Acquired bleeding disorders: the impact of health problems in the developing world.

Several acquired bleeding disorders in the developing world have impacts on health, including late vitamin K deficiency bleeding (VKDB) in infants, dengue haemorrhagic fever (DHF), and malaria. This paper describes their clinical manifestations, mechanisms involved, and treatment.

Genotype and phenotype of haemophilia A in Thai patients.

To study genotype and phenotype correlation of haemophilia A in Thai patients, molecular defects of the factor VIII (FVIII) gene were examined and their correlation with clinical phenotypes were evaluated. The molecular pathologies of FVIII in Thai patients were found to be heterogeneous. The most common mutation was FVIII intron 22 inversion accounting for about 30% of the severe cases while gene deletion was rare. Sixteen point mutations were identified, comprising two nonsense mutations (R-5X and R1966X), five missense mutations (T233I, D542Y, G1850V, W2229S and G2325C), five nucleotide deletions (1145delT, 1187-8delACAC, 1191-4delA, 1458delGA and 1534delA), three nucleotide insertions (1439-41insA, 1934insTA and 2245insACTA) and one splicing defect (IVS15+1G>T). Nine mutations (T233I, D542Y, 1145delT, 1458delGA, 1534delA, 1934insTA, W2229S, 2245insACTA and G2325C) were novel, firstly identified in Thai patients. The genotypes were found to correlate with clinical phenotypes in a majority of cases. However, in five patients the molecular defects did not correlate with clinical severity and FVIII:C level. Cellular and molecular mechanisms were proposed to be responsible in amelioration of clinical severity caused by deleterious mutations. Carrier detection by direct mutation analysis was also demonstrated.

Mutation causing exon 15 skipping and partial exon 16 deletion in factor VIII transcript, and a method for direct mutation detection.

A splicing defect with 201 nucleotide deletion in the factor VIII transcript due to IVS15 + 1G > T mutation inactivating this donor splice site and activating a cryptic acceptor splice site in exon 16 was identified in a severe haemophilia A patient. Allele specific amplification (ASA) method was successfully developed for direct detection of this mutation.

Determination of haemophilia A carrier status by mutation analysis.

A reliable method for determination of carrier status and genetic counselling is required for effective control of haemophilia. Linkage analysis is currently the most widely used method for this purpose; however, in cases where there is no prior family history and/or unavailability of informative polymorphic markers it is less applicable. Detection of a mutation characterized in each family may be an alternative method for determination of the carrier status. In this study, linkage analysis using four polymorphic DNA markers, and direct mutation analysis were compared to determine the carrier status in six unrelated Thai haemophilia A families, two with a family history and four without. In the two families with a family history of haemophilia A, the carrier and noncarrier statuses could readily be determined in eight females by either linkage or direct mutation analysis. In the four families without a family history, the polymorphic DNA markers for linkage analysis were informative in two families and uninformative in the other two. The carrier status could be excluded in all four female siblings of the patients in the former. However, the specific FVIII gene mutation was not observed in the mother of one patient, who should have carried the mutation. In the remaining two families with uninformative polymorphic DNA markers, the carrier and noncarrier statuses of four female members could only be determined by direct mutation analysis. Therefore, direct mutation analysis could circumvent the limitations of linkage analysis in the determination of haemophilia A carrier status in families without a previous history or informative polymorphic markers.

Linear growth in homozygous beta-thalassemia and beta-thalassemia/hemoglobin E patients under different treatment regimens.

The effects on linear growth and development among thalassemic patients under different treatment regimens were compared. Twelve homozygous beta-thalassemia (homozygous beta-thal) and 36 beta-thalassemia/Hb E (beta-thal/Hb E) were studied longitudinally between 1977 and 1998. Eighteen cases (10 homozygous beta-thal and 8 beta-thal/Hb E) received hypertransfusion with iron chelation by desferrioxamine. Another 30 cases (2 homozygous beta-thal and 28 beta-thal/Hb E) were given a low transfusion (depending on their clinical requirement). Their heights were measured serially and are presented as a standard deviation score (SDS). There was no significant difference in initial basic hematological data and ferritin levels between either group. However, the hypertransfused group, seemed to be clinically more severely affected than the other group as evidenced by early age at initial transfusion, the early onset of anemia and diagnosis and also their large acquired iron load after a period of transfusion. The average height SDS of the hypertransfused patients was within the 50th percentile +/- 1 SD during the first decade of life in both sexes and both genotypes. Whereas, in patients who were transfused infrequently, the SDS was always below the -1 SD and decreased gradually. In severe beta-thal/Hb E cases, their growth SDS showed no difference from those with homozygous beta-thal. Normal linear growth in those with homozygous beta thal and severe beta-thal/Hb E was only seen in the group that underwent hypertransfusion and this regimen contributed to normal growth during the first ten years of life. However, adequate iron chelation and hormonal treatment in these patients were also required in order to achieve normal adult height.

Successful bone marrow transplantation in a child with red blood cell pyruvate kinase deficiency.

We report the first successful use of BMT for the treatment of RBC pyruvate kinase (PK) deficiency in a boy who developed neonatal jaundice and severe transfusion-dependent hemolytic anemia a few months after birth. He received a BMT at the age of 5 from an HLA-identical sister who has normal PK activity after conditioning with busulfan and cyclophosphamide. The post-transplant course was uneventful. At present, 3 years after transplant, he is 8 years old and has a normal hemoglobin level and normal RBC PK activity without evidence of hemolysis. DNA analysis has confirmed full engraftment.

Frameshift mutations with severe and moderate clinical phenotypes in Thai hemophilia A patients.

Six frameshift mutations in exon 14 of the factor VIII gene were identified in Thai hemophilia A patients. Although all these mutations created premature stop codons and expected to cause severe disease, the molecular defects and clinical severity were in discrepancy in some patients. Four mutations (delT3490, delACAC3618-21, delGA4429-30, and delA4658) were found in the patients with the severe clinical phenotype while two (delA3629-37 and insA4372-9) were observed in the patients who had moderate severity, with FVIII:C of 4.2 and 2.8%. The frameshift mutations in these two patients were due to deletion and insertion of an 'A' nucleotide in the stretches of 9As and 8As in codons 1191-4 and 1439-41, respectively. This indicates that deletion or insertion in the stretches of poly A nucleotides in exon 14 of the factor VIII gene is a likely cause of the moderate clinical severity in some cases of Thai hemophilia A patients.

Successful prophylaxis of intracranial hemorrhage in infants with severe congenital factor VII deficiency.

During the period 1984-1992, 2 severe cases (1 male, 1 female) of congenital F VII deficiency with intracranial hemorrhage (ICH) were referred to the Department of Pediatrics, Siriraj Hospital Bangkok, Thailand at the ages of 1 and 3 months old. They both responded very well to fresh frozen plasma (FFP) transfusion therapy. Subsequently, both had repeated episodes of ICH (repeated ICH) 5 and 6 times, despite the 10-14 days of replacement therapy for each episode and eventually died at the ages of 11 and 13 months. Since September 1996, another 2 severe cases (2 females) of congenital F VII deficiency who had ICH within their first month of life were referred to us. In order to prevent repeated ICH, we started a prophylactic regime after the second episode of ICH, by giving FFP 10 ml/kg twice a week. The average duration of follow up was 21 months (at 8 and 34 months). All of them (aged 14, and 38 months old) are doing well at this time and free from repeated ICH. From this observation, if there is FFP available, this regime is an effective way to prevent repeated ICH in infants with severe congenital Factor VII deficiency.

Mutations of the factor VIII gene in thai hemophilia A patients.

Hemophilia A is a common X-linked bleeding disorder caused by mutations in the coagulation factor VIII gene. The entire coding and essential sequences of the factor VIII gene were generated by a combination of genomic DNA amplification and long reverse transcription-polymerase chain reaction (long RT-PCR) using factor VIII transcripts prepared from lymphocytes. Mutations were then screened by non-radioactive single strand conformation polymorphism (SSCP) analysis and characterized by DNA sequencing. We have identified six potentially pathogenic mutations in the factor VIII gene in Thai hemophilia A patients, including two nonsense mutations (R-5X and R1966X), three missense mutations (D542Y, G1850V, and G2325C), and a 4-bp insertion (ACTA) at codon 2245. Three of these mutations (D542Y, G2325C, and 4-bp insertion) have never been previously reported, and the ins2245 is the first example of such insertion probably causing factor VIII elongation. R1966X, D542Y, G1850V, and 4-bp insertion were associated with a severe hemophiliac phenotype whereas R-5X and G2325C were observed in moderately affected patients. Mutations in the factor VIII gene in Thai hemophilia A patients are likely to be heterogeneous. This study represents the first attempt to further the understanding of the molecular basis of hemophilia A in Thai.

Compound heterozygosity for one novel and one recurrent mutation in a Thai patient with severe protein S deficiency.

Homozygous or compound heterozygous protein S (PS) deficiency is a very rare disorder in the anticoagulant system, that can lead to life-threatening thrombotic complications shortly after birth. This report describes the results of the genetic analysis of the PROS 1 genes in a Thai girl patient. She was reported in 1990 as the first case with homozygous PS deficiency and neonatal purpura fulminans. In the present report, we identified the mutations in this patient by direct sequencing of PCR products representing all 15 exons of the PROS 1 gene and their flanking intronic regions. The patient turned out to be compound heterozygous for two null mutations. One allele contained a novel sequence variation, an A-insertion in an A5-tract covering codon 146 and 147, that results in a frameshift and a stop codon (TAA) at position 155. The other allele contained a nonsense mutation in exon 12 by a transition at codon 410 CGA (Arg) to TGA (stop). Cosegregation of PS deficiency with these two genetic defects was observed in her family.

Gaucher's disease;thirty-two years experience at Siriraj Hospital.

Gaucher's disease, a lysosomal disorder, is not a common disease in Thailand. During the period 1966-1998 we saw 20 patients with Gaucher's disease at the Department of Pediatrics. Siriraj Hospital. The patients came from different regions of the country but mostly from the central part of Thailand. There were 8 males and 12 females from 13 families of Thai, Thai-Chinese, Thai-Laos and Chinese-Chinese in origin. A history of consanguinity was present in 2 families. The age of onset was 2 months-4 years and the age when they were diagnosed was 4 months-15 years. The most common clinical features included splenomegaly, hepatomegaly, growth retardation, pallor, bleeding disorders and neurological abnormalities. The diagnosis was made by the clinical manifestations, hematologic complications and demonstration of Gaucher cells in the bone marrow and/or other tissues. In one family, the diagnosis was confirmed by evaluation of glucocerebrosidase activities in skin fibroblasts. The management of these patients was symptomatic ie packed red cell and platelet transfusion, splenectomy and other supportive measures. Most patients died of bleeding or infection at an early age.

Malignant lymphoma in Thailand: changes in the frequency of malignant lymphoma determined from a histopathologic and immunophenotypic analysis of 425 cases at Siriraj Hospital.

BACKGROUND: Analysis of malignant lymphoma in a single institution at different periods of time can determine the changing status of the disease in the region. METHODS: To compare with the large series of 1095 lymphoma cases reported between 1957-1971 at Siriraj Hospital, the largest hospital in Thailand, a similar study was performed through histopathologic evaluation of 425 lymphoma cases diagnosed consecutively at the same institution between August 1993 and October 1995. Phenotypic analysis was performed by paraffin section-immunoperoxidase studies. RESULTS: A striking increase in lymphoma cases was noted from 73 cases/year in the first series to 189 cases/year in the second series (an increase of 158.9%). Lymphoma occurred in all age groups, with a peak incidence at the seventh decade of life. The male to female ratio decreased from 2:1 in 1957-1971 to 1.3:1 in the more recent series. The incidence of Hodgkin's disease (HD) was found to have decreased from 28.9% to 8.5%. There were 36 cases (8.5%) of HD and 389 cases (91.5%) of non-Hodgkin's lymphoma (NHL) reported in the second series. The subtypes of HD included 16 cases of mixed cellularity, 13 cases of nodular sclerosis, 6 cases of lymphocyte depletion, and 1 case of lymphocyte predominance. According to the Working Formulation, the 389 NHL cases included low grade (14.1%), intermediate grade (57.3%), high grade (11.3%), and miscellaneous groups (17.2%). They were classified as small lymphocytic (9.5%), follicular (11.1%), diffuse (50.9%), immunoblastic (4.1%), small noncleaved (4.4%), lymphoblastic (2.8%), anaplastic large cell (9.0%), mycosis fungoides (1.8%), hairy cell leukemia (0.3%), true histiocytic (0.5%), and extramedullary plasmacytoma (1.0%). The immunophenotypes of the 359 NHL cases available for paraffin section-immunoperoxidase studies were B-cell (71.0%), T-cell (24.5%), histiocyte (0.6%), and undetermined phenotypes (3.9%). CONCLUSIONS: The incidence of malignant lymphoma is increasing in Thailand, with a high frequency of intermediate to high grade NHL of B-cell phenotype reported.

A novel Asn344 deletion in the core domain of coagulation factor XIII A subunit: its effects on protein structure and function.

In this study a previously undescribed 3 bp deletion, AAT1030-1032, in the factor XIII A subunit gene, has been detected in a Thai patient. The inframe deletion results in the translation of a factor XIII A subunit that lacks Asn344. This is the first inframe deletion to be identified in the factor XIII A subunit gene because six previously reported deletions have all caused frameshifts. The deletion has been introduced into a factor XIII A subunit cDNA and the deleted polypeptide expressed in yeast. The mRNA encoding the mutant enzyme appears to have normal stability but the translated protein is subject to premature degradation. In addition, the mutated enzyme exhibited very little transglutaminase activity compared with the wild-type enzyme. Structural modeling of the deleted enzyme suggests that the absence of Asn344 would have a potent impact on the catalytic activity by reorienting the residues associated with the catalytic center. Thus, the Asn344 deletion strongly confirms the significance of the residues surrounding the catalytic center of the factor XIII A subunit.

Bone marrow, peripheral blood and cord blood stem cell transplantation in children: ten years' experience at Siriraj Hospital.

Stem cell transplantations were performed in 69 children at Siriraj Hospital over a ten year period. The source of stem cells was bone marrow (60), peripheral blood (3), or cord blood (6). The diseases treated included 35 thalassemias, 11 Burkitt's lymphoma, five non-Hodgkin's lymphoma, five aplastic anemia, eight acute leukemia, and one each of neuroblastoma, severe combined immunodeficiency, Wiskott-Aldrich syndrome, myelodysplastic syndrome, and pyruvate kinase deficiency. The success rate of stem cell transplantation in Thai children varied according to the underlying diseases of the patients, ranging from 50% in acute leukemia to 100% in aplastic anemia. The outcome of stem cell transplantation in 35 thalassemic children revealed 23 (79.4%) were cured, whereas three (10.3%) remain alive with disease and the other three (10.3%) died. The incidence of graft-versus-host disease was low hen compared with that of Western countries. It is concluded that bone marrow, peripheral blood and cord blood stem cell transplantation will be the treatment of choice and will be widely used in the future to cure many hematologic and malignant disorders in children.

Pyruvate kinase deficiency in an alpha-thalassemia family: first case report in Thailand.

In Thailand, the most common cause of chronic hemolytic anemia is thalassemia hemoglobinopathy. We report here a 10-year-old girl with pyruvate kinase (PK) deficiency who was initially diagnosed to have Hb H disease, like her sister. The patient had a history of neonatal jaundice which required blood exchange transfusion twice and phototherapy. She became anemic and regular blood transfusion was required since the age of 2 1/2 months. She was very anemic compared to her sister and was transfusion dependent. Besides, she never had red cell inclusion bodies, thus re-evaluation was performed. The diagnosis of red cell pyruvate kinase deficiency and the exclusion of Hb H disease was achieved after cessation of blood transfusion for 3 months. The family study also confirmed the diagnosis. The patient is now on high transfusion and iron chelation. She is doing well with mild splenomegaly.

Glucose-6-phosphate dehydrogenase deficiency in the newborn: its prevalence and relation to neonatal jaundice.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is prevalent in Thailand. This condition can cause acute hemolysis during oxidative stress and also severe hyperbilirubinemia in the newborn in some populations. Our aim was to study the prevalence of G6PD deficiency in relation to neonatal jaundice. We performed quantitative red blood cell (RBC) G6PD assay in the cord blood of 505 male subjects. Observation of jaundice and determination of bilirubin level as well as work up for other causes of jaundice were made in the G6PD deficiency group compared to a G6PD normal group. Questionnaires were also sent for further follow up to both groups. The results of the study were as follows: Sixty-one of 505 male (12.08%) had RBC G6PD deficiency (Group I). The rest (444 cases) had normal G6PD (Group II). In Group I, 49.15% developed neonatal jaundice, of which 28.82% were physiologic and 20.33% were pathologic jaundice. In group II, 23.68% developed jaundice; 16.51% were physiologic and 7.17% were pathologic jaundice, respectively. Onset of jaundice, date of peak bilirubin and peak bilirubin level in Group I and Group II were not statistically different. ABO incompatibility was associated with Group I in 17.24% and with Group II in 9.09%. Hospitalization day in Groups I and II were not statistically different. Other associated diseases were found in both groups, ie infection, congenital malformation, respiratory distress syndrome, but there was no significant difference in terms of jaundice. Phototherapy was required in 18.64% and 10.28% in Group I and II with a duration of 3.91 +/- 1.24 and 3.21 +/- 1.75 days, respectively. One case in Group I who was also premature received one exchange blood transfusion due to severe sepsis but he did not survive. One case in Group II who had polycythemia was successfully treated by partial exchange transfusion with plasma.

Prevalence of hemoglobin E, alpha-thalassemia and glucose-6-phosphate dehydrogenase deficiency in 1,000 cord bloods studied in Bangkok.

Thalassemia hemoglobinopathies and glucose-6-phosphate dehydrogenase (G-6-PD) deficiency are prevalent in Thailand. We studied the prevalence of these disorders from 1,000 cord bloods collected during 14 months period, using EDTA as anticoagulant. Red blood cell G-6-PD quantitative assay was performed in all male subjects. Nine hundred and eighty five specimens were available for hemoglobin (Hb) typing by starch gel electrophoresis. Further evaluation by cellulose acetate electrophoresis and follow up were made in the cases who had Hb E and/or high level of Hb Bart's. It was found that out of 505 males, 61 cases (12.08%) had G-6-PD deficiency. Among 985 cases studied for Hb typing, 61.92% revealed normal Hb type AF while Hb E was present in 18.68% and Hb Bart's designated alpha-thalassemias were present in 25.18% respectively. Of these 985 cases, 18.78% had low Hb Bart's level ie detectable to 8.2% consistent with alpha-thal2, Hb Constant Spring (CS) or alpha-thal1 trait. Ten cases (1.02%) had high levels of Hb Bart's ranging from 16.1-35% without or with Hb CS and E, and further follow-up revealed homozygous Hb CS, Hb A-E-Bart's, Hb H and Hb H with Hb CS disease. The other 53 cases (5.38%) had low level of Hb Bart's with Hb E consistent with alpha-thalassemia trait with Hb E trait. There were 127 cases (12.89%) who had only Hb E trait and 3 cases (0.3%) who had Hb F and E without Hb A initially.

Hemophilia with factor VIII and factor IX inhibitors, incidence, bleeding problems and management.

Among 117 cases of hemophilia, there were 7 hemophilia A and 2 hemophilia B with factor VIII and factor IX inhibitors diagnosed at the Department of Pediatrics, Siriraj Hospital, Bangkok, Thailand. The overall incidence of hemophilia with inhibitors was 7.7%. Eight cases (6 hemophilia A. 2 hemophilia B) were severe hemophilia and 1 moderate hemophilia A. The average age of the inhibitor detection was about 5 years. Of the 9 cases, 7 had high inhibitor titers and 2 had low inhibitor titers. The frequency of bleeding problems before and after inhibitor detection were not different. The bleedings included hemarthrosis, mucosal bleed, hematoma, oozing from wound, hematuria and intracranial hemorrhage. The treatment of hemarthrosis in hemophilia A with low inhibitor titers was the combination of short course of prednisolone and single large dose factor VIII. In high inhibitor titer patients with acute hemarthrosis (both hemophilia A and hemophilia B), the treatment consisted of prednisolone short course and single high dose of PCC. For bleeding control in both high and low inhibitor titer with mucosal bleeds, oozing from wounds, central nervous system bleeding and hematuria, the combination was used of high dose factor VIII or factor IX for 2 days, and tranexamic acid, prednisolone, cyclophosphamide were required. In life-threatening hemorrhage and surgical operation, plasmapheresis and large dosage factor VIII or factor IX were the treatment of choice. All supportive measures were also important in every case of mucosal bleeds, wounds and surgical operations. The result of treatment revealed one death from massive intracranial hemorrhage and 8 survivals, with joint contracture in 2 cases. All still have inhibitor detected, but in low titer.

Evidence of vitamin K deficiency in cord blood.

The prevalence of vitamin K deficiency in the newborns delivered at Siriraj Hospital was studied. The prolongation of one stage prothrombin time and the presence of PIVKA-II (non carboxylated prothrombin antigen) in cord blood were interpreted as the secondary change from vitamin K deficiency state. The most reliable method to diagnose vitamin K deficiency is the detection of vitamin K level in plasma which is not yet available in Thailand. Although the prevalence of vitamin K deficiency in the newborns from our data is not high, only 0.6%, it is shown that some of the apparently normal newborn infants may have bleeding problem from vitamin K deficiency in both newborn and early infancy periods. So, the correction of this deficiency by administration of vitamin K to all newborns is appropriate and reasonable decision.