Not All SCID Pigs Are Created Equally: Two Independent Mutations in the Artemis Gene Cause SCID in Pigs

Abstract

Mutations in >30 genes are known to result in impairment of the adaptive immune system, causing a group of disorders collectively known as SCID. SCID disorders are split into groups based on their presence and/or functionality of B, T, and NK cells. Piglets from a line of Yorkshire pigs at Iowa State University were shown to be affected by T(-)B(-)NK(+) SCID, representing, to our knowledge, the first example of naturally occurring SCID in pigs. In this study, we present evidence for two spontaneous mutations as the molecular basis for this SCID phenotype. Flow cytometry analysis of thymocytes showed an increased frequency of immature T cells in SCID pigs. Fibroblasts from these pigs were more sensitive to ionizing radiation than non-SCID piglets, eliminating the RAG1 and RAG2 genes. Genetic and molecular analyses showed that two mutations were present in the Artemis gene, which in the homozygous or compound heterozygous state cause the immunodeficient phenotype. Rescue of SCID fibroblast radiosensitivity by human Artemis protein demonstrated that the identified Artemis mutations are the direct cause of this cellular phenotype. The work presented in the present study reveals two mutations in the Artemis gene that cause T(-)B(-)NK(+) SCID in pigs. The SCID pig can be an important biomedical model, but these mutations would be undesirable in commercial pig populations. The identified mutations and associated genetic tests can be used to address both of these issues.

Figure 1. Flow cytometry shows very few CD4⁺ CD8⁺ and γδ⁺ cells in SCID pig thymus

The number in each diagram is the percentage of each cell type in thymocyte populations of a SCID pig or a non-SCID carrier littermate (panels A and B). CD8a (x-axis) and CD4 (y-axis; panel A) and γδ (panel B) are T-cell surface markers. The least square means of percentages of each thymocyte sub-population are shown in panel C. Error bars represent the standard error of the estimates. Bars with different letters (a and b) indicate statistically significant (p<0.01) differences between SCID and non-SCID expression within cellular subset.

Figure 2. Effect of ionizing radiation on fibroblasts from SCID and normal pigs

Fibroblasts from SCID piglets (n=10) and normal littermates (n=10) were exposed to increasing doses of gamma-rays. Colonies (≥ 2 mm) were stained and counted after 14 days. Survival proportion was calculated as the average number of surviving colonies for three replicates at each radiation dose divided by the number of colonies from non-irradiated cells for each animal. Error bars represent the standard error of the least squares means. * indicates statistically significant (p<0.0001) difference between SCID and normal pigs within radiation dose.

Figure 3. Manhattan plot of the Genome Wide Association Study for SCID status

Results show the –log(p-value) of the association of ordered SNPs on Sus scrofa chromosomes 1 through 18, X, Y, and unknown (UNK) with SCID status based on the dfam option in PLINK.

Figure 4. Pedigree of SCID ancestors showing carriers of mutated haplotypes based on the Illumina Porcine SNP60 panel

Circles represent females and squares represent males. Green symbols are h16 haplotype carriers and pink symbols are h12 haplotype carriers, with lines of each color tracking the respective SCID haplotype through the pedigree to the founder generation. Beige symbols are pigs that were genotyped with the SNP60 panel and did not carry either SCID haplotype. White symbols in generations 0 through 7 represent non-genotyped individuals. Boxes in generation 8 give information on the numbers of SCID (Affected) and non-SCID (Unaffected) piglets and the number of piglets that died before their SCID phenotype was determined (Unknown) from each parent pair.

Figure 5. Two independent mutations were found in Artemis

(Panel A) The coding region of the Artemis transcript is indicated by slanted lines and genotypes at mutated positions are shown for chromosomes that carry normal, h12, and h16 haplotypes. Mutant alleles that cause SCID are shown in pink lettering. (Panel B) Genomic sequence of the h16 haplotype shows a splice donor site mutation (g.51578763 G→A) responsible for the lack of exon 8 in all h16 transcripts. Capital letters denote exonic sequence while lower case letters denote intronic sequence. (Panel C) A nonsense point mutation (g.51584489 G→A) in exon 10 changes the Tryptophan at position 267 to a stop codon in the h12 haplotype.

Figure 6. Rescue of sensitivity to ionizing radiation

Fibroblasts from compound heterozygous SCID (n=2) and normal (n=3) littermates were transfected with human Artemis-expressing plasmid (5 μg, Artemis), with a molar equivalent of pExodus plasmid without the Artemis gene (3.45 μg, Empty Vector), or shocked without plasmid added. Fibroblasts were exposed to a 4 Gy radiation dose 24 hours after transfection. Colonies (≥ 2 mm) were counted after 14 days of growth. The average number of surviving colonies for three replicates for a given plasmid was divided by the number of colonies from shock only cells for each animal. Error bars represent the standard error of the least squares means. Dots show individual observations. Bars with different letters within affected status (a and b) represent statistical differences between means with p<0.01.