PIG-a Gene Expression Deficiency Association with Reduced DNA Damage Checkpoint Response and Activation

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disease caused by mutation in the PIG-A gene. When expressed, it encodes a protein that is crucial in glycophosphatidylinositol (GPI) anchor biosynthesis. The mutation of this gene causes plasma membrane GPI anchor biosynthesis deficiency, cell membrane structure alteration, signal transduction pathway blockage and vulnerability to complement complex attack. Despite these deficiencies, PNH cells have a clonal advantage and this dominant expansion is the hallmark of PNH progression. The mechanism of this growth, however, is still unknown. Recently, multiple studies demonstrated that malignant cells harboring the PIG-A mutation appear to have a higher degree of genomic instability and disease progression. This led to the proposal of using the PIG-A gene mutation frequency as the biomarker of mutagenesis to evaluate the transgenic animal mutation or mutagenicity of test compounds. The PIG-A gene mutation assay is even considered as a valuable tool for quantifying mutational events in vivo and in vitro despite the mechanism remaining unknown. This study investigated the impacts PIG-A mutation in genomic stability, DNA damage response, and DNA damage checkpoint activity, which as a measure of cellular stability as well as a possible mechanism for clonal expansion. Methods: To investigate the relationship between PIG-A gene mutation status and DNA damage checkpoint activity we looked at 45 high-risk MDS/AML patients, 3 classic PNH patients and 12 healthy controls. Additionally, the TF-1 leukemia cell line was used to evaluate PIG-A wild-type vs PIG-A CRISPR knockout vs PIG-A siRNA transient suppression. H2AX/ɣH2AX, ⍺-pChk1, ⍺-pChk2, ⍺-ubPCNA, and ⍺-pRPA proteins and RNA levels were used as a quantitative indicator of DNA damage repair activity via qTR-PCR and western blot. The comet assay and fiber combing assay were further applied to explore the DNA damage and cellular replication events. RNAseq analysis was used to explore the impact of PIG-A mutation in global gene expression. Results: The in vitro results from the leukemia cell lines with various PIG-A mutation statuses showed that the PIG-A mutation decreases ɣH2AX expression. PIG-A mutation was also associated with down-regulated expression of ⍺-pChk1, ⍺-pChk2, ⍺-ubPCNA, and ⍺-pRPA proteins. The decreased levels of those gene expressions indicate that there is increased genomic stability and less repair activity at