Vichai Laosombat

Molecular basis of β-thalassemia in Thailand: analysis of β-thalassemia mutations using the polymerase chain reaction

Summaryβ-Thalassemia mutations in 71 chromosomes of Thai patients from the northeast, the middle and the south of the country were investigated using dot blot hybridization of PCR (polymerase chain reaction)-amplified DNA with allelespecific oligonucleotide probes. Eight different known molecular defects were detected, at different frequencies. There was an amber mutation in codon 17, a C-T transversion at position 654 of IVS-2, a frameshift mutation between codons 71 and 72, an A-G transition at nucleotide -28 within the TATA box (known as Chinese mutations), a G-T transversion at position 1 of IVS-1 (an Indian mutation), a 4bp deletion in codons 41/42 and a G-C transversion at position 5 of IVS-1 (described as both Chinese and Indian mutations) and a Thai original mutation, an ochre mutation in codon 35. Analysis of the three unknown alleles by DNA sequencing of the cloned DNA fragment amplified by PCR revealed an A-G substitution at the second position of the codon for amino acid 19 (AAC-AGC). The analytic approach used in the present study and the characteristic distribution of mutations in each region of Thailand will prove useful for setting up a prenatal diagnosis program.

Clinical features and molecular analysis in Thai patients with HbH disease

We studied the α-globin gene abnormalities, the clinical features, hematologic values, growth assessment, transfusion therapy, and serum ferritin levels of patients with hemoglobin H (HbH) disease in southern Thailand. HbH disease in 83 of the 147 patients was the deletional type of HbH. The remaining 64 patients was the nondeletional type of HbH disease. All 83 patients with the deletional type were double heterozygotes of α0-thalassemia and α+-thalassemia. The Southeast Asian type of α0-thalassemia accounted for 98% of the Thai patients with HbH disease and the Thai type of α0-thalassemia made up the rest. A 3.7-kb deletion accounted for 91% of α+-thalassemia, and a 4.2-kb deletion made up the rest of the deletional type. In patients with nondeletional type of HbH disease, the Constant Spring variant was the majority of the disease. Newborns with a nondeletional genotype had higher mean corpuscular volume, had higher mean corpuscular hemoglobin, had higher red blood cell distribution width, had lower mean corpuscular hemoglobin concentration, and had higher proportions of Hb Barts than those with a deletional genotype. Twenty-one percent of children with HbH disease had growth deficiency. A genotype–phenotype correlation was found; patients with the nondeletional type of HbH disease had more symptoms at a younger age, more severe hemolytic anemia, more growth deficiency, more dysmorphic facial features, larger spleens, larger livers, and higher serum ferritin levels and required more transfusions than patients with deletional HbH disease.

Novel missense mutation of the UGT1A1 gene in Thai siblings with Gilbert's syndrome

Background : Gilberts syndrome is a common inherited disorder of bilirubin metabolism contributing to the development of neonatal jaundice and causing recurrent jaundice after the neonatal period. In the patients with Gilberts syndrome, mutations have been reported in the promoter and exons of the uridine diphosphate‐glucuronosyl transferase 1 (UGT1A1) gene on chromsome 2q37, which encodes bilirubin uridine diphosphate‐glucuronosyltransferase. However, the genetic basis of Gilberts syndrome, including its inheritance trait, remains to be clarified.

CLINICAL OUTCOME OF FEBRILE NEUTROPENIA IN CHILDREN WITH CANCER USING CEFTAZIDIME AND AMINOGLYCOSIDES

To determine treatment outcome using ceftazidime–aminoglycosides in febrile neutropenic children with cancer, the authors conducted a prospective cohort study in 216 episodes. Early and complete responses to antibiotics were 108/216 (50.0%) and 133/216 (61.6%) episodes, respectively. Death, a modification of antibiotic(s), and resistance to ceftazidime were 2/118 (1.7%), 73/216 (33.8%), and 4/216 (1.9%) episodes, respectively. Primary bacteremia and emerging bacteremia during treatment were 20/216 (9.3%) and 5/216 (2.3%) episodes. Ceftazidime–aminoglycosides was found to be a reasonable initial treatment of febrile neutropenia in the authors’ institution. Imipenem is considered in patients who have clinical sepsis and who fail to respond to initial treatment.

A low dose adrenocorticotropin test (1 microg ACTH) for the evaluation of adrenal function in children with beta-thalassemia receiving hypertransfusion with suboptimal iron-chelating therapy.

A cross-sectional study of adrenal function was carried out in 48 patients with beta-thalassemia who were receiving hypertransfusion with suboptimal desferoxamine. A low dose adrenocorticotropic hormone (1 microg ACTH) stimulation test was performed using the cut-off criteria of peak cortisol for adrenal sufficiency >18 microg/dl. Adrenal impairment was diagnosed in 22 patients, giving a prevalence of 45.8%. The peak cortisol concentrations in normal and impaired adrenal function groups were 26.22 +/- 2.84 and 14.03 +/- 3.12 microg/dl, respectively, and the mean basal morning cortisol was 8.93 +/- 2.97 and 6.52 +/- 2.45 microg/dl, respectively. There was no significant difference in any clinical characteristic between the patients with impaired adrenal function and those with normal adrenal function.

Glucose-6-phosphate dehydrogenase variants associated with favism in Thai children.

In a study conducted at Songklanagarind Hospital in the south of Thailand, the subjects were 225 patients (210 boys and 15 girls) with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Favism was found in 3.6% of the G6PD-deficient children. Approximately one half of the G6PD-deficient patients with favism were younger than 2 years. Sudden onset of anemia was found within 1 to 3 days after ingestion of dried fava beans. The classic features of favism, which are pallor, hemoglobinuria, and jaundice, were detected in all cases. To characterize the known G6PD mutations in Thai children, molecular analysis was performed for 8 G6PD-deficient children with favism by a combination of polymerase chain reaction-restriction fragment length polymorphism analysis and amplification refractory mutation system analysis.The G6PD variants in these children were G6PD Kaiping 1388,G->A; G6PD Mahidol 487,G->A; G6PD Viangchan 871,G→A; and uncharacterized mutation with silent mutation 1311,C→T.

Acquired platelet dysfunction with eosinophilia in children in the south of Thailand.

One hundred and sixty-eight children aged 13 months to 12.6 years with acquired platelet dysfunction with eosinophilia (APDE) were studied. The male to female ratio was 1.15:1. All of the children were in good health and no history of any drug ingestion was detected. All of the children had widespread spontaneous bruising on the extremities, body and face off and on. Severe bleeding symptoms were detected in 8% of these patients. The number of platelets in these children was within the normal range but the platelet morphology was abnormal in all of them. Eosinophilia was detected in 86% of these children. Prolonged bleeding time was detected in 53% of these patients. Abnormal platelet adhesiveness was found in 33% of cases. Abnormal platelet aggregation induced by collagen was the most sensitive test in these patients. Abnormal ADP release from the platelets was detected in these patients by the absence of a second wave of aggregation during stimulation of PRP by ADP or epinephrine. Abnormal or no ATP secretion from the platelets during stimulation by ADP, epinephrine or collagen was detected in these patients. Ristocetin-induced platelet aggregation was normal in these children. Decreased or absence of platelet dense granules by TEM study was detected in some patients. These changes in platelet functions and morphology may be due to acquired storage pool deficiency of the platelet. Parasitic infection was detected in 56% of these children. About 83% of these children with APDE had serum total IgE higher than 100 IU/ml. There was no correlation between the number of eosinophils and serum total IgE and the severity of bleeding symptoms. The majority of children with APDE did not receive any treatment except those who had severe bleeding symptoms which required platelet concentrate to stop bleeding. In more than 90% of the patients, the bruising or ecchymosis disappeared within 6 months and the abnormal platelet functions returned to normal within 4 months. Recurrence of these bleeding syndromes was detected in 7% of the children.

INTERACTION OF THE α2 POLYADENYLATION SIGNAL MUTATION (AATAAA → AATA– –) AND α0-THALASSEMIA (– –SEA), RESULTING IN Hb H DISEASE IN A THAI PATIENT

We report a Thai boy with a compound heterozygosity for the α2 polyadenylation signal mutation (AATAAA → AATA– –) and α0-thalassemia (– –SEA), who suffered from Hb H disease with more severe clinical symptoms than those usually observed with deletional Hb H disease. His Hb H level was as high as 52% of total hemoglobin. The hematologic data of this unusual case of Hb H disease was compared with those of Hb H disease with a homozygosity for the α2 polyadenylation signal mutation, and compound heterozygosity of the α2 polyadenylation signal mutation and α0-thalassemia. A simple DNA assay based on an allele specific polymerase chain reaction for the detection of this polyadenylation signal mutation is described.

Neonatal Anemia Associated with Southeast Asian Ovalocytosis

The purposes of this study were to evaluate the reliability of the previously described diagnostic criteria for Southeast Asian ovalocytosis (SAO) in adults in the diagnosis of SAO in newborns and to describe the role of SAO in newborn infants presenting with pallor and jaundice. The inclusion criteria in this retrospective descriptive study were that the patient be a newborn with pallor or jaundice and with ovalocytes in the peripheral blood smear (PBS). The exclusion criteria were newborn status with other causes of neonatal hemolysis or anemia. Controls were age-matched newborn infants who did not have SAO or other causes of neonatal anemia or hemolysis. Hematological data were assessed with a hematology analyzer. DNA analysis for SAO band 3 was done by polymerase chain reaction. Among 107 newborn infants with SAO, 30 infants were excluded from the study. The exclusions were premature infants, an infant with congenital syphilis, low-birth-weight infants, infants with ABO blood group incompatibility, infants with α-thalassemia, infants with hemoglobin E heterozygote or homozygotes, glucose-6-phosphate dehydrogenase-deficient infants, and infants with fetomaternal hemorrhage. The DNA analysis for SAO band 3 was done in 56 newborns, and 54 had positive results for SAO band 3 gene deletion. Approximately one half of the 54 newborn infants with SAO had hyperbilirubinemia, and 3 had severe hyperbilirubinemia. The mean hemoglobin concentration, packed cell volume, and red blood cell (RBC) count in the infants with SAO in the first week of life were significantly lower than those in control infants. The mean absolute number of reticulocytes, mean corpuscular hemoglobin, and red cell volume distribution width in infants with SAO band 3 in the first week of life were significantly higher than those in control infants. The neonatal diagnosis of SAO can be made by examination of RBC morphology in the PBS with the presence of stomatocytes, theta cells, and ≥25% ovalocytes. SAO plays a role in anemia and hyperbilirubinemia in newborn infants.The purposes of this study were to evaluate the reliability of the previously described diagnostic criteria for Southeast Asian ovalocytosis (SAO) in adults in the diagnosis of SAO in newborns and to describe the role of SAO in newborn infants presenting with pallor and jaundice. The inclusion criteria in this retrospective descriptive study were that the patient be a newborn with pallor or jaundice and with ovalocytes in the peripheral blood smear (PBS). The exclusion criteria were newborn status with other causes of neonatal hemolysis or anemia. Controls were age-matched newborn infants who did not have SAO or other causes of neonatal anemia or hemolysis. Hematological data were assessed with a hematology analyzer. DNA analysis for SAO band 3 was done by polymerase chain reaction. Among 107 newborn infants with SAO, 30 infants were excluded from the study. The exclusions were premature infants, an infant with congenital syphilis, low-birth-weight infants, infants with ABO blood group incompatibility, infants with α-thalassemia, infants with hemoglobin E heterozygote or homozygotes, glucose-6-phosphate dehydrogenase-deficient infants, and infants with fetomaternal hemorrhage. The DNA analysis for SAO band 3 was done in 56 newborns, and 54 had positive results for SAO band 3 gene deletion. Approximately one half of the 54 newborn infants with SAO had hyperbilirubinemia, and 3 had severe hyperbilirubinemia. The mean hemoglobin concentration, packed cell volume, and red blood cell (RBC) count in the infants with SAO in the first week of life were significantly lower than those in control infants. The mean absolute number of reticulocytes, mean corpuscular hemoglobin, and red cell volume distribution width in infants with SAO band 3 in the first week of life were significantly higher than those in control infants. The neonatal diagnosis of SAO can be made by examination of RBC morphology in the PBS with the presence of stomatocytes, theta cells, and ≥25% ovalocytes. SAO plays a role in anemia and hyperbilirubinemia in newborn infants.

Neonatal hyperbilirubinemia associated with Southeast Asian ovalocytosis

We report, herein, an infant who is twin A of a dizygotic twin, with premature birth and both twins having hemoglobin (Hb) E heterozygosity. Twin A who had Southeast Asian ovalocytosis (SAO) developed neonatal jaundice at the age of 2 days and needed phototherapy at the age of 3 days. The microbilirubin level was rapidly rising up to 535.2 μmol/L (31.3 mg/dl) with the hematocrit value of 38% at the age of 4 days prior to exchange blood transfusion. Exchange blood transfusion was done by 220 ml of O, Rh positive packed red blood cell reconstituted with 180 ml of O, Rh positive fresh plasma to lower the bilirubin level. Twin A received phototherapy from about 8 hr prior to exchange blood transfusion until 3 days later. Twin B, who did not have SAO, developed neonatal hyperbilirubinemia and needed only phototherapy. Twin A received a deletion of 27 basepairs in the erythroid band 3 gene and Hb E heterozygosity from his father. Am. J. Hematol. 60:136–139, 1999.