P-305 SHIP1 Deficiency in Adult Inflammatory Bowel Disease Is Associated with Severe Crohn's Disease and Peripheral T Cell Reduction

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Abstract

Background:

SH2-containing Inositol Phophatase1 (SHIP1) is a key component of the PI3K pathway. It is expressed predominantly albeit not exclusively in hematopoietic cells. Germline SHIP1-deficiency in mice causes fatal eosinophillic pneumonia and severe ileitis in a phenotype mimicking human Crohn's Disease (CD). The focus of this study was to determine if SHIP1 is involved in CD or Ulcerative Colitis (UC) in humans.

Methods:

Blood samples were collected from adult IBD patients at UCSF, San Francisco VA Hospital, following enrolment in our study. Cells were isolated from the samples following red blood cell lysis or by gradient density centrifugation. Protein and nucleic acids were extracted from the cells, and aliquots of cells were processed for intracellular flow cytometry (icFlow). Western blotting was used to determine the relative abundance of SHIP1 protein as compared to a standardized control prepared from protein lysates pooled from healthy donors. Samples displaying less than 10% of the SHIP1 protein found in the standard control were termed SHIP1-deficient. This finding was confirmed on secondary samples from the same patients taken at later times.

Results:

The SHIP1 protein level in PBMC was determined in 89 patients and 24 healthy controls. SHIP1-deficiency was found in 14 of these IBD patients (15.7%). IcFlow analysis revealed a general reduction in SHIP1 MFI across all major leukocyte lineages, and significantly lower T cell number in SHIP1-deficient versus sufficient patients or healthy controls. SHIP1-deficiency was stable over sequential blood draws obtained several months apart and was independent of intestinal inflammatory changes or immunosuppressive therapy. Clinically, SHIP1-deficient CD patients were more likely to have a history of intestinal resections compared to SHIP1-sufficient CD patients, suggesting that SHIP1-deficiency may be associated with more aggressive disease. SHIP1 exome sequencing failed to reveal coding mutations that would account for total loss of SHIP1 protein. RNA-Seq, however, identified a novel mRNA fusion transcript combining exon 25 of SHIP1 spliced to exon 2 of ATG16L1 in SHIP1-deficient, but not SHIP1-sufficient patients. This predicts a truncated from of the SHIP1 protein and the loss of the ATG16L1 start codon. RNA-Seq analyses suggest that several potential targets in the Ubiquitin Proteasome System (UPS) may also be dysregulated in this subset of IBD patients. Using icFlow, we observed that brief ex vivo treatment with proteasome inhibitor Mg132 allowed for some recovery of SHIP1 protein in SHIP1-deficient patient samples. This strongly suggests the involvement of the Ubiquitin-Proteasome System in altered regulation of SHIP1 protein in SHIP1-deficient patients. We predict that a truncated SHIP1 protein translated from the fusion transcript may be catalytically active but unstable, leading to dysregulation SHIP1 function and rapid degradation of SHIP1 proteins in these patients.

Conclusions:

Results obtained thus far in our study strongly suggest that a novel SHIP1:ATG16L1 fusion transcript results in altered SHIP1 protein stability. The genetic basis for this novel fusion transcript is currently under investigation. Since SHIP1-deficiency corresponds to a severe reduction, but not an absence of SHIP1 protein, this opens the possibility for rescuing the residual wild type SHIP1 using small molecule SHIP1 agonists.

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