The Glycosylphosphatidylinositol Anchor

Understanding the pathophysiology of PNH requires a consideration of the GPI anchor. Although there are variations between species as well as modifications, the core GPI structure consists of protein ethanoloamine-P-6Manα1–2Manα1–6Manα1 (P-ethanolamine) 4Glucoseamine- phosphatidylinositol (Fig. 1A). Unlike in yeast, GPI is not essential for the survival of mammalian cells but is essential for embryogenesis. The anchor is synthesized in the endoplasmic reticulum (ER) membrane and involves 10 reactions and almost 30 different genes (Table 1). The first two steps take place on the cytoplasmic side and the remaining steps in the lumen of the ER. The first step is the transfer of N-acetylglucosamine (GlcNAc) from uridine diphosphate (UDP)-GlcNAc to phosphatidylinositol (PI) to yield GlcNAc-PI: this is the step that is deficient in PNH. This reaction is catalyzed by GlcNAc-PI α1–6 GlcNAc transferase, an enzyme whose subunits are encoded by seven different genes: PIGA, PIGC, PIGH, PIGQ, PIGY, PIGP, and DPM2 (see Fig.1B). The PIGA gene encodes a membrane protein in the endoplasmic reticulum that has the active site of this enzyme complex; it has a nonfunctional intron-less pseudogene on chromosome 12.

Fig1. (A) The core structure of GPI. A protein is covalently linked to the ethanolamine moiety of this structure by a peptide bond. The diacylglycerol moiety is inserted in cell phospholipid membrane bilayer, which keeps the structure localized to the surface of the cell. This provides lateral mobility in the mem brane for the attached protein. (B) It requires 7 proteins to catalyze the first step in the biosynthesis of GPI in the endoplasmic reticulum. The reaction occurs on the cytoplasmic side but later steps occur on the luminal side. PIGA has the active site of the enzyme complex, indicated with the asterisk. This is the step of GPI-anchor synthesis that is deficient in PNH. (Based on Ferguson MAJ, et al. In Varki A, et al, eds. Essentials of Glycobiology. Cold Spring Harbor, 2015–2017.)

Table1. Enzymatic Steps in the Synthesis of Glycosylphosphatidylinositol

Because of embryonic lethality, the mutations seen in PNH are almost always acquired somatic mutations occurring in a hematopoietic stem cell. Because PIGA is X-linked (Fig. 2), a mutational “single hit” will generate a GPI-negative (PNH) phenotype. Of note, although PIGA is near the pseudo-autosomal region, it is completely silenced on the inactive X-chromosome in female cells. Therefore, on the cellular level, any female with a mutation on the active X chromosome that disrupts PIGA will have an equally severe phenotype as the same mutation in a hemizygous male. Thus females are as severely affected as males.

Fig2. (A) The location of the PIGA gene on the X-chromosome. Large deletions that also involve neighboring genes (shown in green) have been reported occasionally in patients with PNH. (B): A broad spectrum of mutations in the PIG-A gene have been identified in patients with PNH. (Illustration after Luzzatto L, Nafa K. Genetics of PNH. In: Young N, Moss J, eds. PNH and GPI-Linked Proteins. Academic Press; 2000;21–47.)

PIGA Gene Mutations

The PIGA gene was identified in the early 1990s by expression cloning and was found to correct the defect in GPI-anchor synthesis when expressed in cell lines from patients with PNH. Since then, PIGA has been found to be mutated in almost all patients. The PIGA gene encodes a protein of 484 amino acids, with a transmembrane domain inserted in the endoplasmic reticulum. Most of the protein is on the cytoplasmic side (see Fig. 1B), with the GlcNAc transferase homologous region containing the active site near the transmembrane domain. There are 6 exons, the longest is exon 2, which contains the translation initiation codon (see Fig. 2); 600 bp of upstream sequence is required for full gene expression, which has features of a housekeeping gene promoter, and PIGA is listed as a housekeeping gene—as is the case for almost all of the genes required for GPI anchor assembly.

The somatic inactivating PIGA mutations in PNH are quite diverse and include missense mutations, in frame deletions, nonsense and frameshift mutations, splice site mutations, deletions of hundreds of base pairs—and much larger deletions that also involve contiguous genes on the X-chromosome (see Fig. 2). Missense mutations are often seen in patients where there is a partial loss of CD59 (PNH II cells), suggesting that there is some residual enzymatic activity, whereas truncating mutations are generally associated with complete loss of CD59. It is not uncommon to observe 3 distinct populations of cells demonstrated by flow: normal cells, PNH II cells with a partial deficiency, and PNH III cells with a complete deficiency of CD59, corresponding to differing degrees of complement senstitivity. In keeping with this, multiple PIGA mutations have been identified in some patients. The oligoclonality sometimes seen in PNH must be taken into account in any model of how PIGA mutant clones expand.

The PIGA gene has also proven useful to other fields. Particularly, because flow cytometry can identify rare mutants among a much larger population of normal cells, PIGA represents an excellent reporter gene for measuring the frequency and rate of spontaneous and induced mutations in human cells and in animals. This provides an important tool in the preclinical evaluation of the genotoxic effects of medications. Furthermore, because of the ease of cell sorting for GPI-positive and GPI-negative cells, and because mutants can be selected by aerolysin (which selectively kills normal cells in a GPI-dependent manner) PIGA represents a selectable and counter-selectable marker for genome editing and genome writing projects.

Loss of Glycosylphosphatidylinositol in Blood Cells Due to Mutations in Genes Other Than PIGA

There is a recently discovered alternative pathway to produce the PNH phenotype: germline loss of one of the autosomal genes involved in GPI-anchor synthesis and a second acquired event that eliminates the remaining allele in hematopoietic stem cells. This was first reported for the PIGT gene on chromosome 20q, and the resulting syndrome has been termed “PIGT-PNH.” Here the first event is an inherited chain-terminating mutation on the maternal allele and the second mutation is a deletion of chromosome 20q ranging from 8 to 18 Mb on the paternal allele. In addition to features of PNH, these patients also develop recurrent aseptic meningitis, urticaria, arthralgia, and bowel inflammation. Of note, the PIGT mutation affects the 10th step in the pathway, where the protein is transferred to the anchor, and accumulation of the completed but un-conjugated anchor may result in the inflammatory phenotype. Recently, a family has been reported where something similar occurs in the PIGB gene on chromosome 15q. Here there is a germline missense mutation in PIGB as well as a 70 kb deletion that is telomeric to PIGB on the same chromosome that may greatly increase the rate of copy neutral loss of heterozygosity events (CN-LOH). A woman with a history of aplastic anemia was found to have the inherited PIGB mutation, the 70 kb deletion, and CN-LOH resulting in the PNH phenotype in almost 100% of blood cells. Her asymptomatic mother, who inherited the same mutations, was found to have approximately 4% PNH leukocytes. Another individual in the pedigree with the missense mutation but without the deletion did not have PNH blood cells.

A partial loss of GPI is not embryonic lethal, and inherited germ line hypomorphic alleles have been reported for many of the genes involved, including for PIGA. This produces a severe neurologic phenotype, but there is generally sufficient residual GPI anchor production so that these patients do not have thrombosis or hemolysis. An exception to this has been the patient reported by Almeida et al. with a PIGM promoter mutation resulting in Budd Chiari syndrome as well as seizures—which responded to epigenetic modification therapy.

Glycosylphosphatidylinositol Anchored Proteins and Complement-Mediated Hemolysis

The proteins that require the GPI-anchor have in common a lack of a hydrophobic transmembrane domain, the presence of a cleaved N-terminal signal that localizes the protein to the endoplasmic reticulum, and a c-terminal sequence that is cleaved in the process of transfer of the peptide to the anchor. Alkaline phosphatase was the first GPI-linked protein discovered, by virtue of its release from the cell surface by bacterial phospholipase C. GPI–linked proteins are important in the biology of malaria, trypanosomes, and prion diseases. In addition to complement regulators, GPI-anchored proteins in humans can serve as enzymes, blood group antigens, receptors, and adhesion molecules. Certain proteins (e.g., CD58 [LFA3] and CD16 [FcγRIII]) may exist in both GPI-linked and transmembrane forms. Membrane inhibitor of reactive lysis (CD59) and decay accelerating factor (CD55)— both complement regulatory proteins—are the most widely expressed GPI-anchored proteins and can be found on cells belonging to all hematopoietic lineages including CD34+CD38− stem cells. Their function is discussed further in the chapter on complement biology.

The loss of CD59 is most consequential in untreated patients with PNH, as it works to inhibit the assembly of the C5b-9 membrane attack complex; pharmacologic C5 inhibition compensates for this defect. CD55, in contrast, works upstream, by inhibiting the alternative and classic C3 convertases. In patients who are treated with C5 inhibitors, the lack of CD55 becomes more consequential, in that unregulated C3 conversion results in C3 degradation products that opsonize the red cell and result in uptake in the reticuloendothelial system, thus leading to extravascular hemolysis. In untreated patients, C3 activation on these red cells would have instead led to the full progression of the complement cascade, leading to lysis by the membrane attack complex. Flow cytometry demonstrates that this process occurs only on the GPI-negative cell population, and in some patients receiving C5 inhibitors, the DAT (direct Coombs test) is positive. The severity of hemolysis in patients with PNH is likely affected by inter-individual differences in complement activation, for example, as a result of polymorphisms in the CR1 gene. An extreme case of this is inherited deficiency of C9, which can partially mask the hemolytic phenotype.

In patients with partial deficiency in GPI (PNH II populations) the degree of hemolysis is generally less severe. Of note, when there is a limiting amount of GPI, CD55 may take precedence over CD59 for acquiring the anchor. Therefore the PNH II populations are best delineated with antibodies specific for CD59. Interestingly, bi-allelic mutations in CD59 recapitulate PNH and can be treated with C5 inhibition, whereas bi-allelic inactivation of CD55 results in the CHAPLE syndrome (complement hyperactivity, angiopathic thrombosis, and protein-losing enteropathy) rather than hemolysis.

Clonal Expansion of Glycosylphosphatidylinositol Negative Populations in Paroxysmal Nocturnal Hemoglobinuria

Since 1989, the leading hypothesis on the expansion of GPI-negative populations has been the immune escape model, which posits that the PIGA mutation is the first “hit” and immune-mediated mar row failure is the “second hit.” In this model, the PNH stem cells are relatively spared and are able to repopulate the marrow after an immune-mediated injury. According to this hypothesis, in some patients there is a prior history of aplastic anemia, and in others the marrow failure is subclinical. This hypothesis explains several key features of PNH: (1) the fact that platelet and neutrophil counts are often decreased in PNH even without a history of aplastic anemia; (2) the fact that PNH is sometimes oligoclonal; (3) the associations between HLA-DR2 alleles and PNH; and (4) the observation that patients with PNH clones often respond well to immunosuppression when they develop cytopenias.

This model holds that PIGA mutations by themselves do not provide a growth advantage. As predicted, normal individuals harbor tiny PNH cell populations on the order of 1:10⁵ to 1:10⁶, as a result of spontaneous mutations of the PIGA gene—and yet these clones do not go on to expand in normal individuals. Likewise, in a tissue-specific model involving female mice heterozygous for a floxed Pig-A allele, PNH clones arise as a result of the expression of Cre over time, in cells with the deleted Piga on the active X-chromosome. However, the PNH cells that arise do not outcompete GPI-positive cells that have the deleted allele on the inactive X-chromosome. Likewise, in the majority of patients followed over time, PNH clones either remain stable in size or sometimes regress. Of note, PIGA mutations are not turning up in genome wide sequencing analysis of malignancies, and it has long been observed that it is easy to eradicate PNH clones by allogeneic stem cell transplantation.

This model posits subclinical aplasia in patients without a history of PNH, and stem cell numbers are indeed reduced in PNH, with a specific growth defect in the GPI-positive population. As further evidence of the immune escape model, oligoclonal T-cell expansions have been demonstrated. Since the autoantigens in aplastic anemia are still unknown, the escape mechanism has not been completely worked out. An obvious theory is that there is an autoantigen that is GPI-linked. One possibility involves the UL-16 binding proteins (ULBPs), which are NKGD2 ligands. Alternatively, the GPI anchor itself can fit into the groove of CD1d and can be presented to T cells; indeed GPI-reactive T cells have been demonstrated in some patients with PNH. This model of immune selection may therefore be analogous to the loss of HLA allele expression due to chromosome 6 loss of heterozygosity in some patients with aplastic anemia.

The immune escape model does not quite explain the increased relative risk of leukemia and MDS that is seen in PNH, though this can also occur in immune-mediated bone marrow failure without PNH. The hypermutability model, which attempts to explain this feature and account for oligoclonality at the same time posits that PNH patients are prone to mutations. However, an increase in mutant frequency or mutation rate has not been observed in cells from PNH patients, who are not prone to solid tumors or lymphoid neo plasms. On the other hand, some patients with PNH do have a second genetic hit: mutations in TET2, JAK2, HMGA, SUZ12, U2AF1, ASXL1, BCOR, MECOM, and RIT1, the Philadelphia chromosome, as well as other cytogenetic abnormalities (which are sometimes transient) have all been reported. Cytogenetic abnormalities and mutations in some of these genes can be seen in aplastic anemia, and second genetic hits in PNH are not mutually exclusive with the immune escape model. In some cases, the mutation or cytogenetic abnormality occurs within the PNH clone, and in some cases it is within the GPI-positive population. In some cases, the mutant allele burden is sufficiently high and the nature of the mutation is such that these second genetic events are probably contributing to the expansion of the PNH clone. An alternative explanation posits that the PNH clone expands as a result of stochastic effects, which is also not mutually exclusive with immune escape.

Thrombosis

As for hemolysis, the size of the PNH clone is strongly associated with the risk of thrombosis, which is almost never seen in patients who have aplastic anemia and small PNH clones. Historically, there has been a marked geographic disparity, where the rates of thrombosis are higher in Western countries than in Asia. It was observed in the era before routine anticoagulation and before the availability of anti-complement therapy that African-American and Latin American patients had a higher risk of thrombosis; it is not clear if that is still the case. Because vessel wall injury and stasis can occur due to previous occlusions, it can be said here, as in other disorders, that “clot begets clot.” Indeed, those with a prior history of thrombosis have a high risk of second events despite therapeutic anticoagulation. The combination of PNH with another hypercoagulable state such as the JAK2-mutated myeloproliferative syndromes (PNH/MPN) or Factor V Leiden can produce a severe thrombotic phenotype, but these are rarely seen.1

Chronic hemolysis can be associated with thrombosis in some other conditions (e.g., sickle cell anemia and autoimmune hemolytic anemia), but not others (e.g., heart valve dysfunction) and so hemolysis may not be the sole explanation. Other possible mechanisms include: (1) decreased fibrinolysis due to abnormal expression of the GPI-linked uPAR receptor; (2) increased thrombin generation on platelet-derived microparticles; (3) lack of a GPI-linked tissue factor pathway inhibitor; and (4) increased complement mediated activation of CD59-negative platelets. While using flow cytometry to demonstrate GPI-negative platelets is technically difficult, it can be done, and the results correlate with the percentage of PNH granulocytes. The presence of PNH platelets may explain why unusual patients who have large PNH granulocyte populations but small PNH red cell populations are at a particularly high risk of thrombosis. As reviewed in Chapter 23, while complement can activate platelets, coagulation factors can activate complement, and there may be a vicious cycle. Of note, while the explanations above are not mutually exclusive, the success of eculizumab in reducing thrombosis supports a central role for complement.

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مواضيع ذات صلة

Secondary Forms of Pure Red Cell Aplasia

Clinical Manifestations of Paroxysmal Nocturnal Hemoglobinuria

Hypothyroidism: Etiology and Treatment

Introduction and History of Paroxysmal Nocturnal Hemoglobinuria

Diseases of The Central Nervous System

Diseases of The Oral Cavity

Diseases of The Upper Respiratory Tract

Hyperthyroidism: Etiology and Treatment

Clinical Features of Hyper- and Hypothyroidism

Clinical Associations of Aplastic Anemia

Typical and Atypical Presentations of Aplastic Anemia

Pathophysiologic Pathways Leading to Aplastic anemia