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 the DNA damage response checkpoint. The results of the comet assay support this data with the PIG-A mutated cells exhibiting significantly (p<0.01) shorter tail lengths than the wild-type counterparts. The fiber-combing assay showed a reduced helicase activity for DNA replication in PIG-A mutated cells.

In patient samples, subjects with PNH showed significantly lower levels of ɣH2AX expression when compared to both healthy controls and AML/MDS patients. The comet assay revealed that PNH patients had decreased DNA double strand breaks and fiber combing assay showed a reduced DNA repair activity when compared to normal controls and AML patients with intact PIG-A gene expression (p<0.01).

Additionally, RNAseq analysis reveals that the expression of 10/10 genes involved in the DNA repair mechanism were down-regulated in PIG-A mutated cells when compared to wild-type control in both cell lines and patient samples.

Conclusion:

The above-mentioned results showed that there were decreased expressions of both DNA damage response checkpoint genes and repair genes in both the PIG-A CRISPR knockout leukemia cell lines as well as in the PNH patients. PIG-A mutation is globally associated with reduced DNA damage response capability and increased cellular stability. Our finding explains, at least partially, why PIG-A gene mutation status could be seen as a biomarker of mutagenesis and how PNH cells dominantly expend via clonal escape.