Monica Bessler

Natural history of paroxysmal nocturnal hemoglobinuria.

BACKGROUND Paroxysmal nocturnal hemoglobinuria (PNH), which is characterized by intravascular hemolysis and venous thrombosis, is an acquired clonal disorder associated with a somatic mutation in a totipotent hematopoietic stem cell. An understanding of the natural history of PNH is essential to improve therapy. METHODS We have followed a group of 80 consecutive patients with PNH who were referred to Hammersmith Hospital, London, between 1940 and 1970. They were treated with supportive measures, such as oral anticoagulant therapy after established thromboses, and transfusions. RESULTS The median age of the patients at the time of diagnosis was 42 years (range, 16 to 75), and the median survival after diagnosis was 10 years, with 22 patients (28 percent) surviving for 25 years. Sixty patients have died; 28 of the 48 patients for whom the cause of death is known died from either venous thrombosis or hemorrhage. Thirty-one patients (39 percent) had one or more episodes of venous thrombosis during their illness. Of the 35 patients who survived for 10 years or more, 12 had a spontaneous clinical recovery. No PNH-affected cells were found among the erythrocytes or neutrophils of the patients in prolonged remission, but a few PNH-affected lymphocytes were detectable in three of the four patients tested. Leukemia did not develop in any of the patients. CONCLUSIONS PNH is a chronic disorder that curtails life. A spontaneous long-term remission can occur, which must be taken into account when considering potentially dangerous treatments, such as bone marrow transplantation. Platelet transfusions should be given, as appropriate, and long-term anticoagulation therapy should be considered for all patients.

Paroxysmal nocturnal haemoglobinuria (PNH) is caused by somatic mutations in the PIG-A gene.

Paroxysmal nocturnal haemoglobinuria (PNH), an acquired clonal blood disorder, is caused by the absence of glycosyl phosphatidylinositol (GPI)‐anchored surface proteins due to a defect in a specific step of GPI‐anchor synthesis. The cDNA of the X‐linked gene, PIG‐A, which encodes a protein required for this step has recently been isolated. We have carried out a molecular and functional analysis of the PIG‐A gene in four cell lines deficient in GPI‐linked proteins, obtained by Epstein‐Barr virus (EBV) transformation of affected B‐lymphocytes from PNH patients. In all four cell lines transfection with PIG‐A cDNA restored normal expression of GPI‐linked proteins. In three of the four cell lines the primary lesion is a frameshift mutation. In two of these there is a reduction in the amount of full‐length mRNA. The fourth cell line contains a missense mutation in PIG‐A. In each case the mutation was present in the affected granulocytes from peripheral blood of the patients, but not in normal sister cell lines from the same patient. These data prove that PNH is caused in most patients by a single mutation in the PIG‐A gene. The nature of the mutation can vary and most likely occurs on the active X‐chromosome in an early haematopoietic stem cell.

Diagnosing and treating Diamond Blackfan anaemia: results of an international clinical consensus conference

Diamond Blackfan anaemia (DBA) is a rare, genetically and clinically heterogeneous, inherited red cell aplasia. Classical DBA affects about seven per million live births and presents during the first year of life. However, as mutated genes have been discovered in DBA, non‐classical cases with less distinct phenotypes are being described in adults as well as children. In caring for these patients it is often difficult to have a clear understanding of the treatment options and their outcomes because of the lack of complete information on the natural history of the disease. The purpose of this document is to review the criteria for diagnosis, evaluate the available treatment options, including corticosteroid and transfusion therapies and stem cell transplantation, and propose a plan for optimizing patient care. Congenital anomalies, mode of inheritance, cancer predisposition, and pregnancy in DBA are also reviewed. Evidence‐based conclusions will be made when possible; however, as in many rare diseases, the data are often anecdotal and the recommendations are based upon the best judgment of experienced clinicians. The recommendations regarding the diagnosis and management described in this report are the result of deliberations and discussions at an international consensus conference.

Somatic Mutations in Paroxysmal Nocturnal Hemoglobinuria: A Blessing in Disguise?

We are now left with the third paradox unsolved: namely, how can a hematopoietic PNH clone expand? On one hand, one might surmise that such a clone has an intrinsic growth advantage; i.e., it is aggressive, just like a leukemic clone. However, several lines of evidence militate against this notion. First, in patients with PNH, PNH clone(s) can co-exist for years or decades with apparently normal hematopoietic cells: thus, the PNH clone, having expanded, does not take over (as a leukemic clone would do). Second, patients who have had PNH for years may sometimes recover spontaneously; but the PNH clone is still present years after this self-cure has occurred (Hillmen et al. 1995xHillmen, P, Lewis, S.M, Bessler, M, Luzzatto, L, and Dacie, J.V. N. Engl. J. Med. 1995; 333: 1253–1258Crossref | PubMed | Scopus (493)See all ReferencesHillmen et al. 1995). We infer that, when conditions change, the PNH clone not only stops expanding, but may actually regress in relation to non-PNH hematopoietic cells. Third, in chimeric mice produced by knocking out PIG-A in embryonic stem cells, the resulting PNH red blood cells, far from prospering, tend to disappear rather quickly after birth (Kawagoe et al. 1996xKawagoe, K, Kitamura, D, Okabe, M, Taniuchi, I, Ikawa, M, Watanabe, T, Kinoshita, T, and Takeda, J. Blood. 1996; 87: 3600–3606PubMedSee all ReferencesKawagoe et al. 1996).The rate of expansion of a PNH clone might be influenced by the existence of other as yet unknown genetic changes in addition to PIG-A mutations. At any rate, the facts we have listed, seen in the context of the close relationship between PNH ad AA, have led to the hypothesis that PNH cells might have a conditional growth or survival advantage in an environment which is injurious to hematopoietic cells through a GPI-mediated mechanism (Rotoli and Luzzatto 1989xRotoli, B and Luzzatto, L. Baillieres Clin. Haematol. 1989; 2: 113–138Abstract | Full Text PDF | PubMed | Scopus (110)See all ReferencesRotoli and Luzzatto 1989; see Figure 2Figure 2). For instance, if the damage was caused by autoreactive T cells or by natural killer cells, as has been suggested to be the case in AA (Rosenfeld and Young 1991xRosenfeld, S.J and Young, N.S. Blood Rev. 1991; 5: 71–77Abstract | Full Text PDF | PubMed | Scopus (1)See all ReferencesRosenfeld and Young 1991), we could speculate that this happens by virtue of these cells triggering an apoptotic pathway by interacting with a GPI-linked molecule normally present on the surface of hematopoietic stem cells. Under this hypothesis it is obvious that PNH cells, being invulnerable to this special kind of injury, would be at an advantage as long as the offending T cells or natural killer cells are present; whereas they would revert to being neutral or even at a disadvantage once such offending cells are no longer present. In this respect, one cannot help noting that much of the biochemical work on GPI-linked molecules has sprung from the study of the variable surface glycoprotein (VSG) of trypanosomes (14xMasterson, W.J, Doering, T.L, Hart, G.W, and Englund, P.T. Cell. 1989; 56: 793–800Abstract | Full Text PDF | PubMed | Scopus (160)See all References, 8xFerguson, M.A, Homans, S.W, Dwek, R.A, and Rademacher, T.W. Science. 1988; 239: 753–759Crossref | PubMedSee all References). In that instance, the functional role of the GPI anchor seems clearly to consist of making it easy for the parasite to shed its VSG coat and thus evade immune attack by host antibodies (Cross 1990xCross, G.A.M. Annu. Rev. Immunol. 1990; 8: 83–110Crossref | PubMedSee all ReferencesCross 1990). In PNH cells there may be an advantage for the cell in not having GPI-linked molecules on its surface, in order to evade a different sort of immune attack. Thus, PNH and AA might in fact give us a new handle for elucidating the functional role of GPI-linked molecules in mammalian cells which, in spite of a wealth of recent information (see in Cardoso-de-Almeida et al. 1994xCardoso-de-Almeida, M.L, Mendonca-Previato, L, Ramalho-Pinto, F.J, da Silva, A.M, and Travassos, L.R. J. Med. Biol. Res. 1994; 27: 115–562PubMedSee all ReferencesCardoso-de-Almeida et al. 1994), has remained rather elusive. In the meantime, we should consider that a PIG-A mutation may be a blessing in disguise, because it enables a patient who otherwise would have AA to survive, although at the price of having PNH (Luzzatto and Bessler 1996xLuzzatto, L and Bessler, M. Curr. Opin. Hemat. 1996; 3: 101–110Crossref | PubMedSee all ReferencesLuzzatto and Bessler 1996).Figure 2A Model for the Pathogenesis of PNHIn this cartoon the dark red circles are normal hematopoietic stem cells, on which the green prongs represent GPI-linked surface molecules. The pink circles are stem cells in which a PIG-A mutation prevents insertion in the membrane of any of the protein molecules that are normally GPI-linked. A black bracket symbolizes a hypothetical molecule which, upon binding to a GPI-linked protein, can cause damage to the stem cell. Panel 1 represents a normal bone marrow, in which an occasional cell with a PIG-A mutation may exist. In Panel 2, normal cells are suffering damage, whereas cells lacking GPI-linked molecules (i.e., PNH cells) are not affected and may grow. At this stage the patient may manifest symptoms of bone marrow failure (AA), and the PNH clone would be picked up by appropriate tests. In Panel 3, as a result of selection, the majority of stem cells are PNH cells. At this stage the patient has overt PNH. If the PNH clone had not grown, by this time the patient would have instead severe AA. Based on Rotoli and Luzzatto 1989xRotoli, B and Luzzatto, L. Baillieres Clin. Haematol. 1989; 2: 113–138Abstract | Full Text PDF | PubMed | Scopus (110)See all ReferencesRotoli and Luzzatto 1989.View Large Image | View Hi-Res Image | Download PowerPoint SlideIn conclusion, PNH shares with leukemias several features of clonal disorders and, since it can evolve to leukemia, it may still teach us something about leukemogenesis. On the other hand, whereas leukemic cell proliferation is, by definition, unconditionally aggressive, PNH cell proliferation is conditioned by the environment. In this respect PNH can be also seen as a remarkable exception to the golden rule that in every disease process, no matter how protean, we ought to be able to pinpoint a single primary agent. It seems instead that two different causes are required to give the clinical phenotype of PNH: one (A) that we now fully understand, namely a somatic mutation in the PIG-A gene; and one (B) that we can only define as a specific mode of bone marrow failure. The implications of this by now testable model are that A alone would produce PNH clones of no clinical significance, which may be lurking in normal people, whereas B alone would give, of course, the clinical picture of AA. It is only when A and B co-exist in the same person that we see the clinical phenotype of PNH.

Somatic mutations and cellular selection in paroxysmal nocturnal haemoglobinuria

Patients with paroxysmal nocturnal haemoglobinuria (PNH) have in their blood two red-cell populations, one normal and one deficient in proteins anchored to the membrane through a glycan phosphatidylinositol (GPI) structure. The PNH abnormality is due to a somatic mutation in the PIG-A gene, whose product is required for an early step in GPI anchor biosynthesis. We show that in two patients, two PNH clones with different mutations co-exist, and must therefore have arisen independently. This finding supports the concept that PNH develops under the pressures of a positive selection mechanism whereby GPI-anchor-deficient haemopoietic cells have a survival advantage.

The role of human ribosomal proteins in the maturation of rRNA and ribosome production.

Production of ribosomes is a fundamental process that occurs in all dividing cells. It is a complex process consisting of the coordinated synthesis and assembly of four ribosomal RNAs (rRNA) with about 80 ribosomal proteins (r-proteins) involving more than 150 nonribosomal proteins and other factors. Diamond Blackfan anemia (DBA) is an inherited red cell aplasia caused by mutations in one of several r-proteins. How defects in r-proteins, essential for proliferation in all cells, lead to a human disease with a specific defect in red cell development is unknown. Here, we investigated the role of r-proteins in ribosome biogenesis in order to find out whether those mutated in DBA have any similarities. We depleted HeLa cells using siRNA for several individual r-proteins of the small (RPS6, RPS7, RPS15, RPS16, RPS17, RPS19, RPS24, RPS25, RPS28) or large subunit (RPL5, RPL7, RPL11, RPL14, RPL26, RPL35a) and studied the effect on rRNA processing and ribosome production. Depleting r-proteins in one of the subunits caused, with a few exceptions, a decrease in all r-proteins of the same subunit and a decrease in the corresponding subunit, fully assembled ribosomes, and polysomes. R-protein depletion, with a few exceptions, led to the accumulation of specific rRNA precursors, highlighting their individual roles in rRNA processing. Depletion of r-proteins mutated in DBA always compromised ribosome biogenesis while affecting either subunit and disturbing rRNA processing at different levels, indicating that the rate of ribosome production rather than a specific step in ribosome biogenesis is critical in patients with DBA.

Murine embryonic stem cells without pig-a gene activity are competent for hematopoiesis with the PNH phenotype but not for clonal expansion.

Paroxysmal nocturnal hemoglobinuria (PNH) develops in patients who have had a somatic mutation in the X-linked PIG-A gene in a hematopoietic stem cell; as a result, a proportion of blood cells are deficient in all glycosyl phosphatidylinositol (GPI)-anchored proteins. Although the PIG-A mutation explains the phenotype of PNH cells, the mechanism enabling the PNH stem cell to expand is not clear. To examine this growth behavior, and to investigate the role of GPI-linked proteins in hematopoietic differentiation, we have inactivated the pig-a gene by homologous recombination in mouse embryonic stem (ES) cells. In mouse chimeras, pig-a- ES cells were able to contribute to hematopoiesis and to differentiate into mature red cells, granulocytes, and lymphocytes with the PNH phenotype. The proportion of PNH red cells was substantial in the fetus, but decreased rapidly after birth. Likewise, PNH granulocytes could only be demonstrated in the young mouse. In contrast, the percentage of lymphocytes deficient in GPI-linked proteins was more stable. In vitro, pig-a- ES cells were able to form pig-a- embryoid bodies and to undergo hematopoietic (erythroid and myeloid) differentiation. The number and the percentage of pig-a- embryoid bodies with hematopoietic differentiation, however, were significantly lower when compared with wild-type embryoid bodies. Our findings demonstrate that murine ES cells with a nonfunctional pig-a gene are competent for hematopoiesis, and give rise to blood cells with the PNH phenotype. pig-a inactivation on its own, however, does not confer a proliferative advantage to the hematopoietic stem cell. This provides direct evidence for the notion that some additional factor(s) are needed for the expansion of the mutant clone in patients with PNH.

Dyskeratosis congenita -- a disease of dysfunctional telomere maintenance.

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome associated with abnormalities of the skin, fingernails, and tongue. Other clinical manifestations may include epiphora, lung fibrosis, liver cirrhosis, osteoporosis, and a predisposition to develop a variety of malignancies. The clinical picture often resembles that of a premature aging syndrome and tissues affected are those with a high cell turnover. DC has been linked to mutations in at least four distinct genes, three of which have been identified. The product of these genes, dyskerin, the telomerase RNA (TERC), and the catalytic unit of telomerase (TERT) are part of a ribonucleoprotein complex, the telomerase enzyme, that is essential for the elongation and maintenance of chromosome ends or telomeres. All patients with DC have excessively short telomeres, indicating that the underlying defect in these individuals is an inability to maintain the telomeres. The purpose of the current review is to highlight recent insights into the molecular pathogenesis of DC. We discuss the impact these findings have on our current understanding of telomere function and maintenance, and on the diagnosis, management, and treatment of patients with conditions caused by dysfunctional telomeres.

TERC and TERT gene mutations in patients with bone marrow failure and the significance of telomere length measurements

Dyskeratosis congenita (DC) is a rare inherited form of bone marrow failure (BMF) caused by mutations in telomere maintaining genes including TERC and TERT. Here we studied the prevalence of TERC and TERT gene mutations and of telomere shortening in an unselected population of patients with BMF at our medical center and in a selected group of patients referred from outside institutions. Less than 5% of patients with BMF had pathogenic mutations in TERC or TERT. In patients with BMF, pathogenic TERC or TERT gene mutations were invariably associated with marked telomere shortening (<< 1st percentile) in peripheral blood mononuclear cells (PBMCs). In asymptomatic family members, however, telomere length was not a reliable predictor for the presence or absence of a TERC or TERT gene mutation. Telomere shortening was not pathognomonic of DC, as approximately 30% of patients with BMF due to other causes had PBMC telomere lengths at the 1st percentile or lower. We conclude that in the setting of BMF, measurement of telomere length is a sensitive but nonspecific screening method for DC. In the absence of BMF, telomere length measurements should be interpreted with caution.

Effect of eculizumab on haemolysis-associated nitric oxide depletion, dyspnoea, and measures of pulmonary hypertension in patients with paroxysmal nocturnal haemoglobinuria.

Pulmonary hypertension (PH) is a common complication of haemolytic anaemia. Intravascular haemolysis leads to nitric oxide (NO) depletion, endothelial and smooth muscle dysregulation, and vasculopathy, characterized by progressive hypertension. PH has been reported in patients with paroxysmal nocturnal haemoglobinuria (PNH), a life‐threatening haemolytic disease. We explored the relationship between haemolysis, systemic NO, arginine catabolism and measures of PH in 73 PNH patients enrolled in the placebo‐controlled TRIUMPH (Transfusion Reduction Efficacy and Safety Clinical Investigation Using Eculizumab in Paroxysmal Nocturnal Haemoglobinuria) study. At baseline, intravascular haemolysis was associated with elevated NO consumption (P < 0·0001) and arginase‐1 release (P < 0·0001). Almost half of the patients in the trial had elevated levels (≥160 pg/ml) of N‐terminal pro‐brain natriuretic peptide (NT‐proBNP), a marker of pulmonary vascular resistance and right ventricular dysfunction previously shown to indicate PH. Eculizumab treatment significantly reduced haemolysis (P < 0·001), NO depletion (P < 0·001), vasomotor tone (P < 0·05), dyspnoea (P = 0·006) and resulted in a 50% reduction in the proportion of patients with elevated NT‐proBNP (P < 0·001) within 2 weeks of treatment. Importantly, the significant improvements in dyspnoea and NT‐proBNP levels occurred without significant changes in anaemia. These data demonstrated that intravascular haemolysis in PNH produces a state of NO catabolism leading to signs of PH, including elevated NT pro‐BNP and dyspnoea that are significantly improved by treatment with eculizumab.