Identification of novel MHC class I sequences in pig-tailed macaques by amplicon pyrosequencing and full-length cDNA cloning and sequencing

Abstract

Pig-tailed macaques (Macaca nemestrina) provide important animal models in biomedical research, but utility of this species for HIV and other disease pathogenesis research is limited by incomplete knowledge of major histocompatibility complex (MHC) class I genetics. Here, we describe comprehensive MHC class I genotyping of 24 pig-tailed macaques, using pyrosequencing to evaluate a 367 bp cDNA-PCR amplicon spanning the highly polymorphic peptide-binding region of MHC class I transcripts. We detected 29 previously described Mane transcripts, 90 novel class I sequences, and eight shared MHC class IB haplotypes. We used this genotyping data to inform full-length MHC class I cDNA allele discovery, characterizing 66 novel full-length transcripts. These new full-length sequences nearly triple the number of Mane-B cDNA sequences previously characterized. The comprehensive genotypes and full-length Mane transcripts described herein add value to pig-tailed macaques as model organisms in biomedical research; furthermore, the coordinated method for MHC genotyping and allele discovery is extensible to other less well-characterized nonhuman primate species.

Linking Pig-Tailed Macaque Major Histocompatibility Complex Class I Haplotypes and Cytotoxic T Lymphocyte Escape Mutations in Simian Immunodeficiency Virus Infection

“…The resulting sequencing data were binned by multiplex identifier, and sequence reads from each animal were assembled into unidirectional contigs having 100% identity using a custom genotyping analysis work flow (LabKey, USA). The number of sequence reads comprising each unidirectional contig was enumerated, and the resulting consensus sequences were mapped against an in-house database containing known pig-tailed MHC-I allele sequences using the BLASTn program (33)(34)(35). Putative Mane-A and Mane-B haplotypes were inferred by identifying combinations of shared alleles between animals as described previously for rhesus macaques (35).…”

“…Multiple MHC-I alleles and/or haplotypes have been characterized in a limited number of pig-tailed macaques (33,34,40,41). Macaques can express more than 20 MHC-I transcripts from a variable number of Mane-A and Mane-B loci that have undergone gene duplication, in contrast to their human counterparts (34,40).…”

“…Macaques can express more than 20 MHC-I transcripts from a variable number of Mane-A and Mane-B loci that have undergone gene duplication, in contrast to their human counterparts (34,40). Each macaque carries a pair of Mane-A and Mane-B haplotypes inherited from the parents.…”

“…Each macaque carries a pair of Mane-A and Mane-B haplotypes inherited from the parents. Abbreviated haplotype designations for Mane-A were based on the presence of a transcriptionally abundant Mane-A1 allele, while Mane-B haplotypes were assigned on the basis of a diagnostic major Mane-B transcript that is characteristic for a group of linked Mane-B alleles, as previously described for rhesus macaques (34,42). We identified a diverse range of Mane-A and Mane-B haplotypes in our cohort of 44 SIV mac251 -infected pig-tailed macaques, as shown in Table 2.…”

Genomic Sequence Analysis of the MHC Class I G/F Segment in Common Marmoset (Callithrix jacchus)

“…Primers and sequencing were essentially as described by O'Leary et al (18). Obtained sequences were compared by basic local alignment search tool (BLAST) search with Caja-G and Caja-E cDNA sequences available from public databases (IPD), as well as with Caja-B1, Caja-B3, Caja-B4, Caja-B6, and Caja-B7 sequences described by Shiina et al (12).…”