Simvastatin alleviates bone resorption in apical periodontitis possibly by inhibition of mitophagy-related osteoblast apoptosis.

AIM: To assess the connection between mitophagy and hypoxia-induced apoptosis in osteoblasts and whether simvastatin alleviates bone resorption in apical periodontitis through modulation of mitophagy-related apoptosis. METHODOLOGY: Hypoxia-induced generation of reactive oxygen species in mitochondria and changes in mitochondrial membrane potential were evaluated, respectively, by MitoSOX and JC-1 fluorescence dye signalling. Accumulation of mitophagy markers PTEN-induced putative kinase 1 (PINK1) and Parkin in mitochondria was examined by Western blotting and immunofluorescence microscopy. Osteoblast apoptosis was assessed by Western analysis of cleaved-poly (adenosine diphosphate ribose) polymerase (PARP). In a rat model of induced apical periodontitis, the therapeutic effect of simvastatin and its action on osteoblast mitophagy and apoptosis were examined. anova, Fisher's and Student's t-test were used for data analysis. RESULTS: Hypoxia-induced mitochondrial dysfunction and stimulated mitophagy in osteoblasts. Hypoxia also provoked apoptosis in osteoblasts and inhibition of mitophagy decreased hypoxia-augmented apoptotic activity. Simvastatin alleviated hypoxia-induced mitochondrial dysfunction, mitophagy and apoptosis. The protective action of simvastatin against apoptosis was related to its antimitophagy activity. Experiments in the rat model of induced apical periodontitis supported the laboratory findings. Simvastatin treatment mitigated periapical bone loss and reduced the activities of apoptosis and mitophagy in regional osteoblasts. CONCLUSIONS: The results suggest that modulation of osteoblast mitophagy may help diminish bone loss associated with inflammation and has potential as an auxiliary therapy for apical periodontitis.

Sirtuin 6 attenuates periapical lesion propagation by modulating hypoxia-induced chemokine (C-C motif) ligand 2 production in osteoblasts.

AIM: To investigate the attenuating effect of sirtuin 6 (SIRT6) on hypoxia-induced production of chemokine (C-C motif) ligand 2 (CCL2) by osteoblasts and the relevance of this action on the pathogenesis of periapical lesions. METHODOLOGY: Sirtuin 6 was overexpressed in MC3T3-E1 murine osteoblasts by lentivirus-mediated gene transfer. The relationship between the antiglycolytic/antioxidative activities of SIRT6 and its effect on hypoxia-induced CCL2 production were examined. Pathogenetic relevance of the actions of SIRT6 was assessed in a rat model of induced apical periodontitis. The data were analysed statistically using Student's t-test or one-way analysis of variance (anova) and then a Tukey's multiple comparison test. RESULTS: In cultured murine osteoblasts, 24-h hypoxic treatment significantly enhanced the generation of reactive oxygen species (P = 0.003), expression of lactate dehydrogenase A (LDHA) and production of lactate (P = 0.007). A reciprocal effect between hypoxia-induced redox imbalance and hypoxia-enhanced glycolysis was noted which in turn augmented the secretion of CCL2. Through its antiglycolytic and antioxidative effects, SIRT6 blocked the vicious cycle to suppress CCL2 production. In normal periapical tissues of rats, strong expression of SIRT6 and low levels of LDHA and 8-OHdG (a marker of oxidative DNA damage) were found in osteoblasts. In induced apical periodontitis, osteoblastic expression of SIRT6 was significantly suppressed (P = 0.001) which was associated with significantly elevated levels of LDHA (P = 0.003) and 8-OHdG (P = 0.004) and significantly enhanced recruitment of macrophages (P = 0.004). CONCLUSIONS: Sirtuin 6 has a therapeutic effect on periapical lesions through suppression of CCL2 synthesis. The anti-inflammatory action of SIRT6 is closely related to its regulatory activities in cellular metabolism and redox homoeostasis.

Genome-wide approaches to identify pharmacogenetic contributions to adverse drug reactions.

Adverse drug reactions (ADRs) have a major impact on patients, physicians, health care providers, regulatory agencies and pharmaceutical companies. Identifying the genetic contributions to ADR risk may lead to a better understanding of the underlying mechanisms, identification of patients at risk and a decrease in the number of events. Technological advances have made the routine monitoring and investigation of the genetic basis of ADRs during clinical trials possible. We demonstrate through simulation that genome-wide genotyping, coupled with the use of clinically matched or population controls, can yield sufficient statistical power to permit the identification of strong genetic predictors of ADR risk in a prospective manner with modest numbers of ADR cases. The results of a 500,000 single nucleotide polymorphism analysis of abacavir-associated hypersensitivity reaction suggest that the known HLA-B gene region could be identified with as few as 15 cases and 200 population controls in a sequential analysis.

Pharmacogenetics of hypersensitivity to abacavir: from PGx hypothesis to confirmation to clinical utility.

The hypersensitivity (HSR) to abacavir (ABC) pharmacogenetics (PGx) program represents the progression from an exploratory discovery to a validated biomarker. Within the program, two retrospective PGx studies were conducted to identify HIV-1 patients at increased risk for ABC HSR, a treatment-limiting and potentially life-threatening adverse event. A strong statistical association between the major histocompatibility complex allele, HLA-B*5701, and clinically diagnosed ABC HSR was identified but varied between racial populations. Subsequently, ABC skin patch testing was introduced as a research tool to supplement clinical case ascertainment. In a randomized, prospective study evaluating the clinical utility of HLA-B*5701 screening, avoidance of ABC in HLA-B*5701-positive patients significantly reduced clinically diagnosed ABC HSR and eliminated patch test-positive ABC HSR. Finally, a retrospective PGx study supports the generalizability of the association across races. Prospective HLA-B*5701 screening should greatly reduce the incidence of ABC HSR by identifying patients at high risk for ABC HSR before they are treated.

Use of pairwise marker combination and recursive partitioning in a pharmacogenetic genome-wide scan.

The objective of pharmacogenetic research is to identify a genetic marker, or a set of genetic markers, that can predict how a given person will respond to a given medicine. To search for such marker combinations that are predictive of adverse drug events, we have developed and applied two complementary methods to a pharmacogenetic study of the hypersensitivity reaction (HSR) associated with treatment with abacavir, a medicine that is used to treat HIV-infected patients. Our results show that both of these methods can be used to uncover potentially useful predictive marker combinations. The pairwise marker combination method yielded a collection of marker pairs that featured a spectrum of sensitivities and specificities. Recursive partitioning results led to the genetic delineation of multiple risk categories, including those with extremely high and extremely low risk of HSR. These methods can be readily applied in pharmacogenetic candidate gene studies as well as in genome-wide scans.

Further investigation of linkage disequilibrium SNPs and their ability to identify associated susceptibility loci.

There is currently considerable interest in the use of single-nucleotide polymorphisms (SNPs) to map disease susceptibility genes. The success of this method will depend on a number of factors including the strength of linkage disequilibrium (LD) between marker and disease loci. We used a data set of SNP genotypings in the region of the APOE disease susceptibility locus to investigate the likely usefulness of SNPs in case-control studies. Using the estimated haplotype structure surrounding and including the APOE locus, and assuming a codominant disease model, we treated each SNP in turn as if it were a disease susceptibility locus and obtained, for each disease locus and markers, the expected likelihood ratio test (LRT) to assess disease association. We were particularly interested in the power to detect association with the susceptibility polymorphism itself, the power of nearby markers to detect association, and the ability to distinguish between the susceptibility polymorphism and marker loci also showing association. We found that the expected LRT depended critically on disease allele frequencies. For disease loci with a reasonably common allele we were usually able to detect association. However, for only a subset of markers in the close neighbourhood of the disease locus was association detectable. In these cases we were usually, but not always, able to distinguish the disease locus from nearby associated marker loci. For some disease loci, no other loci demonstrated detectable association with the disease phenotype. We conclude that one may need to use very dense SNP maps in order to avoid overlooking polymorphisms affecting susceptibility to a common phenotype.

SNPing away at complex diseases: analysis of single-nucleotide polymorphisms around APOE in Alzheimer disease.

There has been great interest in the prospects of using single-nucleotide polymorphisms (SNPs) in the search for complex disease genes, and several initiatives devoted to the identification and mapping of SNPs throughout the human genome are currently underway. However, actual data investigating the use of SNPs for identification of complex disease genes are scarce. To begin to look at issues surrounding the use of SNPs in complex disease studies, we have initiated a collaborative SNP mapping study around APOE, the well-established susceptibility gene for late-onset Alzheimer disease (AD). Sixty SNPs in a 1.5-Mb region surrounding APOE were genotyped in samples of unrelated cases of AD, in controls, and in families with AD. Standard tests were conducted to look for association of SNP alleles with AD, in cases and controls. We also used family-based association analyses, including recently developed methods to look for haplotype association. Evidence of association (P

Genomewide search for type 2 diabetes susceptibility genes in four American populations.

Type 2 diabetes is a serious, genetically influenced disease for which no fully effective treatments are available. Identification of biochemical or regulatory pathways involved in the disease syndrome could lead to innovative therapeutic interventions. One way to identify such pathways is the genetic analysis of families with multiple affected members where disease predisposing genes are likely to be segregating. We undertook a genomewide screen (389-395 microsatellite markers) in samples of 835 white, 591 Mexican American, 229 black, and 128 Japanese American individuals collected as part of the American Diabetes Association's GENNID study. Multipoint nonparametric linkage analyses were performed with diabetes, and diabetes or impaired glucose homeostasis (IH). Linkage to diabetes or IH was detected near markers D5S1404 (map position 77 cM, LOD = 2.80), D12S853 (map position 82 cM, LOD = 2.81) and GATA172D05 (X-chromosome map position 130 cM, LOD = 2.99) in whites, near marker D3S2432 (map position 51 cM, LOD = 3.91) in Mexican Americans, and near marker D10S1412 (map position 14 cM, LOD = 2.39) in African Americans mainly collected in phase 1 of the study. Further analyses showed evidence for interactions between the chromosome 5 locus and region on chromosome 12 containing the MODY 3 gene (map position 132 cM) and between the X-chromosome locus and region near D12S853 (map position 82 cM) in whites. Although these results were not replicated in samples collected in phase 2 of the GENNID study, the region on chromosome 12 was replicated in samples from whites described by Bektas et al. (1999).

Analysis of association at single nucleotide polymorphisms in the APOE region.

The discussion of the prospects of using a dense map of single nucleotide polymorphisms (SNPs) to identify disease genes with association analysis has been extensive. However, there is little empiric evidence to support this strategy. To begin to examine the practical issues surrounding this methodology, we identified 10 SNPs in the region immediately surrounding the apolipoprotein E locus (APOE), an established susceptibility gene for Alzheimer disease. Our goal was to examine patterns of allelic association to begin to investigate the question of whether APOE could have been identified using SNPs. Our strongest evidence of association was at the 2 SNPs immediately flanking APOE.

From amplification to gene in thyroid cancer: a high-resolution mapped bacterial-artificial-chromosome resource for cancer chromosome aberrations guides gene discovery after comparative genome hybridization.

Chromosome rearrangements associated with neoplasms provide a rich resource for definition of the pathways of tumorigenesis. The power of comparative genome hybridization (CGH) to identify novel genes depends on the existence of suitable markers, which are lacking throughout most of the genome. We now report a general approach that translates CGH data into higher-resolution genomic-clone data that are then used to define the genes located in aneuploid regions. We used CGH to study 33 thyroid-tumor DNAs and two tumor-cell-line DNAs. The results revealed amplifications of chromosome band 2p21, with less-intense amplification on 2p13, 19q13.1, and 1p36 and with least-intense amplification on 1p34, 1q42, 5q31, 5q33-34, 9q32-34, and 14q32. To define the 2p21 region amplified, a dense array of 373 FISH-mapped chromosome 2 bacterial artificial chromosomes (BACs) was constructed, and 87 of these were hybridized to a tumor-cell line. Four BACs carried genomic DNA that was amplified in these cells. The maximum amplified region was narrowed to 3-6 Mb by multicolor FISH with the flanking BACs, and the minimum amplicon size was defined by a contig of 420 kb. Sequence analysis of the amplified BAC 1D9 revealed a fragment of the gene, encoding protein kinase C epsilon (PKCepsilon), that was then shown to be amplified and rearranged in tumor cells. In summary, CGH combined with a dense mapped resource of BACs and large-scale sequencing has led directly to the definition of PKCepsilon as a previously unmapped candidate gene involved in thyroid tumorigenesis.

Inhibition of phosphorylcholine binding to antibodies using synthetic peptides.

The amino-acid sequence Phe-Tyr-Met-Glu is unique to phosphorylcholine (PC)-binding antibodies. It occurs in the first complementarity-determining region (CDR1) of the immunoglobulin heavy chains in 89% of all the anti-PC myeloma and hybridoma proteins but is not present in 490 other immunoglobulin heavy chains, 854 light chains or in 2,260 other unrelated proteins. This unique tetrapeptide therefore seems to be involved in PC binding. Here we compare the effectiveness of Phe-Tyr-Met-Glu and other structurally related peptides in inhibiting the binding of PC to PC-binding proteins McPC603 and HOPC8. We also test a surface-simulation peptide that was constructed to mimic the combining site of McPC603. Our data suggest that all these peptides inhibit the binding of PC to PC-binding proteins non-specifically and we show by computer modelling that the surface-simulation peptide does not duplicate the combining site of McPC603.