Prenatal diagnosis for haemophilia: the Thai experience.

BACKGROUND: Haemophilia is a lifelong X-linked recessive inherited bleeding disorder. Since the haemophilia management in economically less-developed countries is inadequately provided, prevention of new cases of haemophilia is essentially required. SUBJECTS AND METHODS: A total of 42 pregnancies in 37 women at risk for severe and moderate haemophilia (A = 33, B = 4) were enrolled. The prenatal diagnostic (PND) procedure was performed in 32 women, while 10 women refused further PND procedure after knowing their foetuses were female (n = 8) and male (n = 2). The foetal specimen was obtained through chorionic villus sampling (n = 14), amniocentesis (n = 1) and cordocentesis (n = 17). The status of haemophilia was determined using informative RFLP markers and inversion of intron 22 of the F8 gene, and/or foetal FVIII:C or FIX:C. RESULTS: The final diagnosis revealed normal males (n = 18), haemophilia A males (n = 9), normal females (n = 3) and haemophilia A carrier females (n = 2). All women with affected haemophilia sons requested to terminate their pregnancies except one woman. One of 32 pregnancies (3.1%) had spontaneous abortion. At follow-up after birth, the PND was accurately confirmed in one haemophilia A male, three normal females and two carrier females by laboratory testing, and 18 unaffected normal males by history taking of no bleeding manifestations. However, 10 women who continued their pregnancies after knowing foetal sex turned out to have two haemophilia A males, one normal female, one haemophilia A carrier female and six normal or carrier females. CONCLUSION: The PND of haemophilia could be accurately determined but it was not well accepted by all couples at risk.

Hematopoietic stem cell transplantation for homozygous ß-thalassemia and ß-thalassemia/hemoglobin E patients from haploidentical donors.

Thalassemia-free survival after allogeneic stem cell transplantation (SCT) is about 80-90% with either matched-related or -unrelated donors. We explored the use of a mismatched-related ('haplo- ') donor. All patients received two courses of pretransplant immunosuppressive therapy (PTIS) with fludarabine (Flu) and dexamethasone (Dxm). After two courses of PTIS, a conditioning regimen of rabbit antithymocyte globulin, Flu and IV busulfan (Bu) was given followed by T-cell-replete peripheral blood progenitor cells. GvHD prophylaxis consisted of cyclophosphamide (Cy) on days SCT +3 and +4 (post-Cy), and on day SCT +5 tacrolimus or sirolimus was started together with a short course of mycophenolate mofetil. Thirty-one patients underwent haplo-SCT. Their median age was 10 years (range, 2-20 years). Twenty-nine patients engrafted with 100% donor chimerism. Two patients suffered primary graft failure. Median time to neutrophil engraftment was 14 days (range, 11-18 days). Five patients developed mild to moderate, reversible veno-occlusive disease, while nine patients developed acute GvHD grade II. Only five patients developed limited-chronic GvHD. Projected overall and event-free survival rates at 2 years are 95% and 94%, respectively. The median follow up time is 12 months (range, 7-33 months).

Interphase-FISH screening for eight common rearrangements in pediatric B-cell precursor acute lymphoblastic leukemia.

INTRODUCTION: This is the first pilot study to screen multiple common genetic aberrations in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). METHODS: Thirty-two children with BCP-ALL were investigated for chromosomal rearrangements using interphase fluorescence in situ hybridization (FISH). Eight common translocations and rearrangements, including ETV6-RUNX1, TCF3-PBX1, BCR-ABL1, ETV6, TCF3, MLL, IGH@, and PAX5, were tested for using dual-color DNA probes. RESULTS: ETV6-RUNX1 was the most frequent translocation detected in 11 children (34.4%). Two patients with BCR-ABL1 (6.3%) and one with TCF3-PBX1 (3.1%) translocations were also observed. Using break-apart probes, 11 children (34.4%) had a positive FISH result for ETV6, two patients for IGH@ (6.3%), one patient for MLL (3.1%), and one patient for PAX5 rearrangements (3.1%). All patients with the ETV6-RUNX1 fusion were also identified by split signals for ETV6. Other abnormalities, including extra copies and deletion of genes, were observed within the range of 3.1-34.4%. Cytogenetics analysis showed a single case each of BCR-ABL1 fusion, MLL, and IGH@ rearrangements (3.1% each). ETV6-RUNX1 fusion and ETV6 split-apart rearrangements were not visible by cytogenetics. Likewise, one each of cases with TCF3-PBX1 fusion and with PAX5 split signal seen by FISH was not visible by cytogenetics. CONCLUSION: By using 8 FISH probes in conjunction cytogenetics for the detection of common aberrations, interphase FISH enhanced the detection of chromosomal rearrangements in children with BCP-ALL.

Severe nonfebrile dengue infection in an adolescent after postoperative kidney transplantation: a case report.

We herein have reported a case of severe nonfebrile dengue infection complicated with refractory pancytopenia and a large perinephric hematoma with shock in a 16-year-old adolescent during the early postoperative period after kidney transplantation. After the diagnosis of end-stage renal disease she underwent living-related kidney transplantation. Thirteen days after successful transplantation, she exhibited a notable amount of ascites, bilateral pleural effusions, thrombocytopenia, and increased hemoglobin without pre-existent fever. Further investigation revealed positive dengue nonstructural protein 1 antigen (dengue NS1 Ag) and dengue virus serotype 1 by a reverse transcriptase-polymerase chain reaction (RT-PCR) in the patient's serum. She exhibited hemophagocytic syndrome, manifested by refractory pancytopenia and refractory anemia resulting in hypovolemic shock and acute graft failure on day 28 posttransplantation. The anemia was attributed to a large hematoma around the transplanted kidney requiring surgical evacuation of clotted blood. Postoperatively, she gradually recovered with resolution of thrombocytopenia and excellent graft function. Persistent dengue antigenemia and viremia was shown by dengue NS1 Ag and RT-PCR of dengue serotype-1. The viremia was present longer than the dengue antigenemia. Dengue-specific immunoglobulin M (IgM) and IgG by enzyme-linked immunosorbent assay confirmed the primary dengue infection and evidence of a recent donor dengue infection.

Auto-immune haemolytic anaemia concurrent with Plasmodium vivax infection: a case report.

A 7-month-old Myanmar boy was admitted with a 3-day history of fever. He was markedly pale and his temperature was 38·2°C. Peripheral blood smear demonstrated Plasmodium vivax infection with spherocytosis and auto-agglutination of red blood cells. Haematocrit was 16% and reticulocyte count 14·9%. Direct and indirect antiglobulin tests were positive. Antibody analysis was positive for auto-antigen I. P. vivax malaria with auto-immune haemolytic anaemia (AIHA) was diagnosed. He was treated with chloroquine and primaquine for the P. vivax infection, and oral prednisolone for the AIHA. Because of the clinical symptoms of anaemia and mild dyspnoea, blood with the least incompatible red blood cells was transfused. The clinical symptoms and signs improved. At follow-up 3 and 7 weeks after treatment, his haematocrit, reticulocyte count and peripheral blood smear results were within normal limits. Prednisolone was then tapered and stopped. The patient has since been well with no detectable recurrence of AIHA.

Simple multiplex RT-PCR for identifying common fusion transcripts in childhood acute leukemia.

Nonrandom gene rearrangements have been demonstrated in leukemic cells at diagnosis. These genetic abnormalities are associated with specific types, clinical characteristics, and prognosis of acute leukemia. Common fusion transcripts in childhood acute lymphoblastic leukemia (ALL) are TEL-AML1, E2A-PBX, MLL-AF4, and BCR-ABL (p190) and in acute nonlymphoblastic leukemia (ANLL) are AML-ETO, PML-RARA, and CBFB-MYH11. Reverse transcription-polymerase chain reaction (RT-PCR) for detection of each individual fusion transcript is impractical and time consuming. The purpose of this study was to develop simple RT-PCR methods to identify common fusion transcripts of newly diagnosed acute leukemia in children. Total RNA was extracted from bone marrow samples of children diagnosed with acute leukemia. Multiplex RT-PCR panel A (ALL) included primers for TEL-AML1, E2A-PBX, MLL-AF4, and BCR-ABL (p190) whereas panel B (ANLL) composed of primers for AML-ETO, PML-RARA, and CBFB-MYH11. Known leukemic cell lines were used to serve as positive controls. Eighty three children diagnosed with ALL (n = 63) and ANLL (n = 20) were included in this study. Fusion transcripts could be identified using multiplex RT-PCR panel A for ALL and panel B for ANLL in 26/83 (31.3%) cases. In ALL samples, we found TEL-AML1 = 16/63 (25.4%), E2A-PBX = 3/63 (4.8%), MLL-AF4 = 1/63 (1.6%), and BCR-ABL = 1/63 (1.6%). Four cases of AML1-ETO (20%) and one PML-RARA (5%) were found in ANLL samples. In conclusion, our simple multiplex RT-PCR for detection of fusion transcripts in childhood acute leukemia was found to be a rapid, accurate, and effective method.

Rituximab as an adjuvant therapy to immune tolerance in a haemophilia A boy with high inhibitor titre.

We report a haemophilia A boy with high inhibitor titre (170 BU) who experienced five life-threatening bleeding episodes during a one-year period from 9 to 21 months. At the age of 22 months, he received rituximab (375 mg m(-2) per dose) at one- and three-week intervals, three courses each and alternative daily treatment with factor VIII concentrate at doses of 100 units kg(-1) for 24 weeks and 50 units kg(-1) for the following 28 weeks. Although the pretreatment inhibitor level of 4.5 BU showed an anamnestic response reaching the maximum level of 200 BU at the 9th week of treatment, it gradually declined to 30 BU at the 22nd week and was constantly maintained at 25-30 BU for the following 30 weeks. Only three bleeding episodes of two haematomas and one haemarthrosis were found during the one-year treatment period. No opportunistic infection occured during this period.

Combined factor V and factor VIII deficiency in a Thai patient: a case report of genotype and phenotype characteristics.

A Thai woman, with no family history of bleeding disorders, presented with excessive bleeding after minor trauma and tooth extraction. The screening coagulogram revealed prolonged activated partial thromboplastin time and prothrombin time. The specific-factor assay confirmed the diagnosis of combined factor V and factor VIII deficiency (F5F8D). Her plasma levels of factor V and factor VIII were 10% and 12.5% respectively. The medications and blood product treatment to prevent bleeding from invasive procedure included 1-deamino-8-d-arginine vasopressin, cryoprecipitate, factor VIII concentrate, fresh frozen plasma and antifibrinolytic agent. Gene analysis of the proband identified two LMAN1 gene mutations; one of which is 823-1 G --> C, a novel splice acceptor site mutation that is inherited from her father, the other is 1366 C --> T, a nonsense mutation that is inherited from her mother. Thus, the compound heterozygote of these two mutations in LMAN1 cause combined F5F8D.

Outcome of transplantation with unrelated donor bone marrow in children with severe thalassaemia.

SUMMARY: We conducted a study of unrelated donor bone marrow transplantation (BMT) in 11 children with severe thalassaemia. The conditioning regimen consisted of busulphan, cyclophosphamide and antilymphocyte globulin. All received T-cell nondepleted bone marrow. The median marrow-nucleated cell dose was 4.9 x 10(8) /kg (range; 3.5-8.0 x 10(8) /kg). Median time of granulocyte recovery was 16 days (range; 13-21 days), and of platelet recovery was 39 days (range; 14-196). Grade 2-4 acute graft-versus-host disease (GVHD) developed in six patients (54%), and grade 3-4 in one patient (9%). Three (27%) of 11 evaluable patients had chronic GVHD (limited stage). All 11 patients are alive without thalassaemia after a median follow-up time of 397 days (range; 171-814 days). This study lends support to consideration of unrelated donor BMT as an acceptable therapy to cure severe thalassaemia especially in patients who are young and do not yet show irreversible severe complications of iron overload.