What Is in the Field for Genetics and Epigenetics of Diabetic Neuropathy: The Role of MicroRNAs.

Despite the high prevalence of diabetic neuropathy, its early start, and its impact on quality of life and mortality, unresolved clinical issues persist in the field regarding its screening implementation, the understanding of its mechanisms, and the search for valid biomarkers, as well as disease-modifying treatment. Genetics may address these needs by providing genetic biomarkers of susceptibility, giving insights into pathogenesis, and shedding light on how to select possible responders to treatment. After a brief summary of recent studies on the genetics of diabetic neuropathy, the current review focused mainly on microRNAs (miRNAs), including the authors' results in this field. It summarized the findings of animal and human studies that associate miRNAs with diabetic neuropathy and explored the possible pathogenetic meanings of these associations, in particular regarding miR-128a, miR-155a, and miR-499a, as well as their application for diabetic neuropathy screening. Moreover, from a genetic perspective, it examined new findings of polymorphisms of miRNA genes in diabetic neuropathy. It considered in more depth the pathogenetic implications for diabetic neuropathy of the polymorphism of MIR499A and the related changes in the downstream action of miR-499a, showing how epigenetic and genetic studies may provide insight into pathogenetic mechanisms like mitochondrial dysfunction. Finally, the concept and the data of genotype-phenotype association for polymorphism of miRNA genes were described. In conclusion, although at a very preliminary stage, the findings linking the genetics and epigenetics of miRNAs might contribute to the identification of exploratory risk biomarkers, a comprehensive definition of susceptibility to specific pathogenetic mechanisms, and the development of mechanism-based treatment of diabetic neuropathy, thus addressing the goals of genetic studies.

TNFAIP3 Gene Polymorphisms in Three Common Autoimmune Diseases: Systemic Lupus Erythematosus, Rheumatoid Arthritis, and Primary Sjogren Syndrome-Association with Disease Susceptibility and Clinical Phenotypes in Italian Patients.

Autoimmune diseases (AIDs) are complex diseases characterized by persistent or recurrent inflammation, alteration of immune response, and production of specific autoantibodies. It is known that different AIDs share several susceptibility genetic loci. Tumor necrosis factor alpha inducible protein 3 (TNFAIP3) encodes the ubiquitin-modifying enzyme A20, which downregulates inflammation by restricting NF-κB, a transcription factor that regulates expression of various proinflammatory genes. Variants in TNFAIP3 gene have been described as associated with susceptibility to several AIDs. Here, we analyzed two TNFAIP3 polymorphisms in Italian patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and primary Sjogren's syndrome (pSS), to verify if the genetic variability of TNFAIP3 gene is involved in genetic predisposition to AIDs also in the Italian population. We recruited 313 SLE patients, 256 RA patients, 195 pSS patients, and 236 healthy controls. Genotyping of rs2230926 and rs6920220 in TNFAIP3 gene was performed by an allelic discrimination assay. We carried out a case/control association study and a genotype/phenotype correlation analysis. A higher risk to develop SLE was observed for rs2230926 (P = 0.02, OR = 1.92). No association was observed between this SNP and the susceptibility to pSS or RA. However, the rs2230926 variant allele seems to confer a higher risk to develop lymphoma in pSS patients, while in RA patients, the presence of RF resulted significantly associated with the variant allele. Regarding the rs6920220 SNP, we observed a significant association of the variant allele with SLE (P = 0.03, OR = 1.53), pSS (P = 0.016, OR = 1.69), and RA (P = 0.0001, OR = 2.35) susceptibility. Furthermore, SLE patients carrying the variant allele showed a higher risk to develop pericarditis, pleurisy, and kidney complications. Our results support the importance of the TNFAIP3 gene variant role in the development of different autoimmune diseases in the Italian population and furtherly confirm a sharing of genetic predisposing factors among these three pathologies.

Evaluation of ATG5 polymorphisms in Italian patients with systemic lupus erythematosus: contribution to disease susceptibility and clinical phenotypes.

Systemic lupus erythematosus (SLE) is a common heterogeneous autoimmune disease that is caused by the involvement both of genetic and environmental factors. There is evidence that autophagy is involved in several aspects of SLE pathogenesis. In particular, polymorphisms in the ATG5 gene have been observed to be associated with disease susceptibility. Our aim was to verify if ATG5 polymorphisms are involved in the susceptibility to disease and its clinical phenotypes in an Italian cohort of SLE patients. This study involved 315 SLE patients and 265 healthy controls. Three polymorphisms in the ATG5 gene (rs573775, rs6568431 and rs2245214) were investigated by allelic discrimination assay. A case-control association study, a genotype/phenotype correlation analysis and a haplotype study were performed. Moreover, an expression study was conducted in peripheral blood mononuclear cells from 15 SLE patients to verify a possible effect of the three SNPs on the expression of ATG5. Among the three investigated SNPs, only the rs573775 SNP was significantly associated with disease susceptibility with the variant allele conferring a higher risk of developing SLE (OR = 1.50, p = 0.018 and OR = 1.48, p = 0.007 at the genotypic and allelic level, respectively). The variant allele of rs6568431 SNP was more present in patients with anemia (OR = 1.86, p = 0.009) and renal involvement (OR = 1.63, p = 0.06), while the variant allele of rs2245214 SNP was significantly associated with a higher risk of producing anti-DNA autoantibodies (OR = 1.66, p = 0.04). Carriers of the rs6568431 variant allele showed higher messenger RNA levels compared to the carriers of the wild-type allele, suggesting also a potential variant allele dose-dependent effect on gene expression. In conclusion, our study confirms a role for ATG5 polymorphisms both in disease susceptibility and in the modulation of clinical phenotypes in an Italian SLE cohort. These results further suggest that genetic variations in autophagy genes could play a role in autoimmune diseases susceptibility and are worth further investigation.

Assessing individual risk for AMD with genetic counseling, family history, and genetic testing.

PurposeThe goal was to develop a simple model for predicting the individual risk profile for age-related macular degeneration (AMD) on the basis of genetic information, disease family history, and smoking habits.Patients and methodsThe study enrolled 151 AMD patients following specific clinical and environmental inclusion criteria: age >55 years, positive family history for AMD, presence of at least one first-degree relative affected by AMD, and smoking habits. All of the samples were genotyped for rs1061170 (CFH) and rs10490924 (ARMS2) with a TaqMan assay, using a 7500 Fast Real Time PCR device. Statistical analysis was subsequently employed to calculate the real individual risk (OR) based on the genetic data (ORgn), family history (ORf), and smoking habits (ORsm).Results and conclusionThe combination of ORgn, ORf, and ORsm allowed the calculation of the Ort that represented the realistic individual risk for developing AMD. In this report, we present a computational model for the estimation of the individual risk for AMD. Moreover, we show that the average distribution of risk alleles in the general population and the knowledge of parents' genotype can be decisive to assess the real disease risk. In this contest, genetic counseling is crucial to provide the patients with an understanding of their individual risk and the availability for preventive actions.

A polymorphism upstream MIR1279 gene is associated with pericarditis development in Systemic Lupus Erythematosus and contributes to definition of a genetic risk profile for this complication.

Recently, a study has shown that a polymorphism in the region of MIR1279 modulates the expression of the TRAF3IP2 gene. Since polymorphisms in the TRAF3IP2 gene have been described in association with systemic lupus erithematosus (SLE) susceptibility and with the development of pericarditis, our aim is to verify if the MIR1279 gene variability could also be involved. The rs1463335 SNP, located upstream MIR1279 gene, was analyzed by allelic discrimination assay in 315 Italian SLE patients and 201 healthy controls. Moreover, the MIR1279 gene was full sequenced in 50 patients. A case/control association study and a genotype/phenotype correlation analysis were performed. We also constructed a pericarditis genetic risk profile for patients with SLE. The full sequencing of the MIR1279 gene in patients with SLE did not reveal any novel or known variation. The variant allele of the rs1463335 SNP was significantly associated with susceptibility to pericarditis ( P = 0.017 and OR = 1.67). A risk profile model for pericarditis considering the risk alleles of MIR1279 and three other genes (STAT4, PTPN2 and TRAF3IP2) showed that patients with 4 or 5 risk alleles have a higher risk of developing pericarditis ( OR = 4.09 with P = 0.001 and OR = 6.04 with P = 0.04 respectively). In conclusion, we describe for the first time the contribution of a MIR1279 SNP in pericarditis development in patients with SLE and a genetic risk profile model that could be useful to identify patients more susceptible to developing pericarditis in SLE. This approach could help to improve the prediction and the management of this complication.

Polymorphisms in STAT-4, IL-10, PSORS1C1, PTPN2 and MIR146A genes are associated differently with prognostic factors in Italian patients affected by rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease resulting in chronic inflammation of the synovium and consequent cartilage and bone erosion. RA is associated strongly with the presence of rheumatoid factor (RF), and consists of clinical subsets of anti-citrullinated protein antibody (ACPA)-positive and -negative patients. This study was designed to evaluate whether relevant single nucleotide polymorphisms (SNPs) associated with RA and other autoimmune disorders are related to RF, ACPA and clinical phenotype in a cohort of biologic drugs naive Italian RA patients; 192 RA patients and 278 age-matched healthy controls were included. Clinical and laboratory data were registered. We analysed a total of 12 single nucleotide polymorphisms (SNPs) in signal transducer and activator of transcription-4 (STAT-4), interleukin (IL)-10, psoriasis susceptibility 1 candidate 1 (PSORS1C1), protein tyrosine phosphatase, non-receptor type 2 (PTPN2), endoplasmic reticulum aminopeptidase 1 (ERAP1), tumour necrosis factor receptor-associated 3 interacting protein 2 (TRAF3IP2) and microRNA 146a (MIR146A) genes by allelic discrimination assays. Case-control association studies and genotype/phenotype correlation analyses were performed. A higher risk to develop RA was observed for rs7574865 in the STAT-4 gene, while the rs1800872 in the IL-10 gene showed a protective effect. The presence of RF was associated significantly with rs1800872 variant in IL-10, while rs2910164 in MIR146A was protective. ACPA were associated significantly with rs7574865 in STAT-4. The SNP rs2233945 in the PSORS1C1 gene was protective regarding the presence of bone erosions, while rs2542151 in PTPN2 gene was associated with joint damage. Our results confirm that polymorphisms in STAT-4 and IL-10 genes confer susceptibility to RA. For the first time, we described that SNPs in PSORS1C1, PTPN2 and MIR146A genes were associated differently with a severe disease phenotype in terms of autoantibody status and radiographic damage in an Italian RA population.

Direct PCR: a new pharmacogenetic approach for the inexpensive testing of HLA-B*57:01.

One of the most successful applications of pharmacogenetics research is the genetic screening for HLA-B*57:01, strongly associated with an increased risk to develop hypersensitivity reaction in HIV-positive patients following abacavir administration. Taking into consideration the limits of current genotyping methodologies, we have developed and validated (150 buccal swabs) an inexpensive pharmacogenetic approach for HLA-B*57:01 typing. In our assay DNA extraction and amplification are combined in one single step (direct PCR protocol), which is performed directly on the biological sample without the need of extraction and sequencing passages. The amplicons obtained by direct PCR can be easily separated on the agarose gel under ultraviolet. As per our results, the direct PCR represents a good alternative to the traditional methods of HLA-B*57:01 pharmacogenetic test, especially for those laboratories or countries where currently available approaches are often not available or not affordable. Furthermore it is an innovative approach, promoting a personalized, safer and cost-effective therapy.

High warfarin sensitivity in carriers of CYP2C9*35 is determined by the impaired interaction with P450 oxidoreductase.

Cytochrome P450 2C9 (CYP2C9) metabolizes many clinically important drugs including warfarin and diclofenac. We have recently reported a new allelic variant, CYP2C9*35, found in a warfarin hypersensitive patient with Arg125Leu and Arg144Cys mutations. Here, we have investigated the molecular basis for the functional consequences of these polymorphic changes. CYP2C9.1 and CYP2C9-Arg144Cys expressed in human embryonic kidney 293 cells effectively metabolized both S-warfarin and diclofenac in NADPH-dependent reactions, whereas CYP2C9-Arg125Leu or CYP2C9.35 were catalytically silent. However, when NADPH was replaced by a direct electron donor to CYPs, cumene hydroperoxide, hereby bypassing the CYP oxidoreductase (POR), all variant enzymes were active, indicating unproductive interactions between CYP2C9.35 and POR. In silico analysis revealed a decrease of the electrostatic potential of CYP2C9-Arg125Leu-POR interacting surface and the loss of stabilizing salt bridges between these proteins. In conclusion, our data strongly suggest that the Arg125Leu mutation in CYP2C9.35 prevents CYP2C9-POR interactions resulting in the absence of NADPH-dependent CYP2C9-catalyzed activity in vivo, thus influencing the warfarin sensitivity in the carriers of this allele.

Impact of the CYP4F2 p.V433M polymorphism on coumarin dose requirement: systematic review and meta-analysis.

A systematic review and a meta-analysis were performed to quantify the accumulated information from genetic association studies investigating the impact of the CYP4F2 rs2108622 (p.V433M) polymorphism on coumarin dose requirement. An additional aim was to explore the contribution of the CYP4F2 variant in comparison with, as well as after stratification for, the VKORC1 and CYP2C9 variants. Thirty studies involving 9,470 participants met prespecified inclusion criteria. As compared with CC-homozygotes, T-allele carriers required an 8.3% (95% confidence interval (CI): 5.6-11.1%; P < 0.0001) higher mean daily coumarin dose than CC homozygotes to reach a stable international normalized ratio (INR). There was no evidence of publication bias. Heterogeneity among studies was present (I(2) = 43%). Our results show that the CYP4F2 p.V433M polymorphism is associated with interindividual variability in response to coumarin drugs, but with a low effect size that is confirmed to be lower than those contributed by VKORC1 and CYP2C9 polymorphisms.

A study of three polymorphic sites of the ADA gene in children with type 1 diabetes mellitus.

BACKGROUND: Adenosine deaminase is a polymorphic enzyme that has an important role in immune functions and in the regulation of intracellular and extracellular concentrations of adenosine and adenosine receptor activity. AIM: To search for possible association of type 1 diabetes mellitus (DM1) with three loci haplotypes (ADA1, ADA2, ADA6) of the adenosine deaminase gene. PATIENTS: One hundred and eighty-nine consecutive children with DM1 from Sassari, Sardinia, and a control sample of 239 children from the same area were studied. METHODS: ADA loci genotypes were determined by DNA analysis. RESULTS: Compared to controls, diabetic boys show a decrease of the 2(2)/6(1) haplotype while diabetic girls show an increase of the same haplotype. This association was replicated in an independent sample from Continental Italy. CONCLUSIONS: The 2(2)/6(1) haplotype may exert a protective action in males but may increase susceptibility to DM1 in females: OR = 0.398, 95% CI 0.16-0.96 for males, and OR = 2.31, 95% CI 1.32-4.06 for females.

Pharmacogenomics: role in medicines approval and clinical use.

Pharmacogenomics (PGx) and pharmacogenetics (PGt) are emerging interdisciplinary areas recently defined by the regulatory authorities at an international level as 'the investigation of variations of DNA and RNA characteristics as related to drug response' (PGx), and the study of 'the influence of variations in DNA sequence on drug efficacy and toxicity' (PGt). In recent years a number of studies have in fact produced growing evidence that, besides the effects of age, sex, diseases, and different drugs interactions, genetic factors play a role in the inter-individual variability of drugs response. The increasing genomic knowledge has also raised the profile and role of the so called 'genomic biomarkers' (GBs) in drug development, approval, and clinical use. The aims of this review are to (a) revisit the general understanding of the role of genomics and genetics in drug response, (b) provide an update on the definition and classification criteria of GBs as recently defined at a global level by regulatory agencies such as the European Medicines Agency and the Food and Drug Administration, and (c) illustrate with some examples current and potential applications of biomarkers in clinical practice and in drug development.

Increased release and activity of matrix metalloproteinase-9 in patients with mandibuloacral dysplasia type A, a rare premature ageing syndrome.

Mandibuloacral dysplasia type A (MADA; OMIM 248370), a rare disorder caused by mutation in the LMNA gene, is characterized by post-natal growth retardation, craniofacial and skeletal anomalies (mandibular and clavicular hypoplasia, acroosteolysis, delayed closure of cranial sutures, low bone mass and joint contractures), cutaneous changes and partial lipodystrophy. Little is known about the molecular mechanisms by which LMNA mutations produce bone alterations. An altered bone extracellular matrix (ECM) remodelling could play a pivotal role in this disorder and influence part of the typical bone phenotype observed in patients. Therefore, we have focused our investigation on matrix metalloproteinases (MMPs), which are degradative enzymes involved in ECM degradation and ECM remodelling, thus likely contributing to the altered bone mineral density and bone metabolism values seen in five MADA patients. We evaluated the serum levels of several MMPs involved in bone development, remodelling and homeostasis, such as MMP-9, -2, -3, -8 and -13, and found that only the 82 kDa active enzyme forms of MMP-9 are significantly higher in MADA sera compared with healthy controls (n = 16). The serum level of MMP-3 was instead lower in all patients. No significant differences were observed between controls and MADA patients for the serum levels of MMP-2, -8 and -13 and of tissue inhibitor of metalloproteinase 2, a natural inhibitor of MMP-9. Similarly, normal serum levels of tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-6 and IL-1beta were detected. These data suggest a possible involvement of MMP-9 in MADA disease, underlying the potential use in diagnosis and therapy.

[New insights in atherosclerosis research: LOX-1, leading actor of cardiovascular diseases]. / Nuovi approfondimenti nella ricerca su l'ateroslerosi: LOX-1, un primo attore nelle malattie cardiovascolari.

The OLR1 gene encodes a cell-surface endocytosis receptor (LOX-1) for oxidized low density lipoprotein (OxLDL). LDL is oxidized in vascular endothelial cells to a highly injurious product that results in endothelial cell injury, which is implicated in the development of atherosclerosis. Vascular endothelial cells also internalize and degrade oxLDL though the OLR1 receptor. This receptor is upregulated by ox-LDL itself and by angiotensin II, endothelin, cytokines, and shear stress, important factors of atherosclerosis. This receptor is upregulated in the arteries of hypertensive, dyslipidemic, and diabetic animals. Two independent studies have demonstrated genetic association between polymorphisms in the OLR1 gene and myocardial infarction. Based on genetic and functional studies we propose LOX-1 as a novel biomarker and target in cardiovascular disease diagnosis and prevention.

Characterization of a single nucleotide polymorphism in the ZNF9 gene and analysis of association with myotonic dystrophy type II (DM2) in the Italian population.

Myotonic dystrophy type 2 (DM2) is a dominant inherited disorder clinically similar to myotonic dystrophy type 1 (DM1) with a peculiar pattern of multisystemic phenotypic features. The mutation responsible for DM1 is a CTG repeat in the 3' UTR of the dystrophia myotonica protein kinase gene (DMPK) on chromosome 19q13.3, while DM2 is caused by an unstable CCTG expansion in intron 1 of the zinc finger protein 9 gene (ZNF9) on chromosome 3q21.3. Southern blotting analysis is the conventional test used to determinate the size of the repeats in the molecular diagnosis of DM2. However, the large number of CCTG repeats and their somatic instability complicates this diagnostic protocol. In order to improve the DM2 test, we have recently characterised a single nucleotide polymorphism located in the first intron of the ZNF9 gene. This SNP consists in a C to A nucleotide change, which creates or disrupts and ApaI enzyme restriction site, easily detectable by PCR amplification followed by restriction analysis. We genotyped this SNP in 30 unrelated DM2 patients and 70 unrelated Italians healthy individuals. Our results show that this polymorphism is in linkage disequilibrium with the DM2 mutation.

Type 2 diabetes and the genetics of signal transduction: a study of interaction between adenosine deaminase and acid phosphatase locus 1 polymorphisms.

Acid phosphatase locus 1 (ACP1) is a highly polymorphic enzyme that has an important role in flavoenzyme activity and in the control of insulin receptor activity and band 3 protein phosphorylation status. Adenosine deaminase (ADA) is a polymorphic enzyme that catalyses the irreversible deamination of adenosine to inosine and has an important role in regulating adenosine concentration. Based on the hypothesis that ACP1 counteracts insulin signaling by dephosphorylating the insulin receptor and that adenosine has an anti-insulin action, we reasoned that low ACP1 activity (low dephosphorylating action on insulin receptor) when associated with high ADA activity (low adenosine concentration) would result in a cumulative effect towards an increased glucose tolerance. On the contrary, high ACP1 activity when associated with low ADA activity would result in a cumulative effect towards a decreased glucose tolerance. A total of 280 adult subjects with type 2 diabetes from the population of Penne (Italy) were studied. There was a nonsignificant trend toward an increase in the proportion of subjects with the complex type with high ACP1 activity and low ADA activity (ie, *B/*B; *A/*C; *B/*C; *C/*C//ADA*1/*2 and *2/*2) in type 2 diabetes relative to that observed in newborn infants from the same population. High ACP1 activity/low ADA activity joint genotype was positively associated with high glycemic levels and with high body mass index (BMI) values. Low ACP1 activity/high ADA activity joint genotype was also positively associated with dyslipidemia. These findings suggest that both ACP1 and ADA contribute to the clinical manifestations of type 2 diabetes and probably also have a marginal influence on susceptibility to the disease. Both additive and epistatic interactions between the 2 systems seem to be operative.

Genetic control of serum IgE levels: a study of low molecular weight protein tyrosine phosphatase.

Protein tyrosine phosphatases (PTPases) have recently been recognized as important modulators of various signal transduction pathways in immune cells. Genetic polymorphisms have been described in genes codifying for members of this family of enzymes, and the genetics of PTPases is predicted to play an important role in the etiology of immune diseases and of their clinical variability. The low molecular weight protein tyrosine phosphatase (ACP1 or LMPTP) is one of the few PTPases with a known genetic polymorphism, and has been proposed to be associated with atopic dermatitis in a small sample from an Italian population. In this paper we describe the association of the ACP1 polymorphism with total IgE levels in two independent samples from English and Italian populations. In both the samples the mean value of serum IgE is lower among subjects carrying the BC genotype than in other ACP1 genotypes. The BC genotype is associated with the highest total ACP1 enzymatic activity. Our data suggest that one or both of the ACP1 isoforms exert an inhibitory role on some signal transduction pathway relevant for IgE hyperproduction.

Intrauterine development and MNSs blood groups in repeated spontaneous abortion.

OBJECTIVES: To determine whether the maternal MNSs genotype has an effect on the birth weight and gestation duration of the live offspring of women with repeated primary spontaneous abortion (RSA). METHODS: The study sample consisted of 239 healthy white women who had been delivered of a live infant, and 137 women with a history of primary RSA-54 of whom had recently been delivered of a live infant and 83 who had had a spontaneous abortion. Maternal MNSs phenotypes were determined by standard serological methods, and the results were analyzed for relationships between these phenotypes and the mothers' reproductive status and the infants' birth weight and gestational age. Analysis of variance, the chi(2)-test of independence, and the Mantel-Haenszel test for linear association were performed for data analysis. RESULTS: Infants born to mothers with the Ss genotype showed significantly lower birth weight and gestational duration compared with the infants of mothers with other genotypes. Additionally, the MNSs haplotype was found to be associated with birth weight. CONCLUSIONS: Previous studies have shown that the MNSs system influences the gestational age of aborted fetuses in cases of RSA. The present study supports the hypothesis that this genetic factor influences intrauterine growth and development in women experiencing RSA.