Antigen-presenting genes and genomic copy number variations in the Tasmanian devil MHC

Abstract

Background: The Tasmanian devil (Sarcophilus harrisii) is currently under threat of extinction due to an unusual fatal contagious cancer called Devil Facial Tumour Disease (DFTD). DFTD is caused by a clonal tumour cell line that is transmitted between unrelated individuals as an allograft without triggering immune rejection due to low levels of Major Histocompatibility Complex (MHC) diversity in Tasmanian devils.

Results: Here we report the characterization of the genomic regions encompassing MHC Class I and Class II genes in the Tasmanian devil. Four genomic regions approximately 960 kb in length were assembled and annotated using BAC contigs and physically mapped to devil Chromosome 4q. 34 genes and pseudogenes were identified, including five Class I and four Class II loci. Interestingly, when two haplotypes from two individuals were compared, three genomic copy number variants with sizes ranging from 1.6 to 17 kb were observed within the classical Class I gene region. One deletion is particularly important as it turns a Class Ia gene into a pseudogene in one of the haplotypes. This deletion explains the previously observed variation in the Class I allelic number between individuals. The frequency of this deletion is highest in the northwestern devil population and lowest in southeastern areas.

Conclusions: The third sequenced marsupial MHC provides insights into the evolution of this dynamic genomic region among the diverse marsupial species. The two sequenced devil MHC haplotypes revealed three copy number variations that are likely to significantly affect immune response and suggest that future work should focus on the role of copy number variations in disease susceptibility in this species.

Figure 1

Map of Tasmania showing the sampling sites, location of the first citing of DFTD in 1996 (Mt William), and the present location of the disease front. The black segment in the pie charts shows the proportion of individuals with a deletion at Saha-UA, and the grey segment shows the proportion with intact Saha-UA gene.

Figure 2

Schematic diagram of Tasmanian devil genomic regions containing MHC genes. Arrows represent annotated genes. BAC clones used for sequence assembly are indicated by lines below the annotation. For the Class I Region 1, alignment of two haplotypes is shown, as significant differences are found between them. The three gaps within haplotype 2 (and BAC C158O23 and C161A10) represent three segments that are not present in this haplotype as compared to haplotype 1. Asterisks indicate positions of 12 putative LINE fragments.

Figure 3

FISH image showing genomic locations of Tasmanian devil MHC Class I and II genes.

Figure 4

Comparison of MHC regions containing Class I or II genes between Tasmanian devil, grey short-tailed opossum [21]and tammar wallaby [35]. Class I, II and antigen-processing genes are represented with red, blue and purple arrows, respectively. Class II regions of tammar wallaby are not shown due to high complexity.

Figure 5

Phylogenetic analysis of Tasmanian devil MHC Class I sequences variants. The phylogenetic relationship was inferred using the Neighbour-Joining method [46]. The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (1,000 replicates) are displayed next to the branches, indicating the level of reliability of the phylogeny [47]. Bootstrap frequencies lower than 50% are not shown. Phylogenetic analysis was conducted in MEGA5 [45]. Modo-UK [GenBank:EU886686] and Maeu-UK [GenBank:CU463018] are orthologous UK genes in the grey short-tailed opossum and tammar wallaby. Modo-UA [GenBank:DQ067089], UB [GenBank:NM_001079820] and UC [GenBank:NM_001079819] are Class I genes in the opossum.

Figure 6

Putative promoter elements of Tasmanian devil MHC Class Ia genes. The boxed sequences indicate putative sites of an enhancer A, an interferon stimulated response element (ISRE), the S-X-Y motifs and the CAAT and TATA boxes.

Figure 7

Gel image showing two types of Tasmanian devil MHC Class I haplotypes. Gel lanes: A - Spirit's type with intact Class I gene Saha-UA; B - Cedric's type with a deletion in Saha-UA; C - negative control; D - HyperLadder I (Bioline).

Figure 8

Upstream sequences of Tasmanian devil MHC Class II β chain and α chain genes. The boxed sequences indicate putative sites of S-X-Y motifs.

Figure 9

Amino acid alignment of partial α1 domain of Saha-UA, UB and UC alleles. Asterisks indicate putative peptide-binding sites in a Class I molecule [48].

Figure 10

Amino acid alignment of partial α1 domain of Saha-UD alleles. Asterisks indicate putative peptide-binding sites in a Class I molecule [48].