Genetic and environmental effects in paroxysmal nocturnal hemoglobinuria: this little PIG-A goes “Why? Why? Why?”

Paroxysmal nocturnal hemoglobinuria (PNH) has been a medical curiosity since the description of the first case in the mid–19th century by the English physician Sir William Gull. (See Rosse [ref. 1] for an eloquent history of studies on this disease.) Gull’s patient suffered morning crises of “intermittent hematinuria,” which was later recognized as the destruction of red cells in circulation. Intravascular hemolysis is unusual, as erythrocyte destruction occurs in the reticuloendothelial system in most hemolytic anemias. PNH was puzzling in the laboratory as well: red cells were lysed in the presence of acidified serum by a factor that was not an antibody. When this property was utilized to formulate the diagnostic Ham’s test, the symptoms and signs of the disease were broadened, and PNH was more fully characterized by a clinical triad of hemolysis, venous thrombosis, and marrow failure. Whereas thrombotic disease, which is often in unusual anatomical locations, recurrent, and resistant to therapy, is the major cause of death among Western patients, pancytopenia predominates as a manifestation of PNH in the Far East. PNH is closely related to aplastic anemia (AA), in which an autoimmune reaction leads to hematopoietic cell destruction and an empty bone marrow (2). AA usually responds to immunosuppressive therapies (3). Occasional patients who have been successfully treated with immunosuppressive drugs later manifest symptoms of PNH, evidently as a result of clonal expansion of cells prone to hemolysis and thrombosis. Furthermore, since PNH clones can be detected in as many as 25–35% of AA patients at presentation, the late outgrowth of these clones may not necessarily be seen as a complication of the immunosuppression, but rather as a consequence of persistent low-level PNH that predated and possibly contributed to the development of AA. Despite its rarity, PNH has been studied by exceptional clinical and laboratory investigators, every era attracting a cohort of scientists to concentrate on a different aspect of the disease. Perhaps surprisingly, these often disparate lines of research led to the unraveling of many of PNH’s mysteries (Figure ​(Figure1)1) in logical sequence. The emphasis in the early 20th century was the mechanism for red cell lysis by treated sera, shown to be activation of the alternative pathway of complement (4). The erythrocyte defect was identified functionally as an inability to inactivate complement on the cell surface, in turn due to a deficit in specific membrane proteins (decay-accelerating factor [DAF, or CD55] and membrane inhibitor of reactive lysis [MIRL, or CD59]). Not only were these proteins absent, but PNH cells lacked a variety of others, including leukocyte alkaline phosphatase and erythrocyte acetylcholinesterase. Ultimately, demonstration of the enzymatic release of alkaline phosphatase by a specific phospholipase led to the identification of a structural feature common to this functionally diverse group of proteins: a distinctive biochemical linkage to the cell membrane, the glycosylphosphatidylinositol (GPI) anchor. Preformed GPI is attached covalently to proteins of appropriate carboxyl sequence and links them to the plasma membrane through the phosphatidylinositol moiety. GPI-anchored proteins thus lack the cytosolic tail of the more common transmembrane cell surface proteins, and they also appear to cluster on the cell surface in biophysically distinctive regions termed “rafts.” Almost 100 different GPI-anchored proteins, including enzymes, adhesion molecules, receptors, and blood group antigens, have been identified to date in mammalian cells (5). The GPI anchor is evolutionarily conserved, and in parasites, GPI-anchored proteins predominate on the cell surface (6). Figure 1 The pathophysiology of PNH. The genetic basis of the disease is diagrammed at upper left. Somatic mutations in the X-linked PIG-A gene occur in a hematopoietic stem cell. PIG-A consists of six exons with an open reading frame of 1455 bp; the putative ... The biosynthesis of the GPI anchor was determined for trypanosomes and later in deficient mammalian cell lines (7). The genetic defect in PNH, discovered by expression cloning and then phenotypic correction of mutant cell lines, was localized to the X-linked phosphatidylinositol glycan class A (PIG-A) gene (8), whose product is required for the transfer of N-acetylglucosamine to phosphatidylinositol, an early step in the synthesis of the GPI anchor (4). Immortalized cell lines from PNH patients all belong to the class A complementation group; the most frequent PIG-A alterations are small deletions that create stop codons or frameshifts, followed by missense mutations and small insertions. PIG-A mutations in PNH are somatic, occurring in hematopoietic stem cells. Although there are germline polymorphisms in PIG-A, a constitutional form of PNH has not been described, and knockout animals have all been chimeras. Most likely, although the defect is compatible with survival and function of adult hematopoietic cells, embryonal development cannot proceed without GPI-anchored proteins. PIG-A mutations in all cases represent loss-of-function alleles and so would be expected to be recessive. Nevertheless, the disease phenotype is seen not only in men, who are hemizygous for the gene, but also, and with equal prevalence, in women. Due to random inactivation of the X-chromosome in the hematopoietic system, each blood cell in a female carries only a single transcriptionally active copy of PIG-A. A given stem cell and its progeny will express the PNH phenotype only if their active allele is mutated, but the hemolytic and thrombotic phenotype is evident despite the presence of some normal cells. The clonal origin of PNH cells was first inferred from G6PD enzyme analysis of circulating red cells in informative heterozygous females. All defective erythrocytes, leukocytes, and platelets are derived from a single affected hematopoietic progenitor cell carrying a particular PIG-A mutation. However, in some patients, genetic analysis reveals multiple PNH clones, which may persist for many years (9). Healthy individuals also harbor tiny PNH clones. Lymphocytes of PNH phenotype and PIG-A genotype have appeared in lymphoma patients treated with Campath-1, a monoclonal antibody that coincidentally recognizes a GPI-anchored protein (10), and very small numbers (20–60 per million) of GPI-anchored protein–deficient, PIG-A– granulocytes have been detected in the blood of normal persons (11).