Sumalee Jindadamrongwech

Diagnostic Problems Caused by HBsAg Mutants – A Consensus Report of an Expert Meeting

A panel of 16 experts from 9 countries convened on April 14 at Schloss Reinhartshausen near Wiesbaden in Germany, to discuss the diagnostic significance of mutants, variants and genotypes of hepatitis B virus (HBV). Since the description of Australia antigen in 1965 and the subsequent observation that it was the envelope of the HBV and now designated hepatitis B surface antigen (HBsAg), this lipoprotein has been a mainstay in the diagnosis of HBV infections. HBsAg tests are used routinely in the diagnosis of acute and chronic liver disease, the screening of blood or organ donors and the surveillance of persons at risk to acquire or to transmit HBV. Current immunoassays for HBsAg are very specific and sensitive (both 199%) and are usually able to detect !0.5 ng HBsAg/ml serum. Their performance is validated in extensive trials before licensing and their detection limit is assayed with an International Standard for HBsAg. An immanent problem of virology is the variability of viruses. Due to the low fidelity of the viral nucleic acid polymerases and the high replication rates, virtually all nucleotide positions of a viral genome can be mutated within a relatively short time. However, the viability of the virus and its adaptation to the host allow the selection and outgrowth of only a limited number of mutants. The survival strategy of HBV in the population is mainly based on induction of immune tolerance and persistence of high viremia. In this state of infection the existing viral genome is favored whereas mutants are less fit and selected against or may be subject to an immune reaction and preferably eliminated. In fact, most of the HBsAg carriers in Germany have a very similar S gene sequence. However, under selective pressure the virus can express many different viable HBsAg mutants. Summary of the Presentations

Virus Overlay Protein Binding Assay (VOPBA) reveals serotype specific heterogeneity of dengue virus binding proteins on HepG2 human liver cells.

Objective: This study sought to investigate the presence of dengue virus binding proteins expressed on the surface of HepG2 cells and to determine if there were serotype specific differences in binding. Methods: HepG2 cell membrane proteins were extracted and separated by SDS-PAGE, transferred to nitrocellulose membranes and incubated with dengue virus serotypes 2, 3 and 4 under varying hybridization conditions. The positions of dengue virus binding proteins were established with a pan-specific anti-dengue virus monoclonal antibody. Results: Dengue virus binding proteins were seen at approximately 78–80, 90, 98, and 102 kD for dengue serotype 2, 90, 130 and 182 kD for dengue serotype 3, and 90 and 130 kD for dengue serotype 4. Binding of the serotypes 3 and 4 was significantly altered by the hybridization conditions, while serotype 2 was affected to a lesser extent. Conclusions: The virus overlay assay used here provides further evidence that there is a serotype specific component regulating the entry of the dengue virus into cells. Given that several virus binding proteins are seen for each serotype, multiple proteins may be required to facilitate the entry of the virus into some cell types.

Effect of genetic alterations of cytarabine-metabolizing enzymes in childhood acute lymphoblastic leukemia

BACKGROUND Single nucleotide polymorphisms (SNPs) of deoxycytidine kinase (dCK) and cytidine deaminase (CDA) are known to alter their enzymatic activities, which affect the metabolism of cytarabine. Currently, treatment of childhood acute lymphoblastic leukemia (ALL) includes cytarabine, especially in high-risk patients. Therefore, we hypothesized that a genetic variation of dCK and CDA genes may influence the risk of cytarabine-related toxicities and early response to treatment. PATIENTS AND METHODS We included children diagnosed with ALL and lymphoblastic lymphoma (LL) stage III and IV. The patients received a modified St Jude Total Therapy Study XV protocol. Cytarabine was used during induction remission (low-dose cytarabine) and reinduction II (high-dose cytarabine) phases. Genotyping of dCK-360C>G and -201C>t and CDA 79A>C and 208G>A was performed. Minimal residual disease (MRD) at the end of the induction phase was measured using flow cytometry. RESULTS Ninety-four children with ALL (n=90) and LL (n=4) were analyzed. The median age at diagnosis was 5.8 years (range, 0.4-15 years). All four SNPs showed predominant wild type alleles. There was no CDA-208A allele in our population. Children with dCK-360G allele were at risk of mucositis after receiving low-dose cytarabine (OR=3.7; 95%CI, 1.2--11.3). neither dCK nor CDA polymorphisms affected the MRD status at the end of the induction phase. CONCLUSION The dCK-360G allele was found to increase the risk of mucositis after exposure to low-dose cytarabine in childhood ALL therapy.

Elevated erythropoietin and cytokines levels are related to impaired reticulocyte maturation in thalassemic patients.

Serum EPO concentration is related primarily to the rate of erythrocyte production and, under the stimulation of hypoxia, increases exponentially as hemoglobin (Hb) decreased. The level of EPO was determined in 141 subjects including 43 normal, 44 thalassemic patients and 54 thalassemic trait subjects. The EPO level was significantly higher in the thalassemic patients (54.8mU/ml in HbH disease [α thal1/α thal2;], 78.1mU/ml in HbH with Hb CS [α thal 1/CS]; 95.6mU/ml in β-thal/HbE splenectomized [BE(S)]; and 114.8mU/ml in β-thal/HbE non-splenectomized [BE(NS)]as compared with 12.0mU/ml in normal subjects. No significant differences were detected in thalassemic trait subjects. In addition, the levels of EPO in thalassemic patients is correlated significantly with the number of reticulocytes and the reticulocyte fractions especially the fraction of immature reticulocytes. Interestingly, the highest level of EPO/% retic ratio as indicated for EPO non-responder was detected in BE(NS) patients. However, the impaired reticulocytes maturation was found to be related significantly with the levels of TNF-α,IFN-γ,IL-10, and VEGF. Since, TNF-α, IFN-γ, IL-10 and VEGF are reported as the cytokines with erythropoietic inhibitory mediators, the variation of these cytokines in thalassemic environments may be associated to the anemic crisis in these patients.

Hb Tak and Hb Q-Thailand in Thai patients are S-window hemoglobin variants revealed by high performance liquid chromatography.

The S-window hemoglobin (Hb) variants revealed by high performance liquid chromatography (HPLC) were studied in 12 Thai individuals. The variants were identified, using DNA sequencing and multiplex amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), to be six cases of Hb Tak [β147 (+AC)], and six cases of Hb Q-Thailand [α74(EF3)Asp→His], respectively. By using the Capillarys 2-capillary zone electrophoresis (CE), Hb Tak and Hb Q-Thailand co-migrated with Hb F in zone 7. This might pose a problem as the high Hb F conditions suggest a differential diagnosis. The S-window Hb variants are mostly Hb Tak and Hb Q-Thailand in the Thai population rather than Hb S [β6(A3)Glu→Val]. The definite identification of Hb variants detected by HPLC or capillary electrophoresis (CE) requires DNA analysis.

A rare Hb H disease due to the - -(SEA) and 16.6 kb α-thalassemia-2 deletions.

A large deletional α-thalassemia-2 (α-thal-2) allele was identified in a Thai woman with Hb H disease. The proband has α-thal-1 (SEA type) in conjunction with a 16.6 kb deletion affecting the α2-globin allele. The proband had severe anemia and required a blood transfusion during puerperium.

Nondeletional Hb Queens Park [α32(B13)Met→Lys]/Hb H (β4) Disease

A rare nondeletional α-thalassemia-2 (α-thal-2) allele was identified in a Thai boy with Hb H (β4) disease. The proband has α-thal-1 (– –SEA type) together with a non productive Hb Queens Park (HBA1:c.98T>A) [α32(B13)Met→Lys] α1-globin variant. No abnormal hemoglobin (Hb) fraction was detected by high performance liquid chromatography (HPLC). The clinical effect of this mutation in the proband was comparable to that of deletional α-thal-2 present in Hb H disease.

Occurrence of the – –SEA, – –THAI and – –FIL α-Thalassemia-1 Carriers from a 7-Year Study at Ramathibodi Hospital, Bangkok, Thailand

Abstract α-Thalassemia (α-thal) is one of the most common genetic diseases in Thailand. Homozygosity of α-thal-1 (– –/– –) and compound heterozygosity of α-thal-1/α-thal-2 (– –/–α) leads to Hb Barts (γ4) hydrops fetalis and Hb H (β4) disease, respectively. In order to better control and provide prevention of α-thal disease, the prevalence of α-thal-1 carriers and the types of genotypes in the Thai population should be known. A 7-year retrospective study, employing multiplex gap-polymerase chain reaction (gap-PCR) of 31,632 blood samples from Ramathibodi Hospital, Mahidol University, Bangkok, revealed an α-thal-1 carrier rate of 14.40% with the – –SEA (NG_000006.1: g.26264_45564del19301), – –THAI (NG_000006.1: g.10664_44164del33501) and – –FIL (NG_000006.1: g.11684_43534del31851) genotypes, constituting frequencies of 14.21, 0.18 and 0.01%, respectively. Although the – –FIL genotype is rare in the Thailand, its detection should be included in future α-thal screening programs.

Prevalence of Thalassemia and Glucose-6-Phosphate Dehydrogenase Deficiency in Newborns and Adults at the Ramathibodi Hospital, Bangkok, Thailand

Abstract Thalassemias and glucose-6-phosphate dehydrogenase (G6PD) deficiency are the most common inherited blood disorders. They are distributed among populations living in malaria endemic regions resulting in survival advantage from severe malaria disease. The aims of this study were to analyze the prevalence of thalassemias and G6PD deficiency at the Ramathibodi Hospital, Bangkok, Thailand. A total of 616 adult and 174 cord blood samples were collected and analyzed for red blood cell (RBC) parameters, hemoglobin (Hb) typing and DNA analysis for G6PD mutations and α-thalassemia (α-thal). The two most prominent types of thalassemia were heterozygous Hb E (HBB: c.79G>A), (19.5% in newborns and 35.6% in adults) followed by heterozygous α-thal-2 [–α3.7 (rightward) deletion] at 18.7% in newborns and 19.5% in adults. After performing G6PD genotyping using multiplex amplification refractory mutation system-polymerase chain reaction (multiplex ARMS-PCR) for 10 G6PD mutations, the prevalence of G6PD mutation was found in 12.0% of newborns and 11.7% of adults. The G6PD Viangchan [871 (G>A)] is the most common G6PD mutation in newborns (42.9%) and adults (52.8%). In addition, coinheritance of various types of thalassemia with G6PD deficiency were found. The results indicated that heterozygous Hb E and G6PD Viangchan are predominant both in newborns and adults in this study.

Low oxygen saturation and severe anemia in compound heterozygous Hb Louisville [β42(CD1)Phe→Leu] and Hb La Desirade [β129(H7)Ala→Val]

ABSTRACT Objective: To investigate the cause(s) of a Thai male proband presenting low oxygen saturation by pulse oximetry (SpO2) and severe anemia. Methods: As Hb variant was suspected, Hb typing was determined by high-performance liquid chromatography and capillary electrophoresis, and subsequently Hb variant was identified by DNA sequencing. Complete blood counts were performed using automated blood cell counter and oxygen saturation was measured by pulse oximetry. Results: Proband was compound heterozygous for Hb Louisville [β42(CD1)Phe→Leu] and Hb La Desirade [β129(H7)Ala→Val]. Of the proband’s two sons, one was compound heterozygous for Hb Louisville and Hb E and the other for Hb La Desirade and Hb E. The former son had similar clinical features and laboratory findings with those of the proband while the latter showed had no abnormal clinical manifestations. Conclusion: This the first report of compound heterozygosity of Hb Louisville and Hb La Desirade in an individual of Southeast Asian ethnicity. Hb variant identification is crucial for genetic counseling and appropriate treatment in regions where hemoglobinopathies are common.