proinflammatory disorder. Activation of the JAK/STAT pathway is confirmed by the up-regulation of IL-6, STAT1, ISGF3G and IL10RA genes in coronary and carotid plaques. The functional network constructed in our research is an evidence of the central role of STAT protein and the caveolae system to contribute to preserve the plaque. Moreover, Cav-1 is involved in SMC differentiation and dyslipidemia confirming the importance of lipid homeostasis in the atherosclerotic phenotype.

The PlA polymorphism (Leu33Pro) of the platelet glycoprotein (GP) IIIa gene has been suggested to play an important role in coronary thrombosis. In vitro studies have shown differences for this polymorphism in platelet sensitivity towards antiplatelet drugs (aspirin and abciximab), suggesting a pharmacogenetic modulation. The aim of the study was to assess the modulatory effect of the PlA polymorphism on clopidogrel-induced antiplatelet effects in 38 patients undergoing coronary stent implantation receiving a 300 mg clopidogrel loading-dose. Platelet reactivity was assessed as GPIIb/IIIa activation and P-selectin expression in platelets stimulated with 2 μmol/l adenosine diphosphate using whole blood flow cytometry. The distribution of the homozygous PlA1/A1 and heterozygous PlA1/A2 genotypes were 74 and 26%, respectively. PlA2 carriers had a higher degree of GPIIb/IIIa activation (P = 0.05) and P-selectin expression (P = 0.02) during the overall study time course and a lower antiplatelet effect to a 300 mg clopidogrel loading-dose up to 24 h following intervention (P< 0.05). In conclusion, the PlA polymorphism of the GPIIIa gene modulates platelet reactivity towards clopidogrel front loading in patients undergoing coronary stenting. This suggests the need for individualized antithrombotic regimens to optimally inhibit platelet reactivity.

The aim of this study was to assess the association between genetic variants of the insulin receptor substrate (IRS)-1 gene, platelet function, and long-term outcomes in patients with type 2 diabetes mellitus (DM) and stable coronary artery disease while on aspirin and clopidogrel therapy.The effects of pharmacogenetic determinants on platelet function and cardiovascular outcomes in type DM patients are unknown.The association between IRS-1 genetic variants, platelet function, and the risk of major adverse cardiac events (MACE) at 2 years was assessed in 187 patients with type 2 DM and stable coronary artery disease on maintenance aspirin and clopidogrel therapy.Seven tag single nucleotide polymorphisms were selected. Individuals with high platelet reactivity were more frequent among carriers of the C allele (GC and CC genotypes; approximately 20% of population) of the rs956115 marker (44.4% vs. 20.5%; odds ratio: 3.1, 95% confidence interval [CI]: 1.44 to 6.67; p = 0.006). These patients were at higher risk of MACE (28.0% vs. 10.9%; hazard ratio: 2.90, 95% CI: 1.38 to 6.11; p = 0.005). The C allele carriers of the rs956115 marker were more commonly associated with a hyperreactive platelet phenotype. This was confirmed in an external validation cohort of patients with type 2 DM but not in an external validation cohort of patients without DM. Carriers of the C allele of the rs956115 marker also had a significantly higher risk of MACE compared with noncarriers (30.6% vs. 11.4%; hazard ratio: 2.88, 95% CI: 1.35 to 6.14; p = 0.006).Type 2 DM patients who are carriers of the C allele of the rs956115 marker of the IRS-1 gene have a hyperreactive platelet phenotype and increased risk of MACE.

We tested for interactions between body mass index (BMI) and common genetic variants affecting serum urate levels, genome-wide, in up to 42569 participants. Both stratified genome-wide association (GWAS) analyses, in lean, overweight and obese individuals, and regression-type analyses in a non BMI-stratified overall sample were performed. The former did not uncover any novel locus with a major main effect, but supported modulation of effects for some known and potentially new urate loci. The latter highlighted a SNP at RBFOX3 reaching genome-wide significant level (effect size 0.014, 95% CI 0.008-0.02, Pinter= 2.6 x 10-8). Two top loci in interaction term analyses, RBFOX3 and ERO1LB-EDARADD, also displayed suggestive differences in main effect size between the lean and obese strata. All top ranking loci for urate effect differences between BMI categories were novel and most had small magnitude but opposite direction effects between strata. They include the locus RBMS1-TANK (men, Pdifflean-overweight= 4.7 x 10-8), a region that has been associated with several obesity related traits, and TSPYL5 (men, Pdifflean-overweight= 9.1 x 10-8), regulating adipocytes-produced estradiol. The top-ranking known urate loci was ABCG2, the strongest known gout risk locus, with an effect halved in obese compared to lean men (Pdifflean-obese= 2 x 10-4). Finally, pathway analysis suggested a role for N-glycan biosynthesis as a prominent urate-associated pathway in the lean stratum. These results illustrate a potentially powerful way to monitor changes occurring in obesogenic environment.

ABSTRACT Individuals with autism spectrum disorder (ASD) or related neurodevelopmental disorders (NDDs) often carry disruptive mutations in genes that are depleted of functional variation in the broader population. We build upon this observation and exome sequencing from 154,842 individuals to explore the allelic diversity of rare protein-coding variation contributing risk for ASD and related NDDs. Using an integrative statistical model, we jointly analyzed rare protein-truncating variants (PTVs), damaging missense variants, and copy number variants (CNVs) derived from exome sequencing of 63,237 individuals from ASD cohorts. We discovered 71 genes associated with ASD at a false discovery rate (FDR) ≤ 0.001, a threshold approximately equivalent to exome-wide significance, and 183 genes at FDR ≤ 0.05. Associations were predominantly driven by de novo PTVs, damaging missense variants, and CNVs: 57.4%, 21.2%, and 8.32% of evidence, respectively. Though fewer in number, CNVs conferred greater relative risk than PTVs, and repeat-mediated de novo CNVs exhibited strong maternal bias in parent-of-origin (e.g., 92.3% of 16p11.2 CNVs), whereas all other CNVs showed a paternal bias. To explore how genes associated with ASD and NDD overlap or differ, we analyzed our ASD cohort alongside a developmental delay (DD) cohort from the deciphering developmental disorders study (DDD; n=91,605 samples). We first reanalyzed the DDD dataset using the same models as the ASD cohorts, then performed joint analyses of both cohorts and identified 373 genes contributing to NDD risk at FDR ≤ 0.001 and 662 NDD risk genes at FDR ≤ 0.05. Of these NDD risk genes, 54 genes (125 genes at FDR ≤ 0.05) were unique to the joint analyses and not significant in either cohort alone. Our results confirm overlap of most ASD and DD risk genes, although many differ significantly in frequency of mutation. Analyses of single-cell transcriptome datasets showed that genes associated predominantly with DD were strongly enriched for earlier neurodevelopmental cell types, whereas genes displaying stronger evidence for association in ASD cohorts were more enriched for maturing neurons. The ASD risk genes were also enriched for genes associated with schizophrenia from a separate rare coding variant analysis of 121,570 individuals, emphasizing that these neuropsychiatric disorders share common pathways to risk.

Abstract Methylenetetrahydrofolate reductase (MTHFR) balances the pool of folate coenzymes in one-carbon metabolism for DNA synthesis and methylation, both implicated in carcinogenesis. Epidemiologic studies have shown that two functional polymorphisms in MTHFR gene, 677C&amp;gt;T and 1298A&amp;gt;C, are related to increased cancer risk. We aimed to analyze lymphocyte DNA from 198 subjects to evaluate the MTHFR 1298A&amp;gt;C polymorphism and folate status affecting genomic DNA methylation as a possible mechanism underlying the relationship between MTHFR polymorphisms and cancer susceptibility. Carriers of the 1298AA wild-type genotype showed lower genomic DNA methylation compared with 1298AC or 1298CC genotypes [3.72 versus 8.59 or 6.79 ng 5-methyl-2′-deoxycytidine (5-mCyt)/μg DNA, P &amp;lt; 0.0001 and P = 0.007, respectively]. When DNA methylation was evaluated according to plasma folate status, only 1298AA with low folate levels revealed diminished DNA methylation (P &amp;lt; 0.0001). Moreover, when the two MTHFR polymorphisms were concomitantly evaluated at the low folate status, DNA methylation was reduced only in 1298AA/677TT compared with 1298AA/677CC (3.11 versus 7.29 ng 5-mCyt/μg DNA, P = 0.001) and 1298CC/677CC genotypes (3.11 versus 7.14 ng 5-mCyt/μg DNA, P = 0.004). However, the high prevalence of 677TT mutants within the 1298AA group (79%) and the similar biochemical features of 1298AA/677CC and 1298CC/677CC combined genotypes suggest that the gene-nutrient interaction affecting DNA methylation in 1298AA is mainly due to the coexistence of the 677TT genotype and that the 1298A&amp;gt;C polymorphism may convey its protective effect not through this interaction but through another pathway in one-carbon metabolism. Further mechanistic studies are warranted to investigate how single polymorphisms as well as MTHFR combined genotypes exert their effect on cancer susceptibility.

High plasma concentrations of triglycerides (TG) and apolipoprotein C-III (ApoC-III) are well-known risk factors for cardiovascular disease. Two variants of the recently discovered APOA5, 1131 C > T and S19W, have been associated with hypertriglyceridemia, whereas their relation with coronary artery disease (CAD) remains controversial. Nine hundred and thirteen angiografically defined patients (669 CAD and 244 CAD-free) were genotyped for APOA5 −1131 C > T and S19W polymorphisms. Carriership of the APOA5 −1131 C allele was identified, by multiple linear regression models, as a significant independent predictor for both TG (standardized β-coefficient = 0.112; p = 0.010) and ApoC-III variability (standardized β-coefficient = 0.113; p = 0.013). Similarly, APOA5 19W allele carriership was a significant independent predictor for both TG (standardized β-coefficient = 0.113; p = 0.007) and ApoC-III variability (standardized β-coefficient = 0.088; p = 0.045). Despite the association with at-risk lipid profile, no significant difference was detected in the distribution of both APOA5 gene polymorphisms between subjects with or without CAD. Moreover, homozygous carriers of the APOC3 −455 C, another TG- and ApoC-III raising variant, showed a significant increased risk for CAD (OR 1.90 with 95% CI 1.002–3.62; p = 0.049; by multiple logistic regression). Different genotypes, i.e., APOA5 and APOC3 variants, may lead to similar biochemical phenotypes, namely hypertriglyceridemia, but to contrasting clinical phenotypes such as the presence of angiographically proven CAD.

The platelet P2Y12 receptor plays a key role in platelet activation. The H2 haplotype of the P2Y12 receptor gene (P2RY12) has been found to be associated with maximal aggregation response to adenosine diphosphate (ADP) and with increased risk for peripheral arterial disease. No data are available on its association with coronary artery disease (CAD).The H2 haplotype of the P2RY12 was determined in 1378 unrelated patients of both sexes selected according to the presence of significant coronary artery disease (CAD group) or having normal coronary angiogram at cardiac catheterization (CAD-free group). Significant coronary artery disease was angiographically determined, and was defined as a greater than 50% visually estimated luminal diameter stenosis in at least one major epicardial coronary artery.In the studied population 71.9% had CAD (n = 991) and 28.1% had normal coronary angiogram (n = 387). H2 haplotype carriers were more frequent in the CAD group (p = 0.03, OR = 1.36, 95%CI = 1.02-1.82). The H2 haplotype was significantly associated with CAD in non-smokers (p = 0.007, OR = 1.83 95%CI = 1.17-2.87), but not in smokers. The association remained significant after adjustment for other covariates (age, triglycerides, HDL, hypertension, diabetes) by multivariate logistic regression (p = 0.004, OR = 2.32 95%CI = 1.30-4.15).Gene sequence variations of the P2Y12 receptor gene are associated with the presence of significant CAD, particularly in non-smoking individuals.

The role of genetic and environmental factors, as well as their interaction, in the natural history of asthma, allergic rhinitis and chronic obstructive pulmonary disease (COPD) is largely unknown. This is mainly due to the lack of large-scale analytical epidemiological/genetic studies aimed at investigating these 3 respiratory conditions simultaneously. The GEIRD project is a collaborative initiative designed to collect information on biomarkers of inflammation and oxidative stress, individual and ecological exposures, diet, early-life factors, smoking habits, genetic traits and medication use in large and accurately defined series of asthma, allergic rhinitis and COPD phenotypes. It is a population-based multicase-control design, where cases and controls are identified through a 2-stage screening process (postal questionnaire and clinical examination) in pre-existing cohorts or new samples of subjects. It is aimed at elucidating the role that modifiable and genetic factors play in the occurrence, persistence, severity and control of inflammatory airway diseases, by way of the establishment of a historical multicentre standardized databank of phenotypes, contributed by and openly available to international epidemiologists. Researchers conducting population-based surveys with standardized methods may contribute to the public-domain case-control database, and use the resulting increased power to answer their own scientific questions.

Common variants contribute significantly to the genetics of autism spectrum disorder (ASD), although the identification of individual risk polymorphisms remains still elusive due to their small effect sizes and limited sample sizes available for association studies. During the last decade several genome‐wide association studies (GWAS) have enabled the detection of a few plausible risk variants. The three main studies are family‐based and pointed at SEMA5A (rs10513025), MACROD2 (rs4141463) and MSNP1 (rs4307059). In our study we attempted to replicate these GWAS hits using a case‐control association study in five European populations of ASD patients and gender‐matched controls, all Caucasians. Results showed no association of individual variants with ASD in any of the population groups considered or in the combined European sample. We performed a meta‐analysis study across five European populations for rs10513025 (1,904 ASD cases and 2,674 controls), seven European populations for rs4141463 (2,855 ASD cases and 36,177 controls) and five European populations for rs4307059 (2,347 ASD cases and 2,764 controls). The results showed an odds ratio (OR) of 1.05 (95% CI = 0.84–1.32) for rs10513025, 1.0002 (95% CI = 0.93–1.08) for rs4141463 and 1.01 (95% CI = 0.92–1.1) for rs4307059, with no significant P‐values (rs10513025, P = 0.73; rs4141463, P = 0.95; rs4307059, P = 0.9). No association was found when we considered either only high functioning autism (HFA), genders separately or only multiplex families. Ongoing GWAS projects with larger ASD cohorts will contribute to clarify the role of common variation in the disorder and will likely identify risk variants of modest effect not detected previously. Autism Res 2017, 10: 202–211 . © 2016 International Society for Autism Research, Wiley Periodicals, Inc.

Alternative splicing is a regulatory mechanism essential for cell differentiation and tissue organization. More than 90% of human genes are regulated by alternative splicing events, which participate in cell fate determination. The general mechanisms of splicing events are well known, whereas only recently have deep-sequencing, high throughput analyses and animal models provided novel information on the network of functionally coordinated, tissue-specific, alternatively spliced exons. Heart development and cardiac tissue differentiation require thoroughly regulated splicing events. The ribonucleoprotein RBM20 is a key regulator of the alternative splicing events required for functional and structural heart properties, such as the expression of TTN isoforms. Recently, the polypyrimidine tract-binding protein PTBP1 has been demonstrated to participate with RBM20 in regulating splicing events. In this review, we summarize the updated knowledge relative to RBM20 and PTBP1 structure and molecular function; their role in alternative splicing mechanisms involved in the heart development and function; RBM20 mutations associated with idiopathic dilated cardiovascular disease (DCM); and the consequences of RBM20-altered expression or dysfunction. Furthermore, we discuss the possible application of targeting RBM20 in new approaches in heart therapies.

We examined the long arm XY pseudoautosomal region for linkage to asthma, serum IgE, and bronchial hyperresponsiveness. In 57 Caucasian families multipoint nonparametric analyses provide evidence for linkage between DXYS154 and bronchial hyperresponsiveness (P= 0.000057) or asthma (P= 0.00065). This genomic region is approximately 320 kb in size and contains the interleukin-9 receptor gene. These results suggest that a gene controlling asthma and bronchial hyperresponsiveness maybe located in this region and that the interleukin-9 receptor is a potential candidate.

Several polymorphisms in the apolipoprotein C-III (apoC-III) gene have been associated with hypertriglyceridemia, but the link with coronary artery disease risk is still controversial. In particular, apoC-III promoter sequence variants in the insulin responsive element (IRE), constitutively resistant to downregulation by insulin, have never been investigated in this connection. We studied a total of 800 patients, 549 of whom had angiographically documented coronary atherosclerosis, whereas 251 had normal coronary arteriograms. We measured plasma lipids, insulin, apoA-I, apoB, and apoC-III and assessed three polymorphisms in the apoC-III gene, namely, T-455C in the IRE promoter region, C1100T in exon 3, and Sst1 polymorphic site (S1/S2) in the 3′ untranslated region. Each variant influenced triglyceride levels, but only the T-455C (in homozygosity) and S2 alleles influenced apoC-III levels. In coronary artery disease (CAD) patients, 18.6% were homozygous for the −455C variant compared with only 9.2% in CAD-free group (P < 0.001).In logistic regression models, homozygosity for −455C variant was associated with a significantly increased risk of CAD (OR = 2.5 and 2.18 for unadjusted and adjusted models, respectively) suggesting that it represents an independent genetic susceptibility factor for CAD. Several polymorphisms in the apolipoprotein C-III (apoC-III) gene have been associated with hypertriglyceridemia, but the link with coronary artery disease risk is still controversial. In particular, apoC-III promoter sequence variants in the insulin responsive element (IRE), constitutively resistant to downregulation by insulin, have never been investigated in this connection. We studied a total of 800 patients, 549 of whom had angiographically documented coronary atherosclerosis, whereas 251 had normal coronary arteriograms. We measured plasma lipids, insulin, apoA-I, apoB, and apoC-III and assessed three polymorphisms in the apoC-III gene, namely, T-455C in the IRE promoter region, C1100T in exon 3, and Sst1 polymorphic site (S1/S2) in the 3′ untranslated region. Each variant influenced triglyceride levels, but only the T-455C (in homozygosity) and S2 alleles influenced apoC-III levels. In coronary artery disease (CAD) patients, 18.6% were homozygous for the −455C variant compared with only 9.2% in CAD-free group (P < 0.001). In logistic regression models, homozygosity for −455C variant was associated with a significantly increased risk of CAD (OR = 2.5 and 2.18 for unadjusted and adjusted models, respectively) suggesting that it represents an independent genetic susceptibility factor for CAD. Investigators have long disputed whether elevated serum triglyceride (TG) levels are an independent risk factor for coronary artery disease (CAD) (1Bloomfield Rubins H. The trouble with triglycerides.Arch. Intern. Med. 2000; 160: 1903-1904Google Scholar). A major reason for this controversy stems from the heterogeneity of factors measured by triglyceride (TG) determination (TGs are carried in virtually all plasma lipoproteins) and from the high variance and collinearity of TGs with other recognized risk factors (1Bloomfield Rubins H. The trouble with triglycerides.Arch. Intern. Med. 2000; 160: 1903-1904Google Scholar, 2Hodis H.N. Triglyceride-rich lipoprotein remnant particles and risk of atherosclerosis (Editorial).Circulation. 1999; 99: 2852-2854Google Scholar). Alternative, and more reliable, markers of TG metabolism have therefore been proposed (1Bloomfield Rubins H. The trouble with triglycerides.Arch. Intern. Med. 2000; 160: 1903-1904Google Scholar, 2Hodis H.N. Triglyceride-rich lipoprotein remnant particles and risk of atherosclerosis (Editorial).Circulation. 1999; 99: 2852-2854Google Scholar). Perhaps the most important among them is apolipoprotein C-III (apoC-III), a 79 amino acid protein synthesized in the liver and in the intestine, which is an essential constituent of circulating particles rich in triacylglycerol, i.e., chylomicrons and VLDL (3Jong M.C. Hofker M.H. Havekes L.M. Role of apo Cs in lipoprotein metabolism. Functional differences between apoC1, apoC2, and apoC3.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 472-484Google Scholar). The results of large clinical studies have indicated that apoC-III levels are a better predictor of risk for the development and progression of CAD than the traditionally measured TG levels (4Blankenhorn D.H. Alaupovic P. Wickham E. Chin H.P. Azen S.P. Prediction of angiographic change in native human coronary arteries and aortocoronary bypass grafts. Lipid and nonlipid factors.Circulation. 1990; 81: 470-476Google Scholar, 5Hodis H.N. Mack W.J. Azen S.P. Alaupovic P. Pogoda J.M. La Bree L. Hemphill L.C. Kramsch D.M. Blankenhorn D.H. Triglyceride and cholesterol-rich lipoproteins have a differential effect on mild/moderate and severe lesion progression as assessed by quantitative coronary angiography in a controlled trial of lovastatin.Circulation. 1994; 90: 42-49Google Scholar, 6Mack W.J. Krauss R.M. Hodis H.N. Lipoprotein subclasses in the monitored atherosclerosis regression study (MARS). Treatment effects and relation to coronary angiographic progression.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 697-704Google Scholar, 7Luc G. Fievet C. Arveiler D. Evans A.E. Bard J.M. Cambien F. Fruchart J.C. Ducimetiere P. Apolipoproteins et al. Apolipoproteins C–III and E in apo B- and non-apo B-containing lipoproteins in two populations at contrasting risk for myocardial infarction: the ECTIM study.J. Lipid Res. 1996; 37: 508-517Google Scholar, 8Thompson G.R. Angiographic evidence for the role of triglyceride-rich lipoproteins in progression of coronary artery disease.Eur. Heart J. 1998; 19: H31-H36Google Scholar, 9Sacks F.M. Alaupovic P. Moye L.A. Cole T.G. Sussex B. Stampfer M.J. Pfeffer M.J. Braunwald E. VLDL, apolipoproteins B, CIII, and E, and risk of recurrent coronary events in the cholesterol and recurrent events (CARE) trial.Circulation. 2000; 102: 1886-1892Google Scholar). ApoC-III inhibits the hydrolysis of TG-rich particles by lipoprotein lipase and their apoE–mediated hepatic uptake (10McConathy W.J. Gesquiere J.C. Bass H. Tartar A. Fruchart J.C. Wang C.S. Inhibition of lipoprotein lipase activity by synthetic peptides of apolipoprotein C–III.J. Lipid Res. 1992; 33: 995-1003Google Scholar, 11Ginsberg H.N. Le N.A. Goldberg I.J. Gibson J.C. Rubinstein A. Wang-Iverson P. Norum N. Brown W.V. Apolipoprotein B metabolism in subjects with deficiency of apolipoproteins CIII and AI. Evidence that apolipoprotein CIII inhibits catabolism of triglyceride-rich lipoproteins by lipoprotein lipase in vivo.J. Clin. Invest. 1986; 78: 1287-1295Google Scholar). In vitro and transgenic animal studies have demonstrated that overexpression of apoC-III causes delayed clearance of TG-rich lipoproteins from plasma, resulting in overt hypertriglyceridemia (3Jong M.C. Hofker M.H. Havekes L.M. Role of apo Cs in lipoprotein metabolism. Functional differences between apoC1, apoC2, and apoC3.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 472-484Google Scholar). The human apoC-III gene has been mapped on chromosome 11 and several variant alleles have been investigated as possible genetic markers of hypertriglyceridemia, an atherosclerosis-related “intermediate phenotype” (12Talmud P.J. Humphries S.E. Apolipoprotein C–III gene variation and dyslipidemia.Curr. Opin. Lipidol. 1997; 8: 154-158Google Scholar). Biallelic polymorphisms have been described in the 5′ promoter region (five polymorphisms in strong linkage disequilibrium with one another: T-455C, C-482T, T-625 deletion, G-630A, C-641A) (13Dammeman M. Sandkuijl L.A. Halaas J.L. Chung W. Breslow J.L. An apolipoprotein CIII aplotype protective against hypertriglyceridemia is specified by promoter and 3′ untranslated region polymorphisms.Proc. Natl. Acad. Sci. USA. 1993; 90: 4562-4566Google Scholar), in exon 3 (C1100T) and in the 3′ untranslated region (the so called Sstl polymorphic site S1/S2) (12Talmud P.J. Humphries S.E. Apolipoprotein C–III gene variation and dyslipidemia.Curr. Opin. Lipidol. 1997; 8: 154-158Google Scholar). In particular, over the last decade, this latter polymorphism, which is also the one most extensively studied, has consistently been reported to be associated with hypertriglyceridemia (14Ordovas J.M. Civeira F. Genest Jr., J. Craig S. Robbins A.H. Meade T. Pocovi M. Frossard P.M. Masharani U. Wilson P.W. Salem D.N. Ward R.H. Schaefer E.J. Restriction fragment length polymorphisms of the apolipoprotein A-I, C–III, A-IV gene locus. Relationships with lipids, apolipoproteins, and premature coronary artery disease.Atherosclerosis. 1991; 87: 75-86Google Scholar, 15Surgucho P. Page G. Patsch W. Boerwinkle E. Polimorphic markers in apolipoprotein C–III gene flanking regions and hypertriglyceridemia.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 941-947Google Scholar). Despite the expected implications in terms of cardiovascular morbidity, the evidence of an association between S2 allelic variant and risk of CAD is still controversial (14Ordovas J.M. Civeira F. Genest Jr., J. Craig S. Robbins A.H. Meade T. Pocovi M. Frossard P.M. Masharani U. Wilson P.W. Salem D.N. Ward R.H. Schaefer E.J. Restriction fragment length polymorphisms of the apolipoprotein A-I, C–III, A-IV gene locus. Relationships with lipids, apolipoproteins, and premature coronary artery disease.Atherosclerosis. 1991; 87: 75-86Google Scholar, 15Surgucho P. Page G. Patsch W. Boerwinkle E. Polimorphic markers in apolipoprotein C–III gene flanking regions and hypertriglyceridemia.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 941-947Google Scholar, 16Kee F. Amouyel P. Fumeron F. Arveiler D. Cambou J.P. Evans A. Cambien F. Fruchart J.C. Ducimetiere P. Dallongeville J. Lack of association between genetic variations of apo AI-CIII-AIV gene cluster and myocardial infarction in a sample of European male: ECTIM study.Atherosclerosis. 1999; 145: 187-195Google Scholar, 17Russo G. Meigs J.B. Cupples L.A. Demissie S. Otvos J.D. Wilson P.W. Lahoz C. Cucinotta D. Couture P. Mallory T. Schaefer E.J. Ordovas J.M. Association of the Sst-I polymorphism at the APOC3 gene locus with variation in lipids levels, lipoprotein subclass profiles and coronary artery disease risk: the Framingham offspring study.Atherosclerosis. 2001; 158: 173-181Google Scholar). Less information is available regarding the possible coronary risk linked to the other polymorphic variants (18Peacock R.E. Hamsten A. Johansson J. Nilsson-Ehle P. Humphries S.E. Association of genotypes at the apolipoprotein AI-CIII-AIV, apolipoprotein B and lipoprotein lipase gene loci with coronary atherosclerosis and high density lipoprotein subclasses.Clin. Genet. 1994; 46: 273-282Google Scholar, 19Hegele R.A. Small genetic effect in complex diseases: a review of regulatory sequence variants in dyslipoproteinemia and atherosclerosis.Clin. Biochem. 1997; 30: 183-188Google Scholar). This relatively limited interest may be particularly surprising if one considers the expression studies concerning the apoC-III promoter sequence variants in the insulin responsive element (IRE) and their interaction with insulin (20Chen M. Breslow J.L. Li W. Leff T. Transcriptional regulation of the apo C–III gene by insulin in diabetic mice: correlation with changes in plasma triglyceride levels.J. Lipid Res. 1994; 35: 1918-1924Google Scholar). In animals and in cultured cells, the apoC-III gene is transcriptionally downregulated by insulin (20Chen M. Breslow J.L. Li W. Leff T. Transcriptional regulation of the apo C–III gene by insulin in diabetic mice: correlation with changes in plasma triglyceride levels.J. Lipid Res. 1994; 35: 1918-1924Google Scholar). In 1995, Li et al. showed that, unlike the wild-type promoter, the promoter containing variants at positions –455 and –482 remains constitutively active over a 108-fold range of insulin concentrations inasmuch as polymorphic sequences have a reduced affinity for the nuclear transcription factors mediating the insulin response (21Li W.W. Dammeman M. Smith J.D. Metzger S. Breslow J.L. Leff T. Common genetic variation in the promoter of the human apo CIII gene abolishes regulation by insulin and may contribute to hypertriglyceridemia.J. Clin. Invest. 1995; 96: 2601-2605Google Scholar). The variation in the promoter of the apoC-III gene is the first reported example of a genetic polymorphism in an insulin responsive element and of “insulin resistance” at the gene level (21Li W.W. Dammeman M. Smith J.D. Metzger S. Breslow J.L. Leff T. Common genetic variation in the promoter of the human apo CIII gene abolishes regulation by insulin and may contribute to hypertriglyceridemia.J. Clin. Invest. 1995; 96: 2601-2605Google Scholar). Such considerations prompt speculation as to the possible links between these genetic variants and hypothetical related “intermediate” phenotypes, characterized by increased synthesis of apoC-III- and TG-rich lipoproteins, which in turn may imply an increased risk of CAD. In the light of all these elements, we designed a large case-control study in patients with or without angiographically documented CAD to evaluate: i) a possible association between apoC-III gene polymorphisms and circulating levels of apoC-III and/or plasma lipids, and ii) whether the distribution of these polymorphisms was in turn associated with an increased risk of CAD. The criteria for selection of the study population have already been described in detail elsewhere (22Girelli D. Russo C. Ferraresi P. Olivieri O. Pinotti M. Friso S. Manzato F. Mazzucco A. Bernardi F. Corrocher R. Polymorphisms in the factor VII gene and the risk of myocardial infarction in patients with coronary artery disease.N. Engl. J. Med. 2000; 343: 774-780Google Scholar). In brief, we studied a total of 800 unrelated adult patients of both sexes who were recruited from those referred to the Institute of Cardiovascular Surgery or to the Cardiovascular-Hypertension Unit of the Department of Internal Medicine of the University of Verona in Italy. Of these patients, 549 had angiographically documented, severe, often multivessel coronary atherosclerosis and were candidates for coronary-artery bypass grafting. The disease severity was evaluated by counting the number of major epicardial coronary arteries (left anterior descending, circumflex, and right) affected with ⩾1 significant stenosis (⩾50%). As a control group, we considered 251 subjects with angiographically documented normal coronary arteries (CAD-free), examined for reasons other than possible coronary artery disease (in 90% of cases valvular heart disease; the remaining cases were studied for miscellany conditions including atypical chest pain of uncertain origin, congenital heart disease, etc.). The controls were required to have no stenosis in angiogram, no history of atherosclerosis, nor evidence of atherosclerosis in other vascular beds. Since the primary aim of our selection was to provide an objective and clear-cut definition of the atherosclerotic phenotype, subjects with nonsignificant coronary stenosis (<50%) were not included in the study. The angiograms were assessed by two cardiologists unaware that the patients were to be included in the study. All the study participants came from northern Italy and had similar socioeconomic and ethnic backgrounds. At the time of blood sampling, a complete clinical and pharmacological history, including the presence or absence of cardiovascular risk factors such as smoking, hypertension, and diabetes mellitus, was obtained from the patients. Patients who were taking statins or fibrates (n = 266) were excluded from the genotype-phenotype correlation studies because of the documented lowering effects of these drugs on apoC-III and lipids levels (5Hodis H.N. Mack W.J. Azen S.P. Alaupovic P. Pogoda J.M. La Bree L. Hemphill L.C. Kramsch D.M. Blankenhorn D.H. Triglyceride and cholesterol-rich lipoproteins have a differential effect on mild/moderate and severe lesion progression as assessed by quantitative coronary angiography in a controlled trial of lovastatin.Circulation. 1994; 90: 42-49Google Scholar, 9Sacks F.M. Alaupovic P. Moye L.A. Cole T.G. Sussex B. Stampfer M.J. Pfeffer M.J. Braunwald E. VLDL, apolipoproteins B, CIII, and E, and risk of recurrent coronary events in the cholesterol and recurrent events (CARE) trial.Circulation. 2000; 102: 1886-1892Google Scholar, 23Packard C.J. Overview of fenofibrate.Eur. Heart J. 1998; 19: A62-A65Google Scholar). The study was approved by our institutional review boards. Either written or oral informed consent was obtained from all the patients after a full explanation of the study. Samples of venous blood were drawn from each subject after an overnight fast within 10 days from the angiographic procedures. Serum lipids and the other common biochemical parameters were determined as previously described (22Girelli D. Russo C. Ferraresi P. Olivieri O. Pinotti M. Friso S. Manzato F. Mazzucco A. Bernardi F. Corrocher R. Polymorphisms in the factor VII gene and the risk of myocardial infarction in patients with coronary artery disease.N. Engl. J. Med. 2000; 343: 774-780Google Scholar). Insulin was measured by an immunometric sandwich assay (Immulite 2000 Insulin) from Diagnostic Products Corporation, Los Angeles, CA; intra- and interassay CVs of the method were <5%. ApoA-I and apoB were measured by commercially available nephelometric immunoassays; antisera, calibrators, and BNII nephelometer were from Dade Behring, Marburg, Germany. Intraassay CV was calculated on 10 control replicates and interassay on duplicates over 10 days. Imprecision was within manufacturer specifications, i.e., the intraassay CVs were 2.1 and 1.6% and interassay CVs were 3.2 and 2.36 for apoA-I and apoB, respectively. ApoC-III was measured by a fully automated turbidimetric immunoassay. The reagents were obtained from Wako Pure Chemical Industries (Osaka, Japan) and the procedure recommended by the manifacturer was implemented on a RXL Dimension Analyzer (Dade International Inc. Newark, DE). Sample values were determined by interpolation of two spectrophotometric wavelengths measurements on a logit, five-points, calibration curve, covering the range 0.0–20.0 mg/dl; for concentrations of 20–30 mg/dl, a smaller sample volume was automatically rerun by the instrument, whereas for concentrations >30 mg/dl, the sample was diluted manually. Imprecision was assessed on three pools of control sera with low, medium and high concentrations of apoC-III; intraassay CV was 1.84, 2.02, and 1.98% and interassay CV 4.4, 3.4, and 2.29% for low, medium, and high concentration, respectively. Mutation analysis (as well as routine biochemical analysis) was conducted as a study that was blinded as to whether the sample came from CAD or a CAD-free subject. Three polymorphic variants mapping on the promoter (T-455C), on exon 3 in the coding region (C1100T), and on the 3′ untranslated region (S1/S2), were evaluated (Fig. 1). Genomic DNA was prepared from whole blood samples by phenol-chloroform extraction and was then used according to a recently described multilocus genotyping assay protocol (24Cheng S. Grow M.A. Pallaud C. Klitz W. Erlich H.A. Visvikis S. Chen J.J. Pullinger C.R. Malloy M.J. Siest G. Kane J.P. A multilocus genotyping assay for candidate markers of cardiovascular disease risk.Genome Res. 1999; 9: 936-949Google Scholar). Briefly, each sample was amplified by two 33 cycle Multiplex Polymerase Chain Reactions (32 ng of genomic DNA each) and the PCR products were then hybridized to an array of immobilized oligonucleotide probes. The colorimetric detection was based upon streptavidin-horseradish peroxidase method. All computations were performed by using the SPSS 10.0 statistical package (SPSS Inc., Chicago, IL). Distributions of continuous variables in groups were expressed as means ± SD. Logarithmic transformation was performed for skewed variables, i.e., apoC-III and TG, and the statistical differences concerning these parameters were also computed on the corresponding log-transformed values, although, for the sake of simplicity and clarity, crude data are reported in the Results. Statistical significance of differences in quantitative variables was assessed by Student's t-test, and it was also tested by one-way ANOVA adjusted for age and sex (General Linear Model procedure). Qualitative data were analyzed using the chi-square test. Pearson coefficient was used for correlation between variables. In each of the two patient groups (with or without CAD), genotype frequencies were compared by chi-square analysis with the values predicted on the basis of Hardy-Weinberg equilibrium. Intragenic haplotypes for multiple markers within the apoC-III locus were estimated using the EH program (25Terwilliger J. Ott J. Handbook of human genetic linkage. John Hopkins University Press, Baltimore, MD1994: 188-198Google Scholar) and were used to detect pairwise linkage disequilibrium values (D′). Insulin and lipid variables were compared among patients with different polymorphisms by ANOVA, using the Tukey procedure for post hoc multivariate comparison of the means (ANOVA). For T-455C and C1100T polymorphisms, computation was also performed considering the less frequent allele as recessive (homozygous for the less frequent variant vs. all other patients). To assess the extent to which the various genotypes were associated with coronary artery disease, odds ratios with 95% confidence intervals were estimated by logistic-regression analysis. To provide separate odds ratios for each genotype, dummy variables were used, with wild-type genotype used as the reference group. Adjustment for the patients' conventional risk factors (age, gender, smoking status, presence of diabetes and hypertension, cholesterol, triglycerides, apoA-I and apoB) was done by including these covariates in a second set of logistic-regression models. The clinical and biochemical characteristics of the population studied are summarized in Table 1. CAD patients had more conventional cardiovascular risk factors and significantly higher levels of apoC-III than control patients. There were no statistical differences in insulin plasma levels between the two groups (Table 1). In the population as a whole, apoC-III was statistically correlated with total (R = 0.40, P < 0.001), LDL (R = 0.238, P < 0.001), and HDL cholesterol (HDL-C) (R = −0.08, P < 0.05) and, more strongly, with TG levels (R = 0.68, P < 0.001).TABLE 1Characteristics of the study patients and controlsaPlus-minus values are means ± SD.ParametersCAD Patients (n = 549)CAD-free (n = 251)P valueAge (years)60.4 ± 9.457.6 ± 12.6<0.01Male sex (%)81.866.9<0.001BMI (kg/height2)bAge- and sex-adjusted values.26.5 ± 3.325.3 ± 3.4<0.001CholesterolbAge- and sex-adjusted values.Total (mmol/l)5.83 ± 1.125.51 ± 1.050.001LDL (mmol/l)3. 88 ± 0.983.53 ± 0.93<0.001HDL (mmol/l)1.20 ± 0.321.42 ± 0.43<0.001Triglycerides (mmol/l)bAge- and sex-adjusted values.2.01 ± 1.131.50 ± 0.71<0.001ApoA-I (g/l)bAge- and sex-adjusted values.1.31 ± 0.241.42 ± 0.31<0.001ApoB (g/l)bAge- and sex-adjusted values.1.22 ± 0.301.06 ± 0.25<0.001ApoC-III (mg/dl)bAge- and sex-adjusted values.12.31 ± 4.410.7 ± 3.25<0.001Insulin (μIU/ml)bAge- and sex-adjusted values.14.64 ± 7.715.75 ± 12.2NSSmoking (%)69.741.4<0.001Hypertension (%)58.330.8<0.001Diabetes (%)21.913.3<0.01Patients taking lipid-lowering drugs266—Statistical significance for differences was tested by Student's unpaired t-test or by χ2 test when appropriate. P value was considered significant when <0.05.To convert the values for cholesterol to milligrams per deciliter, divide by 0.02586. To convert the values for triglycerides to milligrams per deciliter, divide by 0.01129.a Plus-minus values are means ± SD.b Age- and sex-adjusted values. Open table in a new tab Statistical significance for differences was tested by Student's unpaired t-test or by χ2 test when appropriate. P value was considered significant when <0.05. To convert the values for cholesterol to milligrams per deciliter, divide by 0.02586. To convert the values for triglycerides to milligrams per deciliter, divide by 0.01129. Genotype frequencies of the apoC-III polymorphic variants for CAD and CAD-free groups are described in Table 2. All three polymorphisms were in the Hardy-Weinberg equilibrium in both groups of patients. No homozygous individuals for S2 allele were found. The distribution of C1100T and S1/S2 polymorphisms was similar in CAD and CAD-free patients. In contrast, the frequency of −455C homozygous subjects in CAD group was significantly higher than that observed in individuals free of coronary artery disease (18.6 vs. 9.2%, P < 0.001; Table 2).TABLE 2ApoC-III genotypes in the study patients and controlsGenotype (% of Patients)CAD-free Patients (n = 251)CAD Patients (n = 549)Chi- SquareP valueT-455C−455 TT(%) 43.8 35.3−455 TC (%) 47 46.1−455 CC(%) 9.2 18.613.070.001C1100T1100 CC (%) 50.2 54.31100 CT (%) 43 37.31100 TT (%) 6.8 8.42.529NSS1/S2S1/S1 (%) 85.2 82.4S1/S2 (%) 14.8 17.60.95NS Open table in a new tab Strong linkage disequilibrium between S1/S2 and both T-455C and C1100T (D' = 0.98, D' = 0.97, respectively) was observed, but not between T-455C and C1100T polymorphisms (D' = 0.13) (Fig. 1). It should be noted that, out of a total of 125 homozygotes for −455 variant, there were 55 (44%) subjects with S1/S2 and 13 (10.4%) with 1100TT genotype. In order to estimate the impact of the polymorphisms on apoC-III and/or other lipid metabolism parameters, genotype-phenotype analysis was performed with data from the entire study population. Due to the well known lowering effect of statins and fibrates on apoC-III (5Hodis H.N. Mack W.J. Azen S.P. Alaupovic P. Pogoda J.M. La Bree L. Hemphill L.C. Kramsch D.M. Blankenhorn D.H. Triglyceride and cholesterol-rich lipoproteins have a differential effect on mild/moderate and severe lesion progression as assessed by quantitative coronary angiography in a controlled trial of lovastatin.Circulation. 1994; 90: 42-49Google Scholar, 9Sacks F.M. Alaupovic P. Moye L.A. Cole T.G. Sussex B. Stampfer M.J. Pfeffer M.J. Braunwald E. VLDL, apolipoproteins B, CIII, and E, and risk of recurrent coronary events in the cholesterol and recurrent events (CARE) trial.Circulation. 2000; 102: 1886-1892Google Scholar, 23Packard C.J. Overview of fenofibrate.Eur. Heart J. 1998; 19: A62-A65Google Scholar), patients taking these drugs (n = 266, all CAD patients) were excluded from the analysis. Results for the levels of apoC-III, triglycerides, and insulin, according to each genotype in the remaining population (n = 534), are reported in Table 3. Different genotype groups of such population were similar for age and sex distribution (data not shown). Homozygous individuals for the promoter variant had increased levels of apoC-III as compared with carriers of other genotypes, but a statistical significance was evident only upon assuming recessive allelic transmission as the model (Table 3). Homozygotes for −455C had significantly higher triglyceride values than those observed in heterozygous and wild-type individuals (whether considered separately or as a single group, Table 3).TABLE 3Levels of apoC-III, TGs, and insulin in the study population, according to the ApoC-III genotypes (by ANOVA)ApoC-III GenotypesNo. of Patients (n = 534)ApoC-III (mg/dl)Triglycerides (mmol/l)Insulin (μIU/ml)−455TT20611.47 ± 3.91.79 ± 0.94aStatistically different from patients homozygous for the less common allele.15.7 ± 11−455TC25211.30 ± 3.71.73 ± 0.86aStatistically different from patients homozygous for the less common allele.14.6 ± 8.8−455CC7612.65 ± 5.12.12 ± 1.4014.8 ± 7.2−455TT and −455TC45811.38 ± 3.8aStatistically different from patients homozygous for the less common allele.1.76 ± 0.89aStatistically different from patients homozygous for the less common allele.15.1 ± 9.9−455CC7612.65 ± 5.12.12 ± 1.4014.8 ± 7.21100CC27411.3 ± 3.61.73 ± 0.85aStatistically different from patients homozygous for the less common allele.15.3 ± 10.61100CT21811.8 ± 4.51.85 ± 1.0514.9 ± 8.71100TT4212.1 ± 4.52.13 ± 1.4014.1 ± 5.71100CC and 1100CT49211.5 ± 4.01.79 ± 0.94aStatistically different from patients homozygous for the less common allele.15.1 ± 9.81100TT4212.1 ± 4.52.13 ± 1.4014.1 ± 5.7S1/S143811.3 ± 3.8bStatistically different from S1/S2 patients.1.75 ± 0.92bStatistically different from S1/S2 patients.14.9 ± 9.9S1/S29612.8 ± 4.82.10 ± 1.2215.6 ± 7.7a Statistically different from patients homozygous for the less common allele.b Statistically different from S1/S2 patients. Open table in a new tab C1100T polymorphism was significantly associated with plasma triglycerides (with the raising effect confined to the homozygotes), but this site was not associated with variations in apoC-III levels (Table 3). Different levels of apoC-III and triglycerides were also associated with the S1/S2 polymorphism, with higher values being observed in S2 carriers (Table 3). No substantial effect on fasting insulin values due to the different apoC-III gene polymorphisms was evident. Similarly, none of the other lipid variables analyzed (total, LDL, and HDL-C, apoA-I, apoB) showed any statistically relevant differences according to apoC-III genotype (data not shown). To estimate the disease risk associated with apoC-III genotypes, logistic regression analysis was performed. Crude and adjusted odds ratios for coronary disease in relation to each apoC-III genotype are shown in Table 4. The greatest risk was conferred by homozygosity for the −455C variant, which was associated with a more than 2-fold increased probability of disease. Adjustment for the main vascular risk factors produced no change in the result, indicating that this polymorp

Increased oxidative stress is thought to play a role in the pathogenesis of the atherothrombotic process. Paraoxonases (PONs) are closely related antioxidant enzymes encoded by clustered genes on chromosome 7q. We evaluated three PON polymorphisms (PON1 Leu55Met and Gln192Arg; PON2 Ser311Cys) as possible risk factors for coronary atherosclerotic disease (CAD) and/or its main thrombotic complication, myocardial infarction (MI).We studied 890 subjects with angiographic documentation of coronary vessels (272=CAD-free; 618=CAD). In the CAD group, 341 subjects had a previous MI.Frequencies of various genotypes were not significantly different between CAD-free subjects and the entire CAD population. In the latter group, there were more carriers of the PON2 311Cys variation among those who had suffered a MI than among those who had not (P<0.01 by chi2). The adjusted OR for MI among PON2 311Cys carriers was 1.5 (95%CI, 1.03-2.19). A gene-environmental interaction was found between PON2 Ser311Cys and smoking. Smoking by itself was associated with an increased MI risk. Among smokers, however, the MI risk was related to PON2 genotype: Cys/Cys homozygotes (OR=5.3; 95%CI, 1.7-16.4) and Ser/Cys heterozygotes (OR=2.1; 95%CI, 1.3-3.6) were at greater risk than Ser/Ser subjects (OR=1.2; 95%CI, 0.8-1.8). The PON2 polymorphism did not influence the MI risk among nonsmokers.In CAD subjects, a proportion of the risk of MI may be influenced by the interaction between smoking and a polymorphism in the antioxidant enzyme PON2.

A broad variability in patient response to dual antiplatelet treatment has been described during the first month of treatment. Data on platelet function profiles in patients on dual antiplatelet therapy for a more sustained period are limited. Whether gene sequence variations of the glycoprotein Ia/IIa receptor influence platelet aggregation in these patients is also unknown. The aim of this study was to characterize platelet aggregation profiles in patients on dual antiplatelet treatment (aspirin plus clopidogrel) for >1 month and to assess whether these may be influenced by the C807T polymorphism of the glycoprotein Ia gene. We included 82 patients, who were divided into 2 groups: carriers (CT + TT genotypes; n = 51) and noncarriers (CC genotype; n = 31) of the mutant T allele. Platelet aggregation was assessed using light transmittance aggregometry after stimuli with adenosine diphosphate (20 micromol/L), collagen (6 microg/ml), and epinephrine (20 micromol/L). A significant variability in the distribution of platelet aggregation was observed in the overall study population, as well as in carriers and noncarriers of the T allele. T allele carriers had increased platelet aggregation compared with noncarriers after stimuli with adenosine diphosphate, collagen, and epinephrine (p <0.05 for all platelet aggregation assays). Thus, platelet aggregation varied significantly in patients on long-term dual antiplatelet treatment and was increased in T allele carriers of the 807C/T polymorphism of the glycoprotein Ia gene. These findings may contribute to the increased ischemic risk observed in these patients.

The 677 C-->T polymorphism in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene interacts with folate status in determining elevated total plasma levels of homocysteine, a risk factor for coronary atherosclerotic disease (CAD). The present study had the following goals: 1) to define the 677 C-->T genotype-specific threshold values of both plasma and RBC folate, associated with hyperhomocysteinemia (>15 micro mol/L); and 2) to determine the risk of CAD among subjects with levels of folate below the genotype-specific threshold considered at risk for hyperhomocysteinemia. We examined 655 subjects, with (433) or without (222) angiographically documented CAD. The MTHFR 677 C-->T genotype-specific threshold values of plasma folate corresponded to the 40th, 30th and 10th percentile in the TT, CT and CC genotype, respectively. A multivariate logistic regression analysis showed that the risk of CAD among subjects with plasma folate levels below the genotype-specific thresholds was 1.6 (95% CI, 1.04-2.46). Similar results were obtained when RBC folate was considered as a measure of folate status (odds ratio = 1.8, 95% CI, 1.03-3.15). A gene-nutrient interaction that defines a higher risk for CAD is determined by folate levels below specific thresholds, which differ depending on the MTHFR 677 C-->T genotype.

Intronic variants of TCF7L2 are confirmed genetic risk factors for type 2 diabetes and are associated to alterations in beta cell function in nondiabetic individuals. The objective of the study was to test whether TCF7L2 variability may affect β-cell function also in patients with type 2 diabetes. This was a cross-sectional association study. The study was conducted at a university hospital referral center for diabetes. Patients included 464 (315 males and 149 females) glutamic acid decarboxylase-negative patients [age: median 59 yr (interquartile range: 52–65); body mass index: 29.3 kg/m2 (26.5–32.9); fasting plasma glucose: 7.0 mmol/liter (6.1–8.0)] with newly diagnosed type 2 diabetes. Interventions included frequently sampled oral glucose tolerance test and euglycemic insulin clamp. β-Cell function (derivative control and proportional control); insulin sensitivity; genotypes of the following TCF7L2 single-nucleotide polymorphisms: rs7901695, rs7903146, rs11196205, and rs12255372. Both rs7901695 and rs7903146 diabetes risk alleles were associated with reduced proportional control of β-cell function (P = 0.019 and P = 0.022, respectively). Two low-frequency haplotypes were associated with extreme (best and worst) phenotypes of β-cell function (P < 0.01). No associations between TCF7L2 genotypes and insulin sensitivity were detected. TCF7L2 diabetes risk variants, either as single-nucleotide polymorphisms or as haplotypes, detrimentally influence β-cell function and might play a role in determining the metabolic phenotype of patients with newly diagnosed type 2 diabetes.

Glycoprotein (GP) Ia/IIa is a major platelet-collagen receptor playing a key role in thrombosis following collagen exposure. The 807 C/T polymorphism of the GP Ia gene (ITGA2) has been associated with platelet GP Ia/IIa receptor expression, having T-allele carriers, increased receptor density and thrombotic risk. The aim of the study was to assess the role of the 807 C/T polymorphism on modulating platelet function in patients undergoing coronary stenting receiving a 300 mg clopidogrel loading dose. Platelet aggregation was assessed in 44 patients by light transmittance aggregometry following adenosine diphosphate and collagen stimuli at baseline, and 10 min, 4 h and 24 h after clopidogrel front loading. The T allele was found in 73% of patients. Clopidogrel reduced adenosine diphosphate-induced platelet aggregation (P< 0.01), which was similar in carriers and non-carriers of the T allele throughout the study (P = 0.73). Clopidogrel reduced collagen-induced platelet aggregation only in non-carriers of the T allele (P = 0.03), which resulted in an increase in T allele carriers during the overall study (P = 0.04). In conclusion, the T allele of the GP Ia gene modulates platelet aggregation and clopidogrel antiplatelet effects, suggesting an enhanced reactivity to fibrillar collagens (exposed during coronary stenting) in T allele carriers and might contribute to an increased thrombotic risk in these patients.

Tumor necrosis factor (TNF) is a potent proinflammatory cytokine with increased levels in the sputum of COPD subjects. Two biallelic TNF gene complex polymorphisms have been described: LtalphaNcoI, in the first intron of the lymphotoxin alpha (previously referred to as TNF-beta) gene, and TNF-308, in the promoter region of the TNF-alpha gene. Higher levels of TNF production are associated with allele 1 of LtalphaNcoI (LtalphaNcoI*1) and with allele 2 of TNF-308 (TNF-308*2).To study the frequencies of the two TNF gene complex polymorphisms in patients with COPD and bronchiectasis.Association study.We studied the frequencies of these polymorphisms in 66 subjects with COPD and in 23 subjects with disseminated bronchiectasis and compared them to the frequencies in 98 healthy control subjects and 45 subjects with nonobstructive pulmonary disease. Genomic DNA samples were extracted, and TNF-alpha and LtalphaNcoI polymorphisms were detected after polymerase chain reaction by restriction digestion.We found the following frequencies: the TNF-308*2 allele was detected in 11% of COPD individuals, 15% of bronchiectasis patients, 10% of healthy control subjects, and 18% of subjects with nonobstructive pulmonary disease. The LtalphaNcoI*1 allele was detected in 28% of COPD individuals, 30% of bronchiectasis patients, 29% of healthy control subjects, and 29% of subjects with nonobstructive pulmonary disease. We found evidence of linkage disequilibrium between the two loci (Delta = 0.068).We conclude that the TNF gene complex, at least in Caucasoid individuals and for the considered polymorphisms, does not seem to play a major role as genetic risk factor in COPD and bronchiectasis.

Obstructive pulmonary disease includes asthma, chronic obstructive pulmonary disease (COPD; i.e., pulmonary emphysema and chronic bronchitis), bronchiectasis, and cystic fibrosis (CF). It represents a leading cause of death in developed countries. Both familial clustering of non-CF obstructive pulmonary disease and familial aggregation of impaired lung function have been described. This suggests that genetic factors contribute to non-CF obstructive pulmonary disease, even if it is difficult to determine the relative contribution of environmental factors. 11 refs., 1 tab.

Several genome scans have reported linkage of markers on chromosome 7p with asthma and related phenotypes in different populations. A fine mapping in Finnish and French-Canadian populations has associated the GPR154 gene (also known as G-protein-coupled receptor for asthma susceptibility, GPRA) with elevated IgE or asthma.To confirm chromosome 7p linkage and candidate gene association in Italian families with atopic asthma.In a two-phase approach, we first performed a linkage analysis of chromosome 7, and then a family-based association study on the GPR154 gene for allergic asthma phenotypes in the Italian population.The screening of 117 families with 19 microsatellite markers showed potential linkage for elevated IgE (P<0.002 at 22 cM from p-ter), asthma (P<0.005 at 44 cM), or atopy (P<0.005 at 54 cM). In the second phase of the present study, candidate gene GPR154, which is located in the phase one-linked region, was investigated in 211 families with seven single nucleotide polymorphisms (SNPs) that tag most haplotype variability, by the pedigree disequilibrium test. Elevated IgE levels were associated with two GPR154 gene SNPs (SNP 546333, P=0.0046; rs740 347, P=0.006), and with haplotypes in the global test (P=0.013). Haplotype analysis performed in nuclear families having at least 1 asthmatic parent showed a significant association with asthma (P=0.0173), atopy (P=0.0058), SPT (P=0.0025), and bronchial hyper reactivity (P=0.0163).These results support a susceptibility locus for asthma and related phenotypes on chromosome 7, and are in agreement with recent reports suggesting that a common susceptibility factor for atopic manifestations in asthma is likely conferred by the locus containing the GPR154 gene.

Abstract Background: Apolipoprotein C-III (apo C-III) is a marker of cardiovascular disease risk associated with triglyceride (TG)-rich lipoproteins. The T−455C polymorphism in the insulin-responsive element of the APOC3 gene influences TG and apo C-III concentrations. Long-chain n-3 polyunsaturated fatty acids (PUFAs) contained in fish have well-known apo C-III-lowering properties. Methods: We investigated the possibility of an interactive effect between the APOC3 gene variant and erythrocyte n-3 PUFAs, suitable markers of dietary intake of fatty acids, on apo C-III concentrations in a population of 848 heart disease patients who had coronary angiography. Results: In the population as a whole, apo C-III concentrations were significantly inversely correlated with total erythrocyte PUFAs, but the correlation was not significant when only −455CC homozygous individuals were taken into account. In the total population and in subgroups with the −455TT and −455CT genotypes, the relative proportions of individuals presenting with increased apo C-III (i.e., above the 75th percentile value calculated on the entire population after exclusion of individuals taking lipids-lowering medications) decreased progressively as the n-3 PUFA and docosahexaenoic acid concentrations increased. The opposite situation was observed in the homozygous −455CC subgroup, in whom increasing erythrocyte n-3 PUFA and docosahexaenoic acid concentrations were associated with higher proportions of individuals with high apo C-III. A formal interactive effect between genotype and n-3 PUFAs was confirmed even after adjustment for possible confounding variables [age, sex, body mass index, smoking, coronary artery disease (CAD)/CAD-free status, or use of lipid-lowering medications] by logistic models. Conclusion: Patients homozygous for the −455C APOC3 variant are poorly responsive to the apo C-III-lowering effects of n-3 PUFAs.

The aim of this study was to explore the role of variants of the gene encoding arachidonate 5-lipoxygenase-activating protein (ALOX5AP) as possible susceptibility factors for coronary artery disease (CAD) and myocardial infarction (MI) in patients with or without angiographically proven CAD. A total of 1431 patients with or without angiographically documented CAD were examined simultaneously for seven ALOX5AP single-nucleotide polymorphisms, allowing reconstruction of the at-risk haplotypes (HapA and HapB) previously identified in the Icelandic and British populations. Using a haplotype-based approach, HapA was not associated with either CAD or MI. On the other hand, HapB and another haplotype within the same region (that we named HapC) were significantly more represented in CAD versus CAD-free patients, and these associations remained significant after adjustment for traditional cardiovascular risk factors by logistic regression (HapB: odds ratio (OR) 1.67, 95% confidence interval (CI) 1.04-2.67; P=0.032; HapC: OR 2.41, 95% CI 1.09-5.32; P=0.030). No difference in haplotype distributions was observed between CAD subjects with or without a previously documented MI. Our angiography-based study suggests a possible modest role of ALOX5AP in the development of the atheroma rather than in its late thrombotic complications such as MI.

Relative little attention has been devoted until now to the combined effects of gene polymorphisms of the hemostatic pathway as risk factors for Myocardial Infarction (MI), the main thrombotic complication of Coronary Artery Disease (CAD). The aim of this study was to evaluate the combined effect of ten common prothrombotic polymorphisms as a determinant of MI.We studied a total of 804 subjects, 489 of whom with angiographically proven severe CAD, with or without MI (n = 307; n = 182; respectively). An additive model considering ten common polymorphisms [Prothrombin 20210G>A, PAI-1 4G/5G, Fibrinogen beta -455G>A, FV Leiden and "R2", FVII -402G>A and -323 del/ins, Platelet ADP Receptor P2Y12 -744T>C, Platelet Glycoproteins Ia (873G>A), and IIIa (1565T>C)] was tested. The prevalence of MI increased linearly with an increasing number of unfavorable alleles (chi(2) for trend = 10.68; P = 0.001). In a multiple logistic regression model, the number of unfavorable alleles remained significantly associated with MI after adjustment for classical risk factors. As compared to subjects with 3-7 alleles, those with few (</=2) alleles had a decreased MI risk (OR 0.34, 95%CIs 0.13-0.93), while those with more (>/=8) alleles had an increased MI risk (OR 2.49, 95%CIs 1.03-6.01). The number of procoagulant alleles correlated directly (r = 0.49, P = 0.006) with endogenous thrombin potential.The combination of prothrombotic polymorphisms may help to predict MI in patients with advanced CAD.

In genome-wide association studies, performed mostly in nondiabetic individuals, genetic variability of glucokinase regulatory protein (GCKR) affects type 2 diabetes-related phenotypes, kidney function, and risk of chronic kidney disease (CKD). We tested whether GCKR variability affects type 2 diabetes or kidney-related phenotypes in newly diagnosed type 2 diabetes.In 509 GAD-negative patients with newly diagnosed type 2 diabetes, we 1) genotyped six single nucleotide polymorphisms in GCKR genomic region: rs6717980, rs1049817, rs6547626, rs780094, rs2384628, and rs8731; 2) assessed clinical phenotypes, insulin sensitivity by the euglycemic insulin clamp, and β-cell function by state-of-the-art modeling of glucose/C-peptide curves during an oral glucose tolerance test; and 3) estimated glomerular filtration rate (eGFR) by the Modification of Diet in Renal Disease formula.The major alleles of rs6717980 and rs2384628 were associated with reduced β-cell function (P < 0.05), with mutual additive effects of each variant (P < 0.01). The minor alleles of rs1049817 and rs6547626 and the major allele of rs780094 were associated with reduced eGFR according to a recessive model (P < 0.03), but with no mutual additive effects of the variants. Additional associations were found between rs780094 and 2-h plasma glucose (P < 0.05) and rs8731 and insulin sensitivity (P < 0.05) and triglycerides (P < 0.05).Our findings are compatible with the idea that GCKR variability may play a pathogenetic role in both type 2 diabetes and CKD. Genotyping GCKR in patients with newly diagnosed type 2 diabetes might help in identifying patients at high risk for metabolic derangements or CKD.

The objective of this study was to replicate an association study on a newly collected Italian autism spectrum disorder (ASD) cohort by studying the genetic markers associated with ASDs from recent genome-wide and candidate gene association studies.We have genotyped 746 individuals from 227 families of the Italian Autism Network using allelic discrimination TaqMan assays for seven common single-nucleotide polymorphisms: rs2292813 (SLC25A12 gene), rs35678 (ATP2B2 gene), rs4307059 (between CDH9 and CDH10 genes), rs10513025 (between SEMA5A and TAS2R1 genes), rs6872664 (PITX1 gene), rs1861972 (EN2 gene), and rs4141463 (MACROD2 gene). A family-based association study was conducted.A significant association was found for two of seven markers: rs4307059 T allele (odds ratio: 1.758, SE=0.236; P-value=0.017) and rs35678 TC genotype (odds ratio: 0.528, SE=0.199; P-value=0.0013).A preferential allele transmission of two markers located at loci previously associated with social and verbal communication skill has been confirmed in patients of a new ASD family sample.

The risk of developing adult T-cell leukemia/lymphoma (ATLL) in individuals infected with human T-cell lymphotropic virus 1 (HTLV-1) is about 3–5%. The mechanisms by which the virus triggers this aggressive cancer are still an area of intensive investigation. The viral protein Tax-1, together with additional regulatory proteins, in particular HTLV-1 basic leucine zipper factor (HBZ), are recognized as relevant viral factors required for both viral replication and transformation of infected cells. Tax-1 deregulates several cellular pathways affecting the cell cycle, survival, and proliferation. The effects of Tax-1 on the NF-κB pathway have been thoroughly studied. Recent studies also revealed the impact of Tax-1 and HBZ on microRNA expression. In this review, we summarize the recent progress in understanding the contribution of HTLV-1 Tax- and HBZ-mediated deregulation of NF-κB and the microRNA regulatory network to HTLV-1 pathogenesis.

We investigated 116 Italian atopic families (560 individuals) for linkage with 13 DNA markers on chromosome 12. All the subjects were phenotyped for asthma, total serum IgE, bronchial hyperresponsiveness, skin-prick positivity to common aeroallergens, and atopy. A relative location map of the markers was prepared from Centre d'Etude du Polymorphisme Humain families. Affected sib pair multipoint linkage methods were used to perform the statistical analyses. We report suggestive linkage for asthma with markers on chromosome 12. The region of interest centers around marker D12S390 (maximum logarithm of odds [mlod] = 2.81; p = 0.003). These results provide additional support that asthma susceptibility factors are located on chromosome 12q.

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).

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 (<svg style="vertical-align:-0.17555pt;width:42.912498px;" id="M1" height="10.9125" version="1.1" viewBox="0 0 42.912498 10.9125" width="42.912498" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,10.9125)"> <g transform="translate(72,-63.27)"> <text transform="matrix(1,0,0,-1,-71.95,63.5)"> <tspan style="font-size: 12.50px; " x="0" y="0">𝑛</tspan> <tspan style="font-size: 12.50px; " x="9.6773224" y="0">=</tspan> <tspan style="font-size: 12.50px; " x="21.71771" y="0">8</tspan> <tspan style="font-size: 12.50px; " x="27.969212" y="0">8</tspan> </text> </g> </g> </svg>) or without MS (<svg style="vertical-align:-0.17555pt;width:50.724998px;" id="M2" height="11.1" version="1.1" viewBox="0 0 50.724998 11.1" width="50.724998" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,11.1)"> <g transform="translate(72,-63.12)"> <text transform="matrix(1,0,0,-1,-71.95,63.35)"> <tspan style="font-size: 12.50px; " x="0" y="0">𝑛</tspan> <tspan style="font-size: 12.50px; " x="9.6773224" y="0">=</tspan> <tspan style="font-size: 12.50px; " x="21.71771" y="0">2</tspan> <tspan style="font-size: 12.50px; " x="27.969212" y="0">0</tspan> <tspan style="font-size: 12.50px; " x="34.220711" y="0">5</tspan> </text> </g> </g> </svg>) and with (<svg style="vertical-align:-0.27588pt;width:50.724998px;" id="M3" height="11.225" version="1.1" viewBox="0 0 50.724998 11.225" width="50.724998" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,11.225)"> <g transform="translate(72,-63.02)"> <text transform="matrix(1,0,0,-1,-71.95,63.35)"> <tspan style="font-size: 12.50px; " x="0" y="0">𝑛</tspan> <tspan style="font-size: 12.50px; " x="9.6773224" y="0">=</tspan> <tspan style="font-size: 12.50px; " x="21.71771" y="0">1</tspan> <tspan style="font-size: 12.50px; " x="27.969212" y="0">9</tspan> <tspan style="font-size: 12.50px; " x="34.220711" y="0">5</tspan> </text> </g> </g> </svg>) or without (<svg style="vertical-align:-0.27588pt;width:42.912498px;" id="M4" height="11.0375" version="1.1" viewBox="0 0 42.912498 11.0375" width="42.912498" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,11.0375)"> <g transform="translate(72,-63.17)"> <text transform="matrix(1,0,0,-1,-71.95,63.5)"> <tspan style="font-size: 12.50px; " x="0" y="0">𝑛</tspan> <tspan style="font-size: 12.50px; " x="9.6773224" y="0">=</tspan> <tspan style="font-size: 12.50px; " x="21.71771" y="0">9</tspan> <tspan style="font-size: 12.50px; " x="27.969212" y="0">8</tspan> </text> </g> </g> </svg>) 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&#x25; CI 1.44&#x2013;13.10), while no significant increase of risk was found among those with MS but high DEPCyMCase activity (OR 1.45 with 95&#x25; CI 0.47&#x2013;4.46). Our results suggest that low PON1 concentrations are typical in MS and may modulate the MS-related risk of CAD.

The development of thoracic aortic aneurysms (TAAs) involves a multifactorial process resulting in alterations of the structure and composition of the extracellular matrix (ECM). Recently, modifications in microRNA (miRNA) expression were implicated in the pathogenesis of TAA. This study presents a preliminary miRNA microarray analysis conducted on pooled ascending aorta RNAs obtained from non familial non syndromic TAA patients (five males and five females) compared to matched control pools. Ninety-nine differentially expressed miRNAs with >1.5-fold-up- or down-regulation in TAAs compared to controls were identified, 16.0% of which were similarly regulated in the two sexes. Genes putatively targeted by differentially expressed miRNAs belonged preferentially to focal adhesion and adherens junction pathways. The results indicate an altered regulation of miRNA-mediated gene expression in the cellular interactions of aneurysmal aortic wall.

Notwithstanding several research efforts in the past years, robust and replicable molecular signatures for autism spectrum disorders from peripheral blood remain elusive. The available literature on blood transcriptome in ASD suggests that through accurate experimental design it is possible to extract important information on the disease pathophysiology at the peripheral level. Here we exploit the availability of a resource for molecular biomarkers in ASD, the Italian Autism Network (ITAN) collection, for the investigation of transcriptomic signatures in ASD based on a discordant sibling pair design. Whole blood samples from 75 discordant sibling pairs selected from the ITAN network where submitted to RNASeq analysis and data analyzed by complementary approaches. Overall, differences in gene expression between affected and unaffected siblings were small. In order to assess the contribution of differences in the relative proportion of blood cells between discordant siblings, we have applied two different cell deconvolution algorithms, showing that the observed molecular signatures mainly reflect changes in peripheral blood immune cell composition, in particular NK cells. The results obtained by the cell deconvolution approach are supported by the analysis performed by WGCNA. Our report describes the largest differential gene expression profiling in peripheral blood of ASD subjects and controls conducted by RNASeq. The observed signatures are consistent with the hypothesis of immune alterations in autism and an increased risk of developing autism in subjects exposed to prenatal infections or stress. Our study also points to a potential role of NMUR1, HMGB3, and PTPRN2 in ASD.

Patients with migraine are known to be at risk for stroke. It has been reported that in a group of patients with cerebral ischemia and the Leiden mutation of factor V, 67% had classical migraine. We have studied the frequency of this mutation in a group of Italian children and adolescents affected by migraine with aura. The Leiden mutation was detected in 2 (3.5%) of 57 patients and in 8 (3.7%) of 219 controls. The 2 patients carrying the mutation had no peculiar characteristics as compared with the rest of the migrainous population. In our study, the frequency of the Leiden mutation in patients was not different from that of controls. These data contrast with those collected in the Finnish population and in a group of northwestern Italian adult patients, but agree with results previously reported from The Netherlands.

The human oxidised low-density lipoprotein receptor 1 (OLR1) gene is a functional candidate for atherosclerosis. An association of the OLR1 gene with acute myocardial infarction (AMI) or coronary artery disease (CAD) has recently been reported. In the present study a total of 677 Italian subjects, 327 CAD-free, 350 CAD, of which 190 with AMI and 160 AMI-free, was genotyped for the following four OLR1 single nucleotide polymorphisms: exon 4 K167N, IVS4 -73C>T, IVS4 -14A>G, and 3'UTR 188 C>T. No statistically significant difference was observed in allele or genotype distribution of the exon 4, intron 4, or 3'UTR SNPs in CAD patients compared to CAD-free subjects, or within CAD, in AMI patients compared to AMI-free patients. A correlation was found between the K167N G/G genotype and the increased number of obstructed vessels. Even if the OLR1 genotype frequency distribution data in CAD or AMI subjects here reported do not fully confirm the positive results of some other association studies, an association with a marker of CAD severity was observed.

Abstract Background: Iron may promote coronary atherosclerotic disease (CAD) by increasing lipid peroxidation. Studies on biochemical or genetic markers of body iron stores as risk factors for CAD have yielded conflicting results. Methods: We studied 849 individuals with a clear-cut definition of the CAD phenotype, i.e., with (CAD; n = 546) or without (CAD-free; n = 303) angiographically documented disease. We determined serum ferritin, as a biochemical estimate of iron stores, and the C282Y mutation in the HFE gene, i.e., the main cause of hemochromatosis in Caucasians. The relationships of ferritin with serum markers of either inflammation [C-reactive protein (CRP)] or lipid peroxidation (malondialdehyde) were also investigated. Results: Mean ferritin concentrations were slightly higher in CAD vs CAD-free individuals, but this difference disappeared after adjusting for sex and CRP. Ferritin was significantly correlated with CRP (Spearman’s test, ρ = 0.129; P &amp;lt;0.001). Heterozygotes for Cys282Tyr were 4.8% among the CAD group and 6.6% among the CAD-free group (P = 0.26). The prevalence of high concentrations of stored iron, defined as ferritin concentrations above the sex-specific upper quintiles of the control distribution, was also similar in the two groups. There was a higher prevalence of “iron depletion” in CAD-free vs CAD females (20% vs 8.8%, respectively), but this difference disappeared after adjustment for age and other cardiovascular risk factors (odds ratio, 0.66; 95% confidence interval, 0.21–2.08). No differences in iron markers were found in CAD patients with or without myocardial infarction. Conclusions: Our results do not support a role for biochemical or genetic markers of iron stores as predictors of the risk of CAD or its thrombotic complications.

Genetic variability of the major subunit (CACNA1E) of the voltage-dependent Ca(2+) channel Ca(V)2.3 is associated to risk of type 2 diabetes, insulin resistance and impaired insulin secretion in nondiabetic subjects. The aim of the study was to test whether CACNA1E common variability affects beta cell function and/or insulin sensitivity in patients with newly diagnosed type 2 diabetes.In 595 GAD-negative, drug naïve patients (mean ± SD; age: 58.5 ± 10.2 yrs; BMI: 29.9 ± 5 kg/m(2), HbA1c: 7.0±1.3) with newly diagnosed type 2 diabetes we: 1. genotyped 10 tag SNPs in CACNA1E region reportedly covering ∼93% of CACNA1E common variability: rs558994, rs679931, rs2184945, rs10797728, rs3905011, rs12071300, rs175338, rs3753737, rs2253388 and rs4652679; 2. assessed clinical phenotypes, insulin sensitivity by the euglycemic insulin clamp and beta cell function by state-of-art modelling of glucose/C-peptide curves during OGTT. Five CACNA1E tag SNPs (rs10797728, rs175338, rs2184945, rs3905011 and rs4652679) were associated with specific aspects of beta cell function (p<0.05-0.01). Both major alleles of rs2184945 and rs3905011 were each (p<0.01 and p<0.005, respectively) associated to reduced proportional control with a demonstrable additive effect (p<0.005). In contrast, only the major allele of rs2253388 was related weakly to more severe insulin resistance (p<0.05).In patients with newly diagnosed type 2 diabetes CACNA1E common variability is strongly associated to beta cell function. Genotyping CACNA1E might be of help to infer the beta cell functional phenotype and to select a personalized treatment.

Organoids are self-organized, three-dimensional structures derived from stem cells that can mimic the structure and physiology of human organs. Patient-specific induced pluripotent stem cells (iPSCs) and 3D organoid model systems allow cells to be analyzed in a controlled environment to simulate the characteristics of a given disease by modeling the underlying pathophysiology. The recent development of 3D cell models has offered the scientific community an exceptionally valuable tool in the study of rare diseases, overcoming the limited availability of biological samples and the limitations of animal models. This review provides an overview of iPSC models and genetic engineering techniques used to develop organoids. In particular, some of the models applied to the study of rare neuronal, muscular and skeletal diseases are described. Furthermore, the limitations and potential of developing new therapeutic approaches are discussed.

Background Allergic asthma is a multifactorial disease for which there is a widely assessed, although poorly understood, genetic involvement. Genome‐wide screens reported evidence for linkage of allergic asthma‐related phenotypes to several chromosomal locations. Markers on chromosome 19 have been linked to allergic asthma phenotypes in different populations in independent studies. Objective The aim of this study was to perform a genetic linkage analysis on chromosome 19 to search for DNA markers linked to phenotypes related to allergic asthma. Methods Using non‐parametric multipoint linkage analysis on a total of 22 random DNA markers in 2 stages, a sample of 111 families (542 subjects) from north‐eastern Italy, recruited through an asthmatic allergic proband, was investigated. Phenotypes examined were: clinical asthma, total serum elevated IgE, skin prick test positivity, bronchial hyper‐responsiveness, and atopy defined as skin prick test positivity and/or elevated IgE. Simulation studies were performed to confirm the significance of the results. Results A novel linkage of atopy and skin prick test positivity to marker D19S601 (19q13.3) was found. Modest evidence for linkage of atopy, skin prick test positivity, and IgE was also found to marker D19S591 (19p13.3). Simulation analysis for atopy gave an NPL‐Z &gt; 3.326 in 2 replicates out of 1000 ( P = 0.002) for D19S601, and an NPL‐Z &gt; 2.56 in 16 replicates out of 1000 ( P = 0.016) for D19S591. Conclusions On chromosome 19, suggestive linkage of atopy and skin prick test positivity with marker D19S601 (19q13.3) and modest evidence of linkage of marker D19S591 (19p13.3) to the atopic phenotypes investigated were found. These results suggest that these regions may contain susceptibility loci associated to atopic phenotypes.

Platelet membrane receptors play a pivotal role in thrombus formation. Expression of platelet membrane receptors are under genetic control and gene sequence variations of receptors pivotal to thrombotic formation have been hypothesized to contribute to different degrees of individual response to aspirin. The aim of the present study was to investigate the impact of functional genetic polymorphisms of platelet membrane receptors on aspirin sensitivity assessed by means of the PFA-100 system in patients with coronary artery disease. Gene sequence variations of three platelet membrane receptors (GPIa/IIa, P2Y12, GPIIb/IIIa) pivotal to thrombus formation were assessed in 76 patients with coronary artery disease on chronic aspirin treatment. Patients with reduced sensitivity to aspirin were defined when closure-times of collagen/epinephrine cartridges ≤193 seconds and coined as PFA-100 non-responders. PFA-100 non-responders were observed in 33% of patients. Patients with diabetes mellitus were more frequently PFA-100 non-responders. Closure times of collagen/ADP coated cartridges were reduced in PFA-100 non-responders. The genotype distribution was similar in PFA-100 responder and non-responder patients for all three genotypes and did not vary in contemporaneous carriers of allelic variants. In conclusion, in vitro determined sensitivity to aspirin assessed using PFA-100 is not associated with gene sequence variations of platelet membrane receptors key to thrombus formation.

Abstract 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) ε2/ε3/ε4 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 ε2/ε3/ε4 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 ε2/ε3/ε4 genotype in determining ApoE concentrations and risk of MI in an Italian population.

Previous studies reported linkage between maternally inherited atopy and the beta subunit of the high affinity IgE receptor (Fc epsilon RI-beta) located on chromosome 11q13. We have investigated 45 Italian families with atopic asthmatic children, for a total of 213 subjects, including 148 patients. Genotyping was carried out with two microsatellite DNA markers: one (Fc epsilon RI-beta CA) located inside the gene, and one (CI11-319 CA) closely linked to it. Affected sib-pair analysis in families with several affected children indicated 128 pairs in which either both markers were informative. An excess of maternal allele sharing was observed, although not significant. The allele-specific DNA amplification test for the FcRI-beta Ile181Leu mutation, described previously in 17% of atopic English families by Shirakawa and coworkers, was negative in all our families, as well as in 42 Italian children with atopic asthma and without family histories of the disease.

Iron may promote coronary atherosclerotic disease (CAD) by increasing lipid peroxidation. Studies on biochemical or genetic markers of body iron stores as risk factors for CAD have yielded conflicting results.We studied 849 individuals with a clear-cut definition of the CAD phenotype, i.e., with (CAD; n = 546) or without (CAD-free; n = 303) angiographically documented disease. We determined serum ferritin, as a biochemical estimate of iron stores, and the C282Y mutation in the HFE gene, i.e., the main cause of hemochromatosis in Caucasians. The relationships of ferritin with serum markers of either inflammation [C-reactive protein (CRP)] or lipid peroxidation (malondialdehyde) were also investigated.Mean ferritin concentrations were slightly higher in CAD vs CAD-free individuals, but this difference disappeared after adjusting for sex and CRP. Ferritin was significantly correlated with CRP (Spearman's test, rho = 0.129; P <0.001). Heterozygotes for Cys282Tyr were 4.8% among the CAD group and 6.6% among the CAD-free group (P = 0.26). The prevalence of high concentrations of stored iron, defined as ferritin concentrations above the sex-specific upper quintiles of the control distribution, was also similar in the two groups. There was a higher prevalence of "iron depletion" in CAD-free vs CAD females (20% vs 8.8%, respectively), but this difference disappeared after adjustment for age and other cardiovascular risk factors (odds ratio, 0.66; 95% confidence interval, 0.21-2.08). No differences in iron markers were found in CAD patients with or without myocardial infarction.Our results do not support a role for biochemical or genetic markers of iron stores as predictors of the risk of CAD or its thrombotic complications.

To the Editor: Two recent, independent, large-scale genome-wide association studies described the association of asthma with single nucleotide polymorphisms (SNPs) mapping on chromosome 9 in a region flanking the IL33 gene.1Gudbjartsson D.F. Bjornsdottir U.S. Halapi E. Helgadottir A. Sulem P. Jonsdottir G.M. et al.Sequence variants affecting eosinophil numbers associate with asthma and myocardial infarction.Nat Genet. 2009; 41: 342-347Crossref PubMed Scopus (642) Google Scholar, 2Moffatt M.F. Gut I.G. Demenais F. Strachan D.P. Bouzigon E. Heath S. et al.A large-scale, consortium-based genomewide association study of asthma.N Engl J Med. 2010; 363: 1211-1221Crossref PubMed Scopus (1568) Google Scholar No family-based association study has yet been reported to confirm the association that was found in the case-control studies. We have previously reported the preliminary results of a genome-wide linkage study conducted on a sample of Italian asthmatic families that indicated linkage to the microsatellite marker D9S286, which is located in an extended region of chromosome 9p24 that includes the IL33 gene.3Malerba G, Trabetti E, Patuzzo C, Migliaccio C, Galavotti R, Lauciello MC, et al. A genome scan for allergic asthma and related phenotypes in an Italian population sample. Presented at: the 52nd Annual Meeting of the American Society of Human Genetics; October 15-19, 2002; Baltimore, Md.Google Scholar, 4Bouzigon E. Forabosco P. Koppelman G.H. Cookson W.O. Dizier M.H. Duffy D.L. et al.Meta-analysis of 20 genome-wide linkage studies evidenced new regions linked to asthma and atopy.Eur J Hum Genet. 2010; 18: 700-706Crossref PubMed Scopus (50) Google Scholar We have now investigated whether the SNPs rs1342326 and rs928413, which showed association in the recent study by Moffatt et al,2Moffatt M.F. Gut I.G. Demenais F. Strachan D.P. Bouzigon E. Heath S. et al.A large-scale, consortium-based genomewide association study of asthma.N Engl J Med. 2010; 363: 1211-1221Crossref PubMed Scopus (1568) Google Scholar are associated with childhood asthma or related phenotypes in the abovementioned Italian families. The SNP rs928413 is in absolute linkage disequilibrium (LD; http://www.hapmap.org, rel#24) with rs3939286, which was associated to atopic asthma in the study by Gudbjartsson et al.1Gudbjartsson D.F. Bjornsdottir U.S. Halapi E. Helgadottir A. Sulem P. Jonsdottir G.M. et al.Sequence variants affecting eosinophil numbers associate with asthma and myocardial infarction.Nat Genet. 2009; 41: 342-347Crossref PubMed Scopus (642) Google Scholar Therefore this last marker was not genotyped because it is tagged by the marker rs928413. A family-based association study was performed in 137 family trios (father, mother, and asthmatic child) from northeastern Italy ascertained through an allergic asthmatic child. Details of the enrollment criteria were given elsewhere.5Venanzi S. Malerba G. Galavotti R. Lauciello M.C. Trabetti E. Zanoni G. et al.Linkage to atopy on chromosome 19 in north-eastern Italian families with allergic asthma.Clin Exp Allergy. 2001; 31: 1220-1224Crossref PubMed Scopus (34) Google Scholar Association analysis was conducted by using the transmission disequilibrium test (TDT) with the computer program PLINK.6Purcell S. Neale B. Todd-Brown K. Thomas L. Ferreira M.A.R. Bender D. et al.PLINK: a toolset for whole-genome association and population-based linkage analysis.Am J Hum Genet. 2007; 81: 559-575Abstract Full Text Full Text PDF PubMed Scopus (21543) Google Scholar, 7Purcell S. Package: PLINK (V1.07). Available at: http://pngu.mgh.harvard.edu/purcell/plink/. Accessed November 23, 2010.Google Scholar The following phenotypes were investigated: doctor-diagnosed asthma according to the Global Initiative for Asthma criteria,8Górski P. Global Initiative for Asthma 2002—what concerns occupational medicine.Int J Occup Med Environ Health. 2002; 15: 207-208PubMed Google Scholar increased total serum IgE levels (defined as a qualitative trait with serum concentrations >200 kU/L for adults and >90th percentile in an age-adjusted standard curve value for subjects aged <10 years5Venanzi S. Malerba G. Galavotti R. Lauciello M.C. Trabetti E. Zanoni G. et al.Linkage to atopy on chromosome 19 in north-eastern Italian families with allergic asthma.Clin Exp Allergy. 2001; 31: 1220-1224Crossref PubMed Scopus (34) Google Scholar), bronchial hyperresponsiveness (BHR) to methacholine, positive skin prick test (SPT) responses to common aeroallergens, positive SPT responses to house dust mites, and positive SPT responses to graminae. Table I reports the phenotype distribution in asthmatic/atopic children and in their parents.Table IPhenotype distribution in asthmatic/atopic children and their parentsSubjectsParentsChildrenPhenotypesNo.Prevalence (%)No.Prevalence (%)No.Prevalence (%)Asthma4094327214.3137100BHR+39136.626618.212581.6IgE+34758.523241.811592.2SPT+40859.827240.813697.8SPT+-GMN40841.227222.413678.7SPT+-HDM40837.327221.313669.1Clinical data were not available for all the participants.BHR+, Presence of bronchial hyperresponsiveness to methacholine; IgE+, increased total IgE levels; Parents, subjects without any parent in the family structure; Prevalence, number of subjects with the phenotype on the total number of subjects characterized for the phenotype; SPT+, positive SPT response; SPT+-GMN, positive SPT response to graminae; SPT+-HDM, positive SPT response to house dust mite; Subjects, total number of subjects phenotyped in the sample. Open table in a new tab Clinical data were not available for all the participants. BHR+, Presence of bronchial hyperresponsiveness to methacholine; IgE+, increased total IgE levels; Parents, subjects without any parent in the family structure; Prevalence, number of subjects with the phenotype on the total number of subjects characterized for the phenotype; SPT+, positive SPT response; SPT+-GMN, positive SPT response to graminae; SPT+-HDM, positive SPT response to house dust mite; Subjects, total number of subjects phenotyped in the sample. Genotype data were available for all subjects (parents and offspring), and therefore missing parental genotypes did not represent an issue in this study. The SNPs rs1342326 and rs928413 were in Hardy-Weinberg equilibrium and LD with each other (D′ = 0.95, R2 = 0.59). The observed minor allele frequency was 23% (minor allele, G; major allele, T) and 31% (minor allele, G; major allele, A) for rs1342326 and 928413, respectively. TDT analysis showed a preferential transmission of the rs928413 G allele to subjects with positive SPT responses (transmitted, 65; not transmitted, 43; P = .034) and in particular with positive SPT responses to graminae (transmitted, 54; not transmitted, 33; P = .024) but not with positive SPT responses to house dust mite (transmitted, 41; not transmitted, 33; P = .353). No significant association was observed between rs928413 and the other phenotypes or between rs1342326 and any of the phenotypes investigated. Even if the study had a limited dimension that might affect statistical power, we emphasize that the families were enriched for the genetic risk factor because they were in linkage with the 9p24 marker.3Malerba G, Trabetti E, Patuzzo C, Migliaccio C, Galavotti R, Lauciello MC, et al. A genome scan for allergic asthma and related phenotypes in an Italian population sample. Presented at: the 52nd Annual Meeting of the American Society of Human Genetics; October 15-19, 2002; Baltimore, Md.Google Scholar, 4Bouzigon E. Forabosco P. Koppelman G.H. Cookson W.O. Dizier M.H. Duffy D.L. et al.Meta-analysis of 20 genome-wide linkage studies evidenced new regions linked to asthma and atopy.Eur J Hum Genet. 2010; 18: 700-706Crossref PubMed Scopus (50) Google Scholar A 2-loci haplotype TDT was performed. Table II reports the haplotype frequencies observed. Haplotype rs1342326-rs928413/T-A was the most frequent haplotype observed (Table II). Table III shows the result of the haplotype association test. Haplotype rs1342326-rs928413/T-G was preferentially transmitted to children affected by asthma (P = .018), BHR to methacholine (P = .006), increased IgE levels (P = .022), or positive SPT responses (P = .015), particularly positive SPT responses to graminae (P = .006). The TDT performed on 2 loci confirms the association found in the rs928413 analysis and adds the information that the rs1342326-rs928413/T-G haplotype (frequency, 8.8%) is associated to allergic phenotypes. This haplotype can therefore be considered the at-risk haplotype, and it might contain the susceptibility allele for the phenotypes investigated. It is worth noting that haplotype analysis showed a significant association for 5 of the 6 phenotypes, and this is far superior to what would be expected by chance (P = .00002 when considering 5 associated phenotypes of the 6 phenotypes or P = .001 for 5 significant results of 18 tests). It is therefore reasonable to conclude that the excess of associations might indicate the presence of a true association.Table IIHaplotype frequencies reconstructed on chromosomes of parentsrs1342326-rs928413haplotypeFrequency expected under linkage equilibriumObserved frequenciesT-A0.5470.693G-G0.0660.211T-G0.2340.088G-A0.1530.008 Open table in a new tab Table IIIHaplotype association with allergic phenotypesPhenotypeHaplotypers1342326rs928413T/NTχ2 TestP valueAsthmaG-G48.5/43.50.172.678T-G28.5/12.55.625.018T-A46.5/62.52.867.091BHR+G-G33.5/35.50.058.810T-G22.5/7.57.500.006T-A35.5/47.51.735.188IgE+G-G28.5/31.50.150.699T-G22.5/9.55.281.022T-A35.5/45.51.235.267SPT+G-G47.5/37.50.405.525T-G26.5/11.55.921.015T-A43.5/63.50.017.897SPT+-GMNG-G38.5/33.50.347.556T-G22.5/7.57.500.006T-A33.5/52.54.198.040SPT+-HDMG-G29.5/30.50.172.678T-G17.5/8.53.115.077T-A33.5/41.50.85.356The TDT test was used in the families. Haplotypes showing a significant excess of transmission to affected children are shown in boldface.NT, Number of times the haplotype was not transmitted; T, number of times the haplotype was transmitted. Open table in a new tab The TDT test was used in the families. Haplotypes showing a significant excess of transmission to affected children are shown in boldface. NT, Number of times the haplotype was not transmitted; T, number of times the haplotype was transmitted. These results are therefore in agreement with the previous studies because Moffatt et al2Moffatt M.F. Gut I.G. Demenais F. Strachan D.P. Bouzigon E. Heath S. et al.A large-scale, consortium-based genomewide association study of asthma.N Engl J Med. 2010; 363: 1211-1221Crossref PubMed Scopus (1568) Google Scholar indicated association with the rs938413 allele G and Gudbjartsson et al1Gudbjartsson D.F. Bjornsdottir U.S. Halapi E. Helgadottir A. Sulem P. Jonsdottir G.M. et al.Sequence variants affecting eosinophil numbers associate with asthma and myocardial infarction.Nat Genet. 2009; 41: 342-347Crossref PubMed Scopus (642) Google Scholar with the rs3939286 allele A, which is tagged by the rs928413 allele G. It is noteworthy that we observe a significant association of rs928413 with positive SPT responses but not with asthma, and Gudbjartsson et al1Gudbjartsson D.F. Bjornsdottir U.S. Halapi E. Helgadottir A. Sulem P. Jonsdottir G.M. et al.Sequence variants affecting eosinophil numbers associate with asthma and myocardial infarction.Nat Genet. 2009; 41: 342-347Crossref PubMed Scopus (642) Google Scholar reported the association of rs3939286 with atopic asthma but not with nonatopic asthma. This could suggest that rs928413 (and rs3939286) could be mainly associated with the atopic component of the disease. The association to the most common aeroallergens was in particular because of positive SPT response to graminae, suggesting that rs928413 might be associated with the atopic component of seasonal epidemic asthma. We could not exclude that association with positive SPT responses is independent from asthmatic status because all the children were asthmatic. The 2 studied SNPs map in an intergenic region, and the closest gene is IL33; the 5′ side is located in an LD block containing the SNP rs928413 (http://www.hapmap.org, rel#24). No significant association was observed between genetic markers and total serum IgE levels by using the family-based association test statistic. We can speculate that SNP rs928413 might be in LD with variants affecting gene expression. For its characteristics and functions, IL33 represents an attractive candidate gene for the association with allergic asthma–related traits. The IL33 gene, a member of the IL-1 family, is an inducer of TH2 cytokine immunity and systemic inflammation through a complex including the membrane-bound ST2 protein. Binding of IL-33 to its receptor, which is present on mast cells, basophils, eosinophils, natural killer cells, and natural killer T cells, triggers the release of several proinflammatory mediators, induces systemic TH2-type inflammation in vivo, and contributes to allergen-induced airway inflammation and hyperresponsiveness.9Préfontaine D. Nadigel J. Chouiali F. Audusseau S. Semlali A. Chakir J. et al.Increased IL-33 expression by epithelial cells in bronchial asthma.J Allergy Clin Immunol. 2010; 125: 752-754Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar In conclusion, the results of this family-based association study in an Italian population extends the previously described asthma association with 9p24 markers to allergic childhood asthma traits, which is in agreement with the suggested function of IL33 “as an endogenous alarmin….to alert the immune system of tissue injury or infection.”10Moussion C. Ortega N. Girard J.P. The IL-1-like cytokine IL-33 is constitutively expressed in the nucleus of endothelial cells and epithelial cells in vivo: a novel ‘alarmin’?.PLoS One. 2008; 3: e3331Crossref PubMed Scopus (942) Google Scholar Future studies will be necessary to define the role of this region in the regulation of expression of the IL33 gene or to identify as yet unknown allergic asthma susceptibility genes.

Background Sporadic non-syndromic thoracic aortic aneurysms (SNSTAAs) are less well understood than familial non-syndromic or syndromic ones. The study aimed at defining the peculiar morphologic and molecular changes occurring in the media layer of SNSTAAs. Design This study was based on a single centre design. Methods Media layer samples taken from seven carefully selected SNSTAAs and seven reference patients (controls) were investigated via quantitative polymerase chain reaction, proteomics-bioinformatics, immunoblotting, quantitative histology, and immunohistochemistry/immunofluorescence. Results In SNSTAAs media, aortic smooth muscle cells numbers were halved due to an apoptotic process coupled with a negligible cell proliferation. Cystathionine γ-lyase was diffusely up-regulated. Surviving aortic smooth muscle cells exhibited diverging phenotypes: in inner- and outer-media contractile cells prevailed, having higher contents of smooth-muscle-α-actin holoprotein (45-kDa) and of caspase-3-cleaved smooth-muscle-α-actin 25-kDa fragments; in mid-media, aortic smooth muscle cells exhibited a synthetic/secretor phenotype, down-regulating vimentin, but up-regulating glial fibrillary acidic protein, trans-Golgi network 46 protein, Jagged1 (172-kDa) holoprotein, and Jagged1's receptor Notch1. Extracellular soluble Jagged1 (42-kDa) fragments accumulated. Conclusions In SNSTAAs, there is a relentless aortic smooth muscle cells attrition caused by the up-regulated cystathionine γ-lyase. In mid-media, synthetic/secretor aortic smooth muscle cells intensify Jagged1/NOTCH1 signalling in the attempt to counterbalance the weakened aortic wall, due to aortic smooth muscle cells net loss and mechanical stress. Synthetic/secretor aortic smooth muscle cells are apoptosis-prone, and the accruing thrombin-cleaved Jagged1 fragments counteract the otherwise useful effects of Jagged1/NOTCH1 signalling, thus hampering tissue homeostasis/remodelling, and aortic smooth muscle cells adhesion, differentiation, and migration.

BackgroundRenal artery stenosis (RAS) is associated with high cardiovascular mortality and significant clinical complications, including resistant hypertension and ischemic nephropathy. Despite availability of endovascular revascularization techniques, determining which patients should undergo revascularization and the timing of the procedure still are controversial. Several studies have reported a higher frequency of the DD genotype of the insertion/deletion (I/D) polymorphism of the angiotensin converting enzyme (ACE) gene in patients with RAS, and one study found higher mortality in patients with the DD genotype.Material and methodsWe retrospectively studied 100 patients with documented atherosclerotic RAS and evaluated long-term (median follow-up, 28 months) mortality, blood pressure control, and renal function in relation to the ACE genotype and two therapeutic strategies, that is, endovascular treatment with percutaneous renal transluminal angioplasty or stenting (ET group) versus conservative drug therapy (CT group).ResultsComparison between therapeutic groups showed a higher cumulative probability of survival (86.7% vs 67.1%), better blood pressure control (57.4% vs 29%), and slower decline in renal function (17.9% vs 48.4%) in the ET group. The DD genotype was strongly represented in our study patients (DD, 50%; II, 15.5%; I/D, 34.5%), but bore no relation to mortality, blood pressure control, decline in renal function, or rate of recurrent stenosis.ConclusionsConservative medical treatment of RAS, compared with endovascular treatment, is associated with higher mortality, poorer blood pressure control, and impaired renal function over the long term. Early endovascular treatment enables amelioration of this unfavorable evolution. The DD genotype does not predict clinical outcome of RAS.

A study of two DNA polymorphisms (i2 RsaI, E237G) in the gene for the beta subunit of the IgE high affinity receptor (FcepsilonRIbeta) was performed in 168 Italian families with atopic asthmatic children. The prevalence of the E237G allele in the Italian population was 4%, so this polymorphism was unsuitable for this study. The i2 RsaI polymorphism minor allele frequency was 44%, and it had a PIC value of 0.37. Linkage analysis indicated a significant allele sharing in affected sib pairs for bronchial hyper-responsiveness (BHR, p=0.048), but not for allergic asthma. These data indicate an association of bronchial hyper-responsiveness with the FcepsilonRIbeta gene.

Genome and chromosome screens reported DNA markers on chromosome 14 linked to allergic asthma or intermediate phenotypes in several populations.We sought to perform a linkage study on chromosome 14 and a further association study on candidate genes mapped in the region found to be linked to allergic asthma or intermediate phenotypes.The study consisted of a sample of 189 families (847 genotyped individuals) from a restricted geographic area in northeastern Italy. The subjects were characterized for the following phenotypes: allergic asthma, total serum IgE levels, skin prick test responses, and bronchial hyperresponsiveness (BHR) to methacholine. Genotyping was done with 14 DNA markers and 4 polymorphisms in the genes encoding alpha(1)-anti-trypsin and alpha(1)-antichymotrypsin (ACT).Multipoint analysis indicated a potential linkage of BHR with marker D14S617 (nonparametric linkage z score = 2.32, P =.01). Transmission disequilibrium of Thr -15Ala in the gene encoding ACT was observed with all the phenotypes investigated: allergic asthma, BHR, total IgE levels, or skin prick test responses (P =.041,.02,.0053, or.026, respectively).Chromosome 14 screening and transmission disequilibrium testing on the gene encoding ACT suggest that it or a closely located gene may be involved in susceptibility to allergic asthma in the Italian population.

The independent prognostic impact, as well as the possible causal role, of hyperhomocysteinemia (HHcy) in coronary artery disease (CAD) is controversial. No previous study specifically has addressed the relationship between HHcy and mortality after coronary artery bypass grafting (CABG) surgery. The aim of this study is to evaluate the prognostic impact of HHcy after CABG surgery.We prospectively followed 350 patients who underwent elective CABG between May 1996 and May 1999. At baseline, fasting total homocysteine (tHcy) levels were measured in all participants, and a post-methionine loading (PML) test was performed in 77.7% of them (n = 272). After a median follow-up of 58 months, 33 patients (9.4%) had died, 25 because of cardiovascular events. HHcy, defined by levels higher than the 90th percentile (25.2 micromol/L) of the population's distribution, was significantly associated to total and cardiovascular mortality (P = 0.018 [log-rank test 5.57]; P = 0.002 [log-rank test 9.76], respectively). The PML test had no prognostic value. After multiple adjustment for other univariate predictors by Cox regression, including statin therapy (the most powerful predictor in uni-/multivariate analyses), high-sensitivity C Reactive Protein (hs-CRP) levels, and all known major genetic (MTHFR 677C-->T polymorphism) and non-genetic (B-group vitamin status and renal function) tHcy determinants, HHcy remained an independent prognostic factor for mortality (HRs: 5.02, 95% CIs 1.88 to 13.42, P = 0.001).HHcy is an important prognostic marker after CABG, independent of modern drug therapy and biomarkers.

After allogeneic bone marrow transplantation (BMT) it is important to be able to distinguish between the host and donor origin of cells in order to monitor the engraftment process. However, identifying whether the hematopoietic stem cells are of donor or recipient origin may be a difficult task. DNA studies using Southern blotting techniques or the amplification by PCR of regions in the human genome with high polymorphic neutral sequence variation showing Mendelian inheritance as variable number of tandem repeats (VNTR) can detect the origin of host bone marrow after BMT. We have tried to apply these sensitive systems of detection to the early stages of BMT when small numbers of regenerated cells are available for analysis.We used in vitro polymerase chain reaction (PCR) amplification of three single-locus simple repetitive DNA sequences, all of which vary extensively in their repeat number among different individuals (VNTR D1S80, ApoB, and D17S5), to evaluate post transplant engraftment in six patients who showed no signs of peripheral blood engraftment at 2-3 weeks after transplant. We tested 2 patients with chronic myelogenous leukemia (CML), 2 with B-acute lymphoblastic leukemia (B-ALL), 1 with T-acute lymphoblastic leukemia (T-ALL), and 1 with aplastic anemia (SAA), all in prolonged aplasia following allogeneic bone marrow transplantation (BMT).In a sequential analysis protocol with the different loci, the donor was distinguishable from the recipient in all pairs with at least one of the three markers used. After 16 days (median 16.2; range 15 to 20 days) we found that complete chimerism was present in 5 patients: 4 of donor origin (= engraftment) and one of host origin (= rejection), this last case being one of mixed chimerism. In the 2 cases in which the presence (one complete and one partial) of host DNA was detected, rejection of the donor bone marrow followed, and in 1 patient a second BMT was necessary. The other 4 patients with complete chimerism of donor origin achieved hematological reconstitution and we documented complete engraftment of donor bone marrow a few months later.Utilizing PCR to document early post-transplant engraftment and chimerism in the first month after BMT has the advantage over Southern blotting of being more sensitive and requiring small amounts of sample. It may also be useful for guiding subsequent therapeutic decisions.

P-selectin is an adhesion molecule involved in the pathogenesis of inflammation, thrombosis, and oncogenesis. In this study of 51 polymorphisms in candidate genes for cardiovascular disease in 1561 individuals, we identified a new allelic variant of the SELP gene, g.18196_20704del, that determined the lack of genotyping for one polymorphism in one individual. It is a deletion of 2509 nucleotides which starts in intron 6 and ends in intron 8. Re-genotyping of 1023 apparent homozygotes indicated an overall allele frequency of 0.27%. The inclusion of this allelic variant in genetic association studies will avoid genotyping errors and marginally improve the sensitivity.

Acute coronary syndromes (ACS) and chronic stable angina represent extremes of the clinical spectrum of coronary artery disease (CAD). It is unknown whether genetic determinants affect the first clinical manifestation of CAD. We evaluated the role of the C(-260)T polymorphism in the promoter of the CD14-receptor gene, an important mediator of the inflammatory response to lipopolysaccharide.CD14 C(-260)T polymorphism was assessed in 100 patients with an acute presentation of CAD (group 1), 66 patients with stable presentation (group 2) and 88 healthy people (group 3); all patients were whites. In addition, baseline sCD14 plasma levels, and interleukin-6 production by circulating monocytes after in-vitro stimulation with lipopolysaccharide (1 ng/ml) were assessed. T/T homozygosis was more frequent in group 1 (36%, P < 0.001 versus others). Interleukin-6 production was higher in T/T homozygotes (median 4092.4; range 387-10 582 pg/ml) than in C/T heterozygotes (median 2442, range 40.5-9625 pg/ml, P < 0.001) and C/C homozygotes (median 3277.5; range 374.4-6250 pg/ml, P < 0.001). At multivariate analysis, T/T homozygosis and interleukin-6 production were independent predictors of acute presentation of CAD.The present study shows that genetic factors that influence the reactivity of inflammatory cells may play a role in determining the first clinical presentation of CAD.

Background Sporadic non-syndromic thoracic aortic aneurysms (SNSTAAs) are less well understood than familial non-syndromic or syndromic ones. Here, we focused on morphologic and molecular changes of the extracellular matrix of the tunica media of SNSTAAs. Design Single centre design. Methods Surgical media samples from seven SNSTAAs and seven controls underwent quantitative polymerase chain reaction, proteomics-bioinformatics, immunoblotting, histology and immunohistochemistry analysis. Results A down-regulation of Decorin mRNA with unchanged protein levels associated with a remarkable increase of collagen fibres. A reduced and distorted network of elastic fibres partnered with an attenuated expression of microfibril-associated glycoprotein1 despite the rise of MFAP2 gene-encoded mRNA levels. An increasingly proteolysed paxillin (55 kDa PXN), a focal adhesion protein, combined with an upregulated 62 kDa PXN holoprotein, without changes in amount and phosphorylation of focal adhesion kinase (pp125FAK). The upregulation of SPOCK2-encoded Testican2 proteoglycan and of ectodysplasin (EDA) protein was coupled with a down-regulation of EDA2 receptor (EDA2R). Conclusions Several tunica media extracellular matrix-related changes favour SNSTAA development. A steady level of decorin and a microfibril-associated glycoprotein1 protein shortage cause the assembly of structurally defective collagen and elastic fibres. Up-regulation of PXN holoproteins perturbs PXN/pp125FAK interaction and focal adhesion functioning. Testican2 up-regulation suppresses the membrane-type matrix metalloproteinase inhibiting activities of other SPOCK family members thus enhancing extracellular matrix proteolysis. Finally, the altered EDA•EDA2R signalling would impact on the remodelling of SNSTAA tunica media. Altogether, our results pave the way to a deeper molecular understanding of SNSTAAs necessary to identify their early diagnostic biochemical markers.

Variable number of tandem repeat (VNTR) DNA polymorphisms were studied in a random sample of 100 Italians. The following systems were used: YNH24/MspI, CMM101/Msp1, MLJ14/RsaI, EFD64.2/RsaI, EFD64.2/HinfI, JCZ3.1/HinfI and AW101/EcoRI. For each system, the allele size, number and frequency were determined. The number of alleles, for the different VNTRs, varied from 21 to 44, and the heterozygosity from 70 to 90%. All the VNTRs showed a modal distribution of fragment size and overrepresentation of the lower-molecular-weight DNA fragments. All of them exhibited greater variability than that reported for the North American population. These data will be useful for individual identification purposes and for calculating the probability of random inheritance of a particular allele in the Italian population. The results are discussed in relation with the possible mechanism of generation of new-length alleles.

Objective Patients with unstable angina (UA) and high C-reactive protein (CRP) have increased cardiovascular risk. Whether genetic factors such as the synonymous 1059G/C polymorphism within the exon 2 of the human CRP gene determine CRP levels and outcome is unclear. Methods In 105 consecutive patients with UA, we assessed the CRP 1059G/C polymorphism, CRP plasma levels and interleukin-6 production after in-vitro stimulation of whole blood with lipopolysaccharide (1 ng/ml). Coronary events during a 24-month follow-up were recorded. Results CRP levels (median, range) were significantly lower among C-allele carriers (2.3 mg/l, 0.5–26.9) than among GG homozygotes (5.9 mg/l, 0.8–72.12, P=0.009). Interleukin-6 production was lower in C-allele carriers (1645 pg/ml, 832.0–9522) than in GG homozygotes (3929 pg/ml, 670.8–10 582), (P=0.085). At follow-up, 1059C-allele carriers experienced fewer coronary events than 1059GG homozygotes (13 vs. 47%, P=0.021). At multivariable analysis, a CRP level >3 mg/l, but not the 1059G/C polymorphism, was an independent predictor of coronary events (odds ratio 10.04, 95% confidence interval 2.84–35.44, P=0.0002). Conclusion This study shows that the CRP synonymous 1059G/C polymorphism affects CRP levels. No independent association was, however, observed between this polymorphism and clinical outcome in UA.

Background Poor blood pressure control in renal artery disease patients after percutaneous renal angioplasty (PTRA), with or without stenting (PTRAS), may be due to pre-existing hypertension. Primary hyperaldosteronism is much more frequent than was previously suspected. We hypothesized that residual hypertension observed in some renal artery disease patients after technically successful endovascular treatment may be due to primary hyperaldosteronism. Methods Only patients free of significant residual artery stenosis were included in the study. Aldosterone and renin were measured in 52 renal artery disease patients (8 with fibrodysplastic and 44 with atherosclerotic lesions), in whom successful PTRA/PTRAS had been performed previously. An aldosterone-to-renin ratio ≥ 23 pg/ml per pg/ml was considered as the cut-off value for performing tests to confirm the diagnosis of primary hyperaldosteronism. Results Residual hypertension (blood pressure ≥ 160/90 mmHg) was observed in 24/52 patients (46%) after revascularization. A raised aldosterone-to-renin ratio was found in nine subjects (17.3%), eight of whom had poor blood pressure control (33% of patients with residual hypertension). A diagnosis of primary hyperaldosteronism was confirmed in seven patients (four atherosclerotic, three fibrodysplastic). All fibrodysplastic subjects with unresponsive blood pressure after PTRA were affected by primary hyperaldosteronism. Primary hyperaldosteronism was confirmed in 9% (4/44) of the atherosclerotic patients (19% of subjects with residual hypertension). No specific clinical features were associated with the subsequent blood pressure control. Conclusions Primary hyperaldosteronism is a frequently neglected cause of residual hypertension despite technically successful endovascular treatment of renal artery disease.

GCK gene analysis in an Italian MODY patient revealed a novel synonymous substitution in exon 4 (c.459T>G; p.Pro153Pro) resulting in an aberrant transcript lacking the last eight codons of the same exon. Our findings emphazise the importance of not underestimating synonymous variations when screening for disease-causing mutations.

Several lines of evidence suggest that RBFOX1 is a key regulator of transcriptional and splicing programs in neural cells during development, and that it is expressed in a neuronal module enriched for known autism susceptibility genes. We have investigated its expression by semiquantitative RT-PCR in accessible nonbrain resources in eighteen autism spectrum disorder sib-pairs belonging to the Italian Autism Network cohort. RBFOX1 gene expression was detected in lymphoblastoid cell lines but not in lymphocytes. No significant differences between autism spectrum disorders and non-affected brothers were found. We were not able to replicate in lymphoblastoid cell lines the previously reported RBFOX1 gene downregulation in autism, even if a trend was observed. This might be due to less pronounced transcription level differences in RBFOX1 gene expression in lymphoblastoid cell lines than in brain samples.

Tumour necrosis factor (TNF) is a proinflammatory cytokine that increases human airway tissue responsiveness and is considered a candidate gene for asthma. Two common polymorphisms (LTalphaNcoI and TNFalpha-308) in the TNF gene complex were studied in 600 subjects from 131 Italian families with atopic asthmatic children. Skin prick test (SPT), total IgE levels, atopy (defined as increased IgE levels or SPT positivity or both), bronchial hyperresponsiveness, and clinical asthma were investigated. The observed distribution of the identical by descent alleles at the LTalphaNcoI locus was different from expected for SPT and atopy (p=0.015). The LTalphaNcoI genotype distribution for increased IgE levels was different between males and females (p=0.0011), and an association of the 2.2 genotype with increased IgE levels was observed in females (p=0.0032). The results indicate that the LTalpha gene, or a closely linked locus, is associated with atopy, and suggest a sex difference in the effect of the gene.

The CD14 receptor is an important mediator of inflammatory reactions, and its expression is under genetic control. The allelic variant of the C260T polymorphism located in the promoter region of the CD14 gene is associated with receptor expression and ischemic risk. To date, most studies assessing the functional implications of the C260T polymorphism have been performed under proinflammatory conditions (e.g., acute coronary syndromes), and whether gene sequence variations of the CD14 receptor have any functional effect on systemic inflammation in patients in a stable phase of their atherosclerotic disease process is unknown. Eighty-two patients with stable coronary artery disease were studied. High-sensitivity C-reactive protein (hs-CRP) was used as a measurement of systemic inflammation. The genotype distribution of the C260T polymorphism of the CD14 gene was as follows: CC in 18 of 82 patients (22%), TC in 48 of 82 patients (58.5%), and TT in 16 of 82 patients (19.5%). TT subjects had increased hs-CRP levels compared with carriers of the C allele (p = 0.04). A higher percentage of T allele homozygotes had hs-CRP levels >0.3 mg/dl (p = 0.01). Homozygosis status of the T allele was independently associated with hs-CRP levels >0.3 mg/dl (p = 0.004). In conclusion, these observations may support the findings in large-scale studies that T homozygotes of this functional polymorphism are at increased ischemic risk. The CD14 receptor is an important mediator of inflammatory reactions, and its expression is under genetic control. The allelic variant of the C260T polymorphism located in the promoter region of the CD14 gene is associated with receptor expression and ischemic risk. To date, most studies assessing the functional implications of the C260T polymorphism have been performed under proinflammatory conditions (e.g., acute coronary syndromes), and whether gene sequence variations of the CD14 receptor have any functional effect on systemic inflammation in patients in a stable phase of their atherosclerotic disease process is unknown. Eighty-two patients with stable coronary artery disease were studied. High-sensitivity C-reactive protein (hs-CRP) was used as a measurement of systemic inflammation. The genotype distribution of the C260T polymorphism of the CD14 gene was as follows: CC in 18 of 82 patients (22%), TC in 48 of 82 patients (58.5%), and TT in 16 of 82 patients (19.5%). TT subjects had increased hs-CRP levels compared with carriers of the C allele (p = 0.04). A higher percentage of T allele homozygotes had hs-CRP levels >0.3 mg/dl (p = 0.01). Homozygosis status of the T allele was independently associated with hs-CRP levels >0.3 mg/dl (p = 0.004). In conclusion, these observations may support the findings in large-scale studies that T homozygotes of this functional polymorphism are at increased ischemic risk.

A substantial genetic component accounts for Autism Spectrum Disorders (ASD) aetiology, with some rare and common genetic risk factors recently identified. Large collections of DNAs from thoroughly characterized ASD families are an essential step to confirm genetic risk factors, identify new variants and investigate genotype-phenotype correlations. The Italian Autism Network aimed at constituting a clinical database and a biorepository of samples derived from ASD subjects and first-degree relatives extensively and consistently characterized by child psychiatry centers in Italy. The study was approved by the ethical committee of the University of Verona, the coordinating site, and by the local ethical committees of each recruiting site. Certified staff was specifically trained at each site for the overall study conduct, for clinical protocol administration and handling of biological material. A centralized database was developed to collect clinical assessment and medical records from each recruiting site. Children were eligible for recruitment based on the following inclusion criteria: age 4–18 years, at least one parent or legal guardian giving voluntary written consent, meeting DSM-IV criteria for Autistic Disorder or Asperger’s Disorder or Pervasive Developmental Disorder NOS. Affected individuals were assessed by full psychiatric, neurological and physical examination, evaluation with ADI-R and ADOS scales, cognitive assessment with Wechsler Intelligence Scale for Children or Preschool and Primary, Leiter International Performance Scale or Griffiths Mental Developmental Scale. Additional evaluations included language assessment, the Krug Asperger’s Disorder Index, and instrumental examination such as EEG and structural MRI. DNA, RNA and plasma were collected from eligible individuals and relatives. A central laboratory was established to host the biorepository, perform DNA and RNA extraction and lymphocytes immortalisation. The study has led to an extensive collection of biological samples associated with standardised clinical assessments from a network of expert clinicians and psychologists. Eighteen sites have received ADI/ADOS training, thirteen of which have been actively recruiting. The clinical database currently includes information on 812 individuals from 249 families, and the biorepository has samples for 98% of the subjects. This effort has generated a highly valuable resource for conducting clinical and genetic research of ASD, amenable to further expansion.

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An increased frequency of the angiotensin converting enzyme (ACE) D variant has recently been reported in patients with atheromatous renal artery disease (RAD), whereas controversy exists on the risk of coronary artery disease (CAD) associated with this polymorphism. In spite of the frequent coexistence of RAD and CAD, no studies have specifically compared ACE insertion/deletion (I/D) polymorphism in patients with coronary versus renal artery disease or defined the risk of each disease associated with the D variant.We designed a large case-control study of subjects for whom objective renal or coronary angiographic documentation was available.We analysed ACE I/D genotype and clinical-biochemical data of a total of 942 subjects (123 patients with and 137 without angiographic evidence of RAD, 420 patients with and 262 without angiographic evidence of CAD). Renal (with and without RAD) and coronary patients were similar for most conventional risk factors. There was no difference in DD frequency between CAD and CAD-free patients (38.1 versus 33.6%, NS) and DD homozygosity was not associated with CAD risk (OR = 1.27, 95% CI = 0.82-1.98). In contrast, the DD genotype was more frequent in RAD than in RAD-free patients (54.5 versus 39.4, P < 0.05) and was associated with a 2.25-fold increased risk of RAD in both the univariate and multivariate logistic regression models. Additional predictors of RAD were age and creatinine. In RAD/RAD-free patients with angiographically documented CAD, DD homozygosity was confirmed to be preferentially associated with RAD (P < 0.05).ACE D variant is preferentially associated with atherosclerotic renal rather than with coronary disease, despite similar exposure to atherogenic noxae. DD homozygosity confers a 2.25-fold increased risk of RAD.

The literature contains conflicting reports on the association of common variants of the interleukin (IL)-4 receptor alpha (IL4RA) gene with atopic asthma. The purpose of the present study was to investigate the linkage and association of several gene polymorphisms with atopic asthma in a large series of well-characterized individuals. Analysis of five polymorphisms (I50V, E375A, C406R, S478P and Q551R) of the IL4RA gene was performed in 823 individuals from 182 families with atopic asthmatic children from north-east Italy. The subjects were tested for clinical asthma, total serum IgE level, skin prick test positivity to common aeroallergens, and bronchial hyperresponsiveness to methacholine. The frequency of the polymorphisms was similar to that reported for other populations. The 375, 406, 478 and 551 polymorphisms were in linkage disequilibrium, as previously reported. No linkage or transmission disequilibrium was observed in the families between any mutation and any of the phenotypes investigated. No multipoint haplotype was associated with any phenotype. In conclusion, the IL4RA gene does not seem to play an important role in genetic predisposition to atopic asthma in the population tested.

This study aims to investigate whether renal and cardiovascular phenotypes in Italian patients with type 2 diabetes (T2D) could be influenced by a number of disease risk SNPs recently found in genome-wide association studies (GWAS). In 1591 Italian subjects with T2D: (1) 47 SNPs associated to kidney function and/or chronic kidney disease (CKD) and 49 SNPs associated to cardiovascular disease (CVD) risk were genotyped; (2) urinary albumin/creatinine (A/C) ratio, glomerular filtration rate (eGFR) and lipid profile were assessed; (3) a standard electrocardiogram was performed; (4) two genotype risk scores (GRS) were computed (a renal GRS calculated selecting 39 SNPs associated with intermediate traits of kidney damage and a cardiovascular GRS determined selecting 42 SNPs associated to CVD risk phenotypes). After correction for multiple comparisons, the renal GRS was not associated to A/C ratio (p = 0.33), but it was significantly related to decreased eGFR (p = 0.005). No association between the cardiovascular GRS and electrocardiogram was detected. Thus, in Italian patients with T2D a renal GRS might predict the decline in glomerular function, suggesting that the clock of diabetes associated CKD starts ticking long before hyperglycemia. Our data support the feasibility of gene-based prediction of complications in people with T2D.

The androgenetic origin of hydatidiform moles, due to a monospermic or dispermic mechanism, has been reported, and a possible pathogenetic relation with blighted ova suggested. To evaluate the origin of hydatidiform moles and their genetic relationship with blighted ova we investigated a series of samples, utilizing several hypervariable DNA polymorphisms by Southern blotting or PCR. Seven complete or partial hydatidiform mole and 49 blighted ovum cases were investigated. The results confirm the androgenetic origin of complete hydatidiform moles, which were always due in our sample to a monospermic mechanism. Our data exclude a relationship between hydatidiform moles and blighted ova, as in the latter a mixed paternal and maternal DNA contribution was always shown. A high incidence of chromosomal abnormalities in blighted ova was also found.

A multilocus genotype survey of 190 to 352 chromosomes was performed in Italians. Genomic DNA of five tandem repeat loci was amplified in vitro with the polymerase chain reaction. Variable number of tandem repeat (VNTR) or short tandem repeat loci investigated were: D1S80; AP0B, located in the 3' flanking region of the apolipoprotein B gene; D17S5; F8VWF, located in intron 40 of the von Willebrand factor gene; and D6S89. The repeat motif was from 2 to 70 bp. The polymerase chain reaction product size was from 100 to 1070 bp. Relative allele frequencies exhibited bimodal or complex distributions. The number of alleles detected in the sample varied from 10 for F8VWF to 20 for D1S80. The observed heterozygote frequencies of the loci ranged from 0.75 for D17S5 and F8VWF to 0.83 for D1S80, and were in accord with expected frequencies. No mutations were detected in a total of 566 meioses for the five loci studied. The most frequent genotype for all five loci combined has a frequency of 4.1 x 10(-6). In 90 parental determination cases, D1S80, AP0B, D17S5 and F8VWF gave conclusive information in 39/45 exclusions and in 21/45 attributions.

Genes that influence the renin-angiotensin system have been investigated in recent years as potential etiologic candidates of cardiovascular and renal diseases. In atheromatous renal artery stenosis (RAS), a condition characterized by persistent activation of the renin-angiotensin system, the study of these genes may be of particular relevance. We evaluated angiotensin-converting enzyme (ACE) insertion/deletion, angiotensinogen (AGT) M235T, and angiotensin II receptor (ATR) A1166C polymorphisms in relation to the occurrence of RAS. We studied 58 patients with angiographically documented RAS; 102 normotensive subjects with normal coronary arteries and no history or clinical or instrumental evidence of atherosclerosis in other vascular districts were considered the control group. Patients had a significantly higher D allele frequency (0.70 versus 0.55; chi(2) 6.88, P=0.01; odds ratio [OR] 1. 9, 95% CI 1.17 to 3.07) than did the control population; 48.3% of patients were homozygous for DD (chi(2) 6.62, P<0.05; OR 2.04, 95% CI 1.05 to 3.95); and only 8.6% carried the II genotype (OR 0.34, 95% CI 0.19 to 1.47). No significant association was found for AGT M235T and ATR A1166C. Our results suggest a predisposing role for ACE genetic polymorphism in the development and progression of atheromatous RAS.

Several variable number of tandem repeat (VNTR) DNA polymorphisms detecting different loci (YNH24/MspI or TaqI, CMM101/MspI or MLJ14/MspI, EFD64.2/RsaI or HinfI, YNZ22/TaqI, AW101/EcoRI, EKMDA2.1/PvuII and 3'-HVR/PvuII) were used in the analysis of 27 cases of disputed paternity in the Italian population. Fourteen exclusions and 17 attributions were performed. The results were compared with those obtained with immunohematologic analyses. Four exclusions and 2 attributions were made possible only by the combined use of several DNA polymorphisms, as the analyses of red-blood-cell antigens and isoenzymes, serum proteins and HLA group determinants were inconclusive. With the DNA test, 10 exclusions and 15 attributions were confirmed, with increased overall probability. In conclusion, VNTR polymorphisms were more informative, accurate and sensitive than the immunohematologic tests. Therefore, DNA analysis is the method of choice for testing genetic relationships.

We tested the hypothesis that common genetic variability of beta-cell genes responsible for monogenic diabetes may affect beta cell function in type 2 diabetes mellitus (T2DM). We studied 794 drug- naïve GAD-negative patients with newly diagnosed T2DM (age: median=59 years; I.Q. range: 52-66; body mass index: 29.3 kg/m2; 26.6-32.9). Beta-cell function was assessed by state-of-art mathematical modeling of glucose/C-peptide curves during a 240’-300’ frequently sampled oral glucose tolerance test, to provide the beta-cell responses to the rate of increase in glucose concentration (derivative control: DC) and to glucose concentration (proportional control: PC). Forty-two single nucleotide polymorphism (SNPs), selected to cover over 90% of common genetic variability, were genotyped in nine monogenic diabetes genes: HNF4A, GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, KCNJ11 and ABCC8. Allelic variants of four SNPs (rs1303722 and rs882019 of GCK, rs7310409 of HNF1A and rs5219 of KCNJ11) were significantly associated with DC of beta-cell secretion (all P < 0.036). Allelic variants of four other SNPs (rs2868094 and rs6031544 of HNF4A, and rs1801262 and rs12053195 of NEUROD1) were associated with PC of beta-cell secretion (P < 0.02). In multivariate models, GCK, HNF1A and KCNJ11 SNPs explained 2.5% of the DC variability of beta-cell secretion, whereas HNF4A and NEUROD1 SNPs explained 3.6% of the PC variability of beta-cell secretion. We conclude that common variability of monogenic diabetes genes is significantly associated with an impaired beta-cell function in patients with newly diagnosed T2DM; thereby, these genes might be targeted by specific treatments in T2DM.

Abstract Background To establish whether the frequent finding of a moderate–intermediate increase in plasma total homocysteine (tHcy) causes coronary artery disease (CAD), the authors evaluated the number of coexisting major traditional risk factors, as well as the major tHcy determinants, in patients with the same degree of CAD but different tHcy levels. Materials and methods The authors studied 180 patients with CAD, who were divided into three groups according to tHcy levels: 60 patients with normal tHcy, 60 patients with moderate (15–30 µmol L −1 ) and 60 patients with intermediate hyperhomocysteinaemia (30–100 µmol L −1 ). The patient groups were matched for gender, age and number of affected coronary vessels. All patients were checked for the presence of traditional risk factors for CAD (i.e. hypertension, diabetes, hyperlipidaemia, smoking habit, familial history, obesity), as well as determinants of tHcy levels. The population was subdivided into those having, or not, a substantial burden of traditional risk factors (i.e. &lt; 4 and ≥ 4, respectively). Results There was a significant trend towards a reduced number of subjects within the group with ≥ 4 risk factors across increasing tHcy levels (51·7%, 37·8%, 26%, for normal, moderate, intermediate tHcy, respectively, χ 2 for linear‐trend = 0·006). Folate and vitamin B12 concentrations, estimated glomerular filtration rate (GFR), MTHFR 677C &gt; T polymorphism were the major determinants of tHcy in this population. Conclusions In patients with the same degree of CAD, those with hyperhomocysteinaemia had a reduced burden of traditional risk factors as compared with those with normal tHcy levels. Hyperhomocysteinaemia was significantly associated with an emerging non‐traditional risk factor such as lower GFR.

Abstract While the genetics of autism spectrum disorders (ASD) has been intensively studied, resulting in the identification of over 100 putative risk genes, the epigenetics of ASD has received less attention, and results have been inconsistent across studies. We aimed to investigate the contribution of DNA methylation (DNAm) to the risk of ASD and identify candidate biomarkers arising from the interaction of epigenetic mechanisms with genotype, gene expression, and cellular proportions. We performed DNAm differential analysis using whole blood samples from 75 discordant sibling pairs of the Italian Autism Network collection and estimated their cellular composition. We studied the correlation between DNAm and gene expression accounting for the potential effects of different genotypes on DNAm. We showed that the proportion of NK cells was significantly reduced in ASD siblings suggesting an imbalance in their immune system. We identified differentially methylated regions (DMRs) involved in neurogenesis and synaptic organization. Among candidate loci for ASD, we detected a DMR mapping to CLEC11A (neighboring SHANK1 ) where DNAm and gene expression were significantly and negatively correlated, independently from genotype effects. As reported in previous studies, we confirmed the involvement of immune functions in the pathophysiology of ASD. Notwithstanding the complexity of the disorder, suitable biomarkers such as CLEC11A and its neighbor SHANK1 can be discovered using integrative analyses even with peripheral tissues.