Mutations at the activated protein C cleavage sites Arg336 and Arg562 of factor VIII in Thai patients with venous thrombosis.

Venous thrombosis is a multicausal disease, more than one genetic risk factor may cooperate to effect thrombotic risk. Factor V Leiden is found to be an important hereditary risk factor for venous thromboembolism. Analogous to factor V Leiden, a point mutation at amino acid positions Arg336 and Arg562 in factor VIII may predispose patients to thrombosis. Eighty-one Thai patients with venous thrombosis and 100 Thai healthy volunteers have been studied. Neither heterozygous nor homozygous mutations were detected both thrombosis patients or normal volunteers. However, further studies with larger samples of venous thrombosis patients are recommended.

Detection of factor V Leiden in Thai patients with venous thrombosis.

The molecular defect underlying activated protein C resistance (APC-R) is caused by a G to A point mutation in the codon for arginine 506 in the factor V gene (factor V Leiden) which is a major risk factor for venous thrombosis, especially in Caucasian populations. This study is an analysis of the Thai population to determine the prevalence of the factor V Leiden mutation. Twenty-seven patients with apparent venous thrombosis were divided into two groups according to APC-R test. Thirteen patients were diagnosed as positive for n-APC-SR, ratio < 0.8 and fourteen patients were diagnosed as negative for n-APC-SR, ratio > 0.8. Two of thirteen APC-R positive patients and one of fourteen APC-R negative patients were found to have the heterozygous allele for the factor V Leiden mutation but the homozygous allele was not detected in these groups of patients. Neither the heterozygous nor homozygous Leiden mutation was detected in 200 healthy volunteer blood donors. In conclusion, our findings indicate that factor V Leiden mutation is related to venous thrombosis in Thai people. Moreover, a further study of other mutations at the activated protein C cleavage sites of factor V and factor VIII is recommended.

Analysis of HLA-DRB1 alleles using PCR-RFLP and PCR-MPH.

In this study we compare the results of HLA-DRB1 genotyping by PCR-RFLP and PCR-MPH. HLA-DR specificities were also performed by LCT. Samples were obtained from 20 Thai patients who were on the waiting list for kidney transplant. DNA was extracted by phenol-chloroform extraction. It was found that the results gave complete agreement with two methods of DNA typing, however, there were 3 discrepancies in assigning serologic DR specificities and DNA subtypes (p = 0.0001) which were due to the cross reactive antibodies and the lack of potent antisera to define proper HLA-DR subtypes by LCT. These PCR techniques can be applied to identify other alleles such as HLA-DPB1 and HLA-DQB1 which will improve the standard histocompatibility testing in the future.

Prenatal diagnosis of Hb Bart's hydrops fetalis by PCR technique: Pramongkutklao experience.

Hb Bart's hydrops fetalis is very common in Southeast Asia, especially in Thailand. As the mother of such an infant may suffer from toxemia of pregnancy, ante- or post-partum hemorrhage as well as the psychological burden for carrying a nonviable fetus to term, so prenatal diagnosis is indicated and the family should be given the choice of early termination of the pregnancy. Seven high risk pregnancies with Hb Bart's hydrops fetalis (homozygous alpha-thalassemia 1) were studied. Amniocentesis was done at 16-33 weeks of gestation. DNA analysis was performed by polymerase chain reaction (PCR) using 2 techniques, 1) three nucleotide primers and 2) four nucleotide primers. After either therapeutic abortion or birth, heart blood or cord blood was drawn to confirm the diagnosis by Hb electrophoresis and DNA analysis. Of 7 high risk fetuses, 3 were recognized as Hb Bart's hydrops fetalis, 2 showed the alpha-thal 1 trait, 1 showed alpha-thal 2 trait and 1 was a normal fetus. The technique was entirely suitable for prenatal diagnosis of Hb Bart's hydrops fetalis. This technique was a rapid, simple non-radioactive method, less expensive and available in most PCR laboratories.

Clinical phenotypes and genotypes diagnosis of thalassemia in children.

Thalassemia is a relatively common hemolytic anemia in Southeast Asia. Alpha and beta thalassemia, hemoglobin (Hb) E, and Hb Constant Spring (CS) are prevalent in Thailand. Different gene combinations lead to over 60 thalassemic syndromes. One hundred and forty-nine thalassemia families were retrospectively studied. They were 4 homozygous beta-thalassemia (beta-thal/ beta-thal), 79 beta-thal/Hb E, 22 Hb H disease, 32 Hb with Hb CS, and 6 AE Bart's disease. The first clinical manifestation and hematologic data including hemoglobin electrophoresis were analysed. Most homozygous beta-thalassemia and beta-thal/Hb E presented with anemia (100% vs 81%), hepatomegaly (40% vs 21%), and splenomegaly (20% vs 27%). In Hb H disease and Hb H with Hb CS, the clinical findings were anemia (74% vs 79%), hepatomegaly (9% vs 8%), splenomegaly (9% vs 13%), jaundice (24% vs 13%), and fever (18% vs 25%). The 317 hematologic data and hemoglobin types of the patients, their parents and relative were also analyzed. These findings can be used as reference values for childhood thalassemia and heterozygous states.