Paroxysmal nocturnal haemoglobinuria

Abstract

Paroxysmal nocturnal haemoglobinuria (PNH) is a clonal haematopoietic stem cell (HSC) disease that presents with haemolytic anaemia, thrombosis and smooth muscle dystonias, as well as bone marrow failure in some cases. PNH is caused by somatic mutations in PIGA (which encodes phosphatidylinositol N-acetylglucosaminyltransferase subunit A) in one or more HSC clones. The gene product of PIGA is required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; thus, PIGA mutations lead to a deficiency of GPI-anchored proteins, such as complement decay-accelerating factor (also known as CD55) and CD59 glycoprotein (CD59), which are both complement inhibitors. Clinical manifestations of PNH occur when a HSC clone carrying somatic PIGA mutations acquires a growth advantage and differentiates, generating mature blood cells that are deficient of GPI-anchored proteins. The loss of CD55 and CD59 renders PNH erythrocytes susceptible to intravascular haemolysis, which can lead to thrombosis and to much of the morbidity and mortality of PNH. The accumulation of anaphylatoxins (such as C5a) from complement activation might also have a role. The natural history of PNH is highly variable, ranging from quiescent to life-threatening. Therapeutic strategies include terminal complement blockade and bone marrow transplantation. Eculizumab, a monoclonal antibody complement inhibitor, is highly effective and the only licensed therapy for PNH.

Figure 1 |. Clonal expansion in paroxysmal nocturnal haemoglobinuria.

Paroxysmal nocturnal haemoglobinuria is caused by somatic mutations (denoted by white stars) in the X-linked PIGA gene in one or more clones of multipotent haematopoietic stem cells (HSCs). For clinical manifestations to develop, the mutated HSC clone must expand, thereby generating many affected peripheral blood cells. PIGA mutations are not sufficient to lead to clonal expansion. Clonal expansion can arise from clonal selection by extrinsic factors (for example, aplastic anaemia) that preferentially target normal HSCs and, therefore, confer a conditional growth advantage to the mutated HSCs. Clonal expansion can also arise from intrinsic clonal evolution, by which HSCs acquire additional mutations that provide an intrinsic survival and growth advantage. Both mechanisms can coexist. NK, natural killer.

Figure 2 |. Biosynthesis of glycosylphosphatidylinositol-anchored proteins.

Precursor glycosylphosphatidylinositol (GPI)-anchored proteins have a GPI-attachment signal peptide at their carboxyl terminus, where preassembled GPI is attached as a post-translational modification. GPI-transamidase cleaves off the GPI-attachment signal peptide and attaches GPI to the newly generated C terminus by transamidation. Paroxysmal nocturnal haemoglobinuria (PNH) cells lack or have severely reduced activity of phosphatidylinositol (PI)-N-acetylglucosamine (GlcNAc) transferase, the enzyme encoded by PIGA that mediates the first step of the GPI biosynthetic pathway: the transfer of GlcNAc from uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) to PI that generates PI-GlcNAc. Thus, GPI molecules that are competent for attachment to proteins are not generated, the C-terminal GPI-attachment signal peptide in precursor proteins is not cleaved by GPI-transamidase and the precursor proteins are degraded intracellularly, most likely by proteasomes. For each reaction step, the genes involved are shown; a question mark indicates that the corresponding genes have not been identified. From step 5 onwards, diacyl glycerol might be replaced by 1-alkyl,2-acyl glycerol (denoted by the change in colour). ER, endoplasmic reticulum.

Figure 3 |. Intravascular and extravascular haemolysis in paroxysmal nocturnal haemoglobinuria.

Complement decay-accelerating factor (also known as CD55) and CD59 glycoprotein (CD59) are glycosylphosphatidylinositol (GPI)-anchored self-protective complement regulatory factors. CD55 is a widely expressed membrane protein that accelerates the decay of C3 convertases (C3 con) bound to the cell surface, thereby limiting the formation of C5 convertases. CD59 is also widely expressed: it blocks the generation of the membrane attack complex (MAC) and is, therefore, the major inhibitor of terminal complement^,. a | Normal red blood cells (RBCs) are protected from activated complement. b | By contrast, the CD55-defective and CD59-defective paroxysmal nocturnal haemoglobinuria (PNH) RBCs are highly sensitive to complement activation, which causes intravascular haemolysis. c | Upon eculizumab treatment, CD59 deficiency is compensated for and intravascular haemolysis is prevented owing to inhibition of C5 activation and subsequent MAC formation. However, CD55 deficiency remains on unlysed PNH RBCs, which causes inefficient downregulation of C3 convertases and in turn could lead to an accumulation of C3b and its processed forms iC3b and C3dg. iC3b and C3dg are ligands of integrin αM, β2 (CR3), a receptor expressed on macrophages in the spleen and liver^,; thus, these macrophages can recognize PNH RBCs, leading to extravascular haemolysis. C5b-8, complex of C5b, C6, C7 and C8; Hb, haemoglobin.

Figure 4 |. Complement cascade inhibition.

The lectin, classical and alternative pathways converge at the step of complement component 3 (C3) activation. Haemolysis in paroxysmal nocturnal haemoglobinuria (PNH) is usually chronic because the alternative pathway is always in a state of low-level activation through a process known as tickover. Terminal complement is initiated by C5 convertases, leading to cleavage of C5 to C5a and C5b. C5b oligomerizes with C6, C7, C8 and multiple C9 molecules to form the membrane attack complex (MAC). The complement decay-accelerating factor (CD55) inhibits proximal complement activation by accelerating the decay of C3 convertases; CD59 glycoprotein (CD59) inhibits terminal complement activation by preventing the incorporation of C9 into the MAC. There is a potent amplification loop within the alternative pathway. The absence of CD55 and CD59 on PNH cells leads to haemolysis, inflammation, platelet activation and thrombosis. Eculizumab prevents C5 convertases from cleaving C5 into C5a and C5b. C5 activation promotes coagulation via various mechanisms, including activating thrombin. Thrombin cleaves C3 (REFS 84,85) and also generates C5a in the absence of C3 (REF. 86). The fibrinolytic factors plasmin and kallikrein also directly cleave C3 (REF. 87). MASP, mannose-binding lectin-associated serine protease.