Although commonly regarded as a type of hemolytic anemia, paroxysmal nocturnal hemoglobinuria (PNH) is properly categorized as a hematopoietic stem cell disorder. PNH arises as a result of clonal expansion of one or several hematopoietic stem cells that have acquired a somatic mutation of the X-chromosome gene PIGA (phosphatidylinositol glycan class A). As a consequence of mutant PIGA, any progeny of affected stem cells (erythrocytes, granulocytes, monocytes, platelets, and lymphocytes) are deficient in all glycosylphosphatidylinositol-anchored proteins (GPI-APs) that are normally expressed on hematopoietic cells. The clinical manifestations of PNH are hemolytic anemia, thrombophilia, and marrow failure, but only the hemolytic anemia is unequivocally a consequence of somatic mutation of PIGA. It is not a malignant neoplasm in the classical sense of uncontrolled proliferation of cells, spread to tissues other than marrow, or spatial replacement of hematopoiesis. Its effects can be lethal and it can uncommonly undergo clonal evolution to acute myelogenous leukemia.

Comprehensive, scholarly reviews of the history of PNH have been published.^1,2,3,4 The first published clinical description of PNH is attributed to William Gull in 1866, but he failed to distinguish definitively PNH from paroxysmal cold hemoglobinuria. Paul Strübing, in 1882, clearly recognized PNH as a distinct entity and undertook prescient experiments designed to test his hypothesis that the nocturnal hemoglobinuria was a consequence of acidification of plasma that occurred when carbon dioxide and lactic acid accumulated because of slowing of respiration during sleep. In 1911, A.A. Hijmans van den Berg demonstrated that the hemolysis of PNH is caused by a defect in the red cell rather than by the presence of an abnormal plasma factor (as is the case with paroxysmal cold hemoglobinuria; Chap. 54). Thomas Hale Ham is credited with discovering, in the late 1930s, that complement mediates the hemolysis of PNH erythrocytes, although it was not until the alternative pathway of complement was identified and characterized in the mid-1950s by Louis Pillemer that the basis of Ham’s original observations became apparent. Ham developed the acidified serum lysis test (Ham test) that, along with the sucrose lysis test (sugar water test) of Robert Hartmann and David Jenkins, was used as the standard diagnostic test for PNH until being supplanted in the early 1990s by flow cytometry. Both Hartmann and William Crosby brought attention to the important role that thrombosis (particularly the Budd-Chiari syndrome) plays in the natural history of PNH, and John Dacie and his student and colleague S.M. Lewis were the first to systematically characterize the relationship between PNH and marrow failure.

The prevalence of PNH is not known with certainty. Prevalence estimates are influenced by bias in study design and results differ considerably, in large part, because of the heterogeneous nature of the disease. The blood of patients with PNH is a mosaic of normal and abnormal cells, and the extent of the mosaicism varies widely among patients (see “Phenotypic Mosaicism is Characteristic of Paroxysmal Nocturnal Hemoglobinuria” below). Patients with small PNH clones have few or no symptoms related to hemolysis. Thus an argument can be made that asymptomatic patients with small clones do not have clinically significant PNH and should be excluded from prevalence estimates. Others, however, may argue that any patient with flow cytometric evidence of a population of GPI-AP–deficient cells, regardless of clone size, has PNH and should be included in prevalence estimates. Well-designed, rigorous studies of prevalence that address the issue of disease heterogeneity are needed, but, by any definition, PNH is a rare disease. The prevalence of clinically significant PNH (i.e., classic PNH) plus patients with relatively large clones that arise in the setting of another marrow failure syndrome, (see “Clinical Features” and Table 40–2 below) is likely in the order of less than 1 case per 200,000 population, easily fulfilling criteria (<1 case per 50,000 population) for classification as an ultraorphan disease.⁵ There is a close association between PNH and aplastic anemia, and environmental factors, drugs, and toxins, that cause aplastic anemia, concordantly increase the risk of developing PNH. Although PNH is reported in all age groups, the peak incidence is in the third and fourth decades of life, similar to that of aplastic anemia (Chap. 35). PNH is an acquired disorder, and there is no known inherited risk for developing the disease. A number of cases have been reported in which only one of a pair of identical twins was affected.

COMPLEMENT AND PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

The chronic intravascular hemolysis that is the hallmark clinical manifestation of PNH is mediated by the alternative pathway of complement (APC) (Fig. 40–1).⁶ The APC is a component of innate immunity (Chap. 20).⁷ This ancient system evolved to protect the host against invasion by pathogenic microorganisms. Unlike the classical pathway of complement that is part of the system of acquired immunity and requires antibody for initiation of activation, the APC is in a state of continuous activation, armed at all times to protect the host (Chap. 19 provides a detailed review of the complement system). The APC cascade can be divided into two functional components: the amplification C3 and C5 convertases and the membrane attack complex (MAC). The C3 and C5 convertases (Fig. 40–1, top panel) are enzymatic complexes that initiate and amplify the activity of the APC. Generation of C5b by enzymatic cleavage of C5 by the APC C5 convertase activates the terminal pathway of complement that results ultimately in assembly of the cytolytic MAC.

Because the APC is primed for attack at all times, elaborate mechanisms for self-recognition and for protection of the host against APC-mediated injury have evolved. Both fluid-phase and membrane-bound proteins are involved in these processes. Normal human erythrocytes are protected against APC-mediated cytolysis primarily by decay-accelerating factor (DAF, CD55)^8,9,10 and membrane inhibitor of reactive lysis (MIRL, CD59).¹¹ These proteins act at different steps in the complement cascade (see Fig. 40–1, top panel). CD55 regulates the formation and stability of the C3 and C5 convertases, whereas CD59 blocks the formation of the MAC. Deficiency of CD55 and CD59 on the erythrocytes of PNH is the pathophysiologic basis of the Coombs-negative, intravascular hemolysis that is the clinical hallmark of the disease (see Fig. 40–1, bottom panel). But why are PNH erythrocytes deficient in the two complement regulatory proteins?

THE MOLECULAR PATHOGENESIS AND GENETIC BASIS OF PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

PNH is a consequence of clonal expansion of one or more hematopoietic stem cells with mutant PIGA(located on Xp22.1).¹² The protein product of PIGA is a glycosyl transferase^{12,13,14,15,16} that is an obligate constituent of a complex biochemical pathway required for synthesis of the glycosylphosphatidylinositol (GPI) moiety that anchors individual proteins belonging to diverse functional groups to the cell surface (Fig. 40–2). As a result of mutant PIGA, progeny of the affected stem cells are deficient in all GPI-APs. Although more than 20 GPI-APs are expressed by hematopoietic cells, it is deficiency on red blood cells (RBCs) of the two GPI-anchored complement regulatory proteins, CD55 and CD59, that underlies the hemolytic anemia of PNH.¹⁷ RBCs lacking CD55 and CD59 undergo spontaneous intravascular hemolysis as a consequence of unregulated activation of the APC (see Fig. 40–1, bottom panel). Thus, the hallmark clinical manifestation of PNH (intravascular hemolysis and the resultant hemoglobinuria) occurs because the two proteins that regulate complement on erythrocytes happen to be GPI-anchored.

Hypothetically, the PNH phenotype would result from inactivation of any of the more than 25 genes involved in synthesis of the GPI-anchor (see Fig. 40–2), but, with one exception,¹⁸ somatic mutation of no gene involved in GPI-AP synthesis other than PIGA has been reported in patients with PNH. This phenomenon is accounted for by the fact that, of the genes involved in the GPI-anchor synthesis pathway, only PIGA is located on the X-chromosome. Therefore, somatic mutation of only one allele is required for expression of the phenotype as males have one X-chromosome and, as a consequence of X-inactivation during embryogenesis, females have only one functional X-chromosome in somatic tissues (Chap. 10). On the other hand, mutation of two alleles would be required for inactivation of any of the autosomal genes involved in the GPI-anchor synthesis pathway.¹⁸

Cells with PIGA mutations do not appear to have a proliferative advantage in vitro or in hybrid animal models made with PIGA knockouts.¹⁹ They have been found to be relatively resistant to apoptosis in some studies,^20,21,22,23 but not in others.^24,25 Thus, the basis of clonal selection and clonal expansion of PIGA mutant stem cells in patients with PNH remains largely enigmatic²⁶ although a number of hypotheses have been proposed (reviewed in Ref. 17).

PHENOTYPIC MOSAICISM IS CHARACTERISTIC OF PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

The blood of patients with PNH is a mosaic of normal and abnormal cells (Fig. 40–3). Although PNH is a clonal disease, the extent to which the PIGA-mutant clone expands varies widely among patients.¹⁷ As an example, in some cases, greater than 90 percent of the blood cells may be derived from the PIGA-mutant clone, whereas in others, less than 10 percent of the blood cells may be GPI-AP deficient. This unique feature (variability in extent of mosaicism) is clinically relevant because patients with relatively small PNH clones have minimal or no symptoms and require no PNH-specific treatment, whereas those with large clones are often debilitated by the consequences of chronic complement-mediated intravascular hemolysis and respond dramatically to complement inhibitory therapy.

Another remarkable feature of PNH is phenotypic mosaicism (see Fig. 40–3A) based on PIGA genotype²⁷ (see Fig. 40–3B) that determines the degree of GPI-AP deficiency.¹⁷ PNH III cells are completely deficient in GPI-APs, PNH II cells are partially (approximately 90 percent) deficient and PNH I cells express GPI-APs at normal density (putatively, these cells are progeny of residual normal stem cells; see Fig. 40–3A). Phenotype varies among patients (Fig. 40–4). Some patients have only type I and type III cells (the most common phenotype), some have type I, type II, and type III (the second most common phenotype), and some patients have only type I and type II cells (the least-common phenotype). Furthermore, the contribution of each phenotype to the composition of the blood varies. Phenotypic mosaicism is clinically important because PNH II cells are relatively resistant to spontaneous hemolysis, and patients with a high percentage of type II cells have a relatively benign clinical course with respect to hemolysis (Fig. 40–4).

The anemia of PNH is multifactorial as an element of marrow failure is present in all patients, although the degree of marrow dysfunction is variable.²⁸ In some patients, PNH arises in the setting of aplastic anemia. In this case, marrow failure is the dominant cause of anemia. In other patients with PNH, evidence of marrow dysfunction may be subtle (e.g., an inappropriately low reticulocyte count) with the degree of anemia being determined primarily by the rate of hemolysis that is, in turn, determined by PNH clone size.

The primary clinical manifestations of PNH are hemolysis, thrombosis, and marrow failure.²⁸ Constitutional symptoms (fatigue, lethargy, malaise, asthenia) dominate the history, with nocturnal hemoglobinuria being a presenting symptom in only approximately 25 percent of patients.²⁹ Direct questioning frequently elicits a history of episodic dysphagia and odynophagia, abdominal pain, and male impotence. Venous thrombosis, often occurring at unusual sites (Budd-Chiari syndrome, mesenteric, dermal, or cerebral veins), may complicate PNH. Arterial thrombosis is less common.

PNH should be suspected in all patients with nonspherocytic, Coombs-negative intravascular hemolysis (Table 40–1).

Although the clinical manifestations of PNH depend in large part on the size of the PIGA-mutant clone, the extent of the associate marrow failure also contributes significantly to disease manifestations. Thus, PNH is not a binary process and based on clinical features, marrow characteristics, and the size of the mutant clone as determined by the percentage of GPI-AP–deficient polymorphonuclear cells (PMNs), the International PNH Interest Group recognizes three disease subcategories (Table 40–2).²⁸

Reticulocytosis reflects the response to hemolysis, although the reticulocyte count may be lower than expected for the degree of anemia because of underlying marrow failure (see Table 40–1

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