Background/Purpose:  Systemic lupus erythematosus (SLE) is a multi-system, autoimmune disease characterized by autoantibodies to nucleic acids and nucleosomal proteins. The type I interferon pathway is dysregulated in SLE and IFN-α levels are high in patients. We performed a genome-wide association study and found a missense SNP (C297T:gly51ser) in the PNP gene that associates with high IFN levels in SLE (rs1049564; P=1.24 x10^-7). PNP is a key enzyme of purine metabolism. PNP deficiency leads to dysregulated deoxynucleotide levels, a block in DNA synthesis, and defective immunity.  Previously, the rs1049564 SNP was thought not to impact PNP function.  To show a functional link between rs1049564 and SLE, we performed in vitro experiments using HAPMAP cell lines and patient leukocyte samples with and without the PNP variant.

Methods:  To determine if the rs1049564 variant alters PNP function, we exposed 6 HAPMAP cell lines (2 homozygous (CC), 2 heterozygous, and 2 homozygous (TT)) to a dose curve of deoxyguanosine (dGuo; nucleotide precursor and a substrate for PNP). One day later cells were analyzed for cell cycle phase using Click-iT plus EdU chemistry combined with FxCycle violet stain and flow cytometry.  Various pharmacological agents were tested for their ability to reverse the cell cycle block caused by the PNP variant. A similar approach was used to study PNP function in SLE patient samples with and without the rs1049564 variant. Colorimetric enzyme and ELISA assays were used to measure the functional activity of the different PNP isoforms.

Results:  We find that the rs1049564 variant causes increased S phase block and cell death in lymphoblastoid cells. Cell lines homozygous for rs1049564 (TT) had 2 fold increases in S phase block compared to cell lines without the PNP variant (CC). The cell cycle block caused by the PNP variant could be reversed pharmacologically and similar findings were observed in SLE patient cells.

Conclusion: These results suggest that the rs1049564 PNP polymorphism is a loss of function variant that leads to altered PNP function and subsequent S-phase block in select cell subsets within the lymphocyte compartment. This may lead to increased frequencies of circulating apoptotic lymphocytes, and higher type I IFN levels in human SLE. These findings have pharmacogenomic implications, as the S-phase block can be rescued in our in vitro experiments, suggesting a potential for personalized therapeutics.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-lupus-associated-variant-in-purine-nucleoside-phosphorylase-pnp-causes-cell-cycle-abnormalities/