Genotypic, immunophenotypic and clinical features of Thai patients with paroxysmal nocturnal haemoglobinuria

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haematological disorder characterized by complement-mediated haemolytic anaemia caused by deficiency of glycosylphosphatidylinositol (GPI) anchored proteins. Somatic mutation of an X-linked gene, PIG-A, is responsible for the defect in biosynthesis of GPI-anchor. It appears that frequency of PNH differs geographically, and seems to be more frequent in some Asian countries, such as Thailand and China. We studied a group of 34 Thai patients with PNH to see whether the somatic mutations in PIG-A, extent of deficiency of GPI-anchored proteins (complete or partial) and complication with aplastic anaemia among Thai patients are different from those in other regions. We determined 37 PIG-A mutations in 33 patients (10 base substitutions, 14 single-base deletions, five multiple-base deletions, three single-base insertions, two multiple base insertions and three others) which were found to be similar to those found in European, American and Japanese patients. Most patients had cells with a complete deficiency of CD59 (type III cells), whereas 19% and 33% of the patients with reliable data for CD59 expression had partially deficient granulocytes and erythrocytes (type II cells), respectively. Most mutations resulted in a complete loss of function of PIG-A in accordance with the prevalent PNH III phenotype. 19 patients (51%) had aplastic anaemia; their PIG-A mutations were not different from those without pre-existing aplastic anaemia. These characteristics of Thai patients are similar to patients from other regions. There was some negative correlation between mean basal Hb concentration and percentage of CD59-negative granulocytes (r = -0. 374; P = 0.0476). In addition, patients with severe anaemia (basal Hb <7 g/dl) had a significantly higher percentage of affected granulocytes than those with mild anaemia (88.5 +/- 9.4 v 64.9 +/- 25.9; P = 0.01). The data suggest that the severity of anaemia in PNH depends partly on the size of the PNH clone.     

Immunophenotypic discrepancies between granulocytic and erythroid lineages in peripheral blood of patients with paroxysmal nocturnal haemoglobinuria

Abstract: In paroxysmal nocturnal haemoglobinuria (PNH), somatic mutation of the PIG‐A gene is thought to result in altered expression of glycosylphosphatidylinositol (GPI)‐anchored proteins. This study was performed to determine if there were any heterogeneities of cellular phenotypes between two major peripheral blood cells, erythrocytes and granulocytes. Using CD59‐based immunocytometry, the patterns of CD59 expression were shown to be conserved in the circulating erythroid cells (reticulocytes and mature erythrocytes) in all 29 patients with PNH. Twenty‐one patients had distinct combinations of PNH type I, II, and III cells in different lineages. Only eight patients exhibited similar patterns of CD59 expression between the two lineages. Approximately one third of the patients had PNH type II cells in either or both of the two lineages indicating variable lineage involvement. The proportion of abnormal granulocutes was higher than those of abnormal reticulocytes and erythrocytes. In patients with appropriate erythropoietic responses to haemolysis (RPI>2.0), shift reticulocytes display predominantly PNH phenotypes. These immature erythroid cells with altered expression of GPI‐anchored proteins may dominate the peripheral blood during periods of increased marrow activity resulting in greater phenotypic mosaicism in such patients. Discrepancies in expression of GPI‐anchored proteins in PNH which are highly variable between the two lineages may be the result of their different life spans and the influence of complement‐mediated cytolysis. The phenomena also indicated the possible occurrence of more than one PNH clones with variable clonal dominance.     

Increased serum levels of macrophage colony-stimulating factor (M–CSF) in α-and β-thalassaemia syndromes: correlation with anaemia and monocyte activation

Abstract: Serum levels of M–CSF were determined by an ELISA method in 29 and 34 patients with HbH disease (α₁/α₂ or α₁/HbCS) or β⁰-thal/HbE, respectively, in 28 haematologically normal subjects and in five patients with anaemia due to iron deficiency or myelodysplasia. In HbH disease and β⁰-thal/HbE, M–CSF concentrations were significantly higher than those in the normal subjects [986 ± 138 and 1385 ± 133, respectively, vs. 500 ± 33 pg/ml (mean ± SEM); p <0.01, and p <0.001, respectively]. By contrast, in patients with anaemia due to iron deficiency, M–CSF levels were within the normal range. In HbH disease and in β⁰-thal/HbE, M–CSF levels correlated inversely with mean basal Hb values (r = –0.39, p = 0.05 and r = –0.60, p <0.001, respectively). In addition, in some of the HbH and β⁰-thal/HbE patients, monocyte ADCC activities towards red cells were tested and found to be approximately twice as high as those in normal controls [38.3±5.7 and 30.7 ± 4.6 vs. 17.8 ± 1.8 % specific lysis (mean ± SEM), respectively; p <0.01 and p <0.02, respectively]. When thalassaemic patients and normal controls were considered together there was a significant correlation between M–CSF levels and monocyte ADCC activities (r = 0.51, p <0.02). The results suggest that in HbH disease and in β⁰-thal/HbE, raised serum M–CSF contributes to the anaemia by enhancing the effector function of mononuclear phagocytes towards red cells.     

Role of FcγRI (CD64) in erythrocyte elimination and its up-regulation in thalassaemia

To examine any role of the high affinity Fcgamma class I receptor (FcgammaRI) (CD64) in erythrocyte elimination by mononuclear phagocytes (MP) in thalassaemia (thal), we investigated the in vitro interaction of beta-thalassaemic erythrocytes with monocytes (Mo) whose FcgammaR expression had been modulated by cytokines. Treatment of Mo with interferon (IFN)-gamma or interleukin (IL)-10 which up-regulate FcgammaRI, caused a dose-dependent increase in binding of beta-thalassaemic erythrocytes, whereas stimulation with IL-4 which down-regulates the receptor, reduced this interaction, in a dose-dependent manner, to that of normal erythrocytes. Binding of thalassaemic erythrocytes by IFN-gamma or IL-10-treated Mo was inhibited by FcgammaRI-specific reagents. In addition, Mo expression of FcgammaRI and HLA class II DR was determined by flow cytometry in Thai patients with HbH disease (alpha1/alpha2 or alpha1/Hb Constant Spring) (n = 15) or beta degrees -thal/HbE (n = 16). In both groups of patients FcgammaRI expression was increased as compared to normal controls (n = 14): mean fluorescence intensity (+/-SD) 124.79 +/- 38. 77 in HbH disease and 121.86 +/- 18.23 in beta degrees -thal/HbE versus 91.94 +/- 17.36 in normal controls (P < 0.01 and P < 0.001, respectively). In contrast, HLA class II DR expression was similar in patients and controls. The results suggest that, in thalassaemia, up-regulated FcgammaRI on mononuclear phagocytes plays a role in their interaction with erythrocytes and that this process can be modified by cytokines.     

Full‐length sequence of genotype 3 hepatitis E virus derived from a pig in Thailand

Hepatitis E virus (HEV) infection in pigs was investigated in two principal swine farming areas in Thailand. Anti‐HEV antibodies and HEV RNA in sera were examined in 258 pigs reared on five commercial farms from age 1 to 6.5 months and sows. Overall, 167 of 258 (64.7%) pigs were positive for anti‐HEV IgG, while 20 of 258 (7.75%) had detectable HEV RNA. Sequence analysis of 20 HEV isolates obtained from viremic pigs revealed that they were 92.3–100% identical to each other and had 82.2–88.2% nucleotide similarity to other reported genotype 3 isolates in 415 nucleotide sequences within ORF2 region. Further characterization by sequencing the complete genome of the Thai swine HEV isolate (named Thai‐swHEV07) and phylogenetic analysis showed that Thai‐swHEV07 segregated into a cluster consisting of swine isolates from Japan, Mongolia, and Kyrgyzstan within the HEV genotype 3. The Thai‐swHEV07 had a genomic length of 7,229 nt excluding the polyadenylated region at 3′ terminus of the genome. Comparison of Thai‐swHEV07 and 27 reported strains of genotype 3 revealed 80.4–85.9% nucleotide identity, with the highest identity of 85.9% to the novel swHEV strain from Mongolia. These findings suggest that genotype 3 HEV isolates are markedly heterogeneous. J. Med. Virol. 81:657–664, 2009 © 2009 Wiley‐Liss, Inc.     

Serum levels of tumor necrosis factor-alpha, interleukin-1, and interferon-gamma in beta(o)-thalassemia/HbE and their clinical significance.

Serum levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), and interferon-gamma (IFN-gamma) were estimated by conventional ELISA kits in 60, 42, and 58 Thai patients, respectively, with beta(o)-thalassemia HbE and found to be above the normal range in 13%, 21%, and 33% of the patients, respectively. Using high-sensitivity ELISA systems, an additional 10 beta(o)-thal/HbE patients were compared with 9 controls for concentrations of circulating TNF-alpha and IL-1beta, and 9 and 5 patients, respectively, but only 1 and none of the controls, respectively, showed values above the normal ranges. In patients with abnormally high IFN-gamma levels, basal hemoglobin values were significantly lower than in those with normal levels of the cytokine (mean +/- SEM: 6.03+/-0.24 vs. 7.08+/-0.18, p < 0.05), although circulating concentrations of soluble transferrin receptors (sTrF) and absolute reticulocyte counts were similar in the two groups. Patients with raised or normal levels of TNF-alpha, IL-1alpha, or IL-1beta had similar basal hemoglobin values. In a phagocytosis assay, monocytes of patients with raised serum levels of IFN-gamma showed significantly more attached or ingested IgG-coated red cells than those of patients with normal concentrations of the cytokine (mean +/- SEM: 192+/-22 vs. 140+/-14 per 100 monocytes, p < 0.05). Moreover, in 3 of 4 of the former patients, the number of attached or ingested IgG-coated red cells per 100 monocytes was above the 95% reference limit for the latter patients. The results suggest that IFN-gamma aggravates the anemia of beta(o)-thal/HbE by activating mononuclear phagocytes for destruction of red cells but not by inhibiting erythropoiesis. The elevated serum levels of TNF-alpha and IL-1 could contribute to complications of the disease, such as cachexia and thromboembolic phenomena.     

A cohort study of the nature of paroxysmal nocturnal hemoglobinuria clones and PIG-A mutations in patients with aplastic anemia

Abstract:  Background:  Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the clonal expansion of blood cells, which are deficient in glycosylphosphatidylinositol anchored proteins (GPI-APs). As PNH frequently occurs during the clinical course of acquired aplastic anemia (AA), it is likely that a process inducing bone marrow failure in AA is responsible for the selection of GPI-AP deficient blood cells or PNH clone. Objective:  To explore the nature and mutation of a PNH clone in AA. Methods:  We performed regular repeated flow cytometric analyses of CD59 expression on peripheral blood cells from a cohort of 32 patients with AA. Mutation of phosphatidylinositol glycan class A (PIG-A) was also studied. Results:  Fifty-one episodes of occurrences of CD59 negative granulocytes out of a total cohort 167 flow cytometric analyses (31%) were observed in 22 patients (69%). CD59 negative erythrocytes were less apparent than the granulocytes. Repeated occurrences of PNH clones were observed in 16 patients. Most of the emerging PNH clones were transient in nature. They were more frequently detected during episodes of lower white blood cell and platelet counts. Persistence and expansion of the GPI-AP deficient blood cell populations to the level of clinical PNH were seen in only four patients (12.5%). Analysis of PIG-A gene demonstrated eight mutations among the four patients, with two and four independent mutations in two patients. Conclusions:  Our study indicates that PIG-A mutations of hematopoietic stem cells with resultant PNH clones, are relatively common among AA patients. It also supports the hypothesis of selection of the PNH clone by a process or condition associated with or responsible for bone marrow failure in AA. However, there must be an additional factor favoring expansion or growth of the clone to the level of clinical or florid PNH.     

Activation of monocytes for the immune clearance of red cells in β°-thalassaernia/HbE

Summary We have recently provided evidence that IgG antibodies play a role in the destruction of red cells in thalassaemia syndromes. In order further to delineate factors involved in the clearance of thalassaemic cells, monocytes of 30 Thai patients with β°-thal/HbE (17 non-splenectomized and 13 splenectomized) and 16 normal controls were examined for their ability to bind and phagocytose normal red cells coated with IgG anti-Rh(D). In β°-thal/HbE. the mean number of red cells attached to the monocytes was approximately 3-fold greater than in normal controls and the number ingested 30% higher. Among the non-splenectomized patients, the number of red cells attached to and ingested by the monocytes, correlated inversely with mean basal Hb levels, suggesting that activation of mononuclear phagocytes for the immune clearance of red cells is a factor in determining the severity of the anaemia. As Fc-gamma-RI is of primary importance in the recognition of IgG-coated red cells by monocytes, leucocytes from 10 β°-thallHbE patients (four non-splenectomized and six splenectomized) and five normal controls were investigated for their expression of Fc-gamma-RI by flow cytometry. In β°-thallHbE there was an approximately 3-fold increase in the percentage of leucocytes expressing this receptor: the receptor was up-regulated on monocytes and induced on granulocytes. The up-regulation of Fc-gamma-RI in β°-thallHbE is likely to be an important component in the activation of monocytes and in mediating their enhanced effector function towards antibody-coated cells.