A Military Community Cohort Study Reveals Single Nucleotide Polymorphisms in Inflammation Mediator Genes That Associate With Type 2 Diabetes.

INTRODUCTION: Type 2 diabetes mellitus (T2DM) is a prevalent metabolic disorder characterized by hyperglycemia of varying degrees. Genetic and lifestyle variations are known to influence the onset and severity of T2DM. Among the genetic variations reported to confer susceptibility to the disease are certain single nucleotide polymorphisms (SNPs). Here, we report the analysis of 18 such SNPs in a military community cohort of 716 subjects, comprising 477 diabetic and 239 control subjects. The population studied included active-duty military personnel, veterans, and their families. The SNPs analyzed in this work occur in nine different genes, comprising six interleukin (IL) genes (IL1A, IL1B, IL4, IL6, IL10, and IL18), fatty acid amide hydrolase (FAAH) gene, and cannabinoid receptors 1 and 2 genes (CNR1, CNR2). The products of these genes are players in different conditions, including inflammation, a process linked with diabetes. MATERIALS AND METHODS: The T2DM and control (no diabetes) DNA samples were acquired from an archived sample repository (Center for Advanced Molecular Detection, 59th Medical Wing, U.S. Air Force, Joint Base San Antonio [JBSA]-Lackland, TX). The blood samples had been previously collected from gender- and race-mixed cohorts under a protocol approved by the 59th Medical Wing Institutional Review Board. Single nucleotide polymorphism (SNP) genotyping was done by real-time Polymerase Chain Reaction (PCR) using TaqMan assay reagents. The statistical analysis software 9.3 (SAS 9.3) was used for statistical analyses to reveal associations between the SNP genotypes and T2DM. RESULTS: Out of the 18 SNPs analyzed, six showed statistically significant association with T2DM in the overall cohort (P < .05). The odds ratio for these associations varied from 1.57 to 3.16. The rs16944 T/T homozygous genotype (IL1B) showed the strongest association with T2DM, with P = .005. In the White cohort, five of these six SNPs and one other, rs806368 (cannabinoid receptor 1), associate with T2DM. However, the gender-specific analysis of the White cohort revealed only two SNP associations with T2DM in the female cohort, rs16944 (IL1B) and rs2295632 (FAAH), both also showing association in the overall mixed cohort. Likewise, four SNPs showed T2DM association in the White male cohort, with rs187238 (IL18) being uniquely significant in this group. CONCLUSIONS: The IL1B SNP rs16944 showed consistent statistically significant association with T2DM and therefore is likely a promising biomarker for T2DM. We note, however, that this association in a generic sense may be with the inflammatory process that accompanies T2DM and not per se with T2DM.

Identification of Novel Protein Expression Changes Following Cisplatin Treatment and Application to Combination Therapy.

Determining the effect of chemotherapeutic treatment on changes in protein expression can provide important targets for overcoming resistance. Due to challenges in simultaneously measuring large numbers of proteins, a paucity of data exists on global changes. To overcome these challenges, we utilized microwestern arrays that allowed us to measure the abundance and modification state of hundreds of cell signaling and transcription factor proteins in cells following drug exposure. HapMap lymphoblastoid cell lines (LCLs) were exposed to cisplatin, a chemotherapeutic agent commonly used to treat testicular, head and neck, non-small cell lung, and gynecological cancers. We evaluated the expression of 259 proteins following 2, 6, and 12 h of cisplatin treatment in two LCLs with discordant sensitivity to cisplatin. Of these 259 proteins, 66 displayed significantly different protein expression changes (p < 0.05). Fifteen of these proteins were evaluated in a second pair of LCLs with discordant sensitivities to cisplatin; six demonstrated significant differences in expression. We then evaluated a subset of 63 proteins in a second set of LCLs with discordant sensitivity, and 40% of those that were significant in the first pair were also significant in the second part with concordant directionality (p < 0.05). We functionally validated one of the top proteins identified, PDK1, and demonstrated a synergistic relationship between cisplatin and a PDK1 inhibitor in multiple lung cancer lines. This study highlights the potential for identifying novel targets through an understanding of cellular changes in protein expression and modification following drug treatments.

Identification and validation of genetic variants that influence transcription factor and cell signaling protein levels.

Many genetic variants associated with human disease have been found to be associated with alterations in mRNA expression. Although it is commonly assumed that mRNA expression changes will lead to consequent changes in protein levels, methodological challenges have limited our ability to test the degree to which this assumption holds true. Here, we further developed the micro-western array approach and globally examined relationships between human genetic variation and cellular protein levels. We collected more than 250,000 protein level measurements comprising 441 transcription factor and signaling protein isoforms across 68 Yoruba (YRI) HapMap lymphoblastoid cell lines (LCLs) and identified 12 cis and 160 trans protein level QTLs (pQTLs) at a false discovery rate (FDR) of 20%. Whereas up to two thirds of cis mRNA expression QTLs (eQTLs) were also pQTLs, many pQTLs were not associated with mRNA expression. Notably, we replicated and functionally validated a trans pQTL relationship between the KARS lysyl-tRNA synthetase locus and levels of the DIDO1 protein. This study demonstrates proof of concept in applying an antibody-based microarray approach to iteratively measure the levels of human proteins and relate these levels to human genome variation and other genomic data sets. Our results suggest that protein-based mechanisms might functionally buffer genetic alterations that influence mRNA expression levels and that pQTLs might contribute phenotypic diversity to a human population independently of influences on mRNA expression.

Protein quantitative trait loci identify novel candidates modulating cellular response to chemotherapy.

Annotating and interpreting the results of genome-wide association studies (GWAS) remains challenging. Assigning function to genetic variants as expression quantitative trait loci is an expanding and useful approach, but focuses exclusively on mRNA rather than protein levels. Many variants remain without annotation. To address this problem, we measured the steady state abundance of 441 human signaling and transcription factor proteins from 68 Yoruba HapMap lymphoblastoid cell lines to identify novel relationships between inter-individual protein levels, genetic variants, and sensitivity to chemotherapeutic agents. Proteins were measured using micro-western and reverse phase protein arrays from three independent cell line thaws to permit mixed effect modeling of protein biological replicates. We observed enrichment of protein quantitative trait loci (pQTLs) for cellular sensitivity to two commonly used chemotherapeutics: cisplatin and paclitaxel. We functionally validated the target protein of a genome-wide significant trans-pQTL for its relevance in paclitaxel-induced apoptosis. GWAS overlap results of drug-induced apoptosis and cytotoxicity for paclitaxel and cisplatin revealed unique SNPs associated with the pharmacologic traits (at p<0.001). Interestingly, GWAS SNPs from various regions of the genome implicated the same target protein (p<0.0001) that correlated with drug induced cytotoxicity or apoptosis (p ≤ 0.05). Two genes were functionally validated for association with drug response using siRNA: SMC1A with cisplatin response and ZNF569 with paclitaxel response. This work allows pharmacogenomic discovery to progress from the transcriptome to the proteome and offers potential for identification of new therapeutic targets. This approach, linking targeted proteomic data to variation in pharmacologic response, can be generalized to other studies evaluating genotype-phenotype relationships and provide insight into chemotherapeutic mechanisms.

Association between MUC5B and TERT polymorphisms and different interstitial lung disease phenotypes.

TERT and MUC5B polymorphisms have been associated consistently with idiopathic pulmonary fibrosis (IPF) in recent genomewide genetic studies. However, it remains unclear how both loci contribute to the susceptibility to different entities of sporadic interstitial lung disease (ILD). We sought to test the associations of the 2 polymorphisms with IPF and non-IPF ILD entities in a white population. Associations between 2 polymorphisms in TERT (rs2736100) and MUC5B (rs35705950) and IPF or non-IPF sporadic ILD were tested using 227 patients with ILD and 689 control subjects. Genotypic data were also correlated with pulmonary functions measured in patients with ILD. As a result, rs2736100 and rs35705950 were associated significantly and independently with ILD as a single phenotype (Odds ratio [OR], 1.29; 95% confidence interval [CI], 1.04-1.60; P = 2 × 10(-2); and OR, 2.22; 95% CI, 1.69-2.92; P = 7 × 10(-9); respectively). When considering IPF and "other ILD" (non-IPF) separately, rs35705950 had a stronger association with IPF (OR, 3.2; 95% CI, 2.21-4.63; P = 1.2 × 10(-10)) than with other ILD (OR, 1.72; 95% CI, 1.22-2.42; P = 1.2 × 10(-3)). In contrast, rs2736100 was associated with other ILD (OR, 1.43; 95% CI, 1.11-1.85; P = 6.2 × 10(-3)) but not with IPF (OR, 1.08; 95% CI, 0.78-1.49; P > 0.05). Rs35705950 correlated significantly with increased pulmonary function (P < 0.05). It was also associated with ILD without airflow obstruction in both the IPF and other ILD groups (P < 0.01 for both), and conferred the highest risk for IPF without airflow obstruction (OR, 4.46; 95% CI, 2.60-7.66; P = 4.5 × 10(-9)). Our study suggests that although both loci confer independent risks for ILD, rs35705950 may, in particular, contribute differentially to IPF and other ILD entities. Our study further highlights the genetic and phenotypic heterogeneity of ILD.

A pharmacogenetic study of aldehyde oxidase I in patients treated with XK469.

XK469 (NSC 697887) is a selective topoisomerase II ß inhibitor eliminated mainly by aldehyde oxidase I (AOX1). We performed a candidate gene study to investigate whether AOX1 genetic variation contributes to interindividual variability in XK469 clearance. Forty-one AOX1 single nucleotide polymorphisms (SNPs) and seven liver expression quantitative trait loci were genotyped in White patients with advanced refractory solid tumors (n=59) and leukemia (n=33). We found a significant decrease in clearance (τ=-0.32, P=0.003) in solid tumor patients with rs10931910, although it failed to replicate in the leukemia cohort (τ=0.18, P=0.20). Four other AOX1 SNPs were associated with clearance (P=0.01-0.02) in only one of the two cohorts. Our study provides a starting point for future investigations on the functionality of AOX1 SNPs. However, variability in XK469 clearance cannot be attributed to polymorphisms in AOX1.

Linkage and association of successful aging to the 6q25 region in large Amish kindreds.

Successful aging (SA) is a multidimensional phenotype involving living to older age with high physical function, preserved cognition, and continued social engagement. Several domains underlying SA are heritable, and identifying health-promoting polymorphisms and their interactions with the environment could provide important information regarding the health of older adults. In the present study, we examined 263 cognitively intact Amish individuals age 80 and older (74 SA and 189 "normally aged") all of whom are part of a single 13-generation pedigree. A genome-wide association study of 630,309 autosomal single nucleotide polymorphisms (SNPs) was performed and analyzed for linkage using multipoint analyses and for association using the modified quasi-likelihood score test. There was evidence for linkage on 6q25-27 near the fragile site FRA6E region with a dominant model maximum multipoint heterogeneity LOD score = 3.2. The 1-LOD-down support interval for this linkage contained one SNP for which there was regionally significant evidence of association (rs205990, p = 2.36 × 10(-5)). This marker survived interval-wide Bonferroni correction for multiple testing and was located between the genes QKI and PDE10A. Other areas of chromosome 6q25-q27 (including the FRA6E region) contained several SNPs associated with SA (minimum p = 2.89 × 10(-6)). These findings suggest potentially novel genes in the 6q25-q27 region linked and associated with SA in the Amish; however, these findings should be verified in an independent replication cohort.

SNPs affecting serum metabolomic traits may regulate gene transcription and lipid accumulation in the liver.

OBJECTIVE: Circulatory metabolites are important biomarkers for many diseases, especially metabolic disorders. The biological mechanism regulating circulatory levels of metabolites remains incompletely understood. Focusing on the liver as the central organ controlling metabolic homeostasis, we investigated the potential function of nine polymorphisms associated with serum metabolomic traits in a recent GWAS. MATERIALS/METHODS: The mRNA levels of the associated genes were measured by real-time PCR and correlated with genotypes in normal liver tissue (n=42). Genotype and mRNA data were also correlated with total hepatic lipid content (HLC). Our findings were also compared with the previously published gene expression quantitative traits loci (eQTL) data in the liver. RESULTS: We found that seven out of nine genes were highly expressed in hepatic tissue, while expression of four genes was significantly or marginally associated with genotypes (SPTLC3 vs rs168622, P=.002; ACADS vs rs2014355, P=.016; PLEKHH1 vs rs7156144, P=.076; ACADL vs rs2286963, P=.068). The SNP rs168622 at the SPTLC3 locus was also significantly correlated with HLC (P=.02). HLC was significantly correlated with FADS1 (r=-0.45; P=.003) and ETFDH (r=0.33; P=.037) expression. When compared with published eQTL data, SNPs in SPTLC3, ACADS, ELOVL2 and FADS1 were also in strong linkage disequilibrium (R(2)≥0.41, D'≥0.96) with eQTLs significantly affecting expression of these genes (P≤1.74×10(-5)). CONCLUSIONS: Our study suggests that genetic variants affecting serum metabolites levels may play a functional role in the liver. This may help elucidate the mechanism by which genetic variants are involved in metabolic diseases.

MCP1 SNPs and pulmonary tuberculosis in cohorts from West Africa, the USA and Argentina: lack of association or epistasis with IL12B polymorphisms.

The monocyte chemotactic protein-1 (MCP-1) is a chemokine that plays an important role in the recruitment of monocytes to M. tuberculosis infection sites, and previous studies have reported that genetic variants in MCP1 are associated with differential susceptibility to pulmonary tuberculosis (PTB). We examined eight MCP1 single nucleotide polymorphisms (SNPs) in a multi-ethnic, case-control design that included: 321 cases and 346 controls from Guinea-Bissau, 258 cases and 271 controls from The Gambia, 295 cases and 179 controls from the U.S. (African-Americans), and an additional set of 237 cases and 144 controls of European ancestry from the U.S. and Argentina. Two locus interactions were also examined for polymorphisms in MCP1 and interleukin 12B (IL12B), another gene implicated in PTB risk. Examination of previously associated MCP1 SNPs rs1024611 (-2581A/G), rs2857656 (-362G/C) and rs4586 (+900C/T) did not show evidence for association. One interaction between rs2857656 and IL12B SNP rs2288831 was observed among Africans but the effect was in the opposite direction in Guineans (OR = 1.90, p = 0.001) and Gambians (OR = 0.64, p = 0.024). Our data indicate that the effect of genetic variation within MCP1 is not clear cut and additional studies will be needed to elucidate its role in TB susceptibility.

Interleukin 12B (IL12B) genetic variation and pulmonary tuberculosis: a study of cohorts from The Gambia, Guinea-Bissau, United States and Argentina.

We examined whether polymorphisms in interleukin-12B (IL12B) associate with susceptibility to pulmonary tuberculosis (PTB) in two West African populations (from The Gambia and Guinea-Bissau) and in two independent populations from North and South America. Nine polymorphisms (seven SNPs, one insertion/deletion, one microsatellite) were analyzed in 321 PTB cases and 346 controls from Guinea-Bissau and 280 PTB cases and 286 controls from The Gambia. For replication we studied 281 case and 179 control African-American samples and 221 cases and 144 controls of European ancestry from the US and Argentina. First-stage single locus analyses revealed signals of association at IL12B 3' UTR SNP rs3212227 (unadjusted allelic p = 0.04; additive genotypic p = 0.05, OR = 0.78, 95% CI [0.61-0.99]) in Guinea-Bissau and rs11574790 (unadjusted allelic p = 0.05; additive genotypic p = 0.05, OR = 0.76, 95% CI [0.58-1.00]) in The Gambia. Association of rs3212227 was then replicated in African-Americans (rs3212227 allelic p = 0.002; additive genotypic p = 0.05, OR = 0.78, 95% CI [0.61-1.00]); most importantly, in the African-American cohort, multiple significant signals of association (seven of the nine polymorphisms tested) were detected throughout the gene. These data suggest that genetic variation in IL12B, a highly relevant candidate gene, is a risk factor for PTB in populations of African ancestry, although further studies will be required to confirm this association and identify the precise mechanism underlying it.

Variants in toll-like receptors 2 and 9 influence susceptibility to pulmonary tuberculosis in Caucasians, African-Americans, and West Africans.

Tuberculosis (TB) is a global public health problem and a source of preventable deaths each year, with 8.8 million new cases of TB and 1.6 million deaths worldwide in 2005. Approximately, 10% of infected individuals develop pulmonary or extrapulmonary TB, suggesting that host defense factors influence development of active disease. Toll-like receptor' (TLR) polymorphisms have been associated with regulation of TLR expression and development of active TB. In the present study, 71 polymorphisms in TLR1, TLR2, TLR4, TLR6, and TLR9 were examined from 474 (295 cases and 179 controls) African-Americans, 381 (237 cases and 144 controls) Caucasians, and from 667 (321 cases and 346 controls) Africans from Guinea-Bissau for association with pulmonary TB using generalized estimating equations and logistic regression. Statistically significant associations were observed across populations at TLR9 and TLR2. The strongest evidence for association came at an insertion (I)/deletion (D) polymorphism (-196 to -174) in TLR2 that associated with TB in both Caucasians (II vs. ID&DD, OR = 0.41 [95% CI 0.24-0.68], p = 0.0007) and Africans (II vs. ID&DD, OR = 0.70 [95% CI 0.51-0.95], p = 0.023). Our findings in three independent population samples indicate that variations in TLR2 and TLR9 might play important roles in determining susceptibility to TB.

NOS2A, TLR4, and IFNGR1 interactions influence pulmonary tuberculosis susceptibility in African-Americans.

Tuberculosis (TB) has substantial mortality worldwide with 5-10% of those exposed progressing to active TB disease. Studies in mice and humans indicate that the inducible nitric oxide synthase (iNOS) molecule plays an important role in immune response to TB. A mixed case-control association study of individuals with TB, relatives, or close contact controls was performed in 726 individuals (279 case and 166 control African-Americans; 198 case and 123 control Caucasians). Thirty-nine single nucleotide polymorphisms (SNPs) were selected from the NOS2A gene for single SNP, haplotype, and multilocus interaction analyses with other typed candidate genes using generalized estimating equations. In African-Americans, ten NOS2A SNPs were associated with TB. The strongest associations were observed at rs2274894 (odds ratio (OR) = 1.84, 95% confidence interval (CI) [1.23-2.77], p = 0.003) and rs7215373 (OR = 1.67, 95% CI [1.17-2.37], p = 0.004), both of which passed a false discovery rate correction for multiple comparisons (q* = 0.20). The strongest gene-gene interactions were observed between NOS2A rs2248814 and IFNGR1 rs1327474 (p = 0.0004) and NOS2A rs944722 and IFNGR1 rs1327474 (p = 0.0006). Three other SNPs in NOS2A interacted with TLR4 rs5030729 and five other NOS2A SNPs interacted with IFNGR1 rs1327474. No significant associations were observed in Caucasians. These results suggest that NOS2A variants may contribute to TB susceptibility, particularly in individuals of African descent, and may act synergistically with SNPs in TLR4 and IFNGR1.

Adaptation of cyanobacteria to the sulfide-rich microenvironment of black band disease of coral.

Black band disease (BBD) is a cyanobacteria-dominated microbial mat that migrates across living coral colonies lysing coral tissue and leaving behind exposed coral skeleton. The mat is sulfide-rich due to the presence of sulfate-reducing bacteria, integral members of the BBD microbial community, and the sulfide they produce is lethal to corals. The effect of sulfide, normally toxic to cyanobacteria, on the photosynthetic capabilities of five BBD cyanobacterial isolates of the genera Geitlerinema (3), Leptolyngbya (1), and Oscillatoria (1) and six non-BBD cyanobacteria of the genera Leptolyngbya (3), Pseudanabaena (2), and Phormidium (1) was examined. Photosynthetic experiments were performed by measuring the photoincorporation of [(14)C] NaHCO(3) under the following conditions: (1) aerobic (no sulfide), (2) anaerobic with 0.5 mM sulfide, and (3) anaerobic with 0.5 mM sulfide and 10 microM 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU). All five BBD cyanobacterial isolates tolerated sulfide by conducting sulfide-resistant oxygenic photosynthesis. Five of the non-BBD cyanobacterial isolates did not tolerate sulfide, although one Pseudanabaena isolate continued to photosynthesize in the presence of sulfide at a considerably reduced rate. None of the isolates conducted anoxygenic photosynthesis with sulfide as an electron donor. This is the first report on the physiology of a culture of Oscillatoria sp. found globally in BBD.

Molecular detection and ecological significance of the cyanobacterial genera Geitlerinema and Leptolyngbya in black band disease of corals.

Black band disease (BBD) is a pathogenic, sulfide-rich microbial mat dominated by filamentous cyanobacteria that infect corals worldwide. We isolated cyanobacteria from BBD into culture, confirmed their presence in the BBD community by using denaturing gradient gel electrophoresis (DGGE), and demonstrated their ecological significance in terms of physiological sulfide tolerance and photosynthesis-versus-irradiance values. Twenty-nine BBD samples were collected from nine host coral species, four of which have not previously been investigated, from reefs of the Florida Keys, the Bahamas, St. Croix, and the Philippines. From these samples, seven cyanobacteria were isolated into culture. Cloning and sequencing of the 16S rRNA gene using universal primers indicated that four isolates were related to the genus Geitlerinema and three to the genus Leptolyngbya. DGGE results, obtained using Cyanobacteria-specific 16S rRNA primers, revealed that the most common BBD cyanobacterial sequence, detected in 26 BBD field samples, was related to that of an Oscillatoria sp. The next most common sequence, 99% similar to that of the Geitlerinema BBD isolate, was present in three samples. One Leptolyngbya- and one Phormidium-related sequence were also found. Laboratory experiments using isolates of BBD Geitlerinema and Leptolyngbya revealed that they could carry out sulfide-resistant oxygenic photosynthesis, a relatively rare characteristic among cyanobacteria, and that they are adapted to the sulfide-rich, low-light BBD environment. The presence of the cyanotoxin microcystin in these cultures and in BBD suggests a role in BBD pathogenicity. Our results confirm the presence of Geitlerinema in the BBD microbial community and its ecological significance, which have been challenged, and provide evidence of a second ecologically significant BBD cyanobacterium, Leptolyngbya.

The presence of the cyanobacterial toxin microcystin in black band disease of corals.

Black band disease (BBD) is a migrating, cyanobacterial dominated, sulfide-rich microbial mat that moves across coral colonies lysing coral tissue. While it is known that BBD sulfate-reducing bacteria contribute to BBD pathogenicity by production of sulfide, additional mechanisms of toxicity may be involved. Using HPLC/MS, the cyanotoxin microcystin was detected in 22 field samples of BBD collected from five coral species on nine reefs of the wider Caribbean (Florida Keys and Bahamas). Two cyanobacterial cultures isolated from BBD, Geitlerinema and Leptolyngbya sp. contained microcystin based on HPLC/MS, with toxic activity confirmed using the protein phosphatase inhibition assay. The gene mcyA from the microcystin synthesis complex was detected in two field samples and from both BBD cyanobacterial cultures. Microcystin was not detected in six BBD samples from a different area of the Caribbean (St Croix, USVI) and the Philippines, suggesting regional specificity for BBD microcystin. This is the first report of the presence of microcystin in a coral disease.

Black band disease microbial community variation on corals in three regions of the wider Caribbean.

Black band disease (BBD) is a pathogenic consortium of microorganisms that primarily affects massive framework-building scleractinian corals on reefs worldwide. There has been considerable debate concerning the microbial community composition of BBD. The aim of this study was to utilize microbial profiling to assess overall patterns of variation in the BBD bacterial community with respect to geographic location, host coral species, time, and nutrient regime. Length heterogeneity polymerase chain reaction (LH-PCR) was employed to differentiate BBD communities based on the natural variation in the sequence lengths within hypervariable domains of the 16S rRNA gene. Analysis of LH-PCR profiles of 97 BBD samples using multivariate ordination methods and analysis of similarity revealed significant clustering with respect to geographic region when comparing BBD sampled from reefs near Lee Stocking Island in the Bahamas' Exuma Chain, the Northern Florida Keys (NFK), and St. John in the US Virgin Islands. There was much variability in BBD community composition on a regional basis, between sites in the NFK, and in terms of coral host species. The observed differences among BBD microbial community profiles were driven primarily by variation in relative abundance of 313-316-bp amplicons, which correspond to cyanobacteria and alpha-proteobacteria. The results obtained in this study support previous reports of intrinsic variability and complexity of the BBD microbial community but also suggest that this variability has biogeographic patterns.