Characterization of 100 extended major histocompatibility complex haplotypes in Indonesian cynomolgus macaques

Abstract

Many medical advancements-including improvements to anti-rejection therapies in transplantation and vaccine development-rely on preclinical studies conducted in cynomolgus macaques (Macaca fascicularis). Major histocompatibility complex (MHC) class I and class II genes of cynomolgus macaques are orthologous to human leukocyte antigen complex (HLA) class I and class II genes, respectively. Both encode cell-surface proteins involved in cell recognition and rejection of non-host tissues. MHC class I and class II genes are highly polymorphic, so comprehensive genotyping requires the development of complete databases of allelic variants. Our group used PacBio circular consensus sequencing of full-length cDNA amplicons to characterize MHC class I and class II transcript sequences for a cohort of 293 Indonesian cynomolgus macaques (ICM) in a large, pedigreed breeding colony. These studies allowed us to expand the existing database of Macaca fascicularis (Mafa) alleles by identifying an additional 141 MHC class I and 61 class II transcript sequences. In addition, we defined co-segregating combinations of allelic variants as regional haplotypes for 70 Mafa-A, 78 Mafa-B, and 45 Mafa-DRB gene clusters. Finally, we defined class I and class II transcripts that are associated with 100 extended MHC haplotypes in this breeding colony by combining our genotyping analyses with short tandem repeat (STR) patterns across the MHC region. Our sequencing analyses and haplotype definitions improve the utility of these ICM for transplantation studies as well as infectious disease and vaccine research.

Keywords: Haplotype; Macaca fascicularis; Major histocompatibility complex; Short tandem repeat; Transplantation.

Fig. 1

Relative location of STR loci versus MHC class I and class II genes investigated in this study within the full genomic MHC region on chromosome 4

Fig. 2

An example of an extended MHC haplotype defined by the MHC-MAFA-NIAID1-000061 STR pattern. For Mafa-A, Mafa-B, and Mafa-DRB, multiple transcripts with a wide range of steady-state RNA levels as indicated by the intensity of the color of the locus bubble, together make up the regional haplotype in bold. For the remaining class II loci, each gene is indicated by a single transcript in bold font

Fig. 3

PacBio sequencing results for four of the 293 macaques in this study. MHC class I results are shown on the left and class II data is on the right side. For each macaque, we list the animal ID, sire ID, dam ID, and number reads mapped with PacBio sequencing. Only major Mafa-B reads are listed for illustrative purposes, but complete data including minor transcripts can be found in Supplemental Figure 8. Each of these animals shares a common sire and inherited the paternal haplotype containing the MHC-MAFA-NIAID1-000048 STR pattern (yellow). Each of the progeny expresses a series of class I and class II transcripts in common (yellow) that are represented by the number of identical PacBio sequences reads that were detected in each animal. These co-segregating transcripts are used to define the abbreviated regional haplotypes listed below the STR profiles. The remaining transcripts expressed by each animal are highlighted in a different color and inferred to segregate as the maternal haplotype inherited from their respective dams. Diagonally shaded boxes indicate that a transcript such as Mafa-B*030:04 is included on both the paternal and maternal extended MHC haplotypes. If PacBio read support is low, but a sequence is observed in related animals and/or MiSeq data, it is noted as inferred, e.g., Mafa-DPA1*17:01:02 for the extended haplotype defined by the shared MHC-MAFA-NIAID1-000048 STR pattern

Fig. 4

STR patterns for extended MHC haplotypes that share Mafa-A004.01.02 or Mafa-A010.01.05 haplotypes. STR loci are displayed from telomere (left) to centromere (right) and STR allele sizes that define each extended haplotype are listed. Shading and boldface highlights the STR alleles that are shared between multiple extended haplotypes that include Mafa-A004.01.02 (a) or Mafa-A010.01.05 (b) haplotypes