Single Nucleotide Polymorphism within the Promoter Region of Cynomolgus Monkey LDLR Gene

The cynomolgus monkey (Macaca fascicularis) is one of the most common nonhuman primates used as an animal model in biomedical research related to atherosclerosis. However, little is known about genetic variation in the low density lipoprotein receptor (LDLR) gene, its effect on cholesterol levels, and associated risks of atherosclerosis. Therefore this study aimed to identify genetic polymorphisms, namely single nucleotide polymorphisms (SNPs), within the promoter region of LDLR and their relationship with animal responsiveness to hypercholesterolemia due to an atherogenic diet. In this research, SNPs were studied using DNA isolated from 22 cynomolgus monkeys obtained from a previous study, consisting of two hyporesponders, 19 hyperresponders, and one extreme case. The result showed that two SNPs existed in the promoter region, namely g.−169T>C and g.−265G>A. SNP g.−265G>A showed linkages with extreme responsiveness and can be used as a potential genetic marker for extreme animals. The result of this study has extended our knowledge of polymorphism in the LDLR gene and its use in the selection of cynomolgus monkeys as animal models in research on hypercholesterolemia and atherosclerosis.


Introduction
Macaques are commonly used in scientific research as animal models of human behavior and disease. Although less expensive and more abundant animal models, such as rodents and zebrafish, overshadow macaque use in research in part due to their easier husbandry, such animal models do not always provide the best March 2018 Vol. 22  No. 1 translational medicine when trying to elucidate human pathologies [1]. The long-tailed macaque (Macaca fascicularis), also known as the crab-eating or cynomolgus monkey, one of the monkeys (or macaques) most commonly used in biochemical research, particularly as an animal model for human atherosclerosis research, due to the general response of its plasma cholesterol to an atherogenic diet [2]. Even though most cynomolgus monkeys show a strong response to dietary cholesterol (hyperresponder), there are individuals in the population that show only a small response (hyporesponder) and still others that show a particularly strong response (extreme) [3]. A previous study on the development of an atherogenic diet to induce lipid plasma, conducted at the Primate Research Center of Bogor Agricultural University (PSSP IPB), reported that two of 22 cynomolgus monkeys fed an atherogenic diet for three months were hyporesponders and that one was extreme. It is hypothesized that variations in responsiveness to an atherogenic diet may be caused by genetic variability [4].
As with humans, cynomolgus monkeys have a diverse genetic background, as evidenced by a number of genetic polymorphisms reported previously [5,6], including several functional polymorphisms in genes involved in metabolic and inflammatory pathways [7,8] and in the immune system [9]. However, few studies have focused on polymorphisms in genes that are involved in cholesterol metabolism, particularly the LDLR gene, which codes for the low density lipoprotein receptor protein (LDL-R), and its implications for cholesterol levels and risk of atherosclerosis. Our previous study showed the existence of common genetic polymorphisms within the exon 6, intron 5, and 3'UTR regions of the cynomolgus monkey LDLR gene [10,11] but did not focus on the promoter region.
The LDLR gene encoding human LDL-R is 45 kb and is localized on chromosome 19 p13.1-13.3. The gene is composed of 18 exons and 17 introns, encoding an mRNA of 5.3 kb and a protein of 860 amino acids. The LDLR gene encodes a signal peptide (exon 1), a ligand binding domain (exons 2-6), an epidermal growth factor homology domain (exons [7][8][9][10][11][12][13][14], an O-linked sugar domain (exon 15), a transmembrane domain (exons 16,17), and a cytoplasmic domain (exons 17 and 18) [12]. LDL-R, a transmembrane cellular protein, plays a crucial role in the receptor-mediated pathway of lipoprotein metabolism. LDL-R modulates plasma levels of low density lipoprotein cholesterol (LDL-C) by regulating the liver's uptake of LDL particles and by delivering cholesterol to the adrenal glands and gonads for steroid hormone synthesis and to the liver for bile acid synthesis [13].
In this study, an attempt was made to identify polymorphisms within the promoter region of the cynomolgus monkey LDLR gene. The objective of this study was to identify single nucleotide polymorphisms (SNPs) within the promoter region of LDLR and their relationship with animal responsiveness to hypercholesterolemia due to an atherogenic diet. Results from this study on polymorphism are expected to improve efficiency in the use of cynomolgus monkeys as animal models in research on hypercholesterolemia and atherosclerosis.

Methods
Animals. The 22 cynomolgus monkeys (Macaca fascicularis) used in this study were adult males held at the animal facility of the Primate Research Center of Bogor Agricultural University, Indonesia. The monkeys ranged from six to eight years old, as estimated through their dentition, their body weights averaged between four and five kg. All monkeys were subject to the standards set forth by the Institutional Animal Care and Use Committee (ACUC) with protocol number 12-B009-IR. Blood samples were obtained from each of the 22 monkeys, which, as previously described, were identified as hyporesponder, hyperresponder, or extreme after following a three-month, high cholesterol diet [4]. Animals that had plasma cholesterol concentrations within a range of 1.5 standard deviations from the mean (250 to 900 mg/dL) were classified as hyperresponders, whereas animals with cholesterol levels below or above this range (<250 mg/dL or > 900 mg/dL) were classified as hyporesponder or extreme, respectively (Table 1). PCR and Sequencing. The promoter regions within the cynomolgus monkey LDLR genes were amplified based on primers from a previous study [14]. The primers were F: 5'-GGATCCCGACCTGCTGTGTCCTAGC TGGAAA-3' and R: 5'-GGATCCAATCAAGTCGCCT GCCCTG GCGACA-3'. PCR reactions were conducted in a 25 L volume and contained 5 L of genomic DNA, 1 L of each primer (10 pmol

Results and Discussion
Amplification and Sequencing. Amplification using forward and reverse primers for the promoter region resulted in product sizing of 263 bp ( Figure 1). Aligning the consensus sequences to the reference showed that the amplicon contained part of the promoter region of the cynomolgus monkey LDLR gene from position −296 to −34, relative to the major transcriptional initiation site (nucleotide +1 is the A of the ATG-translation codon, and nucleotide 5' of the ATG-translation codon is −1). This region contains three 16-bp repetitions which have been shown to be important for basal level expression and sterol regulation of human LDLR and transcription initiation sites [15].    T  G  --I  T  G  20  FE7777, T3700, T3536, FC9015, T3303, T3278, FC8501, T3535,  T3307, C4927, 9596, T3049, FG7909, FG7998, C0750, T3300, C4939,  FC9113, T3707, K30  II  C  G  1  C2480  III  T  A  1  C0613 were non functional; a functional polymorphism is defined as a site that causes binding site appearance or disappearance of new transcription factors [16].
The two polymorphic sites produced three haplotypes, i.e. haplotype I (TG), II (CG), and III (TA). Haplotype I is similar to that belonging to the reference cynomolgus monkey in the GenBank; almost all (20 of 22) animal samples contained this haplotype (Table 2). Haplotype grouping becomes particularly interesting when the results are compared to the cynomolgus monkeys' responsiveness to an atherogenic diet, shown in Table 1. Namely, haplotype III (TA) belongs to cynomolgus monkey C0613, which is also the only study animal that demonstrates an extreme response to the atherogenic diet. The similarity of the SNP and responsiveness groupings shows a relationship between polymorphism and responsiveness to an atherogenic diet in cynomolgus monkeys.
This research demonstrated the existence of common genetic polymorphisms within the promoter regions of the cynomolgus monkey LDLR gene. Linking haplotype III to extreme responsiveness in the cynomolgus monkey gives the identified polymorphic sites the potential to be used as genetic markers, although the results still need to be confirmed using a larger sample of extreme responders. The existence of non functional SNPs within the promoter region suggests the possibility that these SNPs are exhibiting linkage disequilibrium in relation to other functional SNPs, influencing the responsiveness to an atherogenic diet.
The function of polymorphisms identified in the LDLR gene of cynomolgus monkeys as a genetic marker for responsiveness has been studied before. Our previous study [10] reported the existence of a specific haplotype GCGC within the 3'UTR of the cynomolgus monkey LDLR gene that belonged only to an animal with extreme responsiveness. Moreover, within the regions of exon 6 and intron 5, the study found a specific haplotype associated with hyporesponders [11]. Thus, the present study supports the important principle that a commom genetic variant can be used as a potential genetic marker of responsiveness. Previous reports identifying SNPs or haplotypes of the cynomolgus monkey have been mainly related to origin or geographical distribution [17,18]. Investigations of individual genetic variations that affect susceptibility to diseases or other disorders are still few when compared to the total number of biomedical research studies conducted on cynomolgus monkeys. Some such studies have included genetic variations related to malaria susceptibility [16], drug safety [19], and neurobiological reactivity due to stress [20].
Identification of genetic variations within the promoter region of the LDLR gene as genetic markers of responsiveness to an atherogenic diet is an important breakthrough, as it makes the preliminary selection of animals simpler and more efficient. To date, in primate centers the selection of animals for research on responsiveness to diet requires an atherogenic diet intervention for two months [21,22]. This selection process is inefficient, because it requires strict control of the diet. Using the presence of genetic markers as a basis for the selection of extreme animals eliminates the need for treatment and control of the diet. In terms of time, the analysis of genetic variation is faster, while in terms of budget, genetic variation analysis is cheaper than animal feeding and care for the two month selection period in primate centers.
Selection of test animals prior to conducting research related to atherosclerosis is crucial, as it can improve the accuracy and efficiency of the scientific studies that will eventually support research success. Initial selection also reduces the number of test animals, thereby supporting the principles of the 3Rs (reduction, refinement, and replacement). Furthermore, the selection of animals based on genetic variation may reduce current limitations of using primates as models in genetic studies of complex diseases. Using animals that are genetically uniform will give greater power to the statistical analysis of tested variables, especially when using small samples [23]. The work presented here addresses the genetic variation in a functional gene and its influence on experimental outcomes. This result can also improve knowledge of biomarkers for disease, genetic disorders, and toxicology.

Conclusion
This research has identified two SNPs in the promoter region of the cynomolgus monkey LDLR gene, which can be used as a potential genetic marker for animals with extreme responsiveness to an atherogenic diet. Results from this study contribute to the efficient use of the cynomolgus monkey as an animal model in research related to hypercholesterolemia and atherosclerosis.