Identification of MHC class I sequences in Chinese-origin rhesus macaques

Abstract

The rhesus macaque (Macaca mulatta) is an excellent model for human disease and vaccine research. Two populations exhibiting distinctive morphological and physiological characteristics, Indian- and Chinese-origin rhesus macaques, are commonly used in research. Genetic analysis has focused on the Indian macaque population, but the accessibility of these animals for research is limited. Due to their greater availability, Chinese rhesus macaques are now being used more frequently, particularly in vaccine and biodefense studies, although relatively little is known about their immunogenetics. In this study, we discovered major histocompatibility complex (MHC) class I cDNAs in 12 Chinese rhesus macaques and detected 41 distinct Mamu-A and Mamu-B sequences. Twenty-seven of these class I cDNAs were novel, while six and eight of these sequences were previously reported in Chinese and Indian rhesus macaques, respectively. We then performed microsatellite analysis on DNA from these 12 animals, as well as an additional 18 animals, and developed sequence specific primer PCR (PCR-SSP) assays for eight cDNAs found in multiple animals. We also examined our cohort for potential admixture of Chinese and Indian origin animals using a recently developed panel of single nucleotide polymorphisms (SNPs). The discovery of 27 novel MHC class I sequences in this analysis underscores the genetic diversity of Chinese rhesus macaques and contributes reagents that will be valuable for studying cellular immunology in this population.

Fig. 1. Phylogenetic analysis of 41 MHC class I sequences identified in Chinese-origin rhesus macaques

Neighbor-joining tree based on 1068 aligned nucleotide sites. Numbers on branches are percentages of 1,000 bootstrap samples supporting the branch; only values ≥50% are shown

Fig. 2. Putative shared microsatellite haplotypes and Mamu-A and Mamu-B cDNAs for six animals

a. ChRh10, ChRh11, and ChRh12; b ChRh03, ChRh05, and ChRh07. The microsatellite profiles of each set of animals with a shared haplotype are shown with their respective microsatellite allele sizes. Designation of MHC cDNAs to a shared haplotype is inferred. For Fig. 2b, one or more of the shared cDNAs could be associated with the shared haplotype, despite not being observed in all three animals due to the limited number of cDNA clones examined. cDNAs listed in bold were identified in three or more clones while cDNAs listed in grey were present in only one or two clones

Fig. 3. PCR-SSP results for eight Chinese rhesus macaque MHC class I sequences

The primer sequences and gel images for the eight cDNAs selected for PCR-SSP assay development are shown. Numbers above each lane represent the animal name (ChRh01 through ChRh12). The plus signs indicate animals shown to be positive for each sequence by cDNA cloning and sequencing. The minus sign indicates an animal positive for Mamu-B*8701, which is occasionally amplified by the primers for Mamu-A1*5702. The brightest band of the ladder corresponds to 500 bp

Fig. 4. SNP analysis of ChRh01-ChRh12

SNPs used for the analysis are identified on the x-axis and animal IDs are on the y-axis. Animals shaded in blue are of known Chinese origin and those in orange are of known Indian origin. Rhesus macaques from our cohort that are either Chinese/Indian hybrids or are derived from a population of Chinese rhesus macaques originating in a previously unsampled region of China are shaded in gray. SNP genotypes are color-coded with green representing homozygous major alleles, red representing homozygous minor alleles, and yellow representing heterozygous alleles. A single Indian allele in a putative Chinese animal falls within our definition of pure ancestry, which allows for the presence of a single allele that may be extremely rare in the defined ancestry group (minor allele frequency <0.02). However, statistical likelihood of having two such rare alleles is low (<0.0004) and suggests that the animal is from an uncharacterized geographic region or is a hybrid. It is also possible that all animals in the cohort may be from the same geographic region, explaining the presence of the rare alleles in multiple animals