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LRP5 is known to have an important relationship with bone density and a variety of other biological processes. Mapping to human chromosome 11q13.2, LRP5 shows considerable evolutionary conservation. Orthologs of this gene exist in many species, although comparison of human LRP5 with other non-human primates has not been performed until now.
We report the complementary DNA (cDNA) sequence and deduced amino acid sequence for baboon LRP5, and compare the baboon and human sequences. cDNA sequences for 21 baboons are examined to identify single nucleotide polymorphisms (SNPs).
Sequences of coding regions in human and baboon LRP5 show 97% to 99% homology. Twenty-five SNPs are identified in the coding region of baboon LRP5.
The observed degree of coding sequence homology in LRP5 leads us to expect that the baboon may serve as a useful model for future research into the role(s) of this gene in primate metabolic diseases.
Numerous studies of quantitative trait loci (QTLs) and single nucleotide polymorphisms (SNPs) in humans and mice demonstrate that low-density lipoprotein receptor-related protein 5 (LRP5), a member of the LDL receptor superfamily, plays an important role in several biological processes and that mutations in LRP5 underlie a variety of inherited diseases. Mutations in the gene have been linked to Type I diabetes [12, 41], hypercholesterolemia , atherosclerosis , Familial Exudative Vitreoretinophathy (FEVR) [22,35], and osteosarcoma , as well as several conditions involving variation in bone mineral density (BMD).
The genomic structure of LRP5 is well-characterized in humans. The LRP5 gene contains 23 exons and encodes a single-span transmembrane protein of 1,615 amino acid residues. The first 24 amino acids encode a signal peptide sequence. Four β- propeller domains separated by Epidermal Growth Factor-like (EGF) repeats are located immediately downstream of the signal peptide sequence followed by three LDL-receptor type A (LA) domains, a single-pass transmembrane domain, and a cytoplasmic domain at the C-terminal end. Each of these regions has been characterized in some detail (In particular, see Springer, 1998; Hey, 1998; Rudenko et al, 2002; and Johnson and Summerfield, 2005). The four β- propeller domains contain six conserved YWTD (Tyr-Trp-Thr-Asp) repeats each. While YWTD is the common motif in these domains, structurally similar sequences such as LFAN and FFTN serve the same function . The YWTD repeats form the blades of each β- propeller, which bring the EGF-like regions into close proximity to each other, and may play a role in ligand binding .
Considerable research interest in LRP5 during the past ten years has been motivated largely by the results of whole genome linkage screens and candidate gene association studies that have strongly implicated the gene – and variants therein – in the regulation of bone mass and density [4, 7,17,24, 27-29,31,42,44]. Studies of the relationship between LRP5 polymorphisms and skeletal phenotypes [2,14,22,28,32,43] reveal that mutations in LRP5 in humans are responsible for skeletal disorders of both high and low BMD.
Due to the large number of people affected by bone fragility diseases (i.e. osteoporosis) the extent to which variation in the LRP5 gene contributes to normal variation in BMD in the general human population is of considerable interest. Recent studies suggest that LRP5 polymorphisms in humans may indeed contribute to population-level BMD variation [11,30,34,45]. Studies of inbred mice have provided important insight into the molecular mechanism of LRP5 effects, but substantial differences in processes of skeletal maintenance and turnover between primate and non-primate species [3,21] make the development of a non-human primate model in which to study the role of LRP5 in bone metabolism particularly relevant.
Genome-wide linkage screens using data from captive, pedigreed and genetically characterized baboons show a QTL affecting normal variation in forearm BMD in a region of the baboon chromosome 14 (PHA 14) that may be orthologous to the chromosomal region that harbors the LRP5 gene in humans (HSA 11q13.2) . Localization of bone-related QTLs to likely orthologous regions in both humans and baboons motivated us to further study and characterize PHA14 and its component genes in the latter species. Given the demonstrated relevance of one of these component genes, LRP5, to variation in bone mass and BMD in humans [2,4,11,14,22,24,28,30,32,34,42,43,45], we initiated a study to sequence this gene in baboons from the captive breeding population in which the PHA14 QTL was localized and genetic studies of age-related changes and pathology in bone and other organ systems are ongoing.
We report the results of a study with three main objectives. The first objective is to determine the DNA sequence of the coding regions of baboon LRP5 and the predicted amino acid sequence of its protein product. The second, is to establish the degree of sequence similarity between humans and this potentially valuable non-human primate model species. The third, and last, objective of this study is to identify single nucleotide polymorphisms (SNPs) in the LRP5 coding regions in the baboon.
The baboon shares physiological and developmental characteristics that make it particularly well-suited to studies of skeletal maintenance and turnover in humans. This animal shares with humans a relatively long lifespan (an important point because animals with shorter life spans do not maintain their skeletons in the same way humans do ). Further, baboons and other Old World Monkeys have been shown to undergo bone loss with advancing age [1,10,18,26] as do humans. Baboon reproductive physiology and endocrinology are also quite similar to that of humans  in that the baboon experiences similar timing and phases of the menstrual cycle, parallels humans with regard to hormonal changes accompanying pregnancy , and undergoes a natural menopause [3,6,18]. These physiological similarities and the close genetic proximity between baboons and humans suggest that results of studies investigating baboon LRP5 may be directly relevant to humans. Moreover, examination of specific LRP5 domains across multiple species (i.e., mouse, baboon, and human) will likely reveal highly conserved regions of the gene that are potentially very important to skeletal maintenance.
All animals from which data were obtained are part of the breeding colony at the Southwest National Primate Research Center (SNPRC)/Southwest Foundation for Biomedical Research (SFBR). The sample consists of olive baboons (Papio hamadryas anubis), yellow baboons (Papio hamadryas cynocephalus) and their hybrids. All procedures related to their treatment during their lives at SNPRC/SFBR were approved by the Institutional Animal Care and Use Committee in accordance with established guidelines. The current study involves isolation and sequencing of DNA from stored baboon liver specimens acquired from the SNPRC Blood, Tissue and DNA Repository. SNPRC archived baboon tissues were originally collected during routine necropsy. Animals were euthanized according to methods compliant with the AVMA panel on Euthanasia.
Tissue samples for this study are from a subset of the same group of animals in which a QTL affecting forearm BMD was detected near the location of the LRP5 gene . For these whole genome screens, animals with BMD data were organized into 11 large, extended pedigrees. To improve our chances of capturing all sequence variation in LRP5 likely to be relevant to the previously localized BMD QTL, we used RT-PCR to amplify LRP5 cDNA from 21 baboons (3 males, 18 females) discordant for BMD and representing ten pedigrees. All pedigree founders for which liver tissue was available were included. Additional animals were selected to ensure that one animal of relatively high BMD and one of relatively low BMD was represented in each pedigree (Table 2).
In order to characterize genetic diversity in baboon LRP5, SNPs were identified and examined for any resulting amino acid changes (non-synonymous). Animal ID number, sex, age, and forearm BMD z-scores for each individual are reported in Table 2. LRP5 SNPs, exon location and resulting amino acid changes, if any, are listed in Table 3. Table 4 reports the alleles present at each location in individual baboons.
Messenger RNA was extracted from baboon liver using the Ambion Trizol reagents and extraction protocol (Ambion Inc, Austin, TX). First strand cDNA synthesis used 2.5ug mRNA with Superscript III reverse transcriptase (Invitrogen Corporation, San Diego, CA). Complementary DNA for baboon LRP5 was amplified in three overlapping fragments using rTaq polymerase according to manufacturer’s instructions (Takara Shuzo Co., Kyoto, Japan). Primers for cDNA amplification were designed using Oligo software (Molecular Biology Insights, Inc.) and are listed in Table 1. Fragment one included LRP5 exons 1-8. Fragment two included LRP5 exons 7-18. Fragment three included exons 16-3′UTR.
PCR conditions for DNA amplification were: 1 min 30s denature at 96°C, followed by 35 cycles of 30s denature at 94°C, 30s annealing at 62°C and 5 min extension at 72°C with a final extension of 10 min at 72°C. Excess primers were eliminated using 5.3 units of shrimp alkaline phosphatase (SAP), 27 units of exonuclease I (Exo I). The SAP/Exo I reaction was carried out at 37°C for 30 min followed by a 15-min heat inactivation at 80°C. The DNA from the SAP/Exo I reaction was used directly for DNA cycle sequencing [1min @ 96°C; 25 times (10sec @ 96°C; 4min @ 60°C)] using Big Dye Chemistry (v 3.1, Applied Biosystems, Inc., Foster City, CA) followed by analysis using an automated ABI DNA sequencer (Model 3730). The baboon cDNA sequence was aligned with human LRP5 sequence (Genbank ID: AP002366) using Sequencher software (Gene Codes, Inc., Ann Arbor, MI) and Codon Code Aligner Software (CodonCode Corporation, Dedham, MA). Predicted protein structure was determined using ExPASy translate tool of the Swiss Institute of Bioinformatics (https://www.expasy.ch/). Protein alignment was conducted using ClustalW (www.ebi.ac.uk/clustalw/).
Comparison of baboon and human LRP5 cDNA nucleotide sequences shows a high level of conservation (97% identity). The partial baboon LRP5 cDNA sequence can be found in the publicly accessible databank, GenBank (accession # FJ009119). Human sequence AP002366, used in this comparison, was obtained from GenBank.
Deduced amino acid sequence alignment between baboon and human shows a high degree of conservation with 99% amino acid identity. Figure 1 shows the baboon LRP5 amino acid sequence aligned with the human sequence. The LRP5 signal sequence, encoded by the first 24 amino acids, is not highly conserved and there is a difference in length between humans and baboons due to the deletion of three amino acids in baboon LRP5. Of 1,615 amino acids in LRP5, only four amino acid differences between human and baboon were identified, all of which are non-conserved: S315R, located in the first EGF-like domain; Y932H, located in the third EGF-like domain; and I1162V and I1191V, both located in the fourth β- propeller region. No differences were noted in the 5′ region. The 3′ region of baboon LRP5 shares 94% identity with the human sequence and a CpG island, as well as three predicted miRNA sites in the 3′ region, are conserved in the baboon.
Analysis of baboon LRP5 cDNA sequence from 21 baboons discordant for BMD, showed 25 polymorphisms (Table 3). Two of these SNPs were nonsynonymous: G1469R and M1584L. Both of these are located in the cytoplasmic domain of LRP5 and are not conserved.
In spite of the high level of sequence homology exhibited between human and baboon LRP5, some differences warrant discussion. Two of the four amino acid differences identified between human and baboon LRP5 are located in the epidermal growth factor-like (EGF) regions. Little is known about specific interactions of the LRP5 EGF domains. Mice in which these regions are knocked out usually also show removal of the surrounding β- propeller domains, making it difficult to identify the independent effects of these two regions . In humans, mutations in these domains, which are associated with particular bone density phenotypes, usually result in a LRP5 truncated protein. This is not the case for the amino acid residues that differ between human and baboon where changes in the first and third EGF domains of baboon LRP5 (S315R and Y932H, respectively) are predicted to have no impact on the protein structure. The amino acid changes in the fourth β-propeller region (Ile>Val) are also neutral changes and do not affect any of the conserved YWTD repeats located in the β-propeller region. Additionally, the conserved 5′ and 3′UTR suggest that regulation of mRNA stability and translation are conserved. These results, predicting functional conservation of LRP5 between human and baboon, support the hypothesis that LRP5 may plays a similar role in the regulation of bone density in the baboon.
Identification of SNPs provides important information about gene sequence diversity and can be used to determine the relationship between genetic variation and phenotype. Twenty-five SNPs were identified in the coding regions of baboon LRP5.
The current human SNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/index.html) records 18 coding-region SNPs for human LRP5. This is substantially fewer than the 25 SNPs detected in the coding region of the baboon gene, suggesting less variation in the human gene. Only two of the SNPs identified in baboon LRP5 resulted in amino acid changes (G1469R and L1584M). Non-synonymous amino acid changes in human LRP5, identified in the current human SNP database, are located in exons 2 (rs41494349), 9 (rs4988321),16 (rs11607268),18 (rs3736228) and 22 (rs11574422). The two non-synonymous SNP amino acid changes identified in baboon LRP5 are located in exons 21 and 23. These exons encode for the cytoplasmic portion of LRP5, as does exon 22, which contains human SNP rs11574422.
In general, previous research on human or mouse LRP5 has shown that mutations leading to a phenotype of high BMD seem to be confined to the first β-propeller region. Most of these appear within or near the conserved YWTD repeats. Not all mutations found in this region, however, lead to increased BMD and mutations leading to a phenotype of low BMD have also been identified in the first β- propeller region [40,43]. Current research shows that mutations in the second, third and fourth β- propeller domains are primarily associated with osteoporosis pseudoglioma syndrome and FEVR conditions resulting in both BMD and vision problems [9,14,22,23,40]. FEVR has also been associated with mutations in the first and third LA domains. [9,22,40]. No non-synonymous SNPs were identified in these extracellular regions of baboon LRP5.
Both baboon LRP5 residue changes are located in the cytoplasmic region of LRP5 and may result in a change in protein function. The cytoplasmic portion of LRP5 contains three PP(T/S)P motifs that bind axin . All three repeats are conserved in the baboon LRP5, thus, axin binding should not be affected. However, one of the residues, (L1584M), lies in a region that acts as a binding site for FRAT1 , a protein that interacts with LRP5 to inhibit phosphorylation of β-catenin by GSK-3, resulting in TCF1 transcriptional activation. Recent studies indicate that LRP5/FRAT1 interaction is required for activation of the canonical Wnt signaling pathway . It is possible that a SNP located in this particular region of the cytoplasmic portion of LRP5 may affect LRP5/FRAT1 interaction. If LRP5/FRAT1 interaction and subsequent β-catenin signaling are inhibited, the expected result is a low BMD phenotype due to reduced Wnt-signaling. Indeed, the SNP reported in this region (4789 C>A) was identified in several individuals with low BMD (Individuals 2, 4, 8, 10, 13, 14, 15, 16 and 17), however, it is also present in individuals exhibiting high BMD (individuals 1, 11, 12, and 19), and one individual with moderate BMD (Individual 9). Individuals homozygous for the C allele at this location (individuals 3, 5 and 7) also exhibit a wide range of BMD phenotypes, suggesting that the SNP is not strongly associated with any particular BMD phenotype in these animals. Interestingly, however, individuals exhibiting heterozygosity at this location all show relatively high BMD (individuals 6, 18, 20, 21). It is possible that a single copy of one of the alleles at this location (as exhibited by individuals 6, 18, 20, and 21) may create a conformational change in the protein leading to prolonged Wnt-signaling and, subsequently, higher BMD. The sample size presented here, however, is too small to allow for statistical analyses to identify statistically significant differences. The current study is solely aimed at identifying SNPs for further examination, thus, the issue of specific SNPs and association with BMD phenotypes will require further investigation in a larger sample.
None of the SNPs discovered in baboon LRP5 correspond to polymorphisms in human LRP5 that are reported to cause disorders of high or low BMD. Bone density data, however, are available for the 21 subjects used in this study (Table 2) and specific care was taken to include individuals representing both relatively high and low bone density phenotypes. The next step in our investigations of LRP5 is to genotype these SNPs in a larger sample, for which BMD data are available, in order to ascertain the relationship between bone phenotypes and sequence variation in the baboon. Although it is unlikely that the precise functional variants will match those in humans, it is highly likely that the exons or domains in which they occur and their mechanisms of action will correspond to the human condition.
Though rodent models have been used to generate and test hypotheses regarding the involvement of LRP5 in bone maintenance, these animals are not ideal due to important differences in bone metabolism between rodents and humans . The current study addresses the need for a well-characterized non-human primate model and provides evidence that the baboon is an appropriate animal model for studies of the role of this gene in primate skeletal maintenance, and in other metabolic processes in which LRP5 plays a role.
The results seen here have general implications for conservation and homology between baboons and humans. We acknowledge that variation in a gene’s expression, as well as the processing and modification of its transcripts and the translation of its products may be affected by DNA-level variation yet unmeasured by us in this study: e.g., introns, promoters, enhancers, miRNAs, epistatic genes, etc. However, we hypothesize that the observed degree of coding sequence homology between baboon and human LRP5 also may be predictive of similar functions for this gene in these two species.
The LRP5 sequence data generated by this study were obtained from analyses of DNA from the same pedigreed baboons in which genetics of bone metabolic disorders and disorders of other metabolic systems are ongoing, and which also contributed genetic data on which the baboon genome linkage map is based. Therefore, SNPs identified in this study have several immediate research applications. Not the least of these is using LRP5 SNP genotypes to map LRP5 to the genetic linkage map for PHA14. Doing so will improve resolution for one of the seven baboon chromosomes exhibiting rearrangements relative to their human orthologs [8, 36]. Additionally, LRP5 SNP genotypes can be used in pedigree-based association  and linkage analyses to test for effects of this gene on variation in BMD and a host of other bone-related phenotypes currently under study in these baboons.
Biomaterials collection for this study was supported in part by the following research grant from the U.S. National Institutes of Health (NIH): P01 HL028972. This investigation was conducted in part in facilities constructed with support from Research Facilities Improvement Program Grants C06 RR017515, C06 RR013556, C06 RR014578 from the National Center for research Resources (NCRR), NIH. The NIH NCRR base grant, P51 RR013986, supports the Southwest National Primate Research Center. The authors would like to thank the anonymous reviewers for helpful comments and suggestions.
This research was supported by a Southwest National Primate Research Center (SNPRC) Pilot Study Grant: 2 P51 RR013986-08. Biomaterials collection for this study was supported in part by the following research grant from the U.S. National Institutes of Health (NIH): P01 HL028972. This investigation was conducted in part in facilities constructed with support from Research Facilities Improvement Program Grants C06 RR017515, C06 RR013556, C06 RR014578 from the National Center for research Resources (NCRR), NIH. The NIH NCRR base grant, P51 RR013986, supports the Southwest National Primate Research Center.