Rosario Liguori

miR-519d overexpression is associated with human obesity.

Obesity is a consequence of imbalance of food intake and energy expenditure that results in storage of energy as fat, primarily in adipose tissue. MicroRNAs are non‐coding RNAs that regulate gene expression in metabolic pathways and they are also involved in fat‐cell development. The aim of this study was to evaluate whether microRNA dysfunction contributes to obesity. We analyzed, by microarray, the expression profile of 1,458 microRNAs in subcutaneous adipose tissue (SAT) from nondiabetic severely obese (n = 20) and nonobese adults (n = 8). Among 42 differently expressed microRNAs, we confirmed by reverse‐transcription PCR (RT‐PCR) that miR‐519d was overexpressed whereas the protein levels of peroxisome proliferator‐activated receptor‐α (PPARA) (a predicted miR 519d target) were lower, at western analysis, in severely obese vs. nonobese subjects. We also show that miR‐519d specifically and dose‐dependently suppressed translation of the PPARA protein, and increased lipid accumulation during preadipocyte differentiation. Because PPARA plays a central role in fatty acid homeostasis, and in the transcriptional regulation of genes that are necessary for maintenance of the redox balance during the oxidative catabolism of fatty acids, we suggest that PPARA loss and miR‐519d overexpression could be associated with metabolic imbalance and subsequent adipocyte hypertrophy in SAT during obesity.

Multiple Sclerosis and Hepatitis C Virus Infection Are Associated with Single Nucleotide Polymorphisms in Interferon Pathway Genes

We have studied 35 single nucleotide polymorphisms (SNPs) in the interferon (IFN) pathway to determine their contribution to multiple sclerosis (MS) and hepatitis C virus (HCV) infection. A total of 182 patients with MS, 103 patients with chronic hepatitis C, and 118 control subjects were enrolled in the study. Of the 35 SNPs studied, 3 were in IFN-alpha receptor (IFNAR-1), 10 in IFN-alpha/beta receptor (IFNAR-2), 9 in Stat1, 5 in Stat2, and 8 in IFN regulatory factor-1 (IRF-1). Compared to controls, Stat1 gene polymorphisms were significantly more frequent in MS patients (rs# 2066802 OR = 7.46, 95% CI = 2.22-25.10; rs# 1547550 OR = 1.69, 95% CI = 1.01-2.81) and in HCV patients (rs# 2066802 OR = 5.95, 95% CI = 1.55-22.81; rs# 1547550 OR = 2.30, 95% CI = 1.24-4.24). Also one IRF-1 gene SNP was associated with MS (rs# 2070721 OR = 2.05, 95% CI = 1.03-4.09), and four IRF-1 gene SNPs were associated with HCV infection (rs# 2070721 OR = 2.59, 95% CI = 1.23-5.43; rs# 2070723 OR = 4.8, 95% CI = 1.26-18.20; rs# 2070728 OR = 9.81, 95% CI = 1.21-79.4; rs# 2070729 OR = 3.6, 95% CI = 1.23-10.48; rs# 839 OR = 4.67, 95%CI = 1.29-16.87). Characteristic nucleotide combinations on single chromosomes (haplotype) generated block structures, including SNPs, that differed between patients and controls. Using a permutation test to detect differences in haplotype distribution between groups, the CCATTGA and the CCGAA haplotypes in the IRF-1 gene were more frequent in MS (p = 0.03) and in HCV patients (p = 0.001) than in controls. In conclusion, our data show that genetic variants in the IRF-1 and Stat1 genes of the IFN pathway are associated with MS and HCV infection.

Haplogroup T Is an Obesity Risk Factor: Mitochondrial DNA Haplotyping in a Morbid Obese Population from Southern Italy

Mitochondrial DNA (mtDNA) haplogroups have been associated with the expression of mitochondrial-related diseases and with metabolic alterations, but their role has not yet been investigated in morbid obese Caucasian subjects. Therefore, we investigated the association between mitochondrial haplogroups and morbid obesity in patients from southern Italy. The mtDNA D-loop of morbid obese patients (n = 500; BMI > 40 kg/m2) and controls (n = 216; BMI < 25 kg/m2) was sequenced to determine the mtDNA haplogroups. The T and J haplogroup frequencies were higher and lower, respectively, in obese subjects than in controls. Women bearing haplogroup T or J had twice or half the risk of obesity. Binomial logistic regression analysis showed that haplogroup T and systolic blood pressure are risk factors for a high degree of morbid obesity, namely, BMI > 45 kg/m2 and in fact together account for 8% of the BMI. In conclusion, our finding that haplogroup T increases the risk of obesity by about two-fold, suggests that, besides nuclear genome variations and environmental factors, the T haplogroup plays a role in morbid obesity in our study population from southern Italy.

Catechol-O-methyltransferase (COMT) gene polymorphisms as risk factor in temporomandibular disorders patients from Southern Italy.

Objectives:To evaluate the role of COMT gene variants as potential risk factors in a group of patients affected with chronic temporomandibular disorder (TMD) pain. Methods:We sequenced COMT gene in 182 Italian subjects (50 affected by TMD and 132 controls). The study population consisted of patients affected by myogenous and/or arthrogenous pain (RDC/TMD: Ia, Ib, IIIa, IIIb diagnostic categories). Results:We detected 40 single nucleotide polymorphisms (SNPs) variants (18 novel). Three SNPs, all located in the promoter regions, were more frequently present in cases than in controls (rs 4646310 P=0.018, rs165656 P=0.001, rs 165722 P=0.007). After the False Discovery Rate (FDR) correction rs165656 remained significantly associated with TMD (P=0.049). In addition, the rs 4646310 (AG vs GG, P=0.015) and rs 165656 (GG vs CC, P=0.001) were at binary logistic regression analysis independently associated with TMD, conferring a risk disease of 2.6 (CI= 1.2-5.6) and of 5.3 (CI= 2.0-13.7) respectively. Discussion:Our data extend the number of SNPs present in the promoter region that could play a regulatory role in COMT gene and suggest that the genetic polymorphisms rs 165656 and rs 4646310 exert a role in TMD susceptibility.

Warfarin Anticoagulant Therapy: A Southern Italy Pharmacogenetics-Based Dosing Model

Background and Aim Warfarin is the most frequently prescribed anticoagulant worldwide. However, warfarin therapy is associated with a high risk of bleeding and thromboembolic events because of a large interindividual dose-response variability. We investigated the effect of genetic and non genetic factors on warfarin dosage in a South Italian population in the attempt to setup an algorithm easily applicable in the clinical practice. Materials and Methods A total of 266 patients from Southern Italy affected by cardiovascular diseases were enrolled and their clinical and anamnestic data recorded. All patients were genotyped for CYP2C9*2,*3, CYP4F2*3, VKORC1 -1639 G>A by the TaqMan assay and for variants VKORC1 1173 C>T and VKORC1 3730 G>A by denaturing high performance liquid chromatography and direct sequencing. The effect of genetic and not genetic factors on warfarin dose variability was tested by multiple linear regression analysis, and an algorithm based on our data was established and then validated by the Jackknife procedure. Results Warfarin dose variability was influenced, in decreasing order, by VKORC1-1639 G>A (29.7%), CYP2C9*3 (11.8%), age (8.5%), CYP2C9*2 (3.5%), gender (2.0%) and lastly CYP4F2*3 (1.7%); VKORC1 1173 C>T and VKORC1 3730 G>A exerted a slight effect (<1% each). Taken together, these factors accounted for 58.4% of the warfarin dose variability in our population. Data obtained with our algorithm significantly correlated with those predicted by the two online algorithms: Warfarin dosing and Pharmgkb (p<0.001; R2 = 0.805 and p<0.001; R2 = 0.773, respectively). Conclusions Our algorithm, which is based on six polymorphisms, age and gender, is user-friendly and its application in clinical practice could improve the personalized management of patients undergoing warfarin therapy.

Mitochondrial diabetes in children: seek and you will find it.

Maternally Inherited Diabetes and Deafness (MIDD) is a rare form of diabetes due to defects in mitochondrial DNA (mtDNA). 3243 A>G is the mutation most frequently associated with this condition, but other mtDNA variants have been linked with a diabetic phenotype suggestive of MIDD. From 1989 to 2009, we clinically diagnosed mitochondrial diabetes in 11 diabetic children. Diagnosis was based on the presence of one or more of the following criteria: 1) maculopathy; 2) hearing impairment; 3) maternal heritability of diabetes/impaired fasting glucose and/or hearing impairment and/or maculopathy in three consecutive generations (or in two generations if 2 or 3 members of a family were affected). We sequenced the mtDNA in the 11 probands, in their mothers and in 80 controls. We identified 33 diabetes-suspected mutations, 1/33 was 3243A>G. Most patients (91%) and their mothers had mutations in complex I and/or IV of the respiratory chain. We measured the activity of these two enzymes and found that they were less active in mutated patients and their mothers than in the healthy control pool. The prevalence of hearing loss (36% vs 75–98%) and macular dystrophy (54% vs 86%) was lower in our mitochondrial diabetic adolescents than reported in adults. Moreover, we found a hitherto unknown association between mitochondrial diabetes and celiac disease. In conclusion, mitochondrial diabetes should be considered a complex syndrome with several phenotypic variants. Moreover, deafness is not an essential component of the disease in children. The whole mtDNA should be screened because the 3243A>G variant is not as frequent in children as in adults. In fact, 91% of our patients were mutated in the complex I and/or IV genes. The enzymatic assay may be a useful tool with which to confirm the pathogenic significance of detected variants.

The FTO gene polymorphism (rs9939609) is associated with metabolic syndrome in morbidly obese subjects from southern Italy.

Gene variants in MC4R, SIRT1 and FTO are associated with severe obesity and metabolic impairment in Caucasians. We investigated whether common variants in these genes are associated with metabolic syndrome (MetS) in a large group of morbidly obese young adults from southern Italy. One thousand morbidly obese subjects (62% women, mean body mass index 46.5 kg/m(2), mean age 32.6 years) whose families had lived in southern Italy for at least 2 generations were recruited. Single-nucleotide polymorphisms (SNPs) rs12970134, rs477181, rs502933 (MC4R locus), rs3818292, rs7069102, rs730821, rs2273773, rs12413112 (SIRT1 locus) and rs1421085, rs9939609, 9930506, 1121980 (FTO locus) were genotyped by Taqman assay; blood parameters were assayed by routine methods; the Fat Mass, Fat Free Mass, Respiratory Quotient, Basal Metabolic Rate (BMR) and waist circumference were also determined. Binomial logistic regression showed that the TA heterozygous genotype of SNP rs9939609 in the FTO gene was associated with the presence of MetS in our population [OR (95% CI): 2.53 (1.16-5.55)]. Furthermore, the FTO rs9939609 genotype accounted for 21.3% of the MetS phenotype together with total cholesterol, BMR and age. Our results extend the knowledge on genotype susceptibility for MetS in relation to a specific geographical area of residence.

Nonsynonimous mutation of catechol-O-methyl-transferase (COMT) gene in a patient with temporomandibular disorder.

We report a case of temporomandibular disorder patient with disc displacement without reduction, myofascial pain, limited opening and a novel, never described, nonsynonimous mutation of catechol-O-methyl-transferase (COMT) gene. COMT is one of the enzymes that metabolizes catecholamines, thereby acting as a key modulator of dopaminergic and adrenergic/noradrenergic neurotransmissions, which play a key role in pain modulation. This novel mutation, p.R58S, changed a codon (58 from arginine to serine) in the COMT protein. The introduction of a serine residue in a highly organised secondary structure, in critical regions of the protein, results in a structural alteration. Therefore, we speculate an influence of the mutation on the high pain sensitivity of the patient.