Walter T. Klimecki

Single-nucleotide polymorphisms in the Toll-like receptor 9 gene (TLR9): frequencies, pairwise linkage disequilibrium, and haplotypes in three U.S. ethnic groups and exploratory case-control disease association studies.

TLR9 is a mammalian Toll-like receptor homologue that appears to function as an innate immune pattern recognition protein for motifs that are far more common in bacterial than in mammalian DNA. The gene was sequenced in 71 subjects from three self-identified U.S. ethnic groups to identify single-nucleotide polymorphisms (SNPs). A total of 20 SNPs were found of which only 20% were in the public dbSNP database. Four SNPs were relatively common in all three ethnic samples. Using these four SNPs, seven distinct haplotypes were statistically inferred, of which four accounted for 75% or more chromosomes. These four haplotypes could be distinguished from each other by the alleles of two SNPs (-1237 and 2848). Five exploratory nested case-control disease-association studies (asthma, DVT, MI, and COPD in European Americans and asthma in African Americans) were performed by genotyping DNA collected from four ongoing cohort studies. There was evidence suggesting increased risk for asthma with a C allele at -1237 (odds ratio 1.85, 95%CI 1.05 to 3.25) among European Americans (genotypes available from 67 cases and 152 controls). No other significant disease associations were detected. Replication of this finding in other, larger samples is needed. This study suggests that there is substantial diversity in human TLR9, possibly associated with asthma in Europeans but not African Americans. No association was detected with three other diseases potentially related to innate immunity.

5-Aza-2′-deoxycytidine-mediated reductions in G9A histone methyltransferase and histone H3 K9 di-methylation levels are linked to tumor suppressor gene reactivation

The epigenetic silencing of tumor suppressor genes is a common event during carcinogenesis, and often involves aberrant DNA methylation and histone modification of gene regulatory regions, resulting in the formation of a transcriptionally repressive chromatin state. Two examples include the antimetastatic, tumor suppressor genes, desmocollin 3 (DSC3) and MASPIN, which are frequently silenced in this manner in human breast cancer. Treatment of the breast tumor cell lines MDA-MB-231 and UACC 1179 with 5-aza-2′-deoxycytidine (5-aza-CdR) induced transcriptional reactivation of both genes in a dose-dependent manner. Importantly, DSC3 and MASPIN reactivation was closely and consistently linked with significant decreases in promoter H3 K9 di-methylation. Moreover, 5-aza-CdR treatment also resulted in global decreases in H3 K9 di-methylation, an effect that was linked to its ability to mediate dose-dependent, post-transcriptional decreases in the key enzyme responsible for this epigenetic modification, G9A. Finally, small interfering RNA (siRNA)-mediated knockdown of G9A and DNMT1 led to increased MASPIN expression in MDA-MB-231 cells, to levels that were supra-additive, verifying the importance of these enzymes in maintaining multiple layers of epigenetic repression in breast tumor cells. These results highlight an additional, complimentary mechanism of action for 5-aza-CdR in the reactivation of epigenetically silenced genes, in a manner that is independent of its effects on DNA methylation, further supporting an important role for H3 K9 methylation in the aberrant repression of tumor suppressor genes in human cancer.

Developmentally restricted genetic determinants of human arsenic metabolism: Association between urinary methylated arsenic and CYT19 polymorphisms in children

We report the results of a screen for genetic association with urinary arsenic metabolite levels in three arsenic metabolism candidate genes, PNP, GSTO, and CYT19, in 135 arsenic-exposed subjects from the Yaqui Valley in Sonora, Mexico, who were exposed to drinking water concentrations ranging from 5.5 to 43.3 ppb. We chose 23 polymorphic sites to test in the arsenic-exposed population. Initial phenotypes evaluated included the ratio of urinary inorganic arsenic(III) to inorganic arsenic(V) and the ratio of urinary dimethylarsenic(V) to monomethylarsenic(V) (D:M). In the initial association screening, three polymorphic sites in the CYT19 gene were significantly associated with D:M ratios in the total population. Subsequent analysis of this association revealed that the association signal for the entire population was actually caused by an extremely strong association in only the children (7–11 years of age) between CYT19 genotype and D:M levels. With children removed from the analysis, no significant genetic association was observed in adults (18–79 years). The existence of a strong, developmentally regulated genetic association between CYT19 and arsenic metabolism carries import for both arsenic pharmacogenetics and arsenic toxicology, as well as for public health and governmental regulatory officials.

Epigenetic Inactivation of the HOXA Gene Cluster in Breast Cancer

Using an integrated approach of epigenomic scanning and gene expression profiling, we found aberrant methylation and epigenetic silencing of a small neighborhood of contiguous genes-the HOXA gene cluster in human breast cancer. The observed transcriptional repression was localized to approximately 100 kb of the HOXA gene cluster and did not extend to genes located upstream or downstream of the cluster. Bisulfite sequencing, chromatin immunoprecipitation, and quantitative reverse transcription-PCR analysis confirmed that the loss of expression of the HOXA gene cluster in human breast cancer is closely linked to aberrant DNA methylation and loss of permissive histone modifications in the region. Pharmacologic manipulations showed the importance of these aberrant epigenetic changes in gene silencing and support the hypothesis that aberrant DNA methylation is dominant to histone hypoacetylation. Overall, these data suggest that inactivation of the HOXA gene cluster in breast cancer may represent a new type of genomic lesion-epigenetic microdeletion. We predict that epigenetic microdeletions are common in human cancer and that they functionally resemble genetic microdeletions but are defined by epigenetic inactivation and transcriptional silencing of a relatively small set of contiguous genes along a chromosome, and that this type of genomic lesion is metastable and reversible in a classic epigenetic fashion.

Association between exposure to farming, allergies and genetic variation in CARD4/NOD1

Background:  Caspase recruitment domain protein (CARD) 4 has been recently identified as an intracellular pattern recognition receptor that interacts with muropeptides found in common Gram‐negative bacteria. We therefore aimed to explore whether the previously observed inverse association between exposure to microbial products and asthma and allergies in childhood is modified by genetic variation in CARD4.

Analysis of Global and Absorption, Distribution, Metabolism, and Elimination Gene Expression in the Progressive Stages of Human Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is characterized by a series of pathological changes that range from simple fatty liver to nonalcoholic steatohepatitis (NASH). The objective of this study is to describe changes in global gene expression associated with the progression of human NAFLD. This study is focused on the expression levels of genes responsible for the absorption, distribution, metabolism, and elimination (ADME) of drugs. Differential gene expression between three clinically defined pathological groups—normal, steatosis, and NASH—was analyzed. Genome-wide mRNA levels in samples of human liver tissue were assayed with Affymetrix GeneChip Human 1.0ST arrays. A total of 11,633 genes exhibited altered expression out of 33,252 genes at a 5% false discovery rate. Most gene expression changes occurred in the progression from steatosis to NASH. Principal component analysis revealed that hepatic disease status was the major determinant of differential ADME gene expression rather than age or sex of sample donors. Among the 515 drug transporters and 258 drug-metabolizing enzymes (DMEs) examined, uptake transporters but not efflux transporters or DMEs were significantly over-represented in the number of genes down-regulated. These results suggest that uptake transporter genes are coordinately targeted for down-regulation at the global level during the pathological development of NASH and that these patients may have decreased drug uptake capacity. This coordinated regulation of uptake transporter genes is indicative of a hepatoprotective mechanism acting to prevent accumulation of toxic intermediates in disease-compromised hepatocytes.

Toll-like receptor 6 gene (TLR6): single-nucleotide polymorphism frequencies and preliminary association with the diagnosis of asthma

Toll-like receptor 6 (TLR6) is one of a series of highly conserved innate immune receptors. We resequenced TLR6 in DNA samples from 24 African Americans, 23 European Americans, and 24 Hispanic Americans, identifying 53 SNPs, 22 with an allele frequency >5%. Significant differences in SNP frequencies among the three populations were noted. In all, 11 SNPs caused amino-acid changes, including one with a frequency >5% in all three populations. Utilizing this SNP (Ser249Pro), we performed exploratory nested case–control disease-association studies, including one involving 56 African Americans with asthma and 93 African American controls. The minor allele of this SNP was associated with decreased risk for asthma (odds ratio 0.38, 95% CI 0.16–0.87, P=0.01), an effect consistent with the known biology of the toll-like receptors. Although replication of this finding in other, larger samples is needed, variation in TLR6 may have relevance to the pathogenesis of immunologically mediated diseases.

Cross-sectional analysis of oncogenic HPV viral load and cervical intraepithelial neoplasia

In human papillomavirus (HPV)‐associated carcinogenesis, HPV infection characteristics such as viral load may play an important role in lesion development. The purpose of this study was to determine the association between quantitative assessment of oncogenic HPV viral load, and abnormal cytology among women residing along the United States–Mexico border. A cross‐sectional study of 2,319 women was conducted between 1997 and 1998. Viral load of oncogenic HPV types (16, 18, 31, 39, 45, 51, 52, and 58) was measured among 173 HPV (+) women using quantitative real‐time PCR. Overall, HPV 16, 31, 52 and 58 showed the highest viral load. Single type infection had higher viral loads compared to multiple type infections. HPV viral load declined significantly (p = 0.04) with age. No significant association was observed with other known HPV risk factors such as oral contraceptive use, parity, sexual and STD history. Viral load was independently associated with degree of cervical lesions. An adjusted odds ratio (AOR) of 4.7 for the association between increasing total viral load and Atypical Squamous Cells of Undetermined Significance (ASCUS)/Atypical Glandular Cells of Undetermined Significance (AGUS) was observed (p for trend <0.01). Increased risk of low‐grade SIL was observed with higher viral load compared with HPV negative women (AOR = 47.7 for total viral load; AOR = 37.1 for HPV viral load not including HPV16, and AOR = 25.9 for HPV16 viral load). Likewise, increased risk of high‐grade SIL with higher viral loads was observed (AOR = 58.4 for high total viral load compared with HPV negative women, AOR = 58.1 for HPV viral load not including HPV16, and AOR = 69.8 for HPV16 high viral load). Results from this study suggest a dose–response relationship between increasing oncogenic HPV viral load and risk of LSIL and HSIL.

Genetic regulation of rejection and survival following human lung transplantation by the innate immune receptor CD14.

We have developed the hypothesis that genetic polymorphisms which alter the expression or function of innate immune receptors contribute to the marked interindividual differences in the onset and severity of lung transplant rejection. In this analysis, we considered the effects of a common promotor polymorphism of the lipopolysaccharide receptor CD14 associated with increased transcriptional activity upon the development of posttransplant rejection and graft survival. Genotyping was performed in 226 lung transplant recipients well characterized with regards to clinical outcomes. An earlier onset of acute rejection, bronchiolitis obliterans syndrome (BOS) and worse posttransplant graft survival due to greater BOS related deaths was evident in patients with the CD14 –159 TT genotype (TT). The adverse effect upon graft survival of the TT genotype remained significant in a multivariate Cox model (Hazard Ratio 1.65, 95% CI, 1.03–2.64, p‐value = 0.04) after adjusting for other important covariates. Furthermore, TT patients have significantly greater sCD14, TNF‐α and IFN‐γ in the peripheral blood implying a heightened state of innate immune activation drives the development of increased post‐transplant rejection. Inhibition of innate immune activation through CD14 represents a novel and potentially important therapeutic target to prevent post‐transplant rejection and improve outcomes after human lung transplantation.

The Human IL-13 Locus in Neonatal CD4+ T Cells Is Refractory to the Acquisition of a Repressive Chromatin Architecture

The Th2 cytokine IL-13 is a major effector molecule in human allergic inflammation. Notably, IL-13 expression at birth correlates with subsequent susceptibility to atopic disease. In order to characterize the chromatin-based mechanisms that regulate IL-13 expression in human neonatal CD4+ T cells, we analyzed patterns of DNase I hypersensitivity and epigenetic modifications within the IL-13 locus in cord blood CD4+ T cells, naive or differentiated in vitro under Th1- or Th2-polarizing conditions. In naive CD4+ T cells, hypersensitivity associated with DNA hypomethylation was limited to the distal promoter. Unexpectedly, during both Th1 and Th2 differentiation, the locus was extensively remodeled, as revealed by the formation of numerous HS sites and decreased DNA methylation. Obvious differences in chromatin architecture were limited to the proximal promoter, where strong hypersensitivity, hypomethylation, and permissive histone modifications were found selectively in Th2 cells. In addition to revealing the locations of putative cis-regulatory elements that may be required to control IL-13 expression in neonatal CD4+ T cells, our results suggest that differential IL-13 expression may depend on the acquisition of a permissive chromatin architecture at the proximal promoter in Th2 cells rather than the formation of locus-wide repressive chromatin in Th1 cells.