Werasak Sasanakul

Frequency of thiopurine S-methyltransferase genetic variation in Thai children with acute leukemia

BACKGROUND Thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation (inactivation) of mercaptopurine, azathioprine, and thioguanine, and exhibits genetic variation. About 11% of Caucasians have intermediate TPMT activity because of heterozygosity, and about 1 in 300 inherits TPMT deficiency as an autosomal codominant trait. If patients who have intermediate or deficient TPMT activity receive the standard dose of thiopurine medications, they can accumulate excessive thiopurine nucleotides in hematopoietic tissue, which could lead to severe and possibly fatal myelosuppression. There is very little information about TPMT genetic variation among Asian populations. We investigated the frequency of TPMT genetic variation among Thai children with acute leukemia. PROCEDURE Fresh whole blood was obtained from 75 Thai children with acute leukemia at the time of remission. Genomic DNA was isolated from total peripheral white blood cells. We performed polymerase chain reaction (PCR) to detect 3 types of variant of the human TPMT gene. RESULTS Among 75 patients, the frequency of heterozygotes for the TPMT gene among Thai children with acute leukemia was approximately 11%. However, the TPMT*3C was the only variant TPMT allele found among Thai children. This is different from the North American Caucasian populations, in which TPMT*3A is the predominant variant allele, and TPMT*3C is rare (approximately 5% of variant alleles). CONCLUSIONS There is no difference in the frequency of this genetic variation between Asian and North American Caucasian populations. Determination of the TPMT genotype by PCR method before antileukemic therapy is practical and may have clinical relevance. This knowledge could be applied towards organ transplant recipients who require these medications for immunosuppression.

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

Summary.  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.

Tumour necrosis factor gene polymorphism in dengue infection: association with risk of bleeding

Abstract Background: A single nucleotide polymorphism located at the promoter -308A of tumour necrosis factor-alpha (TNF-α) gene may affect transcription and increase cytokine production, leading to the severe manifestation of dengue virus infection. Aim: To study the association of the TNF-α -308A allele and the severity of patients with dengue infection. Methods: 112 patients suspected of having dengue infection and 106 normal controls were enrolled in the study. Mean (SD) age was 10·4 (3·6) years. In all, 19 and 82 patients were diagnosed with dengue fever (DF) and dengue haemorrhagic fever (DHF), respectively, while 11 were diagnosed with other febrile illnesses (OFIs). They were tested for the polymorphisms at the promoter -308 position of the TNF-α gene and their TNF-α levels were measured. Results: In the patients with dengue infection (14/202, 6·9%) with OFIs (1/22, 4·5%) and in normal controls (17/212, 8·0%), the frequency of the TNF-α -308A allele was not significantly different. Moreover, no statistically significant difference was found in patients with various clinical manifestations of dengue infection involving DF (5·3%, 2/38), DHF grade I (5·0%, 2/40), DHF grade II (9·5%, 4/42), DHF grade III (2·5%, 1/40) and DHF grade IV (11·9%, 5/42). However, patients with dengue infection and significant bleeding manifestations, apart from petechiae and ecchymosis, tended to have a higher frequency of the TNF-α -308A allele (11·8%, 9/76) than those without significant bleeding manifestations (5/126, 4·0%) (P = 0·056). The levels of TNF-α were additionally measured in 67 patients but the results failed to demonstrate a functional effect in the transcriptional rate of the TNF-α -308A allele. Conclusion: In patients with dengue infection there is an association between the TNF-α -308A allele and the risk of bleeding. The test may be used as one of the predictors of the severity of dengue infection.

A comparison of the allelic frequencies of ten DNA polymorphisms associated with factor VIII and factor IX genes in Thai and Western European populations

The frequency of five factor VIII gene intragenic and linked DNA polymorphisms and five factor IX gene intragenic polymorphisms was studied in Thai females. The polymorphisms in the FVIII gene were detected by restriction enzymes BelI, XbaI, BglI and at linked loci DX13 (DXS15) and Stl4 (DXS52) by BqlII and TaqI, respectively, and in the FIX gene by MseI, DdeI, XmnI, TaqI and HhaI. With the exception of the BglI restriction fragment length polymorphism (RFLP), which is absent in Thais, factor VIII polymorphism frequencies were similar in Thais and Caucasians. Combined use of XbaI and TaqI/St14 resulted in a heterozygosity rate of greater than 90% in Thai females. For FIX, the recently described MseI RFLP in the 5‘ flanking region was the most informative polymorphism in Thais, 43% of females being heterozygous. The other four polymorphisms added little to the overall heterozygosity rate. The appropriate polymorphisms were used to track defective factor VIII and IX genes through 22 Thai pedigrees with haemophilia to enable carrier status to be assigned to female family members. The information obtained during this study will form the basis for carrier detection and prenatal diagnosis of haemophilia A and B by DNA polymorphism analysis in Thailand.

Combined Chelation Therapy with Daily Oral Deferiprone and Twice-Weekly Subcutaneous Infusion of Desferrioxamine in Children with β-Thalassemia: 3-Year Experience

Objective: To study the efficacy of combined treatment with oral and subcutaneous iron chelators. Material and Methods: 50-100 mg/kg/day of oral deferiprone (DFP) combined with 40 mg/kg/dose s.c. desferrioxamine (DFO) twice weekly were given to transfusion-dependent β-thalassemia children. Results: Enrolled patients (9 with β-thalassemia major and 33 with β-thalassemia hemoglobin E), ranging from 3 to 18 years in age, were divided into 3 groups; group 1 ferritin ≥1,000-2,500 ng/ml (n = 10), group 2 ferritin >2,500-4,000 ng/ml (n = 23) and group 3 ferritin >4,000 ng/ml (n = 9). Of the 42 patients, 28 reached the 36-month follow-up. Ten patients whose ferritin declined <15% while receiving 100 mg/kg/day of DFP were considered nonresponders. The median age and previous transfusion duration before enrollment were significantly higher in nonresponders than responders (p = 0.04 and 0.003, respectively). The responders exhibited a significant fall in median ferritin levels from 2,954.6 to 936.6 ng/ml (p < 0.001). Time to a significant decrease in serum ferritin among responders was 6 months. In 13 patients, 16 episodes of adverse events occurred: hemophagocytosis with cytopenia (n = 1), neutropenia (n = 2), thrombocytopenia (n = 2), elevated alanine aminotransferase (n = 5), elevated serum creatinine (n = 1), proteinuria (n = 1) and gastrointestinal discomfort (n = 4). Conclusion: Combination therapy with daily oral DFP and subcutaneous DFO twice weekly is a safe and effective alternative to chelation monotherapy in β-thalassemia children.

Activation of a cryptic splice site in a potentially lethal coagulation defect accounts for a functional protein variant

Changes at the invariable donor splice site + 1 guanine, relatively frequent in human genetic disease, are predicted to abrogate correct splicing, and thus are classified as null mutations. However, their ability to direct residual expression, which might have pathophysiological implications in several diseases, has been poorly investigated. As a model to address this issue, we studied the IVS6 + 1G > T mutation found in patients with severe deficiency of the protease triggering coagulation, factor VII (FVII), whose absence is considered lethal. In expression studies, the IVS6 + 1G > T induced exon 6 skipping and frame-shift, and prevented synthesis of correct FVII transcripts detectable by radioactive/fluorescent labelling or real-time RT-PCR. Intriguingly, the mutation induced the activation of a cryptic donor splice site in exon 6 and production of an in-frame 30 bp deleted transcript (8 ± 2%). Expression of this cDNA variant, lacking 10 residues in the activation domain, resulted in secretion of trace amounts (0.2 ± 0.04%) of protein with appreciable specific activity (48 ± 16% of wt-FVII). Altogether these data indicate that the IVS6 + 1G > T mutation is compatible with the synthesis of functional FVII molecules (~ 0.01% of normal, 1 pM), which could trigger coagulation. The low but detectable thrombin generation (352 ± 55 nM) measured in plasma from an IVS6 + 1G > T homozygote was consistent with a minimal initiation of the enzymatic cascade. In conclusion, we provide experimental clues for traces of FVII expression, which might have reverted an otherwise perinatally lethal genetic condition.

Detection of known haemophilia B mutations and carrier testing by microarray

The molecular basis of haemophilia B is heterogeneous and many mutations of the Factor IX (FIX) gene have been characterised. Using the allele-specific arrayed primer extension (AS-APEX) technology, we have designed a FIX array to simultaneously analyse 69 mutations found in British, Thai and Chinese patients. This technology overcomes the problem of multiple reverse dot-blot analysis and has a 100% accuracy in the detection of both affected subjects and carriers in families with known mutations. In seven unknown mutations from Thailand, the array could detect the specific mutation in five and in the remainders the normal primer at specific spots failed to extend due to a mutation a few nucleotides upstream, thus allowing their identification. Hence this FIX array can detect 53% of the 2891 mutation entries in the FIX database. Each of the microarray slide can be used for three different test samples and would be useful for carrier testing for common mutations and prenatal diagnosis. It is simpler and more cost effective than genome sequencing and would be particularly useful in laboratories with limited technical capabilities.

Safety profile of a liquid formulation of deferiprone in young children with transfusion-induced iron overload: a 1-year experience

Background: Data on the use of deferiprone in young children with iron overload are limited. Objective: To study the safety profile of a liquid formulation of deferiprone in chelating young children with transfusion-induced iron overload. Patients and methods: A daily dose of 50–100 mg/kg BW in three divided doses of oral deferiprone was given to young patients who had received at least ten packed red cell transfusions and achieved a serum ferritin level >1000 μg/L during a 12-month period from 2011 to 2012. Results: Nine children (four males) diagnosed with various types of thalassaemia (n = 8) and hereditary spherocytosis (n = 1) were enrolled. Their mean (SD) age was 4.5 (1.9) years. The patients received 15–20 ml/kg BW of packed red cell transfusions every 4–8 weeks from a mean (SD) age of 2.1 (1.7) years to maintain a pre-transfusion haematocrit at 27%. A mean (SD) total of packed red cells of 5132 (2725) ml were given within a mean (SD) duration of 2.4 (1.1) years before the study. During the 1-year study period, they received a mean (SD) total of packed red cells of 2194 (680) ml or 138 (50) ml/kg BW with a mean (SD) daily iron load of 0.29 (0.12) mg/kg BW. The pre-treatment geometric mean of serum ferritin of 1863.8 μg/L decreased to 1279.7 μg/L after 1 year of treatment (P = 0.05). All patients tolerated the liquid formulation well and did not experience any gastro-intestinal discomfort, nausea or vomiting. Conclusion: The liquid formulation of deferiprone is safe in young children with transfusion-induced iron overload.BACKGROUND Data on the use of deferiprone in young children with iron overload are limited. OBJECTIVE To study the safety profile of a liquid formulation of deferiprone in chelating young children with transfusion-induced iron overload. PATIENTS AND METHODS A daily dose of 50-100 mg/kg BW in three divided doses of oral deferiprone was given to young patients who had received at least ten packed red cell transfusions and achieved a serum ferritin level >1000 μg/L during a 12-month period from 2011 to 2012. RESULTS Nine children (four males) diagnosed with various types of thalassaemia (n = 8) and hereditary spherocytosis (n = 1) were enrolled. Their mean (SD) age was 4.5 (1.9) years. The patients received 15-20 ml/kg BW of packed red cell transfusions every 4-8 weeks from a mean (SD) age of 2.1 (1.7) years to maintain a pre-transfusion haematocrit at 27%. A mean (SD) total of packed red cells of 5132 (2725) ml were given within a mean (SD) duration of 2.4 (1.1) years before the study. During the 1-year study period, they received a mean (SD) total of packed red cells of 2194 (680) ml or 138 (50) ml/kg BW with a mean (SD) daily iron load of 0.29 (0.12) mg/kg BW. The pre-treatment geometric mean of serum ferritin of 1863.8 μg/L decreased to 1279.7 μg/L after 1 year of treatment (P = 0.05). All patients tolerated the liquid formulation well and did not experience any gastro-intestinal discomfort, nausea or vomiting. CONCLUSION The liquid formulation of deferiprone is safe in young children with transfusion-induced iron overload.

The Association Between HLA Class II Alleles and the Occurrence of Factor VIII Inhibitor in Thai Patients with Hemophilia A.

Objective: This study aimed to investigate the association between HLA class II alleles and the occurrence of FVIIIinhibitor in Thai hemophilia A patients. Material and Methods: The distribution of HLA-DRB1 alleles and DQB1 alleles in 57 Thai hemophilia A patientsand 36 blood donors as controls was determined using the PCR sequence-specific primer (PCR-SSP) method, and theassociation between the occurrence of factor VIII (FVIII) inhibitor and the presence of certain HLA class II alleles wasinvestigated. Results: The frequency of HLA-DRB1*15 was higher in the hemophilia A patients with and without FVIII inhibitor,whereas that of DRB1*14, DRB1*07, and DQB1*02 was lower in the hemophilia A patients with FVIII inhibitor, ascompared to controls. Interestingly, only the frequency of DRB1*15 was significantly higher in the patients with inhibitorthan in the controls (P = 0.021). Moreover, the frequency of DRB1*15 in the patients with inhibitor was higher than inthose without inhibitor (P = 0.198). Conclusion: The study’s findings show that the DRB1*15 allele might have contributed to the occurrence of inhibitorin the Thai hemophilia A patients; however, additional research using larger samples and high-resolution DRB1 typingis warranted.

Protein C deficiency in Thai children with thromboembolism: A report of clinical presentations and mutation analysis.

Protein C, a natural anticoagulant, has been reported to be a common thrombophilic risk factor in Thai and other Asian populations [1,2]. Patients with protein C deficiency mainly present with venous rather than arterial thromboembolism [3,4]. Protein C deficiency has an autosomal dominant pattern of inheritance. It is classified into two types; type 1, characterized by decreased plasma concentrations of both protein C activity and antigen and type 2, characterized by decreased protein C activity but normal plasma concentration of protein C antigen. The prevalence of protein C deficiency in healthy Thai population and patients with venous thromboembolism was reported as 2% and 8.9%, respectively, higher than Caucasians [5–7]. To date, over 300 mutations have been reported [8]. Significant differences in protein C mutations identified in different ethnicities were seen, for example, the R230C, Q132X, R306X and 3363insC mutations were commonly found in Caucasian populations [9,10] while R147W was a common mutation among Taiwanese Chinese [11]. We report the clinical presentations and mutation analysis of six Thai children with protein C deficiency. Protein C activity and antigen were tested at the time of diagnosis and repeated three months after thromboembolic events or at the time of anticoagulant discontinuation and at the age of five years. The polymerase chain reaction (PCR) amplifications of exon 1 to 9 were performed using our own designed primers (Table 1), and then mutations were screened by using conformation sensitive gel electrophoresis (CSGE), a heteroduplexbased method for nucleotide mismatch detection of all exons [12]. Samples displaying abnormal CSGE pattern were subjected to sequencing. Nucleotide and amino acid numbering was referenced to Foster et al [13].