Comparative analyses of single-nucleotide polymorphisms in the TNF promoter region provide further validation for the vervet monkey model of obesity

Abstract

Tumor necrosis factor is a cytokine that plays critical roles in inflammation, the innate immune response, and a variety of other physiologic and pathophysiologic processes. In addition, TNF has recently been shown to mediate an intersection of chronic, low-grade inflammation and concurrent metabolic dysregulation associated with obesity and its comorbidities. As part of an ongoing initiative to further characterize vervet monkeys originating from St Kitts as an animal model of obesity and inflammation, we sequenced and genotyped the human ortholog vervet TNF gene and approximately 1 kb of the flanking 3' and 5' regions from 265 monkeys in a closed, pedigreed colony. This process revealed a total of 11 single-nucleotide polymorphisms (SNPs) and a single 4-bp insertion-deletion, with minor allele frequencies of 0.08 to 0.39. Many of these polymorphisms were in strong or complete linkage disequilibrium with each other, and all but 1 were contained within a single haplotype block, comprising 5 haplotypes with frequencies of 0.075 to 0.298. Using sequences from humans, chimpanzees, vervets, baboons, and rhesus macaques, phylogenetic shadowing of the TNF promoter region revealed that vervet SNPs, like the SNPs in related species, were clustered nonrandomly and nonuniformly around conserved transcription factor binding sites. These data, combined with previously defined heritable phenotypes, permit future association analyses in this nonhuman primate model and have great potential to help dissect the genetic and nongenetic contributions to complex diseases like obesity. More broadly, the sequence data and comparative analyses reported herein facilitates study of the evolution of regulatory sequences of inflammatory and immune-related genes.

Figure 1.

Linkage disequilibrium structure of the St-Kitts-origin vervet TNF locus. The single haplotype block (outlined triangle) was determined with the confidence interval algorithm of HaploView. Numbers indicate pairwise r² values × 100. Darker blocks denote higher correlation, and black squares with no numbers represent complete linkage disequilibrium (r² = 1). Relative positions of polymorphisms are indicated on diagram of the human ortholog St-Kitts-origin vervet TNF gene and flanking sequence. Colored regions denote promoter–enhancer region (green), untranslated regions (blue), exons (red), and introns (black).

Figure 2.

Sequence comparison of TNF 5′ promoter region between humans (H. sapiens), chimpanzee (P. troglodytes), St-Kitts-origin vervet (C. a. sabaeus), rhesus monkey (M. mulatta), and baboon (P. hamadrayas); SNPs are underlined and bolded with adjacent nucleotide position for the species in which they have been reported. Nucleotide positions are relative to the human transcription start site (+1). The single-letter code is used to identify SNPs: M, A or C; R, A or G; W, A or T; S, C or G; Y, C or T; K, G or T. Transcription factor binding sites are underlined, with names above the sequence. NFAT (nuclear factor of activated T cells), Ets, κ3, CRE (cyclic AMP response element), and SP-1 are critical for TNF gene regulation;^,,,- κ1 and κ2 are reported to bind NFκB proteins and are not essential for gene regulation.^,

Figure 3.

(A) Phylogenetic shadowing of the TNF promoter region in 5 primate species. The multiple-sequence alignment of TNF (including the promoter region) for humans, chimpanzees, St-Kitts-origin vervet, rhesus monkey, and baboon was analyzed with eShadow. The top x axis is nucleotide position relative to human transcription start site (+1). The relative positions of polymorphisms in 4 of the 5 species studied here are indicated along the bottom x axis (H, human; C, chimpanzee; G, St Kitt vervet; R, rhesus monkey); no SNP information for baboon was available. Percentage of nucleotide variation (inverse of conservation) along a 50-nucleotide (nt) sliding window is on the y axis, and darker shading of peaks represents regions of higher conservation. (B) Clustering of SNP in NHP. Plot of the number of SNP in each of 4 primate species (human, chimpanzee, vervet monkey, rhesus macaque) which cluster along a sliding 50-nt window of the TNF promoter–enhancer region (y axis). SNP in each of the 4 species is designated by color (human, blue; chimpanzee, red; vervet, green; rhesus, purple). The x axis is nucleotide position relative to the human transcription start site (+1).