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

Abstract

Mutations in over 30 genes are known to result in impairment of the adaptive immune system, causing a group of disorders collectively known as severe combined immunodeficiency (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 the first example of naturally occurring SCID in pigs. Here, 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 two mutations were present in the Artemis gene, which in 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 here 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.

Restoration of ATM Expression in DNA-PKcs–Deficient Cells Inhibits Signal End Joining

“…The authors suggest that these phenotypes were associated with defects in AIRE expression, which requires DNA-PKcs for appropriate expression. It is noteworthy that of the six spontaneous VDJ associated SCID mutations described in animals (41, 51-54), three are the result of kinase inactivating mutations in DNA-PKcs, whereas none of the hundreds of VDJ associated SCID patients studied completely lacks DNA-PK activity. For this reason, we have long postulated that a completely null DNA-PKcs mutation would be lethal in humans (23, 52).…”

NK cells are intrinsically functional in pigs with Severe Combined Immunodeficiency (SCID) caused by spontaneous mutations in the Artemis gene

“…In total, 33 piglets were processed within 24 hours of farrowing and tissue samples were collected for identification of the SCID affected animals. The SCID genotype was determined through a PCR test (Waide et al, 2015); 18 SCID and 15 non-SCID pigs were used in these experiments. All animal handling protocols and standard operating procedures (SOPs) were approved by and met all IACUC requirements.…”

“…Upon necropsy of piglets that died unexpectedly early in the study, abnormal lymph node and thymus structures as well as very low antibody titers were detected, indicating a SCID-like phenotype. A genome-wide association analysis of 172 pigs within the selection line pedigree that was segregating the SCID phenotype identified a 5.6 Mb region on Sus scrofa chromosome 10, which contains the Artemis ( DCLRE1C ) gene (Waide et al, 2015). Defects in Artemis are known to specifically affect the mechanism of recombination of the T-Cell Receptor (TCR) and B-Cell Receptor (BCR) complexes (Cossu, 2010).…”

“…Due to the clear relevance of this gene for the SCID phenotype observed, molecular genetic analyses of this gene was performed, revealing two independent mutations in Artemis . Both alleles have a Mendelian recessive mode of inheritance and cause the SCID phenotype in either the homozygous or compound heterozygous state (Waide et al, 2015). …”

“…As seen in other species with a defect in Artemis (Schuetz et al, 2014), these pigs lack B-lymphocytes and T-lymphocytes but produce Natural Killer (NK) cells (Ewen et al 2015; Waide et al, 2015). NK cells are innate lymphocytes cytolytic to cells not presenting ‘self’ Major Histocompatibility Complex I (MHC class I), including virally infected cells or tumor cells with down-regulated MHC class I (Vivier et al, 2011).…”