Epigenetic characteristics in inflammatory candidate genes in aggressive periodontitis.

BACKGROUND: Periodontitis is a chronic inflammatory disease triggered by the host immune response. Epigenetic modifications also affect the immune response. We assessed CpG methylation in 22 inflammatory candidate genes (ATF2, CCL25, CXCL14, CXCL3, CXCL5, CXCL6, FADD, GATA3, IL10RA, IL12A, IL12B, IL13, IL13RA1, IL15, IL17C, IL17RA, IL4R, IL6R, IL6ST, IL7, INHA, and TYK2) with respect to the occurrence of aggressive periodontitis (AgP). PATIENTS AND METHODS: In this study 15 AgP patients (53.3% males, 41.4±10.5 years) and 10 controls (40.0% males, 36.9±17.5 years) were included. The methylation patterns of gingival biopsies were quantified using EpiTect® Methyl Signature PCR Array Human Inflammatory Response. RESULTS: In gingival biopsies taken from patients with AgP, CpG methylation of CCL25 (1.73% vs. 2.59%, p=0.015) and IL17C (6.89% vs. 19.27%, p=0.002) was significantly reduced as compared with periodontally healthy tissues. DISCUSSION: We showed for the first time a differential methylation pattern for CCL25 and IL17C in periodontitis. CCL25 plays an important role in T-cell development, whereas IL17C regulates innate epithelial immune responses. The decrease in CpG methylation is presumably accompanied by an increase in gene expression. This could lead to a greater availability of CCL25 and interleukin 17C and support periodontal loss of attachment.

Variable expression of Alagille syndrome in a family with a new JAG1 gene mutation.

We report the case of a patient with tetralogy of Fallot with absent pulmonary valve and familial Alagille syndrome who successfully underwent cardiac repair. The patient's sister had liver and congenital heart disease. The father had undergone liver transplantation but showed no significant cardiac abnormalities. A yet-unknown mutation of the JAG1 gene was discovered in this family with variable expression of Alagille syndrome.

Tumor necrosis factor-α and oral inflammation in patients with Crohn disease.

BACKGROUND: Crohn disease (CD) is a chronic inflammatory bowel disease often accompanied by periodontal symptoms. Based on its function in immune response, tumor necrosis factor (TNF)-α and its genetic variants have been discussed as risk indicators in inflammatory processes. Therefore, the aim of the present study is to investigate the impact of TNF-α polymorphisms on periodontal parameters and inflammatory lesions of oral mucosa as a characteristic of CD. METHODS: A total of 142 patients with CD were included in the study. Oral soft tissue alterations and periodontal parameters were assessed. Genotypes, alleles, and haplotypes of TNF-α polymorphisms (rs1800629, cDNA-308G > A; and rs361525, cDNA-238G > A) were determined by polymerase chain reaction with sequence-specific primers (PCR-SSP). RESULTS: Patients with CD who exhibit more severe oral soft tissue alterations were significantly more often A allele carriers of rs361525 than G allele carriers (14.2% versus 2.2%; P <0.001). Furthermore, A allele carriers had a higher mean periodontal probing depth (P <0.05), mean clinical attachment level (P <0.05), and sites with bleeding on probing (not significant). Similar results were obtained when evaluating A allele-containing genotypes (AG + AA) and haplotypes (GA). In multivariate analyses considering age, sex, smoking, and medication as confounders, the A allele was proven to be an independent risk indicator for oral soft tissue alterations in patients with CD. No genotype-dependent influence of rs1800629 was observed. CONCLUSION: The TNF-α A allele of rs361525 represents a significant risk indicator for oral soft tissue alterations in patients with CD.

The importance of genetic variants in TNFα for periodontal disease in a cohort of coronary patients.

AIM: The aim of this analysis was to evaluate the importance of genetic variants of TNFα for the severity of periodontal disease and periodontal risk factors with respect to periodontal risk factors in a cohort of coronary patients. SUBJECTS: A total of 942 consecutive patients with angiographic proven coronary heart disease were prospectively included in the study entitled "Periodontitis and Its Microbiological Agents as Prognostic Factors in Patients With Coronary Heart Disease" (ClinicalTrials.gov identifier:NCT01045070). METHODS: After including of patients, an extensive periodontal examination also involving PCR-sampling for 11 periodontal bacteria was performed. In this subanalysis, single nucleotide polymorphisms (SNPs) c.-308G>A, c.-238G>A and haplotypes for TNFα were analysed by CTS-PCR-SSP Tray kit (Heidelberg, Germany). RESULTS: The AG+AA genotype of SNP c.-238G>A of TNFα gene was associated with the amount of clinical attachment loss in patients with coronary heart disease in multivariate regression analysis. Moreover, Prevotella intermedia occurred more frequently in carriers who were positive for the AG+AA genotype and A-allele of SNP c.-308G>A in bivariate and multivariate analyses. Furthermore, only in bivariate analyses significant associations of genetic variants of TNFα with intensified bleeding on probing and with higher plasma level of interleukin 6 could be shown. CONCLUSIONS: Genetic variants of TNFα gene, namely c.-308G>A and c.-238G>A, are associated with periodontal conditions in patients with coronary heart disease.

Single nucleotide polymorphisms in interleukin-1gene cluster and subgingival colonization with Aggregatibacter actinomycetemcomitans in patients with aggressive periodontitis.

Periodontitis is initiated by the subgingival occurrence of periodontopathogens. It is triggered by a specific host-dependent immune response that is influenced by genetic predisposition. Polymorphisms in the interleukin-1 (IL-1) gene cluster have been suggested to influence the pathogenesis of periodontitis. A total of 159 periodontitis patients (chronic disease: n = 73, aggressive disease: n = 86) and 89 periodontitis-free controls were included in the study. Polymorphisms IL-1α (rs1800587), IL-1ß (rs16944, rs1143634), IL-1 receptor (rs2234650), and IL-1 receptor antagonist (rs315952) were determined by polymerase chain reaction with sequence-specific primers (PCR-SSP). Subgingival bacterial colonization was assessed using a polymerase chain reaction/DNA probe test (micro-Ident). Haplotype block structure was determined using Haploview 4.2. Statistical analyses were performed applying SPSS 17.0 considering dominant, recessive, and codominant genetic models. In this case-control study, no association between genomic variants of the IL-1 gene cluster and the incidence of severe periodontitis could be shown. Carriers of the rare genotypes of rs1800587 (p(corr) = 0.009), rs1143634 (p(corr) = 0.009) and composite genotype (rs1800587+rs1143634) (p(corr) = 0.031) had a twofold higher risk for subgingival occurrence of Aggregatibacter actinomycetemcomitans. In forward stepwise binary logistic regression analyses considering age, gender, smoking, and approximal plaque index as potential confounders these significant associations were demonstrated. Despite the genetic background of IL-1 gene cluster could be shown to be associated with subgingival colonization of A actinomycetemcomitans, there is no evidence that it is an independent risk indicator for periodontitis.

Interferon-gamma and interleukin-12 gene polymorphisms and their relation to aggressive and chronic periodontitis and key periodontal pathogens.

BACKGROUND: The gene polymorphisms interferon-gamma (IFN-gamma) 874 T/A and interleukin (IL)-12 1188 A/C have been associated with the altered production of cytokines. Therefore, they might be indicative of the occurrence of chronic periodontitis (CP) or aggressive periodontitis (AgP) and the prevalence of key periodontal pathogens. For this purpose, we analyzed these polymorphisms in subjects with generalized AgP or generalized CP. Moreover, we assessed the relationship between these polymorphisms and five periodontopathic bacteria. METHODS: A total of 124 unrelated German white subjects with periodontitis (AgP=72 and CP=52) and 74 periodontitis-free subjects were studied. Gene polymorphisms were determined by polymerase chain reaction with sequence-specific primers. Subgingival bacteria were molecular biologically analyzed using multiplex polymerase chain reaction and reverse hybridization. The distributions of alleles and genotypes were calculated by the chi(2) test with Yates correction. Risk factor analyses were carried out by logistic regression considering established confounders for periodontitis. RESULTS: Allele and genotype frequencies of both investigated polymorphisms were not significantly different between subjects with periodontitis and periodontitis-free controls. However, in the total study group, IL-12 AA-positive subjects had a significantly higher bleeding index than individuals who expressed IL-12 CC (68.2% versus 50.0%, P=0.025). Moreover, IFN-gamma AA carriers had a decreased odds ratio (OR) for the individual presence of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) (OR=0.39, P=0.012) after adjustment for age, gender, smoking, and probing depth. IFN-gamma TA predisposed an individual to infection with Prevotella intermedia (OR=2.15, P=0.019). CONCLUSION: Although a relationship between the bleeding index and the presence of bacteria was shown, IFN-gamma and IL-12 polymorphisms are not suitable diagnostic features for AgP and CP.

Genetic markers of tumour necrosis factor alpha in aggressive and chronic periodontitis.

AIM: Tumour necrosis factor alpha (TNFalpha) plays an important role in the pathogenesis of periodontitis. TNFalpha production is influenced by gene polymorphisms. The aim of this study was to evaluate links between genetic variants and chronic/aggressive periodontitis in a multivariate model. SUBJECTS: One hundred and twenty-three periodontitis patients (chronic: n=54, aggressive: n=69) and 52 healthy controls without periodontitis were included in the study. MATERIAL AND METHODS: Single nucleotide polymorphisms (SNPs) c.-308G>A, c.-238G>A and haplotypes were analysed by a polymerase chain reaction with sequence-specific primers (PCR-SSP). The clinical investigation included smoking status, plaque and bleeding indexes, pocket depth and attachment loss. RESULTS: Prevotella intermedia occurred more frequently in individuals positive for the -308GG/-238GG haplotype combination (Odds Ratio=2, 95% Confidence interval: 1.1-3.7, p=0.037, 1-beta=61%). In binary logistic regression analyses, this TNFalpha haplotype could not be shown to be associated with periodontitis considering smoking, age, gender and approximal plaque index or subgingival bacterial colonization as confounding factors. CONCLUSIONS: Although the genetic background of TNFalpha could be shown to be associated with subgingival colonization with P. Intermedia, there is no evidence that it is an independent risk factor for periodontitis in multivariate models.

Genetic impact of TNF-beta on risk factors for coronary atherosclerosis.

BACKGROUND: Inflammatory processes are considered to play an important role in the development of coronary atherosclerosis. The proinflammatory cytokine, tumor necrosis factor beta (TNF-beta), is thought to contribute to the pathogenesis of atherosclerosis. STUDY DESIGN: In this clinical study, the influence of genetic variants of TNF-beta (c.7G>A, IVS1+90G>A, C13R, T60N) on major coronary risk factors, including gender, smoking, history of cardiovascular diseases, biochemical data (inflammatory markers, factors of lipid metabolism, coagulation/fibrinolysis balance), and angiographically-proven coronary state, was investigated in 176 European Caucasian probands (130 males, mean age: 51.9 +/- 8.9 y). RESULTS: The most frequent combinations of the polymorphisms investigated were significantly associated with four of the coronary risk factors evaluated: hypertension, body mass index, the common inflammatory marker TNF-alpha (mRNA expression), and fibrinogen (p < 0.05). However, on testing the impact of the genetic background on the incidence of coronary stenosis in this sample of European Caucasians, no significant influence of these polymorphisms (stepwise binary logistic regression analysis) could be proven. These findings emphasise a distinct influence of TNF-beta polymorphisms on important modulators of the development of coronary atherosclerosis, but exclude its genetic background, investigated in this study as an independent coronary risk factor.

Polymyxin B-conjugated alpha 2-macroglobulin as an adjunctive therapy to sepsis: Modes of action and impact on lethality.

A drug targeting both the inflammatory initiators (lipopolysaccharide; LPS) and mediators [tumor necrosis factor-alpha (TNF-alpha)] should have advantage over a "single-factor targeting strategy" in sepsis prevention trials. We have prepared conjugates of polymyxin B (PMB) and the cytokine binding protein alpha2-macroglobulin (A2M). The conjugate binds TNF-alpha as well as LPS as studied by electrophoresis and phase partitioning. Compared with free PMB, the conjugate is nontoxic to cells and does not affect the viability of human monocytes. The A2M-PMB conjugate binds to the A2M receptor (CD91/low-density lipoprotein receptor-related protein 1) with affinity similar to that of the nonmodified protein. Fluorescein isothiocyanate-labeled LPS in the presence of A2M-PMB is rapidly transported into fibroblasts for degradation via receptor-mediated endocytosis. In vitro, A2M-PMB demonstrated inhibition of LPS-induced secretion of TNF-alpha from isolated monocytes as well as in the whole blood assay. The efficacy of the drug was tested in mice after induction of acute inflammation (LPS model) and after induction of a polymicrobial sepsis by cecal ligation and puncture (CLP) model. Treatment of mice with A2M-PMB up to 250 microg/g body weight was not toxic to the animal. When the drug was administered 30 min before or 30 min after the LPS challenge, a survival rate of 90 and 70%, respectively, was obtained compared with the placebo control group (5%). A2M-PMB also protected mice after induction of polymicrobial sepsis when administered 30 min before CLP. These results support our hypothesis that A2M-PMB acts as a polyvalent drug to target different host mediators as well as sepsis inducer at the same time.

Minimal phenotype in a girl with trisomy 15q due to t(X;15)(q22.3;q11.2) translocation.

Few cases of de novo unbalanced X;autosome translocations associated with a normal or mild dysmorphic phenotype have been described. We report a 3-year-old dizygotic female twin with prenatally ascertained increased nuchal translucency. Prenatal chromosome studies revealed nearly complete trisomy 15 due to a de novo unbalanced translocation t(X;15)(q22;q11.2) confirmed postnatally. A mild phenotype was observed with normal birth measurements, minor facial dysmorphic features (hypertelorism, short broad nose, and a relatively long philtrum), and moderate developmental delay at the age of 3 years in comparison to her male fraternal twin. Replication timing utilizing BrdU and acridine-orange staining showed that the der(X) chromosome was late-replicating with variable spreading of inactivation into the translocated 15q segment. The der(X) was determined to be of paternal origin by analyses of polymorphic markers and CGG-repeat at FMR1. Methylation analysis at the SNRPN locus and analysis of microsatellites on 15q revealed paternal isodisomy with double dosage for all markers and the unmethylated SNRPN gene. The Xq breakpoint was mapped within two overlapping BAC clones RP11-575K24 and RP13-483F6 at Xq22.3 and the 15q breakpoint to 15q11.2, within overlapping clones RP11-509A17 and RP11-382A4 that are all significantly enriched for LINE-1 elements (36.6%, 43.0%, 26.6%, 22.0%, respectively). We speculate that the attenuated phenotype may be due to inactivation spreading into 15q, potentially facilitated by the enrichment of LINE-1 elements at the breakpoints. In silico analysis of breakpoint regions revealed the presence of highly identical low-copy repeats (LCRs) at both breakpoints, potentially involved in generating the translocation.

Renal failure and hypertension in Alagille syndrome with a novel JAG1 mutation.

Renal failure and hypertension in Alagille syndrome with a novel JAG1 mutation: Alagille syndrome is an autosomal dominant disorder involving liver, heart, eyes, face, skeleton, and other organs. Various renal abnormalities have also been associated with Alagille syndrome, whereas renal vascular hypertension combined with renal insufficiency has been reported in several cases. We describe a patient with a novel frameshift mutation (c.1880_1881insA) in the JAG1 gene who presented with chronic renal failure and hypertension but without evidence of renal vascular or aortic stenosis. The patient's chronic renal failure had persisted for several years. His high blood pressure seemed to be due to renal parenchymal changes and was treated with ACE-inhibitors without worsening his renal function. This novel JAG1 mutation revealed great variability of the phenotype. The patient's daughter suffered from severe paucity of intrahepatic bile ducts and received a liver transplant at the age of two years. These findings are discussed including a review of the literature.

Twelve novel JAG1 gene mutations in Polish Alagille syndrome patients.

Alagille syndrome (AGS) is an autosomal dominant disorder with developmental abnormalities of the liver, heart, eyes, vertebrae, and face. Mutations in the JAG1 (Jagged 1) gene, coding a ligand in the evolutionarily conserved Notch signaling pathway, are responsible for AGS. Here we present sixteen different JAG1 gene mutations, among them twelve novel, not described previously. Seven frameshift: c. 172_178del7 (p.Ala58fs), c.509delT (p.Leu170fs), c.1197delG (p.Val399fs), c.1485_1486delCT (p.Pro495fs), c.1809_1810insTGGG (p.Lys604fs), c.2122_2125delCAGT (p.Gln708fs), c.2753delT (p.Ile918fs); five nonsense: c.383G>A (p.Trp128X), c.496C>T (p.Glu166X), c.841C>T (p.Gln281X), c.1207C>T (p.Gln403X), c.1603C>T (p.Gln535X); two splice site: c.388-1G>C, c.3048+1_3048+2insG and two missense mutations: c.359T>A (p.Ile120Asn), c.560G>A (p.Cys187Tyr) were found. Forty percent of the changes were identified in exons 2 and 4, the remaining mutations are distributed along the entire coding sequence of the gene. Seventy-five percent of the mutations lead to creation of premature termination codons. Family studies revealed that the specific mutations were inherited in 3 out of 11 investigated cases. No correlation between genotype and phenotype was observed.

Relation between the tumor necrosis factor-alpha (TNF-alpha) gene and protein expression, and clinical, biochemical, and genetic markers: age, body mass index and uric acid are independent predictors for an elevated TNF-alpha plasma level in a complex risk model.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) has been implicated in the pathogenesis of numerous complex diseases. The plasma level of this pro-inflammatory cytokine is associated with a variety of different risk factors, but little is known about the genetic background and the complex interactions. METHODS: in this clinical study, correlations were studied between plasma levels of circulating TNF-alpha protein (ELISA), its mRNA expression in monocytes (RT-PCR) and genetic variants of TNF-alpha gene (SSCP), with several diseases, including obesity, atherosclerosis, diabetes mellitus, hypertension, as well as risk factors such as age, gender, inflammatory markers, the coagulation\fibrinolysis balance, and lipid metabolism. One hundred and ninety four clinically and biochemically well-characterized patients were enrolled. RESULTS: At the transcriptional level, measured in monocytes, no association with any clinical or biochemical parameter investigated was found, including TNF-alpha protein level. Investigating the influence of genetic variants of the TNF-alpha gene on mRNA and protein levels, only one promoter polymorphism, namely c.-238G > A, was shown to be associated with transcriptional but not with translational expression. However, at the translational level, significant positive, but weak associations were determined for obesity (P -/+ 0.037), age (P -/+ 0.038), uric acid (P < 0.001), body mass index (P -/+ 0.01), plasminogen (P -/+ 0.013), and fibrinogen (P -/+ 0.002) in bivariate regression analyses, whereas HDL-cholesterol (P -/+ 0.005) was shown to be negatively correlated. However, investigating confounding effects in stepwise multivariate regression analysis, body mass index (P -/+ 0.009), uric acid (P -/+ 0.026) and age (P -/+ 0.037) turned out to be significantly associated with plasma levels of circulating TNF-alpha (adjusted R(2) -/+ 0.117; SE: 0.688).

Genetic and functional characteristics of the human in vivo LRP1/A2MR receptor suggested as a risk marker for Alzheimer's disease and other complex (degenerative) diseases.

LDL receptor-related protein/alpha2-macroglobulin receptor (LRP1/A2MR) a multiligand receptor is considered as not only being a possible risk factor of neurodegenerative diseases like Alzheimer's disease but also as determining the progression of other complex diseases like atherosclerosis and cancer. Although a large number of in vitro studies have highlighted its functional importance, as yet not enough is known about the clinical importance of the genetic background of LRP1 in human diseases. The aim of this ex vivo/in vivo study of 448 subjects was to present data on genetic LRP1 variants of healthy European Caucasians from Central Germany. Genotype-dependent LRP1 expression was analyzed in a representative subgroup (gene expression: n = 127, protein expression: n = 44). These data were evaluated in comparison to other published clinical LRP1 studies. For 15 functionally interesting genetic variants the genotype and allele distributions of the German Caucasians were presented in relation to their in vivo LRP1 gene and protein expression. A direct influence of the LRP1 promoter polymorphism c.1-25C>G on the human in vivo LRP1 expression level was demonstrated. In an analysis of 48 further studies genomic and functional results were evaluated. The analysis especially on Alzheimers's disease partly highlighted contradictory results, but suggested that ethnic as well as genomic characteristics determine LRP1 expression and must be considered in clinical investigations on human LRP1.

The E-selectin SER128ARG gene polymorphism and restenosis after successful coronary angioplasty.

OBJECTIVES: Coronary angioplasty remains plagued by the problem of restenosis. Genetic polymorphisms may contribute to the development of restenosis by mediating exaggerated inflammatory responses of the endothelium to angioplasty-induced injury. BACKGROUND: The serine (Ser)-128-arginine (Arg) gene polymorphism of E-selectin has been implicated in the pathogenesis of coronary artery disease (CAD). We sought to explore whether allelic variants relate to post-angioplasty restenosis. METHODS: The 128Arg allele was analyzed by PCR in 101 (derivation study, age 54+/-1 years, all mean+/-S.E.M.) and 92 (validation study, age 62+/-1 years) patients with CAD who underwent successful angioplasty. RESULTS: Restenosis, defined as >50% luminal diameter reduction at the target lesion at follow-up angiography, was found in 54/101 (53%) and 43/92 (47%) patients during follow-up. The 128Arg allele frequency in the derivation study was 10.39% and was 11.96% in the validation study. The 128Arg allele was more prevalent in the restenosis groups (14.81% and 17.44%, respectively) than in the restenosis-free groups (5.32% and 7.14%, respectively, p=0.027 and p=0.031). In multivariate logistic regression, the 128Arg allele emerged as a predictor of restenosis in both studies (p<0.05). There were no differences in the level of soluble E-selectin according to genotype, gender, age (p>0.20), and between patients with restenosis and those without (43.8+/-3.2 vs. 47.4+/-3.1 ng/ml, p>0.20). CONCLUSIONS: The 128Arg allele of E-selectin may be related to increased endothelial responses to injury, thereby potentially serving as a risk factor for post-angioplasty restenosis in patients with CAD.The development of restenosis remains a problem in patients with CAD. The Ser128Arg polymorphism of E-selectin was analyzed in 101 (derivation) and 92 (validation) CAD patients. Patients with restenosis (54/101 and 43/92) had a higher frequency of the 128Arg allele (14.81 and 17.44%) than those without (5.32%, p=0.027 and 7.14%, p=0.031). In logistic regression, the 128Arg allele emerged as a predictor of restenosis in both studies (p<0.05). The E-selectin 128Arg allele may serve as a risk factor for the development of restenosis.