Disease relevance of PIGA

• As a clinical entity, deficiency of GPI has been recognized as paroxysmal nocturnal hemoglobinuria, an acquired clonal disorder associated with somatic mutations of the X-linked PIGA gene in hematopoietic cells [1].
• This benign clonal disease is caused by abnormalities of the X-linked phosphatidylinositol glycan class A (PIGA) gene and is associated with cytopenias and thrombosis [2].
• We have carried out a molecular and functional analysis of the PIG-A gene in four cell lines deficient in GPI-linked proteins, obtained by Epstein-Barr virus (EBV) transformation of affected B-lymphocytes from PNH patients [3].
• By isolating genomic clones from a human phage library we now show that the PIG-A gene consists of six exons (the first of which is non-coding) spanning 17 kb of DNA, and we have mapped the gene to chromosomal position Xp22 [4].
• Somatic mutation of PIGA in hematopoietic stem cells causes deficiency of glycosyl phosphatidylinositol-anchored proteins in paroxysmal nocturnal hemoglobinuria (PNH) that underlies the intravascular hemolysis but does not account for expansion of the PNH clone [5].

High impact information on PIGA

• The structure of the cloned complementary DNA was analyzed by nucleotide sequencing, and its function was assessed on the basis of its ability to restore to normal the abnormal phenotype of a PIG-A-deficient cell line after transfection [6].
• Abnormalities of PIG-A transcripts in granulocytes from patients with paroxysmal nocturnal hemoglobinuria [6].
• Recently, we demonstrated that a gene termed PIG-A (for phosphatidylinositol glycan class A), a component of glycosyl-phosphatidylinositol biosynthesis, was responsible for PNH in two patients [6].
• Here we report that PIG-A, which participates in the early step of GPI anchor biosynthesis, is the gene responsible for paroxysmal nocturnal hemoglobinuria [7].
• We localized PIG-A to the X chromosome, which accounts for expression of the recessive phenotype of the somatic mutation and the fact that the same one of the multiple biosynthetic steps is affected in all patients so far characterized [7].

Chemical compound and disease context of PIGA

• A somatic mutation in the X-linked phosphatidylinositol glycan class A (PIGA) gene causes the loss of glycosyl phosphatidylinositol (GPI)-linked proteins on blood cells from patients with paroxysmal nocturnal hemoglobinuria [8].

Biological context of PIGA

• In this study, we investigated whether mutation of the PIGA gene could be exploited to identify mutator (Mut) phenotypes in cancer [9].
• The X-linked gene PIGA has been found to be mutated in all patients with PNH [10].
• Human FIGF: cloning, gene structure, and mapping to chromosome Xp22.1 between the PIGA and the GRPR genes [11].
• The presence of a processed pseudogene is a common feature of PIGA, PIGF, and PIGC [10].
• In all four cell lines transfection with PIG-A cDNA restored normal expression of GPI-linked proteins [3].

Anatomical context of PIGA

• Single cell cloning of purged and sorted GPI-anchor- HCT116 cells and sequencing of the PIGA gene in each clone uniformly showed mutations [9].
• Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell (HSC) disorder in which an acquired somatic mutation of the X-linked PIGA gene results in a deficiency in GPI-anchored surface proteins [12].
• The PIGA mutation results in absence or marked deficiency of more than a dozen proteins on PNH blood cells [13].
• In PNH, haematopoietic stem cells that harbour PIGA mutations give rise to blood elements that are unable to synthesize glycosylphosphatidylinositol (GPI) anchors [14].
• Affected granulocytes and B lymphocytes had the same somatic mutation of PIG-A, indicating their clonal origin from a multipotential hematopoietic stem cell [7].

Associations of PIGA with chemical compounds

• Previously, an X-linked gene PIGA (phosphatidylinositol glycan class A), which participates in the first step of the biosynthesis, was shown to be mutated in abnormal cells from all 60 patients with PNH [15].
• Here we demonstrate 1) that PIG-A is an ER transmembrane protein with a large cytoplasmic domain that has homology to a bacterial GlcNAc transferase and a small lumenal domain; 2) that PIG-H is a cytoplasmically oriented, ER-associated protein; and 3) that they form a protein complex [16].
• The cytosolic part of PIG-A/GPI3 has a two-domain alpha/beta/alpha-layered structure; it is suggested that its C-terminal subsegment binds UDP-GlcNAc whereas the N-terminal domain interacts with the phosphatidylinositol moiety [17].
• All patients with PNH studied so far have a somatic mutation in an X-linked gene, called PIG-A (phosphatidyl inositol glycan complementation group A), which encodes for a protein involved in the biosynthesis of the glycosyl phosphatidylinositol (GPI) molecule, that serves as an anchor for many cell surface proteins [18].
• The PIG-A gene maps to Xp22.1 and is required for the transfer of N-acetyl glucosamine to phosphoinositol, an early step in the production of the GPI anchor [19].

Regulatory relationships of PIGA

• There were no differences in major histocompatibility (MHC) class I expression or sensitivity to either purified perforin or to interleukin-2-activated NK cells between PIG-A mutant and control cells [20].
• These experiments demonstrated that the pattern of gene expression was different between the two cells lines in that the PIG-A mutant cells failed to express antiquitin mRNA [21].

Other interactions of PIGA

• GPI-GnT is a uniquely complex glycosyltransferase, consisting of at least four proteins, PIG-A, PIG-H, PIG-C and GPI1 [22].
• YAC contig construction allowed the following locus order to be established: Xpter-DXS16-DXS69E-DXS414-XE59 - DXS9 - (GLRA2, DXS987) - (PIGA, DXS207) - DXS1053-DXS197-(GRPR,DXS43)-CALB3-DXS14 16- DXS1317 - DXS1195 - DXS418 - DXS257 - (PHKA2, DXS999)-DXS443-DXS1229-Xcen [23].
• We report pairs of oligonucleotide primers for polymerase chain reaction that should be useful to amplify and analyze various regions of the PIG-A gene in patients with PNH [24].
• Immunoselection by natural killer cells of PIGA mutant cells missing stress-inducible ULBP [25].
• The purpose of our study was to clarify the distribution of PIG-A gene abnormalities among various cell lineages during differentiation and maturation in PNH patients.The expression of CD16b or CD59 in peripheral blood granulocytes or cultured erythroblasts from three Japanese PNH patients was analyzed using flow cytometry [26].

Analytical, diagnostic and therapeutic context of PIGA

• Expansion of purged GPI-anchor+ cells in the Mut lines and analyses using aerolysin in conjunction with flow cytometry yielded PIGA gene mutation frequencies of 10(-5) to 10(-4), values similar to the mutation frequencies of the hprt gene [9].
• This prompted the design of mutation-specific polymerase chain reaction (PCR) primers, which were used to demonstrate the presence of the same mutation in an additional 12 patients, identifying this as a mutational hot spot in the PIG-A gene [27].
• In the current study, using highly purified GPI-deficient granulocytes, we have performed Northern blot and reverse transcriptase polymerase chain reaction (RT-PCR) analysis of Piga in four patients with PNH [28].
• Using affinity chromatography, epitope-tagged PIG-A protein was partially purified [29].
• A heteroduplex analysis detected PIG-A gene abnormality in 10/11 patients tested [30].

References