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The use of bone marrow stem cells for bone tissue engineering

Ng MH, Aminuddin BS, Tan KK, Tan GH, Sabarul Afian M, Ruszymah BH

Med J Malaysia, 2004 May;59 Suppl B:41-2.
PMID: 15468809

Bone marrow stem cells (BMSC), known for its multipotency to differentiate into various mesenchymal cells such as chodrocyte, osteoblasts, adipocytes, etc, have been actively applied in tissue engineering. BMSC have been successfully isolated from bone marrow aspirate and bone marrow scraping from patients of various ages (13-56 years) with as little as 2ml to 5ml aspirate. BMSC isolated from our laboratory showed the presence of a heterogenous population that showed varying prevalence of surface antigens and the presence of telomerase activity albeit weak. Upon osteogenic induction, alkaline phosphatase activity and mineralization activity were observed.
Optimization of palm oil physical refining process for reduction of 3-monochloropropane-1,2-diol (3-MCPD) ester formation

Zulkurnain M, Lai OM, Tan SC, Abdul Latip R, Tan CP

J Agric Food Chem, 2013 Apr 3;61(13):3341-9.
PMID: 23464796 DOI: 10.1021/jf4009185

The reduction of 3-monochloropropane-1,2-diol (3-MCPD) ester formation in refined palm oil was achieved by incorporation of additional processing steps in the physical refining process to remove chloroester precursors prior to the deodorization step. The modified refining process was optimized for the least 3-MCPD ester formation and acceptable refined palm oil quality using response surface methodology (RSM) with five processing parameters: water dosage, phosphoric acid dosage, degumming temperature, activated clay dosage, and deodorization temperature. The removal of chloroester precursors was largely accomplished by increasing the water dosage, while the reduction of 3-MCPD esters was a compromise in oxidative stability and color of the refined palm oil because some factors such as acid dosage, degumming temperature, and deodorization temperature showed contradictory effects. The optimization resulted in 87.2% reduction of 3-MCPD esters from 2.9 mg/kg in the conventional refining process to 0.4 mg/kg, with color and oil stability index values of 2.4 R and 14.3 h, respectively.
Nutrient composition, antioxidant properties, and anti-proliferative activity of Lignosus rhinocerus Cooke sclerotium

Yap YH, Tan N, Fung S, Aziz AA, Tan C, Ng S

J Sci Food Agric, 2013 Sep;93(12):2945-52.
PMID: 23460242 DOI: 10.1002/jsfa.6121

Lignosus rhinocerus (tiger milk mushroom) is an important medicinal mushroom used in Southeast Asia and China, and its sclerotium can be developed into functional food/nutraceuticals. The nutrient composition, antioxidant properties, and anti-proliferative activity of wild type and a cultivated strain of L. rhinocerus sclerotia were investigated.
Effects of keratinocyte growth factor on skin epithelial differentiation of human amnion epithelial cells

Fatimah SS, Tan GC, Chua K, Tan AE, Nur Azurah AG, Hayati AR

Burns, 2013 Aug;39(5):905-15.
PMID: 23273814 DOI: 10.1016/j.burns.2012.10.019

The aim of the present study was to determine the effects of KGF on the differentiation of cultured human amnion epithelial cells (HAECs) towards skin keratinocyte. HAECs at passage 1 were cultured in medium HAM's F12: Dulbecco's Modified Eagles Medium (1:1) supplemented with different concentrations of KGF (0, 5, 10, 20, 30 and 50 ng/ml KGF). Dose-response of KGF on HAECs was determined by morphological assessment; growth kinetic evaluation; immunocytochemical analysis; stemness and epithelial gene expression quantification with two step real time RT-PCR. KGF promotes the proliferation of HAECs with maximal effect observed at 10 ng/ml KGF. However, KGF decreased the stemness genes expression: Oct-3/4, Sox-2, Nanog3, Rex-1, FGF-4, FZD-9 and BST-1. KGF also down-regulates epithelial genes expression: CK3, CK18, CK19, Integrin-β1, p63 and involucrin in cultured HAECs. No significant difference on the gene expression was detected for each Nestin, ABCG-2, CK1 and CK14 in KGF-treated HAECs. Immunocytochemical analysis for both control and KGF-treated HAECs demonstrated positive staining against CK14 and CK18 but negative staining against involucrin. The results suggested that KGF stimulates an early differentiation of HAECs towards epidermal cells. Differentiation of KGF-treated HAECs to corneal lineage is unfavourable. Therefore, further studies are needed to elucidate the roles of KGF in the differentiation of HAECs towards skin keratinocytes.
Investigation of class characteristics in English handwriting of the three main racial groups: Chinese, Malay and Indian in Singapore

Cheng N, Lee GK, Yap BS, Lee LT, Tan SK, Tan KP

J Forensic Sci, 2005 Jan;50(1):177-84.
PMID: 15831016

This paper investigated the class characteristics in English handwriting of the Chinese, Malays and Indians in Singapore, many of whom learned their native language as a second language. One hundred and fifty-four handwriting exemplars were collected and features such as letter designs, pen-lifts, letter spacing and embellishments were studied. A number of characteristic features peculiar to the individual racial group were identified, which confirmed the impact of their native language writing systems on English handwriting.
Comparative evaluation of three purification methods for the nucleocapsid protein of Newcastle disease virus from Escherichia coli homogenates

Tan YP, Ling TC, Yusoff K, Tan WS, Tey BT

J Microbiol, 2005 Jun;43(3):295-300.
PMID: 15995649

In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional methods for the recovery of NP proteins involved multiple steps, such as centrifugation, precipitation, dialysis, and sucrose gradient ultracentrifugation. For the PBA, clarified feedstock was used for column loading, while in EBA, unclarified feedstock was used. Streamline chelating immobilized with Ni2+ ion was used as an affinity ligand for both PBA and EBA. The final protein yield obtained in conventional and PBA methods was 1.26% and 5.56%, respectively. It was demonstrated that EBA achieved the highest final protein yield of 9.6% with a purification factor of 7. Additionally, the total processing time of the EBA process has been shortened by 8 times compared to that of the conventional method.
Forming a lutein nanodispersion via solvent displacement method: the effects of processing parameters and emulsifiers with different stabilizing mechanisms

Tan TB, Yussof NS, Abas F, Mirhosseini H, Nehdi IA, Tan CP

Food Chem, 2016 Mar 1;194:416-23.
PMID: 26471574 DOI: 10.1016/j.foodchem.2015.08.045

A solvent displacement method was used to prepare lutein nanodispersions. The effects of processing parameters (addition method, addition rate, stirring time and stirring speed) and emulsifiers with different stabilizing mechanisms (steric, electrostatic, electrosteric and combined electrostatic-steric) on the particle size and particle size distribution (PSD) of the nanodispersions were investigated. Among the processing parameters, only the addition method and stirring time had significant effects (p<0.05) on the particle size and PSD. For steric emulsifiers, Tween 20, 40, 60 and 80 were used to produce nanodispersions successfully with particle sizes below 100nm. Tween 80 (steric) was then chosen for further comparison against sodium dodecyl sulfate (SDS) (electrostatic), sodium caseinate (electrosteric) and SDS-Tween 80 (combined electrostatic-steric) emulsifiers. At the lowest emulsifier concentration of 0.1%, all the emulsifiers invariably produced stable nanodispersions with small particle sizes (72.88-142.85nm) and narrow PSDs (polydispersity index<0.40).
Shotgun proteomic analysis of tiger milk mushroom (Lignosus rhinocerotis) and the isolation of a cytotoxic fungal serine protease from its sclerotium

Yap HY, Fung SY, Ng ST, Tan CS, Tan NH

J Ethnopharmacol, 2015 Nov 4;174:437-51.
PMID: 26320692 DOI: 10.1016/j.jep.2015.08.042

The sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden (tiger milk mushroom) has been traditionally used as a complementary and alternative medicine for cancer treatment by the local communities of Southeast Asia. Despite the continuous research interest in its antiproliferative activity, the identity of the bioactive compound(s) responsible has yet to be determined. This study aims to bridge the gap in existing research literature by using proteomics approach for investigation of the nature of the anticancer substance of L. rhinocerotis.
Fulltext Relationships between cancer pattern, country income and geographical region in Asia

Ng CJ, Teo CH, Abdullah N, Tan WP, Tan HM

BMC Cancer, 2015;15:613.
PMID: 26335225 DOI: 10.1186/s12885-015-1615-0

BACKGROUND: Cancer incidence and mortality varies across region, sex and country's economic status. While most studies focused on global trends, this study aimed to describe and analyse cancer incidence and mortality in Asia, focusing on cancer site, sex, region and income status.
METHODS: Age-standardised incidence and mortality rates of cancer were extracted from the GLOBOCAN 2012 database. Cancer mortality to incidence ratios (MIRs) were calculated to represent cancer survival. The data were analysed based on the four regions in Asia and income.
RESULTS: Cancer incidence rate is lower in Asia compared to the West but for MIR, it is the reverse. In Asia, the most common cancers in men are lung, stomach, liver, colorectal and oesophageal cancers while the most common cancers in women are breast, lung, cervical, colorectal and stomach cancers. The MIRs are the highest in lung, liver and stomach cancers and the lowest in colorectal, breast and prostate cancers. Eastern and Western Asia have a higher incidence of cancer compared to South-Eastern and South-Central Asia but this pattern is the reverse for MIR. Cancer incidence rate increases with country income particularly in colorectal and breast cancers but the pattern is the opposite for MIR.
CONCLUSION: This study confirms that there is a wide variation in cancer incidence and mortality across Asia. This study is the first step towards documenting and explaining the changing cancer pattern in Asia in comparison to the rest of the world.
Causative Pathogens of Febrile Neutropaenia in Children Treated for Acute Lymphoblastic Leukaemia

Lam JC, Chai JY, Wong YL, Tan NW, Ha CT, Chan MY, et al.

Ann Acad Med Singap, 2015 Nov;44(11):530-4.
PMID: 27089960

INTRODUCTION: Treatment of acute lymphoblastic leukaemia (ALL) using intensive chemotherapy has resulted in high cure rates but also substantial morbidity. Infective complications represent a significant proportion of treatment-related toxicity. The objective of this study was to describe the microbiological aetiology and clinical outcome of episodes of chemotherapy-induced febrile neutropaenia in a cohort of children treated for ALL at our institution.
MATERIALS AND METHODS: Patients with ALL were treated with either the HKSGALL93 or the Malaysia-Singapore (Ma-Spore) 2003 chemotherapy protocols. The records of 197 patients who completed the intensive phase of treatment, defined as the period of treatment from induction, central nervous system (CNS)-directed therapy to reinduction from June 2000 to January 2010 were retrospectively reviewed.
RESULTS: There were a total of 587 episodes of febrile neutropaenia in 197 patients, translating to an overall rate of 2.98 episodes per patient. A causative pathogen was isolated in 22.7% of episodes. An equal proportion of Gram-positive bacteria (36.4%) and Gram-negative bacteria (36.4%) were most frequently isolated followed by viral pathogens (17.4%), fungal pathogens (8.4%) and other bacteria (1.2%). Fungal organisms accounted for a higher proportion of clinically severe episodes of febrile neutropaenia requiring admission to the high-dependency or intensive care unit (23.1%). The overall mortality rate from all episodes was 1.5%.
CONCLUSION: Febrile neutropaenia continues to be of concern in ALL patients undergoing intensive chemotherapy. The majority of episodes will not have an identifiable causative organism. Gram-positive bacteria and Gram-negative bacteria were the most common causative pathogens identified. With appropriate antimicrobial therapy and supportive management, the overall risk of mortality from febrile neutropaenia is extremely low.
Production of the virus-like particles of nipah virus matrix protein in Pichia pastoris as diagnostic reagents

Joseph NM, Ho KL, Tey BT, Tan CS, Shafee N, Tan WS

Biotechnol Prog, 2016 Jul 08;32(4):1038-45.
PMID: 27088434 DOI: 10.1002/btpr.2279

The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus-like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti-myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high-performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1038-1045, 2016.
Stability evaluation of lutein nanodispersions prepared via solvent displacement method: The effect of emulsifiers with different stabilizing mechanisms

Tan TB, Yussof NS, Abas F, Mirhosseini H, Nehdi IA, Tan CP

Food Chem, 2016 Aug 15;205:155-62.
PMID: 27006226 DOI: 10.1016/j.foodchem.2016.03.008

The stability of lutein nanodispersions was evaluated during storage and when exposed to different environmental conditions. Lutein nanodispersions were prepared using Tween 80, sodium dodecyl sulfate (SDS), sodium caseinate (SC) and SDS-Tween 80 as the emulsifiers. During eight weeks of storage, all samples showed remarkable physical stability. However, only the SC-stabilized nanodispersion showed excellent chemical stability. Under different environmental conditions, the nanodispersions exhibited a varied degree of stability. All nanodispersions showed constant particle sizes at temperatures between 30 and 60°C. However, at pH 2-8, only the SC-stabilized nanodispersion was physically unstable. The addition of NaCl (300-400mM) only caused flocculation in nanodispersion stabilized by SDS-Tween 80. All nanodispersions were physically stable when subjected to different centrifugation speeds. Only Tween 80-stabilized nanodispersion was stable against the effect of freeze-thaw cycles (no phase separation observed). In this study, there was no particular emulsifier that was effective against all of the environmental conditions tested.
Fulltext Non-invasive prenatal diagnosis using fetal DNA in maternal plasma: a preliminary study for identification of paternally-inherited alleles using single nucleotide polymorphisms

Chen JJ, Tan JA, Chua KH, Tan PC, George E

BMJ Open, 2015 Jul 22;5(7):e007648.
PMID: 26201722 DOI: 10.1136/bmjopen-2015-007648

OBJECTIVES: Single nucleotide polymorphism (SNP) with a mutation can be used to identify the presence of the paternally-inherited wild-type or mutant allele as result of the inheritance of either allele in the fetus and allows the prediction of the fetal genotype. This study aims to identify paternal SNPs located at the flanking regions upstream or downstream from the β-globin gene mutations at CD41/42 (HBB:c.127_130delCTTT), IVS1-5 (HBB:c.92+5G>C) and IVS2-654 (HBB:c.316-197C>T) using free-circulating fetal DNA.
SETTING: Haematology Lab, Department of Biomedical Science, University of Malaya.
PARTICIPANTS: Eight couples characterised as β-thalassaemia carriers where both partners posed the same β-globin gene mutations at CD41/42, IVS1-5 and IVS2-654, were recruited in this study.
OUTCOME MEASURES: Genotyping was performed by allele specific-PCR and the locations of SNPs were identified after sequencing alignment.
RESULTS: Genotype analysis revealed that at least one paternal SNP was present for each of the couples. Amplification on free-circulating DNA revealed that the paternal mutant allele of SNP was present in three fcDNA. Thus, the fetuses may be β-thalassaemia carriers or β-thalassaemia major. Paternal wild-type alleles of SNP were present in the remaining five fcDNA samples, thus indicating that the fetal genotypes would not be homozygous mutants.
CONCLUSIONS: This preliminary research demonstrates that paternal allele of SNP can be used as a non-invasive prenatal diagnosis approach for at-risk couples to determine the β-thalassaemia status of the fetus.
Immunological cross-reactivity and neutralization of the principal toxins of Naja sumatrana and related cobra venoms by a Thai polyvalent antivenom (Neuro Polyvalent Snake Antivenom)

Leong PK, Fung SY, Tan CH, Sim SM, Tan NH

Acta Trop, 2015 Sep;149:86-93.
PMID: 26026717 DOI: 10.1016/j.actatropica.2015.05.020

The low potency of cobra antivenom has been an area of concern in immunotherapy for cobra envenomation. This study sought to investigate factors limiting the neutralizing potency of cobra antivenom, using a murine model. We examined the immunological reactivity and neutralizing potency of a Thai polyvalent antivenom against the principal toxins of Naja sumatrana (Equatorial spitting cobra) venom and two related Asiatic cobra venom α-neurotoxins. The antivenom possesses moderate neutralizing potency against phospholipases A2 (P, potency of 0.98mg/mL) and moderately weak neutralizing potency against long-chain α-neurotoxins (0.26-0.42mg/mL) but was only weakly effective in neutralizing the short-chain α-neurotoxins and cardiotoxins (0.05-0.08mg/mL). The poor neutralizing potency of the antivenom on the low molecular mass short-chain neurotoxins and cardiotoxins is presumably the main limiting factor of the efficacy of the cobra antivenom. Our results also showed that phospholipase A2, which exhibited the highest ELISA reactivity and avidity, was most effectively neutralized, whereas N. sumatrana short-chain neurotoxin, which exhibited the lowest ELISA reactivity and avidity, was least effectively neutralized by the antivenom. These observations suggest that low immunoreactivity (low ELISA reactivity and avidity) is one of the reasons for poor neutralization of the cobra venom low molecular mass toxins. Nevertheless, the overall results show that there is a lack of congruence between the immunological reactivity of the toxins toward antivenom and the effectiveness of toxin neutralization by the antivenom, indicating that there are other factors that also contribute to the weak neutralization capacity of the antivenom. Several suggestions have been put forward to overcome the low efficacy of the cobra antivenom. The use of a 'proper-mix' formulation of cobra venoms as immunogen, whereby the immunogen mixture used for hyperimmunization contains a mix of various types of α-neurotoxins and cardiotoxins in sufficient amount, may also help to improve the efficacy and broaden the neutralization spectrum of the antivenom.
Fulltext Ganoderma neo-japonicum Imazeki revisited: Domestication study and antioxidant properties of its basidiocarps and mycelia

Tan WC, Kuppusamy UR, Phan CW, Tan YS, Raman J, Anuar AM, et al.

Sci Rep, 2015;5:12515.
PMID: 26213331 DOI: 10.1038/srep12515

Mushroom cultivation benefits humankind as it deliberately encourages wild mushrooms to be commercially propagated while recycling agricultural wastes. Ganoderma neo-japonicum is a rare polypore mushroom found growing on decaying Schizostachyum brachycladium (a tropical bamboo) clumps in Malaysia. The Malaysian indigenous tribes including the Temuans and Temiars use the basidiocarps of G. neo-japonicum to treat various ailments including diabetes. In this study, the domestication of G. neo-japonicum in artificial logs of different agricultural residues was investigated. Sawdust promoted the mycelia spawn colonisation in the shortest period of 38 ± 0.5 days. However, only sawdust and bamboo dust supported the primodia formation. Complex medium supported mycelium growth in submerged cultures and 27.11 ± 0.43 g/L of mycelia was obtained after 2 weeks of cultivation at 28 °C and 200 rpm. Antioxidant potential in mushroom may be influenced by different cultivation and extraction methods. The different extracts from the wild and cultivated basidiocarps as well as mycelia were then tested for their antioxidant properties. Aqueous and ethanol extracts of mycelia and basidiocarps tested had varying levels of antioxidant activities. To conclude, domestication of wild G. neo-japonicum using agroresidues may ensure a continuous supply of G. neo-japonicum for its medicinal use while ensuring the conservation of this rare species.
Fulltext The Association Between Bullous Pemphigoid and Neurological Disorders in A Selected Malaysian Population

Kwan Z, Lai YN, Ch'ng CC, Tan AH, Tan LL, Robinson S, et al.

Med J Malaysia, 2015 Apr;70(2):81-5.
PMID: 26162382 MyJurnal

BACKGROUND: An association of bullous pemphigoid with neurological disorders has been reported. The objectives of this study were to review the clinical characteristics of patients with bullous pemphigoid and compare the association between bullous pemphigoid and various neurological disorders and comorbidities.

METHODS: This was a retrospective case-control study involving 43 patients with bullous pemphigoid and 43 age-, sex- and ethnicity-matched controls.

RESULTS: There was a statistically significant association between bullous pemphigoid and neurological disorders [Odds Ratio (OR) = 3.5, 95% Confidence Interval (CI) 1.3 to 9.2, p=0.011 and adjusted OR=3.5, 95% CI 1.2-10.3, p=0.026], in particular for dementia (p=0.002). Although stroke was more common among patients with bullous pemphigoid, this association was not statistically significant with OR of 1.9 (95% CI 0.7 to 5.2) and adjusted OR of 2.1 (95% CI 0.6 to 7.2). Similarly both ischaemic stroke (OR 1.5, 95% CI 0.5 to 4.2) and haemorrhagic stroke (OR 1.5, 95% CI 0.2 to 9.7) were more common. Other neurological disorders more common among patients with bullous pemphigoid were Parkinson's disease and epilepsy. Dyslipidaemia was significantly less common among patients with bullous pemphigoid (OR 0.4, 95% CI 0.1 to 0.9, p=0.033).

CONCLUSION: A combination of an inflammatory process, prothrombotic state and endothelial activation leads to an increased frequency of neurological disorders among patients with bullous pemphigoid. Thus, a holistic approach to patient care, including screening for dementia and control of comorbidities, should be practised as bullous pemphigoid affects more than just the skin.
The Effect of a Polyvalent Antivenom on the Serum Venom Antigen Levels of Naja sputatrix (Javan Spitting Cobra) Venom in Experimentally Envenomed Rabbits

Yap MK, Tan NH, Sim SM, Fung SY, Tan CH

Basic Clin Pharmacol Toxicol, 2015 Oct;117(4):274-9.
PMID: 25819552 DOI: 10.1111/bcpt.12398

The treatment protocol of antivenom in snake envenomation remains largely empirical, partly due to the insufficient knowledge of the pharmacokinetics of snake venoms and the effects of antivenoms on the blood venom levels in victims. In this study, we investigated the effect of a polyvalent antivenom on the serum venom antigen levels of Naja sputatrix (Javan spitting cobra) venom in experimentally envenomed rabbits. Intravenous infusion of 4 ml of Neuro Polyvalent Snake Antivenom [NPAV, F(ab')2 ] at 1 hr after envenomation caused a sharp decline of the serum venom antigen levels, followed by transient resurgence an hour later. The venom antigen resurgence was unlikely to be due to the mismatch of pharmacokinetics between the F(ab')2 and venom antigens, as the terminal half-life and volume of distribution of the F(ab')2 in serum were comparable to that of venom antigens (p > 0.05). Infusion of an additional 2 ml of NPAV was able to prevent resurgence of the serum venom antigen level, resulting in a substantial decrease (67.1%) of the total amount of circulating venom antigens over time course of envenomation. Our results showed that the neutralization potency of NPAV determined by neutralization assay in mice may not be an adequate indicator of its capability to modulate venom kinetics in relation to its in vivo efficacy to neutralize venom toxicity. The findings also support the recommendation of giving high initial dose of NPAV in cobra envenomation, with repeated doses as clinically indicated in the presence of rebound antigenemia and symptom recurrence.
Disseminated nocardiosis with bilateral intraocular involvement in a renal allograft patient

Tan SY, Tan LH, Teo SM, Thiruventhiran T, Kamarulzaman A, Hoh HB

Transplant Proc, 2000 Nov;32(7):1965-6.
PMID: 11120022
Fulltext The use of the amplification refractory mutation system (arms) in the detection of rare beta-thalassemia mutations in the Malays and Chinese in Malaysia

Chan YF, Tan KL, Wong YC, Wee YC, Yap SF, Tan JAMA

Southeast Asian J. Trop. Med. Public Health, 2001 Dec;32(4):872-9.
PMID: 12041567

Molecular characterization and prenatal diagnosis for beta-thalassemia can be carried out using the Amplification Refractory Mutation System (ARMS). The ARMS is a rapid and direct molecular technique in which beta-thalassemia mutations are visualized immediately after DNA amplification by gel electrophoresis. In the University of Malaya Medical Center, molecular characterization and prenatal diagnosis for beta-thalassemia is carried out using ARMS for about 96% of the Chinese and 84.6% of the Malay patients. The remaining 4% and 15.4% of the uncharacterized mutations in the Chinese and Malay patients respectively are detected using DNA sequencing. DNA sequencing is an accurate technique but it is more time-consuming and expensive compared with the ARMS. The ARMS for the rare Chinese beta-mutations at position -29 (A-->G) and the ATG-->AGG base substitution at the initiator codon for translation in the beta-gene was developed. In the Malays, ARMS was optimized for the beta-mutations at codon 8/9 (+G), Cap (+1) (A-->C) and the AATAAA-->AATAGA base substitution in the polyadenylation region of the beta-gene. The ARMS protocols were developed by optimization of the parameters for DNA amplification to ensure sensitivity, specificity and reproducibility. ARMS primers (sequences and concentration), magnesium chloride concentration, Taq DNA polymerase and PCR cycling parameters were optimized for the specific amplification of each rare beta-thalassemia mutation. The newly-developed ARMS for the 5 rare beta-thalassemia mutations in the Chinese and Malays in Malaysia will allow for more rapid and cost-effective molecular characterization and prenatal diagnosis for beta-thalassemia in Malaysia.
Mild beta-thalassemia intermedia caused by compound heterozygosity for (G)gamma((A)gammadeltabeta)(o)/beta-thalassemia and molecular characterization of the defect in four Chinese families

Tan JAMA, Yap SF, Tan KL, Wong YC, Wee YC, Kok JL

Acta Haematol., 2003;109(4):169-75.
PMID: 12853688 DOI: 10.1159/000070965

Molecular characterization of the compound heterozygous condition - (G)gamma((A)gammadeltabeta)(o)/beta-thalassemia - in four families showing mild beta-thalassemia intermedia was carried out using DNA amplification techniques. Using the Amplification Refractory Mutation System (ARMS) to confirm the beta-mutations and DNA amplification to detect the 100-kb Chinese-specific (G)gamma((A)gammadeltabeta)(o)-deletion, ()two families were confirmed to possess (G)gamma((A)gammadeltabeta)(o)/beta-thalassemia with the IVSII No. 654 beta(+)-allele. In the third family, the (G)gamma((A)gammadeltabeta)(o)-deletion was confirmed in the father and the mother was a beta-thalassemia carrier with the cd 41-42 beta(o)-allele. Their affected child with (G)gamma((A)gammadeltabeta)(o)/beta-thalassemia was found to be transfusion dependent. The same (G)gamma((A)gammadeltabeta)(o)-deletion and beta-thalassemia (cd 41-42) was also confirmed in a fourth family. In addition, the mother was also diagnosed with Hb H disease (genotype -alpha(3.7)/-(SEA)). Both the children were found to possess (G)gamma((A)gammadeltabeta)(o)/beta-thalassemia but they were not transfusion dependent and this could be due to co-inheritance of alpha-thalassemia-2 (genotype-alpha(3.7)/alphaalpha) in the children together with their compound heterozygous condition.

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