Diversity of Immune Response (Major Histocompatibility Complex, MHC) Genes in Free-Ranging Pinnipeds
The majority of studies examining genetic diversity of marine mammal species have focused on non-functional genetic markers. An understanding of the past role and future threat of infectious disease requires an examination of genetic diversity within the major histocompatibility complex (MHC). The MHC is a family of highly polymorphic genes encoding a set of transmembrane proteins that bind processed foreign peptides and present them to T-lymphocytes and therefore, are central to maintaining immunologic vigor in a population. Two classes of MHC genes have been described. Class II is pivotal for generation of all adaptive immune responses, and class I plays a more limited role, specifically of viral immunity. The high level of MHC genetic variation that is found in most natural populations probably reflects the antagonistic co-evolution between host and pathogen. Since adaptation to pathogen exposure essentially determines the repertoire of antigenic determinants to which an individual is capable of recognizing and responding, the repertoire of MHC genes is critical to the generation of immune responses and influences both disease susceptibility and vaccine responses. The ultimate goal of our laboratory is to identify associations between polymorphism and genotype with population health and pathogen/contaminant pressure(s). Thus, we chose to examine the population immunogenetics of four pinniped species, each of which is confronted with different ecological and pathological challenges
The Hawaiian monk seal (Monachus schauinslandi) population is listed as endangered, having shown an inability to rejuvenate above 1200 individuals following the precipitous declines of approximately 60% between 1950 and 1993. In this study, we provide evidence that the remaining monk seal population exhibits an unprecedented uniformity in several vital genes, of which the sequence variation is essential in maintaining immunological diversity. This information is important, particularly in view of recent pathogen associated mass mortality events in seals, and the recent formal declaration of extinction of the closely related Caribbean monk seal (Monachus tropicalis). The lack of immunogenetic diversity in this species was first observed in 5 captive Hawaiian monk seals. Using degenerate primers designed for the 3 major established classical class I loci (HLA-A, -B and -C), we expected to amplify multiple class I MHC gene sequences from each animal. Surprisingly, only 3 full-length MHC class I gene transcripts could be identified in each monk seal using high throughput sequencing. Furthermore, the amount of sequence variation, both within and between individuals, was extremely low. The utility of these primers in amplifying pinniped class I MHC genes was confirmed by analogous sequencing studies in California sea lions (Zalophus californianus -CSL) and harbor seals (Phoca vitulina), in which multiple and variable expressed genes were detected. The possibility of inadvertently amplifying non-classical MHC gene sequences, which, while similar in primary sequence, are less polymorphic than classical MHC genes, was nullified by demonstrating the same results with multiple primer pair combinations from both translated and un-translated gene regions.
The dearth of MHC variability in the monk seals was investigated further by comparing exons encoding the highly polymorphic domains of the putative peptide binding region between all 3 species. Most importantly, the extent of the poverty of class I MHC genomic sequence variation was demonstrated in an additional 80 monk seals (~6.7% of the estimated population). We hypothesize that the paucity of MHC diversity described in this study is more compatible with low-grade and long-term selection pressures exerted on MHC class I gene loci, rather than an historical population bottleneck. While unprecedented and intuitively disturbing, however, information regarding population heterozygote advantage is not available for monk seal MHC genes and therefore, further studies are required to determine the functional relevance of these findings to the immunological health of these animals and to the survivability of this species.
CSLs are excellent candidates to define MHC class II gene polymorphisms in a thriving pinniped population. Initial efforts were directed at the DQ Class II genes as they carry the major polymorphism in canine species. The identification of five unique DQA sequences and eight unique DQB sequences, which are all encoding functional proteins, indicates the presence of multiple DQ loci in this species. Despite the identification of multiple DQA and DQB sequences, the degree of heterogeneity between them was extremely low. This lack of DQ polymorphism was independent of geographical region. Previous studies that identify a similar lack of DQ polymorphism in thriving species have been used to question the importance of MHC diversity in the vulnerability of a population to disease. An alternative explanation, however, is that sea lion DQ-encoded MHC class II proteins play a non-classical role in the immune response to pathogens, and the species utilizes other MHC genes to generate and maintain immunologic vigor. Studies were extended to characterize genes encoding DR molecules, which have been shown to be polymorphic in some terrestrial carnivores. DRB genes were identified that were unique in their high levels of variability. CSL DRB constitutes a gene family comprised of at least 8 loci, each of which exhibit limited allelic variability, but present in variable configurations among individuals. Studies were extended to begin to apply MHC genotyping capabilities to the identification of disease susceptibility. The first application was to study the ongoing epidemic of urogenital cancer in free-ranging populations of CSLs. The sample population for this study was selected from adult sea lions stranding along the Pacific coast. Extensive necropsies were performed and the cancer status of each individual was defined based upon detailed histopathological examination of multiple tissues. The MHC class II genotype was determined for both cancer positive (n = 28) and cancer negative (n = 22) animals using a combination of 8 genes (DRB-A to -H), and compared to that of non-stranded free-ranging sea lion populations (n = 202). The associations and interactions between MHC genotype, gene multiplicity, presence of cancer, gender, and geographic location were examined by discriminant function and logistic regression analyses. CSL MHC class II diversity arises from variable configurations of multiple loci. Thus, the absence of a relationship between the number of DRB genes and the occurrence of cancer indicates that MHC class II multiplicity was not a factor in conferring susceptibility to this disease (odds ratio = 1.67, 95% confidence interval = 0.91-3.088), and neither were gender or geographical location. In contrast, the presence of the MHC class II locus, DRB-A, was strongly associated with an increased risk of cancer (odds ratio = 3.31, 95% confidence interval = 1.023-10.72). Although breeding site fidelity can give rise to intergroup MHC class II genotypic differences, the stranded individuals were genetically indistinguishable from the broader Pacific sample population.
While other genetic, environmental, or pathogenic factors may be involved in this epidemic, the pattern of associations between the presence of specific MHC class II genes and cancer risk emerges from this study. While incidental deviations in genotype frequencies may result in illegitimate associations when studying polymorphic systems, the phenomenon of MHC class II genes conferring susceptibility to cancer is well-established in people, although the potential mechanisms underlying this process are unknown. Based upon the hypothesis that CSLs from different locations are subject to different pathogen pressures, this species presents a natural model to examine the effects of different environmental influences on MHC. In a separate study, genotypic differences in MHC between CSLs inhabiting the Gulf of California and two Pacific Ocean locations also were identified. Additionally, genotypic differences were identified among CSLs from closely located rookeries in the Gulf of California. The differences in MHC genotypes indicated no substantial gene flows between these populations and among nearby rookeries and have implications for potential disease epidemics in this species.
Similar studies have been initiated to begin to define MHC class II gene polymorphism in Alaskan Steller sea lions (Eumetopias jubatus), with the intent of identifying potential geographically-dependent restricted variability and/or environmental influences on population genotype. Preliminary data has demonstrated the Steller sea lion DRB gene consists of multiple loci and may represent a gene family, similar to that described above for the CSL. Efforts are underway to identify additional genes, and their geographical distribution.
In conclusion, delineation of MHC-encoded immune response genes in marine mammals has the potential to: i) serve as an indicator of immunologic vigor and population health, ii) identify potential risk factors for a population, and iii) associate genotype(s) with unique environmental pressures.