Don A. Baldwin

Microarray-based, high-throughput gene expression profiling of microRNAs

MicroRNAs (miRNAs) are small regulatory RNAs that serve fundamental biological roles across eukaryotic species. We describe a new method for high-throughput miRNA detection. The technique is termed the RNA-primed, array-based Klenow enzyme (RAKE) assay, because it involves on-slide application of the Klenow fragment of DNA polymerase I to extend unmodified miRNAs hybridized to immobilized DNA probes. We used RAKE to study human cell lines and brain tumors. We show that the RAKE assay is sensitive and specific for miRNAs and is ideally suited for rapid expression profiling of all known miRNAs. RAKE offers unique advantages for specificity over northern blots or other microarray-based expression profiling platforms. Furthermore, we demonstrate that miRNAs can be isolated and profiled from formalin-fixed paraffin-embedded tissue, which opens up new opportunities for analyses of small RNAs from archival human tissue. The RAKE assay is theoretically versatile and may be used for other applications, such as viral gene profiling.

Global genomic analysis reveals rapid control of a robust innate response in SIV-infected sooty mangabeys

Natural SIV infection of sooty mangabeys (SMs) is nonprogressive despite chronic virus replication. Strikingly, it is characterized by low levels of immune activation, while pathogenic SIV infection of rhesus macaques (RMs) is associated with chronic immune activation. To elucidate the mechanisms underlying this intriguing phenotype, we used high-density oligonucleotide microarrays to longitudinally assess host gene expression in SIV-infected SMs and RMs. We found that acute SIV infection of SMs was consistently associated with a robust innate immune response, including widespread upregulation of IFN-stimulated genes (ISGs) in blood and lymph nodes. While SMs exhibited a rapid resolution of ISG expression and immune activation, both responses were observed chronically in RMs. Systems biology analysis indicated that expression of the lymphocyte inhibitory receptor LAG3, a marker of T cell exhaustion, correlated with immune activation in SIV-infected RMs but not SMs. Our findings suggest that active immune regulatory mechanisms, rather than intrinsically attenuated innate immune responses, underlie the low levels of immune activation characteristic of SMs chronically infected with SIV.

Gene Expression Subtypes in Patients with Chronic Fatigue Syndrome/Myalgic Encephalomyelitis

Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a multisystem disease, the pathogenesis of which remains undetermined. We set out to determine the precise abnormalities of gene expression in the blood of patients with CFS/ME. We analyzed gene expression in peripheral blood from 25 patients with CFS/ME diagnosed according to the Centers for Disease Control and Prevention diagnostic criteria and 50 healthy blood donors, using a microarray with a cutoff fold difference of expression of >or=2.5. Genes showing differential expression were further analyzed in 55 patients with CFS/ME and 75 healthy blood donors, using quantitative polymerase chain reaction. Differential expression was confirmed for 88 genes; 85 were upregulated, and 3 were downregulated. Highly represented functions were hematological disease and function, immunological disease and function, cancer, cell death, immune response, and infection. Clustering of quantitative polymerase chain reaction data from patients with CFS/ME revealed 7 subtypes with distinct differences in Medical Outcomes Survey Short Form-36 scores, clinical phenotypes, and severity.

Evidence of a gene-environment interaction that predisposes to spontaneous preterm birth: a role for asymptomatic bacterial vaginosis and DNA variants in genes that control the inflammatory response

OBJECTIVE We determined whether an environmental exposure to bacterial vaginosis (BV) modified genetic susceptibilities for spontaneous preterm delivery within genes that regulate the inflammatory response. STUDY DESIGN Maternal DNA samples and vaginal smears for Gram staining were collected from 743 women (68 preterm births). We used a 1536-single nucleotide polymorphism (SNP) custom chip to study associations between genotype distributions and preterm birth. RESULTS For 8 SNPs in 3 genes (protein kinase C alpha, fms-like tyrosine kinase 1, and interleukin 6), the odds ratios for preterm birth ranged from 1.9-4.0 among women with susceptible genotypes who were BV positive. The odds ratios for preterm birth were 2.0-5.0 times greater among women who were BV positive than among women who were BV negative. The significance of these differences was demonstrated by logistic regression analyses for genotype/BV interaction. CONCLUSION These results demonstrate that the risk of preterm delivery that is associated with tag SNPs in genes that regulate the inflammatory response is modified by an environmental exposure such as bacterial vaginosis.

Multiorgan engraftment and differentiation of human cord blood CD34+ Lin- cells in goats assessed by gene expression profiling.

To investigate multitissue engraftment of human primitive hematopoietic cells and their differentiation in goats, human CD34+Lin− cord blood cells transduced with a GFP vector were transplanted into fetal goats at 45–55 days of gestation. GFP+ cells were detected in hematopoietic and nonhematopoietic organs including blood, bone marrow, spleen, liver, kidney, muscle, lung, and heart of the recipient goats (1.2–36% of all cells examined). We identified human β2 microglobulin-positive cells in multiple tissues. GFP+ cells sorted from the perfused liver of a transplant goat showed human insulin-like growth factor 1 gene sequences, indicating that the engrafted GFP+ cells were of human origin. A substantial fraction of cells engrafted in goat livers expressed the human hepatocyte-specific antigen, proliferating cell nuclear antigen, albumin, hepatocyte nuclear factor, and GFP. DNA content analysis showed no evidence for cellular fusion. Long-term engraftment of GFP+ cells could be detected in the blood of goats for up to 2 yr. Microarray analysis indicated that human genes from a variety of functional categories were expressed in chimeric livers and blood. The human/goat xenotransplant model provides a unique system to study the kinetics of hematopoietic stem cell engraftment, gene expression, and possible stem cell plasticity under noninjured conditions.

Convergent Evidence that Choline Acetyltransferase Gene Variation is Associated with Prospective Smoking Cessation and Nicotine Dependence

The ability to quit smoking is heritable, yet few genetic studies have investigated prospective smoking cessation. We conducted a systems-based genetic association analysis in a sample of 472 treatment-seeking smokers of European ancestry after 8 weeks of transdermal nicotine therapy for smoking cessation. The genotyping panel included 169 single-nucleotide polymorphisms (SNPs) in 7 nicotinic acetylcholine receptor subunit genes and 4 genes in the endogenous cholinergic system. The primary outcome was smoking cessation (biochemically confirmed) at the end of treatment. SNPs clustered in the choline acetyltransferase (ChAT) gene were individually identified as nominally significant, and a 5-SNP haplotype (block 6) in ChAT was found to be significantly associated with quitting success. Single SNPs in ChAT haplotype block 2 were also associated with pretreatment levels of nicotine dependence in this cohort. To replicate associations of SNPs in haplotype blocks 2 and 6 of ChAT with nicotine dependence in a non-treatment-seeking cohort, we used data from an independent community-based sample of 629 smokers representing 200 families of European ancestry. Significant SNP and haplotype associations were identified for multiple measures of nicotine dependence. Although the effect sizes in both cohorts are modest, converging data across cohorts and phenotypes suggest that ChAT may be involved in nicotine dependence and ability to quit smoking. Additional sequencing and characterization of ChAT may reveal functional variants that contribute to nicotine dependence and smoking cessation.

A genome-wide association study of early spontaneous preterm delivery.

Preterm birth is the leading cause of infant morbidity and mortality. Despite extensive research, the genetic contributions to spontaneous preterm birth (SPTB) are not well understood. Term controls were matched with cases by race/ethnicity, maternal age, and parity prior to recruitment. Genotyping was performed using Affymetrix SNP Array 6.0 assays. Statistical analyses utilized PLINK to compare allele occurrence rates between case and control groups, and incorporated quality control and multiple‐testing adjustments. We analyzed DNA samples from mother–infant pairs from early SPTB cases (200/7–336/7 weeks, 959 women and 979 neonates) and term delivery controls (390/7–416/7 weeks, 960 women and 985 neonates). For validation purposes, we included an independent validation cohort consisting of early SPTB cases (293 mothers and 243 infants) and term controls (200 mothers and 149 infants). Clustering analysis revealed no population stratification. Multiple maternal SNPs were identified with association P‐values between 10 × 10–5 and 10 × 10–6. The most significant maternal SNP was rs17053026 on chromosome 3 with an odds ratio (OR) 0.44 with a P‐value of 1.0 × 10–6. Two neonatal SNPs reached the genome‐wide significance threshold, including rs17527054 on chromosome 6p22 with a P‐value of 2.7 × 10–12 and rs3777722 on chromosome 6q27 with a P‐value of 1.4 × 10–10. However, we could not replicate these findings after adjusting for multiple comparisons in a validation cohort. This is the first report of a genome‐wide case‐control study to identify single nucleotide polymorphisms (SNPs) that correlate with SPTB.

Single-Nucleotide Polymorphisms Associated with Symptomatic Infection and Differential Human Gene Expression in Healthy Seropositive Persons Each Implicate the Cytoskeleton, Integrin Signaling, and Oncosuppression in the Pathogenesis of Human Parvovirus B19 Infection

This study was undertaken to further examine the role of the host response to parvovirus B19 in the development of symptoms and consequences of viral persistence. Genomic DNA from 42 patients with symptomatic B19 infection was analyzed using the HuSNP assay (Affymetrix), and the results were compared with those from analysis of 53 healthy control individuals. Fifty-seven single-nucleotide polymorphisms were identified that were significantly associated with symptomatic infection. Total RNA from peripheral blood mononuclear cells from 57 B19-seropositive and 13 B19-seronegative donors was analyzed by hybridization to a single-color microarray representing 9522 human genes. Ninety-two genes were shown to be differentially expressed. Differential expression was confirmed in 6 of 38 genes (SKIP, MACF1, SPAG7, FLOT1, c6orf48, and RASSF5) tested using real-time quantitative polymerase chain reaction in a different group of healthy subjects. Genes identified in both studies play a functional role in the cytoskeleton, integrin signaling, and oncosuppression, themes that have been shown to be important in parvovirus infections.

Association of the Nicotine Metabolite Ratio and CHRNA5/CHRNA3 Polymorphisms With Smoking Rate Among Treatment-Seeking Smokers

INTRODUCTION Genome-wide association studies have linked single-nucleotide polymorphisms (SNPs) in the CHRNA5/A3/B4 gene cluster with heaviness of smoking. The nicotine metabolite ratio (NMR), a measure of the rate of nicotine metabolism, is associated with the number of cigarettes per day (CPD) and likelihood of cessation. We tested the potential interacting effects of these two risk factors on CPD. METHODS Pretreatment data from three prior clinical trials were pooled for analysis. One thousand and thirty treatment seekers of European ancestry with genotype data for the CHRNA5/A3/B4 SNPs rs578776 and rs1051730 and complete data for NMR and CPD at pretreatment were included. Data for the third SNP, rs16969968, were available for 677 individuals. Linear regression models estimated the main and interacting effects of genotype and NMR on CPD. RESULTS We confirmed independent associations between the NMR and CPD as well as between the SNPs rs16969968 and rs1051730 and CPD. We did not detect a significant interaction between NMR and any of the SNPs examined. CONCLUSIONS This study demonstrates the additive and independent association of the NMR and SNPs in the CHRNA5/A3/B4 gene cluster with smoking rate in treatment-seeking smokers.

Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues

ABSTRACT Screening for thousands of viruses and other pathogenic microorganisms, including bacteria, fungi, and parasites, in human tumor tissues will provide a better understanding of the contributory role of the microbiome in the predisposition for, causes of, and therapeutic responses to the associated cancer. Metagenomic assays designed to perform these tasks will have to include rapid and economical processing of large numbers of samples, supported by straightforward data analysis pipeline and flexible sample preparation options for multiple input tissue types from individual patients, mammals, or environmental samples. To meet these requirements, the PathoChip platform was developed by targeting viral, prokaryotic, and eukaryotic genomes with multiple DNA probes in a microarray format that can be combined with a variety of upstream sample preparation protocols and downstream data analysis. PathoChip screening of DNA plus RNA from formalin-fixed, paraffin-embedded tumor tissues demonstrated the utility of this platform, and the detection of oncogenic viruses was validated using independent PCR and deep sequencing methods. These studies demonstrate the use of the PathoChip technology combined with PCR and deep sequencing as a valuable strategy for detecting the presence of pathogens in human cancers and other diseases. IMPORTANCE This work describes the design and testing of a PathoChip array containing probes with the ability to detect all known publicly available virus sequences as well as hundreds of pathogenic bacteria, fungi, parasites, and helminths. PathoChip provides wide coverage of microbial pathogens in an economical format. PathoChip screening of DNA plus RNA from formalin-fixed, paraffin-embedded tumor tissues demonstrated the utility of this platform, and the detection of oncogenic viruses was validated using independent PCR and sequencing methods. These studies demonstrate that the PathoChip technology is a valuable strategy for detecting the presence of pathogens in human cancers and other diseases. This work describes the design and testing of a PathoChip array containing probes with the ability to detect all known publicly available virus sequences as well as hundreds of pathogenic bacteria, fungi, parasites, and helminths. PathoChip provides wide coverage of microbial pathogens in an economical format. PathoChip screening of DNA plus RNA from formalin-fixed, paraffin-embedded tumor tissues demonstrated the utility of this platform, and the detection of oncogenic viruses was validated using independent PCR and sequencing methods. These studies demonstrate that the PathoChip technology is a valuable strategy for detecting the presence of pathogens in human cancers and other diseases.