Jeremy P. Glenn

Expression of the Placental Transcriptome in Maternal Nutrient Reduction in Baboons Is Dependent on Fetal Sex

Maternal undernutrition increases the risk of perinatal complications and predisposes offspring to obesity, diabetes, and cardiovascular disease later in life. Emerging evidence suggests that changes in placental function play a role in linking altered maternal nutrition in pregnancy to the subsequent development of adult disease. The susceptibility for disease in response to an adverse intrauterine environment differs distinctly between boys and girls, with girls typically having better outcomes. Here, we tested the hypothesis that regulation of the placental transcriptome by maternal nutrient reduction (NR) is dependent on fetal sex. We used a nonhuman primate model of NR in which maternal global food intake was reduced by 30% in baboons starting at gestational day (GD) 30. At GD 165 (term = GD 183), placental genome expression profiling of 6 control (n = 3 females, 3 males) and 6 nutrient restricted (n = 3 females, 3 males) fetuses was carried out followed by bioinformatic analysis. Surprisingly, there was no coordinated placental molecular response to decreased nutrient availability when analyzing the data without accounting for fetal sex. In contrast, female placentas exhibited a highly coordinated response that included upregulation of genes in networks, pathways, and functional groups related to programmed cell death and downregulation of genes in networks, pathways, and functional groups associated with cell proliferation. These changes were not apparent in the male placentas. Our data support the concept that female placentas initiate complex adaptive responses to an adverse intrauterine environment, which may contribute to increased survival and better pregnancy outcomes in girls.

Differential microRNA response to a high-cholesterol, high-fat diet in livers of low and high LDL-C baboons.

BackgroundDysregulation of microRNA (miRNA) expression has been implicated in molecular genetic events leading to the progression and development of atherosclerosis. We hypothesized that miRNA expression profiles differ between baboons with low and high serum low-density lipoprotein cholesterol (LDL-C) concentrations in response to diet, and that a subset of these miRNAs regulate genes relevant to dyslipidemia and risk of atherosclerosis.ResultsUsing Next Generation Illumina sequencing methods, we sequenced hepatic small RNA libraries from baboons differing in their LDL-C response to a high-cholesterol, high-fat (HCHF) challenge diet (low LDL-C, n = 3; high LDL-C, n = 3), resulting in 517 baboon miRNAs: 490 were identical to human miRNAs and 27 were novel. We compared miRNA expression profiles from liver biopsies collected before and after the challenge diet and observed that HCHF diet elicited expression of more miRNAs compared to baseline (chow) diet for both low and high LDL-C baboons. Eighteen miRNAs exhibited differential expression in response to HCHF diet in high LDL-C baboons compared to 10 miRNAs in low LDL-C baboons. We used TargetScan/Base tools to predict putative miRNA targets; miRNAs expressed in high LDL-C baboons had significantly more gene targets than miRNAs expressed in low LDL-C responders. Further, we identified miRNA isomers and other non-coding RNAs that were differentially expressed in response to the challenge diet in both high LDL-C and low LDL-C baboons.ConclusionsWe sequenced and annotated baboon liver miRNAs from low LDL-C and high LDL-C responders using high coverage Next Gen sequencing methods, determined expression changes in response to a HCHF diet challenge, and predicted target genes regulated by the differentially expressed miRNAs. The identified miRNAs will enrich the database for non-coding small RNAs including the extent of variation in these sequences. Further, we identified other small non-coding RNAs differentially expressed in response to diet. Our discovery of differentially expressed baboon miRNAs in response to a HCHF diet challenge that differ by LDL-C phenotype is a fundamental step in understating the role of non-coding RNAs in dyslipidemia.

Molecular pathways mediating differential responses to lipopolysaccharide between human and baboon arterial endothelial cells

1. Vascular inflammation plays a critical role in atherogenesis. Previously, we showed that baboon arterial endothelial cells (BAEC) were hyporesponsive to lipopolysaccharide (LPS) compared with human arterial endothelial cells (HAEC).

Baboon carboxylesterases 1 and 2: sequences, structures and phylogenetic relationships with human and other primate carboxylesterases.

Background  Carboxylesterase (CES) is predominantly responsible for the detoxification of a wide range of drugs and narcotics, and catalyze several reactions in cholesterol and fatty acid metabolism. Studies of the genetic and biochemical properties of primate CES may contribute to an improved understanding of human disease, including atherosclerosis, obesity and drug addiction, for which non‐human primates serve as useful animal models.

A genome resource to address mechanisms of developmental programming: determination of the fetal sheep heart transcriptome

•  The sheep is a valuable model for biomedical research, including the study of developmental programming mechanisms that are critical to better understand gene–environment interactions responsible for each individuals phenotype and predisposition to chronic diseases. •  The lack of genetic and genomic resources for the sheep has limited the power of this model, impeding progress in emerging areas of pregnancy biology and other fields of biomedical research. •  In this study, we examined the expressed fetal sheep heart transcriptome using high‐throughput sequencing technologies. •  We identified 36,737 novel transcripts and describe genes, gene variants and pathways relevant to fundamental developmental mechanisms. •  The data presented in this study provide a foundation of genetic information on the repertoire of genes expressed in the fetal heart transcriptome, which will augment annotation and assembly of the sheep genome.

Identification of candidate genes encoding an LDL-C QTL in baboons

Cardiovascular disease (CVD) is the leading cause of death in developed countries, and dyslipidemia is a major risk factor for CVD. We previously identified a cluster of quantitative trait loci (QTL) on baboon chromosome 11 for multiple, related quantitative traits for serum LDL-cholesterol (LDL-C). Here we report differentially regulated hepatic genes encoding an LDL-C QTL that influences LDL-C levels in baboons. We performed hepatic whole-genome expression profiling for LDL-C-discordant baboons fed a high-cholesterol, high-fat (HCHF) diet for seven weeks. We detected expression of 117 genes within the QTL 2-LOD support interval. Three genes were differentially expressed in low LDL-C responders and 8 in high LDL-C responders in response to a HCHF diet. Seven genes (ACVR1B, CALCOCO1, DGKA, ERBB3, KRT73, MYL6B, TENC1) showed discordant expression between low and high LDL-C responders. To prioritize candidate genes, we integrated miRNA and mRNA expression profiles using network tools and found that four candidates (ACVR1B, DGKA, ERBB3, TENC1) were miRNA targets and that the miRNAs were inversely expressed to the target genes. Candidate gene expression was validated using QRT-PCR and Western blotting. This study reveals candidate genes that influence variation in LDL-C in baboons and potential genetic mechanisms for further investigation.

Identification of baboon microRNAs expressed in liver and lymphocytes

BackgroundMicroRNAs (miRNAs) are small noncoding RNAs (~22 nucleotides) that regulate gene expression by cleaving mRNAs or inhibiting translation. The baboon is a well-characterized cardiovascular disease model; however, no baboon miRNAs have been identified. Evidence indicates that the baboon and human genomes are highly conserved; based on this conservation, we hypothesized that comparative genomic methods could be used to identify baboon miRNAs.MethodsWe employed an in silico comparative genomics approach and human miRNA arrays to identify baboon expressed miRNAs in liver (n = 6) and lymphocytes (n = 6). Expression profiles for selected miRNAs in multiple tissues were validated by RT-PCR.ResultsWe identified in silico 555 putative baboon pre-miRNAs, of which 41% exhibited 100% identity and an additional 58% shared more than 90% sequence identity with human pre-miRNAs. Some of these miRNAs are primate-specific and are clustered in the baboon genome like human miRNA clusters. We detected expression of 494 miRNAs on the microarray and validated expression of selected miRNAs in baboon liver and lymphocytes by RT-PCR. We also observed miRNA expression in additional tissues relevant to dyslipidemia and atherosclerosis. Approximately half of the miRNAs expressed on the array were not predicted in silico suggesting that we have identified novel baboon miRNAs, which could not be predicted using the current draft of the baboon genome.ConclusionWe identified a subset of baboon miRNAs using a comparative genomic approach, identified additional baboon miRNAs using a human array and showed tissue-specific expression of baboon miRNAs. Our discovery of baboon miRNAs in liver and lymphocytes will provide resources for studies on the roles of miRNAs in dyslipidemia and atherosclerosis, and for translational studies.

The Baboon Kidney Transcriptome: Analysis of Transcript Sequence, Splice Variants, and Abundance

The baboon is an invaluable model for the study of human health and disease, including many complex diseases of the kidney. Although scientists have made great progress in developing this animal as a model for numerous areas of biomedical research, genomic resources for the baboon, such as a quality annotated genome, are still lacking. To this end, we characterized the baboon kidney transcriptome using high-throughput cDNA sequencing (RNA-Seq) to identify genes, gene variants, single nucleotide polymorphisms (SNPs), insertion-deletion polymorphisms (InDels), cellular functions, and key pathways in the baboon kidney to provide a genomic resource for the baboon. Analysis of our sequencing data revealed 45,499 high-confidence SNPs and 29,813 InDels comparing baboon cDNA sequences with the human hg18 reference assembly and identified 35,900 cDNAs in the baboon kidney, including 35,150 transcripts representing 15,369 genic genes that are novel for the baboon. Gene ontology analysis of our sequencing dataset also identified numerous biological functions and canonical pathways that were significant in the baboon kidney, including a large number of metabolic pathways that support known functions of the kidney. The results presented in this study catalogues the transcribed mRNAs, noncoding RNAs, and hypothetical proteins in the baboon kidney and establishes a genomic resource for scientists using the baboon as an experimental model.

A custom rat and baboon hypertension gene array to compare experimental models.

One challenge in understanding the polygenic disease of hypertension is elucidating the genes involved and defining responses to environmental factors. Many studies focus on animal models of hypertension; however, this does not necessarily extrapolate to humans. Current technology and cost limitations are prohibitive in fully evaluating hypertension within humans. Thus, we have designed a single-array platform that allows direct comparison of genes relevant to hypertension in animal models and non-human primates/human hypertension. The custom array is targeted to 328 genes known to be potentially related to blood pressure control. Studies compared gene expression in the kidney from normotensive rats and baboons. We found 74 genes expressed in both the rat and baboon kidney, 41 genes expressed in the rat kidney that were not detected in the baboon kidney and 34 genes expressed in the baboon kidney that were not detected in the rat kidney. To begin the evaluation of the array in a pathological condition, kidney gene expression was compared between the salt-sensitive deoxycorticosterone acetate (DOCA) rat model of hypertension and sham animals. Gene expression in the renal cortex and medulla from hypertensive DOCA compared with sham rats revealed three genes differentially expressed in the renal cortex: annexin A1 (up-regulated; relative intensity: 1.316 ± 0.321 versus 2.312 ± 0.283), glutamate-cysteine ligase (down-regulated; relative intensity: 3.738 ± 0.174 versus 2.645 ± 0.364) and glutathione-S transferase (down-regulated; relative intensity: 5.572 ± 0.246 versus 4.215 ± 0.411) and 21 genes differentially expressed in the renal medulla. Interestingly, few genes were differentially expressed in the kidney in the DOCA-salt model of hypertension; this may suggest that the complexity of hypertension may be the result of only a few gene-by-environment responsive events.

Integration of genetic and genomic methods for identification of genes and gene variants encoding QTLs in the nonhuman primate

We have developed an integrated approach, using genetic and genomic methods, in conjunction with resources from the Southwest National Primate Research Center (SNPRC) baboon colony, for the identification of genes and their functional variants that encode quantitative trait loci (QTL). In addition, we use comparative genomic methods to overcome the paucity of baboon specific reagents and to augment translation of our findings in a nonhuman primate (NHP) to the human population. We are using the baboon as a model to study the genetics of cardiovascular disease (CVD). A key step for understanding gene-environment interactions in cardiovascular disease is the identification of genes and gene variants that influence CVD phenotypes. We have developed a sequential methodology that takes advantage of the SNPRC pedigreed baboon colony, the annotated human genome, and current genomic and bioinformatic tools. The process of functional polymorphism identification for genes encoding QTLs involves comparison of expression profiles for genes and predicted genes in the genomic region of the QTL for individuals discordant for the phenotypic trait mapping to the QTL. After comparison, genes of interest are prioritized, and functional polymorphisms are identified in candidate genes by genotyping and quantitative trait nucleotide analysis. This approach reduces the time and labor necessary to prioritize and identify genes and their polymorphisms influencing variation in a quantitative trait compared with traditional positional cloning methods.