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Historical aspects of paroxysmal nocturnal haemoglobinuria: ‘defining the disease’

2002; Wiley; Volume: 117; Issue: 1 Linguagem: Inglês

10.1046/j.1365-2141.2002.03374.x

1365-2141

Charles J. Parker,

Platelet Disorders and Treatments

British Journal of HaematologyVolume 117, Issue 1 p. 3-22 Free Access Historical aspects of paroxysmal nocturnal haemoglobinuria: ‘defining the disease’ First published: 25 March 2002 https://doi.org/10.1046/j.1365-2141.2002.03374.xCitations: 55 Charles J. Parker, MD, Hematology/Oncology Section (111H), VA Medical Center, 500 Foothill Blvd, Salt Lake City, UT 84148, USA. E-mail: Charles.Parker@hsc.utah.edu AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat This history of paroxysmal nocturnal haemoglobinuria (PNH) is divided into two parts. Part I, the Early History, begins with a review of the initial description of the disease (published in the latter part of the 19th century) and concludes with a recounting of the discovery of the alternative pathway of complement by Pillemer in the early 1950s. Part II, the Modern History, begins with a review of the studies of Dacie and Rosse. The results of their experiments, published in the mid-1960s, defined many of the unique features of the disease. This chronicle ends with a summary of the studies that built on the seminal discovery in 1993 by Kinoshita and colleagues of the genetic basis of PNH. Part i. the early history Why is it that such a rare disease has so captured the imagination of haematologists that it would be chosen as a topic for a historical review in this venerable journal? Although doubtfully their final lament, I am certain that many astute clinicians go to their grave having never diagnosed a case of paroxysmal nocturnal haemoglobinuria (PNH). Yet PNH is a regular lecture topic at the annual meeting of the American Society of Hematology, and the auditorium in which the lecture is presented is invariably filled with bright-eyed clinicians and scientists, senior and junior, eagerly awaiting a discussion of the most recent developments in the field. I believe that the enduring fascination with PNH results from the convergence of three factors: (1) the rarity of the disease and its protean clinical manifestations that make diagnosis challenging but particularly gratifying; (2) the intricacy of the pathophysiology; (3) the captivating way in which the fundamental abnormalities have been elucidated systematically over many years (Table I). The elegant complexity of the disease gives it a natural beauty that has stimulated the imagination of biomedical researchers for over a century, and those who have sought to understand the disease have been rewarded often with remarkable new insights (Table I). Table I. PNH – more than an haemolytic anaemia. * Characteristic (date of discovery or seminal observation when identifiable) Haemoglobinuria (1866, 1882) A consequence of intravascular haemolysis (1882) Due to greater sensitivity of the erythrocytes to complement mediated lysis (1939, 1955, 1966) Due to absence of complement regulatory proteins (1983, 1989) Deficient complement regulatory proteins are GPI-anchored (1986) Deficiency of GPI-anchored proteins due to mutation in PIG-A (1993) Phenotypic mosaicism first defined by differences in complement sensitivity of RBC (1963, 1966) Lead to somatic mutation hypothesis (1963) Phenotypic mosaicism due to genotypic mosaicism (1996) Nocturnal paroxysms (1866, 1882) Defined PNH as a distinct entity (1882) Essentially all of the early research on PNH focused on this symptom (1882–1940) Lead to discovery of acidified serum lysis test of Ham (1939) Helped confirm the existence of the alternative pathway of complement (1954, 1955) Thrombophilia (1950) Primary cause of morbidity and mortality Aetiology uncertain† Haematopoietic stem cell disease – WBC and platelets share phenotypic and genetic defects with erythroid element (1969) Bone marrow failure syndrome (1961) Close association with acquired aplastic anaemia (1967) Lead to natural selection hypothesis to explain the clonal dominance of mutant stem cells (1963) Clonal disease (1963, 1970) But not malignant Basis of clonal selection and dominance currently unknown† Deficiency of multiple cell surface proteins on haematopoietic elements (1951, 1959, 1986) Deficient proteins are GPI-anchored (1986) Mutant gene (PIG-A) required for synthesis of GPI-anchor moiety (1993) Phenotypic mosaicism due to genotypic mosaicism (1996) PIG-A is X-linked (1993) Males and females are affected equally because mutations occurs in a somatic tissue (the haematopoietic stem cell) in which only one X chromosome is active in females All cases of PNH due to PIG-A mutation probably because other genes involved in GPI-anchor synthesis are autosomal (1993–present) * PNH has fascinated physicians and scientists for more than 100 years because of its elegant complexity. Defining the fundamental basis of the characteristic signs and symptoms of the disease has lead to a number of remarkable discoveries. † Determining the basis of the clonal selection and dominance and of the thrombophilia remain challenges for the future. Episodic haemoglobinuria is the sine qua non of PNH. The intricacy of the pathophysiology of PNH is illustrated by the seven distinct layers of investigation that were required to understand the fundamental basis of this clinically defining symptom. First, the haemoglobinuria was found to be a consequence of intravascular haemolysis. Second, the intravascular haemolysis was shown to be due to an abnormality of the PNH red cell that resulted in greater sensitivity to lysis by a serum factor. Third, complement was found to be the serum factor that mediates the greater lysis of PNH red cells. Fourth, this greater susceptibility was discovered to be due to aberrant regulation at two distinct sites of the complement cascade. Fifth, deficiency of two membrane-associated inhibitors was shown to underlie the greater complement sensitivity of PNH erythrocytes. Sixth, the complement regulatory proteins were found to share a common post-translational modification [the glycosyl phosphatidylinositol (GPI) anchor]. Seventh, a gene that is required for synthesis of the GPI-anchor (PIG-A) was found to be mutant in PNH. Each of these layers will be reviewed in some detail within this narrative. This history illustrates vividly how the study of PNH has rewarded persistence and vision (and how serendipity is involved in many remarkable discoveries). These discoveries were made steadily over a period of 111 years (1882–1993), and each generation of physicians and scientists during this period made important contributions to the field. The mysteries of PNH have been solved in a particularly satisfying way because the precision and orderliness of the solutions made clearly understandable what had seemed at the time, prior to resolution, to be a problem of nearly insurmountable complexity. Although it isn't true that more people study PNH than have it, it may be true that more people are interested in the disease than have it. If so, it is because learning about PNH is an inspirational reminder of the elegant complexity of nature, the rewards of curiosity, and the power and beauty of science. Overview Although some question the diagnostic importance of nocturnal haemoglobinuria [Dacie and Lewis (1972) reported it to be the presenting symptom in only 26% of patients], it was this symptom that defined PNH as a distinct clinical entity and ignited the curiosity of early investigators (Table I). Further, the nocturnal aspect of the paroxysms suggested to Strübing (1882) a mechanism for the haemoglobinuria. He hypothesized that this symptom was a consequence of the abnormal sensitivity of PNH erythrocytes to systemic acidosis resulting from accumulation of CO2 during sleep. Although the validity is still debated, systematic investigation of this hypothesis by Strübing (1882), Hijmans van den Berg (1911) and Ham (1937) lead directly to the development of a specific diagnostic test for PNH (the acidified serum lysis test of Ham) and to the discovery by Ham of the fundamental role of complement in the lysis of PNH erythrocytes (Ham, 1939; Ham & Dingle, 1939). In a 1953 review, Crosby reported the high incidence of thrombosis-related deaths in PNH, and subsequent clinical studies have confirmed that thromboembolic events are a major cause of morbidity and mortality (Crosby, 1953a). Thus, in addition to being classified as a haemolytic anaemia, PNH is included on the list of thrombophilic conditions. The fundamental basis of the thrombophilia of PNH is undefined and represents one of two major unsolved mysteries of PNH (Table I) (Dacie, 1963). PNH is also included among the bone marrow failure syndromes because, at some point during the course of their illness, almost all patients have (in addition to anaemia) thrombocytopenia, leucopenia or both. Further, there is a clear, albeit incompletely understood, connection between PNH and acquired aplastic anaemia. PNH is also a stem cell disorder because platelets and leucocytes share, with erythrocytes, the deficiency of GPI-anchored proteins (the exact stage of differentiation in which the genetic mutation occurs is undefined, but it must be effected in a very primitive stem cell because mutant PIG-A is found in erythroid, myeloid and lymphoid elements from the same patient). PNH is remarkable because it is a clonal disease but not a malignant disease, and the bone marrow and peripheral blood are mosaics of normal and abnormal cells. Further, individual patients often have multiple abnormal clones that are phenotypically and genotypically discrete. The abnormal stem cells are a consequence of somatic mutation and the mutant gene is located on the X-chromosome. Thus, inactivation of only one gene in somatic tissues is necessary for manifestation of the phenotype, explaining why all cases of PNH are due to mutant PIG-A (this hypothesis assumes that all other genes that could cause the phenotype are autosomal). Genetically, females and males are equally susceptible because only one X-chromosome is active in the somatic tissue of females. The mutant gene, PIG-A, is an essential component of the pathway required for synthesis of the GPI moiety that serves as an anchor for a functionally diverse group of membrane proteins. Of the 20 or so proteins that are deficient on the haematopoietic cells of PNH, only DAF (CD55) and MIRL (CD59) share an obvious functional relationship (they are both complement regulatory proteins). Absence of these two proteins accounts for the marked susceptibility of PNH erythrocytes to complement-mediated lysis. The PIG-A mutation is necessary for the development of PNH, but it appears insufficient to result in clonal expansion in the absence of some other selective pressure. Defining the nature of the selective process that results in the expansion and clonal dominance of the PIG-A mutant stem cells is currently the most active area of investigation and represents Dacie's ultimate problem –‘the aetiology of the disease and its relationship to marrow hypoplasia’ (Dacie, 1963). Whatever the solution to this problem, it must somehow depend on the absence of one or more GPI-anchored proteins. After reviewing Table I, I hope it is apparent why this elegant, complex disease is a source of continuing fascination for haematologists. This history of PNH is intended to chronicle the landmark events that defined the disease over the past century. Early history Both Crosby (1951) and Rosse (2000) have undertaken scholarly studies of the early history of PNH. Although it is likely that descriptions of the disease were published by others [including a noteworthy report by the renown British physician, William Gull (1866)], Paul Strübing of Greifswald, Germany, clearly recognized PNH as an entity distinct from both paroxysmal cold haemoglobinuria and march haemoglobinuria, two similar syndromes also described in the 19th century. Published in 1882, Strübing's paper is a remarkable example of how insightful clinical observations can suggest disease mechanisms and thereby provide direction for laboratory investigation. A junior physician at the time, Strübing studied a 29-year-old-cartwright beginning in 1880. Based upon the observation that after severe attacks the plasma was red, Strübing concluded that the blood cells were dissolved within the vessels and not in either the kidney or the urine. The astuteness of Strübing's observation is underscored by the fact that, as late as 1911, Marchiafava believed that the haemolysis occurred in the kidney (Marchiafava & Nazari, 1911). [Marchiafava–Micheli disease was an eponym for PNH that was popular during the 1930s and 1940s (Crosby, 1951; Dacie, 1963). Although the eponym lingered into the 1960s, rightfully it has not endured because the contributions of Marchiafava and Micheli were too modest to warrant such recognition. The Dutch physician, Ennekin has been credited with first using the term ‘paroxysmal nocturnal haemoglobinuria’ in 1928 (Crosby, 1951; Dacie, 1963; Rosse, 2000)]. Strübing (1882) used spectroscopy to demonstrate that the discolouration of the urine was due to the presence of haemoglobin. He also described the characteristic morphological features of the urine sediment of PNH, although he did not recognize that haemosiderin accounted for the pigmentation of the cellular material. Further, he noted that erythrocytes were absent from the urine except after a very severe attack, when a rare red cell was observed. These findings are relevant today, as both haemosiderinuria and haemoglobinuria in the absence of haematuria remain important components of the clinical criteria used to diagnose PNH. Strübing concluded that sleep played a critically important role in the haemolytic process because only by awakening the patient during the night was the haemoglobinuria observed at a time other than the first voided urine of the morning. He also thought that the destruction of the erythrocytes was a slow, gradual process because there were no signs of rapid haemolysis such as chills, fever or flushing. In developing his eerily prescient hypothesis that the red cells of PNH were destroyed during sleep because they were abnormally sensitive to an acid environment, Strübing drew upon available information that normal red cells were susceptible to lysis in vitro if the cells suspension is acidified using carbon dioxide. He reasoned that, during sleep, carbon dioxide and lactic acid accumulated because of slowing of the circulation. According to Strübing's hypothesis, normal erythrocytes were resistant to lysis under these conditions, but the defective PNH cells were vulnerable to the mildly acidic conditions that occurred during sleep. Conceivably, Leonhard Landois, the Professor of Physiology at Greifswald and a colleague of Strübing, contributed to this remarkably insightful hypothesis (Rosse, 2000). Strübing attempted to support his hypothesis with experimental data by giving the patient acid, but this treatment failed to induce haemoglobinuria. Fifty-five years later, Ham (1937) repeated this experiment by giving patients a single dose of ammonium chloride to acidify the serum in vivo. Under these conditions, haemoglobinaemia and haemoglobinuria increased. Perhaps the results of Strübing's experiment were negative because he failed to give an amount of acid sufficient to lower the plasma pH into the range in which complement activation is enhanced. Apparently, Strübing did not maintain a long-standing interest in diseases associated with haemoglobinuria as he became Director (in 1889) of the Nose and Throat Clinic at the University of Greifswald (Crosby, 1951). Perhaps this career shift contributed to the dearth of recognition that he received for his seminal contributions to the characterization of PNH. There is evidence, however, that his observations were not completely ignored by other investigators. In 1911, the Dutch physician, Hijmans van den Berg, built upon Strübing's remarkably astute report. Hijmans van den Berg (1911) showed that the red cells of PNH haemolysed in vitro in an atmosphere containing carbon dioxide when the cells were suspended in serum from the patient or from either of two normal subjects. He also demonstrated that red cells from normal volunteers did not lyse under the same experimental conditions. These studies are seminal because they demonstrate conclusively that the haemolysis of PNH is due to a defect in the red cell rather than to the presence of an abnormal plasma factor (as is the case with paroxysmal cold haemoglobinuria). PNH and complement A review of the early history of complement is needed to understand why Hijmans van den Berg failed to identify the serum factor required for the lysis of PNH erythrocytes. Jules Bordet is credited with performing the critical experiments that identified complement in 1894 (Ross, 1986). Investigating the killing of vibrio cholera by immune serum, he demonstrated that the activity was dependent upon both a heat-stable factor (that we now know is antibody) present only in immune serum and a heat-labile cytotoxic factor (that we now know is complement) present both in normal (non-immune) and immune serum. He also observed that the cytotoxic effects that were lost as a result of heat inactivation could be restored by the addition of a small amount of fresh normal serum that by itself had no bactericidal activity. In 1899, Paul Ehrlich proposed a scheme of humoral immunity in which he used the term complement for the heat-labile cytotoxic factor of serum, because this factor complemented the activity of the heat-stable immune factor. Thus, at the time of Hijmans van den Berg's study, the following two characteristics of complement were accepted: (1) the activity was lost if the serum were heat inactivated; (2) the activity could be restored by adding a small amount of fresh serum. Consistent with a role for complement in the lysis of PNH erythrocytes, Hijmans van den Berg (1911) reported that haemolysis was no longer observed when the serum source was incubated at 50°C for 30 min. Hijmans van den Berg, however, reached the erroneous conclusion that complement did not mediate the haemolysis of PNH cells in vitro because the haemolytic activity of the heated serum was not restored by the addition of a small amount of fresh human or guinea pig serum. He concluded that haemolysis was caused not by a specific haemolytic substances in the serum but by an abnormal fragility of the erythrocytes to carbon dioxide. Only upon the discovery of the alternative pathway of complement by Pillemer 43 years later (Pillemer et al, 1954) would the reason that the fresh serum failed to restore the haemolytic activity of heat-inactivated serum become apparent. A characteristic feature of processes mediated by the alternative pathway is that the lytic event is no longer observed following modest dilutions of serum (e.g. 1:4 or 1:8). This observation contrasts sharply with classic pathway-mediated lysis that is sustained despite using serum (as the complement source) at relatively high dilutions (commonly > 1:100). This difference explains the observations of early investigators (including Hijmans van den Berg) who dismissed complement as the mediator of lysis of PNH erythrocytes in acidified serum because haemolysis was not observed following dilutions of serum that were known to support antibody-initiated (classic pathway) lysis. The acidified serum test of Ham In 1937, Thomas Hale Ham (Fig 1) reported findings that were remarkably similar to the studies of Strübing. There are no references to Strübing in that paper (Ham, 1937), however, indicating that Ham (like most others) was unaware of Strübing's work. As was Strübing before him, Ham was struck by the relationship between the haemolysis and sleep. This relationship lead him to postulate (just as Strübing had done in 1882) that ‘Because of the elevation in the carbon-dioxide content of the arterial blood and the decrease in pH known to occur during sleep, it was suspected that a change in acid-base equilibrium was related to the increased haemoglobinaemia of the patients during sleep’. Figure 1Open in figure viewerPowerPoint Thomas Hale Ham (1905–87). In 1939, Ham presented evidence that complement mediates the abnormal lysis of PNH erythrocytes (Ham, 1939; Ham & Dingle, 1939). Ham also developed a highly specific diagnostic test for PNH (the acidified serum lysis test). Dr Ham was on the faculty at Case Western Reserve University (Cleveland, OH, USA) at the same time as Louis Pillemer who discovered the alternative pathway of complement in the early to mid 1950s (this photograph of Dr Ham was obtained from the University Archives of Case Western Reserve University. The owner of the copyright could not be identified). Ham challenged his hypothesis that acidification of the plasma induced the haemolysis by giving two of the three study patients sodium bicarbonate. He reported that this treatment caused the haemoglobinaemia and haemoglobinuria to decrease. In a corollary experiment, Ham also reported that, following administration of ammonium chloride (to acidify the plasma), the haemoglobinaemia and haemoglobinuria increased (as noted above, Strübing undertook similar experiments. The results of those experiments were negative, however, perhaps because of differences in experimental design). As further evidence of the role of acidification in the haemolysis of PNH, Ham noted that, in one of his study patients, the arterial pH was 7·3 during ‘natural sleep’ and during that time the ‘usual haemoglobinemia and haemoglobinuria occurred’ (Ham, 1937). When the patient was subjected to hyperventilation using a Drinker artificial respirator, the arterial blood pH was observed to be 7·47 in association with a pCO2 of 28 mm. Under these conditions a decrease in plasma and urine haemoglobin was noted. Armed with the compelling results of the in vivo studies, Ham sought to investigate his hypothesis using in vitro techniques. Although his experimental design was remarkably similar to that of Hijmans van den Berg, it appears that Ham was not familiar with the Dutch physician's work on PNH [Hijmans van den Berg is not referenced in Ham's 1937 study (although his work is cited in Ham's two papers of 1939 (Ham, 1939; Ham & Dingle, 1939)]. Ham made the following observations about the erythrocytes from the three study patients: (1) rapid haemolysis was observed when the serum or plasma was acidified by using either equilibration with CO2 or addition of lactic acid; (2) the effects of CO2 were inhibited by the addition of sodium bicarbonate; (3) the haemolysis was observed if serum or plasma from normal volunteers was substituted for patients' serum or plasma; (4) blood Group O red cells from normal volunteers were not haemolysed when resuspended in patients' serum or plasma that was subsequently acidified with CO2 or lactic acid. Based on these observations, Ham reached the following conclusion: ‘Thus the essential peculiarity in these patients apparently resides in the red blood cells, whereas a factor essential for haemolysis is common to the plasmas or serums of the patients and of all normal subjects investigated’ (Ham, 1937). This statement accurately and concisely summarizes the characteristics of the haemolysis of PNH, and the conclusions closely resemble those of Hijmans van den Berg (1911). As had Hijmans van den Berg 26 years earlier, Ham studied the role of complement in the lytic process. He observed that the haemolysis did not occur if the serum or plasma were heated for 30 min at 50°C or 60°C and, like Hijmans van den Berg, Ham found that the haemolytic activity was not restored by the addition of complement in the form of fresh human serum (20% v/v, a 1:5 dilution) or by a small amount of guinea pig serum (Ham, 1937). He also observed that the haemolytic activity was inhibited by adding sodium citrate, potassium oxalate or potassium cyanide to the plasma or serum. All these salts were known inhibitors of complement-mediated lytic systems. Ham correctly concluded that a thermolabile factor was essential for the haemolysis that was observed when PNH erythrocytes were incubated in acidified serum or plasma (Ham, 1937). He went on to note five reasons why the haemolytic system that defined PNH differed from that of paroxysmal cold haemoglobinuria owing to the Donath–Landsteiner antibody. His fifth reason was that ‘since the addition of complement did not reactivate the heated serums, the haemolysis described above did not depend upon an antigen-amboceptor-complement system’ (Ham used Ehrlich's 1899 terminology to describe the elements of humoral immunity in which amboceptor receptor represents what we now know to be immunoglobulin). Thus, Ham recognized that the mechanism by which PNH erythrocytes are lysed in acidified serum differed from that of known systems. However, as Hijmans van den Berg before him, Ham arrived at the erroneous conclusion that the haemolysis was not mediated by complement because standard reconstitution experiments using dilutions of fresh serum gave negative results. In 1939, Ham and Dingle published a landmark paper that influenced the course of PNH research for the next 50 years. The subtitle of the paper is ‘Certain Immunological Aspects of the Hemolytic Mechanisms with Special Reference to Serum Complement.’ The paper, a model of thoughtful, rigorous investigation, suggested a novel mechanism by which the abnormal erythrocytes of PNH were lysed by an immune mechanism independent of antibody. Building on previous observations that the haemolysis of PNH cells was inhibited by heat inactivation of serum or by the addition of certain salts that were known to inhibit complement (Ham, 1937), Ham and Dingle investigated each facet of the humoral immune system (antigen, antibody and complement) as it was known to them at the time. They divided their report into seven sections (Ham & Dingle, 1939). Although the paper is cited primarily because of the detailed analysis of the role of complement in the lysis of PNH erythrocytes, only section 2 was concerned with this relationship. Equally important were the two sections that dealt with the investigation of the role of antibody in the lytic process. That no evidence of antibody was found either in patient serum (section 1) or associated with patient red cells (section 4) implied the existence of a novel pathway of immune lysis. The authors also demonstrated that PNH red cells were more susceptible to lysis than normal red cells when incubated with antibody [either rabbit anti-human red blood cell (RBC) antiserum or isohaemolysins] and human serum (section 6) (Ham & Dingle, 1939). Lysis of PNH and normal red cells did not differ, however, in non-immunological systems (saponin, sodium taurocholate and hypotonic sodium chloride) (section 7). The antigenic properties of the red cells were examined in section 3 by injecting PNH and normal erythrocytes into rabbits and examining the characteristics of the antisera that were generated. No differences were observed, but clearly the technique was too crude to detect the relatively subtle differences that we now know exist (i.e. absence of GPI-anchored proteins). Further, only ∼15% of the PNH red cells used in this experiment were susceptible to acidified serum lysis. As erythrocytes from patients with PNH are a mosaic of normal and abnormal cells, the relatively low percentage of susceptible cells implies that the majority of the cells from the PNH patient that were used for immunization lacked the disease phenotype. In section 5, the authors demonstrated that the characteristics of lysis of PNH red cells in acidified serum were markedly different from those of lysis of normal cells using isohaemolysins (i.e. anti-blood group A and anti-blood group B antibodies); again, consistent with a novel mechanism of haemolysis different from known antibody-initiated processes. Thus, in addition to their seminal observations on the role of complement in the lysis of PNH red cells, Ham and Dingle (1939) made several other critically important discoveries about the nature of the lysis of PNH cells in acidified serum. Their finding that antibody did not participate in the lytic process was particularly remarkable because such a process was unprecedented. The rigour with which the authors approached the investigation of the role of antibody in the lysis of PNH cells by acidified serum suggests that they realized the uniqueness of their observation and the scepticism that it might evoke. The authors were equally rigorous in their investigation of the role of complement in acidified serum lysis. They used available methods to increase complement concentration, decrease complement concentration, inhibit complement, and remove or inactivate fractions or components of complement (Ham & Dingle, 1939). The bulk of the data suggested that the lytic substance of serum was complement; however, inconsistencies in the results persuaded Ham and Dingle to be conservative (and in some instances contradictory) in their interpretation of the experiments. At one point they concluded the following: ‘Although serum complement cannot be ident