Family History of MI, Smoking, and Risk of Periodontal Disease.

Periodontal disease (PD) shares common risk factors with cardiovascular disease. Our hypothesis was that having a family history of myocardial infarction (FamHxMI) may be a novel risk factor for PD. Risk assessment based on FamHxMI, conditional on smoking status, was examined given the strong influence of smoking on PD. Exploratory analysis with inflammatory biomarkers and genetic determinants was conducted to understand potential mechanistic links. The Women's Genome Health Study (WGHS) is a prospective cohort of US female health care professionals who provided blood samples at baseline in the Women's Health Study, a 2 × 2 factorial clinical trial investigating vitamin E and aspirin in the prevention of cardiovascular disease and cancer. PD was ascertained via self-report over 12 y of follow-up. Prevalence (3,442 cases), incidence (1,365 cases), and survival analysis of PD were investigated for associations of FamHxMI as well as in strata of FamHxMI by smoking. Kruskal-Wallis, chi-square tests, multivariate regression, and Cox proportional hazard models were used for the analyses. In the WGHS, women with FamHxMI showed higher risk of ever having PD. A particularly high-risk group of having both FamHxMI and smoking at baseline was highlighted in the prevalence and risk of developing PD. PD risk increased according to the following strata: no FamHxMI and nonsmokers (reference), FamHxMI and nonsmokers (hazard ratio [HR] = 1.2, 95% CI = 1.0 to 1.5), smokers without FamHxMI (HR = 1.3, 95% CI = 1.2 to 1.5), and smokers with FamHxMI (HR = 1.5, 95% CI = 1.2 to 1.8). An independent analysis by the dental Atherosclerosis Risk in Communities study ( N = 5,552) identified more severe periodontitis cases among participants in the high-risk group (smokers with FamHxMI). Further examination of interactions among inflammatory biomarkers or genetic exploration with FamHxMI did not explain the risk increase of PD associated with FamHxMI in the WGHS. Future efforts based on an integrative-omics approach may facilitate validation of these findings and suggest a mechanistic link between PD and FamHxMI.

Association of interleukin-1 gene variations with moderate to severe chronic periodontitis in multiple ethnicities.

BACKGROUND AND OBJECTIVE: Genetic markers associated with disease are often non-functional and generally tag one or more functional "causative" variants in linkage disequilibrium. Markers may not show tight linkage to the causative variants across multiple ethnicities due to evolutionary divergence, and therefore may not be informative across different population groups. Validated markers of disease suggest causative variants exist in the gene and, if the causative variants can be identified, it is reasonable to hypothesize that such variants will be informative across diverse populations. The aim of this study was to test that hypothesis using functional Interleukin-1 (IL-1) gene variations across multiple ethnic populations to replace the non-functional markers originally associated with chronic adult periodontitis in Caucasians. MATERIAL AND METHODS: Adult chronic periodontitis cases and controls from four ethnic groups (Caucasians, African Americans, Hispanics and Asians) were recruited in the USA, Chile and China. Genotypes of IL1B gene single nucleotide polymorphisms (SNPs), including three functional SNPs (rs16944, rs1143623, rs4848306) in the promoter and one intronic SNP (rs1143633), were determined using a single base extension method or TaqMan 5' nuclease assay. Logistic regression and other statistical analyses were used to examine the association between moderate to severe periodontitis and IL1B gene variations, including SNPs, haplotypes and composite genotypes. Genotype patterns associated with disease in the discovery study were then evaluated in independent validation studies. RESULTS: Significant associations were identified in the discovery study, consisting of Caucasians and African Americans, between moderate to severe adult chronic periodontitis and functional variations in the IL1B gene, including a pattern of four IL1B SNPs (OR = 1.87, p < 0.0001). The association between the disease and this IL1B composite genotype pattern was validated in two additional studies consisting of Hispanics (OR = 1.95, p = 0.04) or Asians (OR = 3.27, p = 0.01). A meta-analysis of the three populations supported the association between the IL-1 genotype pattern and moderate to severe periodontitis (OR 1.95; p < 0.001). Our analysis also demonstrated that IL1B gene variations had added value to conventional risk factors in predicting chronic periodontitis. CONCLUSION: This study validated the influence of IL-1 genetic factors on the severity of chronic periodontitis in four different ethnicities.

Patient stratification for preventive care in dentistry.

Prevention reduces tooth loss, but little evidence supports biannual preventive care for all adults. We used risk-based approaches to test tooth loss association with 1 vs. 2 annual preventive visits in high-risk (HiR) and low-risk (LoR) patients. Insurance claims for 16 years for 5,117 adults were evaluated retrospectively for tooth extraction events. Patients were classified as HiR for progressive periodontitis if they had ≥ 1 of the risk factors (RFs) smoking, diabetes, interleukin-1 genotype; or as LoR if no RFs. LoR event rates were 13.8% and 16.4% for 2 or 1 annual preventive visits (absolute risk reduction, 2.6%; 95%CI, 0.5% to 5.8%; p = .092). HiR event rates were 16.9% and 22.1% for 2 and 1 preventive visits (absolute risk reduction, 5.2%; 95%CI, 1.8% to 8.4%; p = .002). Increasing RFs increased events (p < .001). Oral health care costs were not increased by any single RF, regardless of prevention frequency (p > .41), but multiple RFs increased costs vs. no (p < .001) or 1 RF (p = .001). For LoR individuals, the association between preventive dental visits and tooth loss was not significantly different whether the frequency was once or twice annually. A personalized medicine approach combining gene biomarkers with conventional risk factors to stratify populations may be useful in resource allocation for preventive dentistry (ClinicalTrials.gov, NCT01584479).

A protein-protein interaction map of the Caenorhabditis elegans 26S proteasome.

The ubiquitin-proteasome proteolytic pathway is pivotal in most biological processes. Despite a great level of information available for the eukaryotic 26S proteasome-the protease responsible for the degradation of ubiquitylated proteins-several structural and functional questions remain unanswered. To gain more insight into the assembly and function of the metazoan 26S proteasome, a two-hybrid-based protein interaction map was generated using 30 Caenorhabditis elegans proteasome subunits. The results recapitulate interactions reported for other organisms and reveal new potential interactions both within the 19S regulatory complex and between the 19S and 20S subcomplexes. Moreover, novel potential proteasome interactors were identified, including an E3 ubiquitin ligase, transcription factors, chaperone proteins and other proteins not yet functionally annotated. By providing a wealth of novel biological hypotheses, this interaction map constitutes a framework for further analysis of the ubiquitin-proteasome pathway in a multicellular organism amenable to both classical genetics and functional genomics.

Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum.

The genome sequence of the solvent-producing bacterium Clostridium acetobutylicum ATCC 824 has been determined by the shotgun approach. The genome consists of a 3.94-Mb chromosome and a 192-kb megaplasmid that contains the majority of genes responsible for solvent production. Comparison of C. acetobutylicum to Bacillus subtilis reveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases closer, phylogenetic proximity. This conservation allows the prediction of many previously undetected operons in both bacteria. However, the C. acetobutylicum genome also contains a significant number of predicted operons that are shared with distantly related bacteria and archaea but not with B. subtilis. Phylogenetic analysis is compatible with the dissemination of such operons by horizontal transfer. The enzymes of the solventogenesis pathway and of the cellulosome of C. acetobutylicum comprise a new set of metabolic capacities not previously represented in the collection of complete genomes. These enzymes show a complex pattern of evolutionary affinities, emphasizing the role of lateral gene exchange in the evolution of the unique metabolic profile of the bacterium. Many of the sporulation genes identified in B. subtilis are missing in C. acetobutylicum, which suggests major differences in the sporulation process. Thus, comparative analysis reveals both significant conservation of the genome organization and pronounced differences in many systems that reflect unique adaptive strategies of the two gram-positive bacteria.

Open-reading-frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans.

The genome sequences of Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana have been predicted to contain 19,000, 13,600 and 25,500 genes, respectively. Before this information can be fully used for evolutionary and functional studies, several issues need to be addressed. First, the gene number estimates obtained in silico and not yet supported by any experimental data need to be verified. For example, it seems biologically paradoxical that C. elegans would have 50% more genes than Drosophilia. Second, intron/exon predictions need to be tested experimentally. Third, complete sets of open reading frames (ORFs), or "ORFeomes," need to be cloned into various expression vectors. To address these issues simultaneously, we have designed and applied to C. elegans the following strategy. Predicted ORFs are amplified by PCR from a highly representative cDNA library using ORF-specific primers, cloned by Gateway recombination cloning and then sequenced to generate ORF sequence tags (OSTs) as a way to verify identity and splicing. In a sample (n=1,222) of the nearly 10,000 genes predicted ab initio (that is, for which no expressed sequence tag (EST) is available so far), at least 70% were verified by OSTs. We also observed that 27% of these experimentally confirmed genes have a structure different from that predicted by GeneFinder. We now have experimental evidence that supports the existence of at least 17,300 genes in C. elegans. Hence we suggest that gene counts based primarily on ESTs may underestimate the number of genes in human and in other organisms.

Complete genome sequence of Methanobacterium thermoautotrophicum deltaH: functional analysis and comparative genomics.

The complete 1,751,377-bp sequence of the genome of the thermophilic archaeon Methanobacterium thermoautotrophicum deltaH has been determined by a whole-genome shotgun sequencing approach. A total of 1,855 open reading frames (ORFs) have been identified that appear to encode polypeptides, 844 (46%) of which have been assigned putative functions based on their similarities to database sequences with assigned functions. A total of 514 (28%) of the ORF-encoded polypeptides are related to sequences with unknown functions, and 496 (27%) have little or no homology to sequences in public databases. Comparisons with Eucarya-, Bacteria-, and Archaea-specific databases reveal that 1,013 of the putative gene products (54%) are most similar to polypeptide sequences described previously for other organisms in the domain Archaea. Comparisons with the Methanococcus jannaschii genome data underline the extensive divergence that has occurred between these two methanogens; only 352 (19%) of M. thermoautotrophicum ORFs encode sequences that are >50% identical to M. jannaschii polypeptides, and there is little conservation in the relative locations of orthologous genes. When the M. thermoautotrophicum ORFs are compared to sequences from only the eucaryal and bacterial domains, 786 (42%) are more similar to bacterial sequences and 241 (13%) are more similar to eucaryal sequences. The bacterial domain-like gene products include the majority of those predicted to be involved in cofactor and small molecule biosyntheses, intermediary metabolism, transport, nitrogen fixation, regulatory functions, and interactions with the environment. Most proteins predicted to be involved in DNA metabolism, transcription, and translation are more similar to eucaryal sequences. Gene structure and organization have features that are typical of the Bacteria, including genes that encode polypeptides closely related to eucaryal proteins. There are 24 polypeptides that could form two-component sensor kinase-response regulator systems and homologs of the bacterial Hsp70-response proteins DnaK and DnaJ, which are notably absent in M. jannaschii. DNA replication initiation and chromosome packaging in M. thermoautotrophicum are predicted to have eucaryal features, based on the presence of two Cdc6 homologs and three histones; however, the presence of an ftsZ gene indicates a bacterial type of cell division initiation. The DNA polymerases include an X-family repair type and an unusual archaeal B type formed by two separate polypeptides. The DNA-dependent RNA polymerase (RNAP) subunits A', A", B', B" and H are encoded in a typical archaeal RNAP operon, although a second A' subunit-encoding gene is present at a remote location. There are two rRNA operons, and 39 tRNA genes are dispersed around the genome, although most of these occur in clusters. Three of the tRNA genes have introns, including the tRNAPro (GGG) gene, which contains a second intron at an unprecedented location. There is no selenocysteinyl-tRNA gene nor evidence for classically organized IS elements, prophages, or plasmids. The genome contains one intein and two extended repeats (3.6 and 8.6 kb) that are members of a family with 18 representatives in the M. jannaschii genome.

Multiplex sequencing of 1.5 Mb of the Mycobacterium leprae genome.

The nucleotide sequence of 1.5 Mb of genomic DNA from Mycobacterium leprae was determined using computer-assisted multiplex sequencing technology. This brings the 2.8-Mb M. leprae genome sequence to approximately 66% completion. The sequences, derived from 43 recombinant cosmids, contain 1046 putative protein-coding genes, 44 repetitive regions, 3 tRNAs, and 15 tRNAs. The gene density of one per 1.4 kb is slightly lower than that of Mycoplasma (1.2 kb). Of the protein coding genes, 44% have significant matches to genes with well-defined functions. Comparison of 1157 M. leprae and 1564 Mycobacterium tuberculosis proteins shows a complex mosaic of homologous genomic blocks with up to 22 adjacent proteins in conserved map order. Matches to known enzymatic, antigenic, membrane, cell wall, cell division, multidrug resistance, and virulence proteins suggest therapeutic and vaccine targets. Unusual features of the M. leprae genome include large polyketide synthase (pks) operons, inteins, and highly fragmented pseudogenes.

Population genetic study of the human dopamine transporter gene (DAT1).

The human dopamine transporter gene, DAT1, acts to transport released dopamine into presynaptic terminals of the brain. The possibility that the DAT1 gene plays a role in genetic diseases of the brain has led to studies of DAT1 in several psychiatric and neurological disorders. Previous sequence analysis of DAT1 revealed a 40-bp repeat in the 3' end of the gene. In order to identify all potential alleles for this VNTR marker a population database was established. One thousand seventy-four unrelated individuals were screened by PCR for the region containing the 40 bp repeat. Allele frequency differences were found between black Americans and Caucasians or Hispanics but no differences were observed between Caucasians and Hispanics. A previously unreported allele was detected in all three populations. Thus, we have shown that screening a large population identifies new alleles and generates more accurate allele frequencies.

The isolation of cDNAs within the Huntington disease region by hybridisation of yeast artificial chromosomes to a cDNA library.

Flanking recombination events have defined the Huntington Disease gene candidate region to between D4S10 and D4S98, about 2.2Mb. Because of the large size of the candidate region and the likely large number of genes within it we decided to screen cDNA libraries with probes generated from whole Yeast Artificial Chromosomes (YACs) containing parts of this region. We have thus far used 4 YACs ranging in size from 180kb to 600kb covering 880kb and have isolate 13 cDNA clones, 7 of which are unique. Three of the 13 clones contain parts of the 3' untranslated region of the alpha-adducin gene. One YAC of 600kb could not be purified from two yeast chromosomes, therefore this YAC probe had a net complexity of 1.8Mb. Even so this probe identified a cDNA from the HD candidate region indicating that very large YACs may be used as probes.

Sequence-tagged sites (STSs) spanning 4p16.3 and the Huntington disease candidate region.

The generation of sequence-tagged sites (STSs) has been proposed as a unifying approach to correlating the disparate results generated by genetic and various physical techniques being used to map the human genome. We have developed an STS map to complement the existing physical and genetic maps of 4p16.3, the region containing the Huntington disease gene. A total of 18 STSs span over 4 Mb of 4p16.3, with an average spacing of about 250 kb. Eleven of the STSs are located within the primary candidate HD region of 2.5 Mb between D4S126 and D4S168. The availability of STSs makes the corresponding loci accessibility to the general community without the need for distribution of cloned DNA. These STSs should also provide the means to isolate yeast artificial chromosome clones spanning the HD candidate region.

Site-specific cleavage of human chromosome 4 mediated by triple-helix formation.

Direct physical isolation of specific DNA segments from the human genome is a necessary goal in human genetics. For testing whether triple-helix mediated enzymatic cleavage can liberate a specific segment of a human chromosome, the tip of human chromosome 4, which contains the entire candidate region for the Huntington's disease gene, was chosen as a target. A 16-base pyrimidine oligodeoxyribonucleotide was able to locate a 16-base pair purine target site within more than 10 gigabase pairs of genomic DNA and mediate the exact enzymatic cleavage at that site in more than 80 percent yield. The recognition motif is sufficiently generalizable that most cosmids should contain a sequence targetable by triple-helix formation. This method may facilitate the orchestrated dissection of human chromosomes from normal and affected individuals into megabase sized fragments and facilitate the isolation of candidate gene loci.

Generation and characterization of irradiation hybrids of human chromosome 4.

In recent years investigators have attempted to develop more rapid and precise methods to isolate specific chromosomal DNA regions. In this paper we demonstrate a modification of the method first developed by Goss and Harris for generation of irradiation hybrids. The gene encoding the dominant selectable marker for resistance to neomycin was introduced into human chromosome 4 using retroviral insertion into human fibroblasts. Transfer of these chromosomes via microcells into the mouse cell line NIH3T6 produced a somatic cell line containing chromosome 4 as the only human chromosome. Irradiation of this cell line followed by fusion with the hamster cell line CHTG49 generated hybrids containing only small portions of chromosome 4p on a hamster background. The use of selection produced stable hybrids that retained chromosome 4 fragments over long periods of tissue culture passage. To obtain new polymorphic markers for Huntington's disease, one of these hybrids was to isolate new genomic fragments. We identified 41 single-copy fragments, of which 27 have been mapped to specific regions of chromosome 4; 52% of these fragments map to the region of chromosome 4 containing the HD gene.