Mary Anne Tan Jin Ai

Abstract: Background/Aims: Beta (β)- thalassaemia is a public health problem in Malaysia. The carrier rate is estimated to be 4.5% by micro-mapping studies particularly among Malays who comprise 53.5% of the population in Malaysia (1). The common diagnostic method in Malaysia for mutation detection is by amplification refractory mutation system (ARMS). It allows single mutation detection in each reaction but is labour intensive and time consuming when many mutations need to be identified. The purpose of this study was to develop a diagnostic tool for effective mutation detection of beta thalassaemia in Malay patients and compare its efficacy with ARMS-PCR, the current method in use. Methods: Reverse dot blot hybridization (RDBH) technique was incorporated in the development of two strip assays [RDBH-Strip M(6) and RDBH-Strip C(6)] to identify common beta thalassaemia mutations in the Malays. The panels of selected mutations were based on the mutation frequencies in Malaysia reported in previous studies. RDBH-Strip M(6) was applied as step 1 and RDBH-Strip C(6) was applied as step 2 for unidentified mutations. The strips were validated with the gold standard method, ARMS-PCR. Results: One hundred and thirty seven Malay patients with 274 alleles were studied. In Step 1 mutation detection, 238 alleles (86.9%) were identified in 119 of patients by RDBH-Strip M(6). Step 2 resulted in a further detection of 20 alleles in another 10 patients by RDBH-Strip C(6). The combination of both strips resulted in the identification of 258 alleles in 129 (94.6%) of 137 Malay patients. The strip assays were 100% sensitive and specific when compared with ARMS-PCR method. Conclusion: Two strip assays utilising the RDBH technique were developed to identify common β-thalassaemia mutations in Malays. The RDBH Strip M(6) identified 86.9% of the mutations and the RDBJ-Strip C (6) detected further 7.3% alleles. This two step strategy was found to be rapid and cost effective for the direct diagnosis of β-thalassaemia mutations in the Malays. The remaining unidentified mutations would require DNA sequencing. It can serve as a specific molecular diagnostic tool for effective diagnosis of β-thalassaemia mutations in this ethnic group.

Abstract: Hemoglobin disorders which include thalassemias are the most common heritable disorders. Effective treatment is available, and these disorders can be avoided as identification of carriers is achievable using simple hematological tests. An in-depth understanding of the awareness, attitudes, perceptions, and screening reservations towards thalassemia is necessary, as Malaysia has a multi-ethnic population with different religious beliefs. A total of 13 focus group discussions (70 participants) with members of the general lay public were conducted between November 2008 and January 2009. Lack of knowledge and understanding about thalassemia leads to general confusions over differences between thalassemia carriers and thalassemia major, inheritance patterns, and the physical and psychologically impact of the disorder in affected individuals and their families. Although most of the participants have not been tested for thalassemia, a large majority expressed willingness to be screened. Views on prenatal diagnosis and termination of fetuses with thalassemia major received mixed opinions from participants with different religions and practices. Perceived stigma and discrimination attached to being a carrier emerged as a vital topic in some group discussions where disparity in the answers exhibited differences in levels of participants’ literacy and ethnic origins. The two most common needs identified from the discussion were information and screening facilities. Participants’ interest in knowing the severity of the disease and assessing their risk of getting the disorder may imply the health belief model as a possible means of predicting thalassemia public screening services. Findings provide valuable insights for the development of more effective educational, screening, and prenatal diagnostic services in the multi-ethnic Asian society.

Abstract: Detection and quantification of Hb subtypes of human blood is integral to presumptive identification of thalassaemias. It has been used in neonatal screening of thalassaemia and Hb variants. The use of discarded red blood cells following processing of the cord blood for stem cells provides readily available diagnostic material for thalassaemia screening. In this study, we determined the range of Hb subtypes in 195 consecutive cord blood samples collected for cord blood banking. The ‘cord blood samples’ analysed were those of the remaining red blood cells after the cord blood was processed for stem cell storage. Quantification of Hb subtypes by high performance liquid chromatography (HPLC) was done on BioRad Variant II Hb testing system. Only 73 (36.5%) of the samples could be analyzed neat without dilution. With a 1:300 dilution with wash solution the acceptable area as recommended by the manufacturer for reading of a C-gram within the 1 to 3 million ranges were achieved in all. Eighteen (9%) 12 showed classical Hb Barts (γ4) prerun peaks were confirmed by Sebia Hydrasys automated Hb gel electrophoresis and quantified by Sebia Capillarys 2 capillary electrophoresis. Only 1 (0.5%) was presumptively identified with HbH disease. Due to the limited number of samples no beta-thalassaemia major, Hb E beta-thalassaemia and Hb Barts hydrops fetalis were found. The HPLC assay was possible at a cost US$ 5 per sample and a turnover time of 10 samples per hour without technical difficulties. This study reports an effective and valuable protocol for thalassaemia screening in red blood cells which would otherwise be discarded during cord blood processing. Cord blood with severe and intermediate forms of thalassaemia can be preselected and not stored.

Abstract: Beta-thalassaemia major causes severe anaemia and patients with it may be transfusion-dependent for life. Regular blood transfusions cause iron-overload that leads to oxidative damage which can hasten mortality. The objective of this research was to study the oxidant-antioxidant indices in beta-thalassaemia major patients at the University of Malaya Medical Centre (UMMC) who were on desferrioxamine-chelation or without chelation therapy Blood was collected from 39 Chinese patients and 20 controls. Plasma and peripheral blood mononuclear cell lysates (PBMC) were extracted and biochemical tests to evaluate oxidative stress were performed. Oxidative stress was evident in these patients as advanced oxidized protein products (AOPP) and lipid hydroperoxides were elevated, whereas glutathione peroxidase activity and the ferric reducing antioxidant power (FRAP) were reduced. The catalase activity in the patients ’ PBMC was elevated, possibly as a compensatory mechanism for the reduced glutathione peroxidase activity in both red blood cells and PBMC. The lower FRAP and higher AOPP levels in the non-chelated patients compared with the chelated patients were indicative of a lower oxidative stress level in the chelated patients. The ferritin levels in the chelated and non-chelated patients were high and the mean levels of liver enzyme activities in the majority of patients were elevated regardless of chelation therapy In conclusion, this study indicates that desferrioxamine chelation therapy does not normalize ferritin level but attenuates oxidative damage and improves total antioxidant level in Malaysian Chinese beta-thalassaemia major patients.

Abstract: Background: Thalassaemia is a common public health problem in Malaysia and about 4.5 to 6% of the Malays and Chinese are carriers of this genetic disorder. The major forms of thalassaemia result in death in utero of affected foetuses (alpha-thalassaemia) or life-long blood transfusions for survival in beta-thalassaemia. This study, the first nationwide population based survey of thalassaemia in Malaysia, aimed to determine differences in public awareness, perceptions and attitudes toward thalassaemia in the multi-racial population in Malaysia. Methods: A cross-sectional computer-assisted telephone interview survey of a representative sample of multi-racial Malaysians aged 18 years and above was conducted between July and December 2009. Results: Of a total of 3723 responding households, 2846 (76.4%) have heard of thalassaemia. Mean knowledge score was 11.85 (SD +/- 4.03), out of a maximum of 21, with higher scores indicating better knowledge. Statistically significant differences (P < 0.05) in total knowledge score by age groups, education attainment, employment status, and average household income were observed. Although the majority expressed very positive attitudes toward screening for thalassaemia, only 13.6% of married participants interviewed have been screened for thalassaemia. The majority (63.4%) were unsupportive of selective termination of foetuses diagnosed with thalassaemia major. Conclusion: Study shows that carrier and premarital screening programs for thalassaemia may be more effective and culturally acceptable in the reduction of pregnancies with thalassaemia major. The findings provide insights into culturally congruent educational interventions to reach out diverse socio-demographic and ethnic communities to increase knowledge and cultivate positive attitudes toward prevention of thalassaemia.

Abstract: The haemoglobinopathies and thalassemias represent the most common inherited monogenic disorders in the world. Beta-thalassaemia major is an ongoing public health problem in Malaysia. Prior to 2004, the country had no national policy for screening and registry for thalassemia. In the absence of a national audit, the true figure of the extent of thalassemia in the Malaysian population was largely presumptive from micro-mapping studies from various research workers in the country. The estimated carrier rate for beta-thalassemia in Malaysia is 3.5-4%. There were 4768 transfusion dependent thalassemia major patients as of May 2010 (Data from National Thalassemia Registry).

Abstract: Dystrophinopathy is the commonest form of muscular dystrophy and comprises clinically recognized forms, Duchenne dystrophy and Becker dystrophy. Mutations in the dystrophin gene which consist of large gene deletions (65%), duplications (5%) and point mutations (30%) are responsible for reducing the amount of functional dystrophin protein in skeletal muscle fibres leading to fibre destruction and disease. The aims of this study are to investigate the detection rate, types and distribution of large gene deletions in Malaysian dystrophinopathy patients using the multiplex polymerase chain reaction (MPCR). MPCR of 18 "hot-spot deletion" regions along the dystrophin gene was performed on DNA from 48 muscle biopsy-confirmed cases of dystrophinopathy. A positive detection rate of 58% (28/48) was observed, where 84% (16/19) Indian, 35% (6/17) Chinese and 50% (6/12) Malay ethnic groups showed deletions in their dystrophin genes. The Malaysian Indians appear to have a higher prevalence for large gene deletions compared to the Chinese and Malays. Further analyses of 42 confirmed positive cases (present 28 plus previous 14 cases) by MPCR showed the majority of deletions were in the mid-distal region of the dystrophin gene (81% in exons 45-60). The MPCR is a specific and sensitive method for confirmation of gene deletions responsible for dystrophinopathy.

Abstract: Thalassemia can lead to severe transfusion-dependent anemia, and it is the most common genetic disorder in Malaysia. This paper aims to determine the prevalence of thalassemia in the Kadazandusuns, the largest indigenous group in Sabah, East Malaysia. alpha-and beta-thalassemia were confirmed in 33.6% and 12.8%, of the individuals studied respectively. The high prevalence of alpha- and beta-thalassemia in the Kadazandusuns indicates that thalassemia screening, genetic counseling, and prenatal diagnosis should be included as part of their healthcare system. This preliminary paper serves as a baseline for further investigations into the health and genetic defects of the major indigenous population in Sabah, East Malaysia.

Abstract: Interactions of different hemoglobin variants with thalassemia alleles can result in various clinical phenotypes. HbE-beta-thalassemia generally manifests with severe anemia where individuals exhibit beta-thalassemia major with regular blood transfusions or beta-thalassemia intermedia with periodic blood transfusions. This study presents a unique Malay family with three beta-globin gene defects-HbE, Hb South Florida, and IVS1-1 (G -> A). HbE activates a cryptic splice site that produces non-functional mRNAs. Hb South Florida is a rare beta-hemoglobin variant, and its interactions with other beta-thalassemia alleles have not been reported. IVS1-1 is a Mediterranean mutation that affects mRNA processing giving rise to beta(o)-thalassemia. Fifteen mutations along the beta-globin gene complex were analyzed using the amplification refractory mutation system. Hb South Florida was identified by direct sequencing using genomic DNA The affected child with HbE/IVS1-1 produced a beta-thalassemia major phenotype. Compound heterozygosity for Hb South Florida/IVS1-1 produced a beta-thalassemia carrier phenotype in the mother.

Abstract: Co-inheritance of alpha-thalassemia with homozygosity or compound heterozygosity for beta-thalassemia may ameliorate beta-thalassemia major. A wide range of clinical phenotypes is produced depending on the number of beta-thalassemia alleles (-alpha(alpha alpha–/alpha alpha, –/-alpha). The co-inheritance of beta-thalassemia with a-thalassemia with a single gene deletion (-alpha/alpha) is usually associated with thalassemia major. In contrast, the co-inheritance of beta-thalassemia with two alpha-genes deleted in cis or trans (–/alpha alpha or -alpha/-alpha) generally produces beta-thalassemia intermedia. In Southeast Asia, the most common defect responsible for alpha-thalassemia is the Southeast Asian (SEA) deletion of 20.5 kilobases. The presence of the SEA deletion with Hb Constant Spring (HbCS) produces HbH-CS disease. Co-inheritance of HbH-CS with compound heterozygosity for beta-thalassemia is very rare. This study presents a Malay patient with HbH-CS disorder and beta degrees/beta(+)-thalassemia. The SEA deletion was confirmed in the patient using a duplex-PCR. A Combine-Amplification Refractory Mutation System (C-ARMS) technique to simultaneously detect HbCS and Hb Quong Sze confirmed HbCS in the patient. Compound heterozygosity for CD41/42 and Poly A was confirmed using the ARMS. This is a unique case as the SEA a-gene deletion in cis (–(SEA)/alpha) is generally not present in the Malays, who more commonly posses the two a-gene deletion in trans In addition, the beta-globin gene mutation at CD41/42 is a common mutation in the Chinese and not in the Malays. The presence of both the SEA deletion and CD41/42 in the mother of the patient suggests the possible introduction of these two defects into the family by marriage with a Chinese.

Abstract: A rare case of thalassaemia-intermedia involving a non-deletion alpha thalassemia point mutation in the alpha1-globin gene CD59 (GGC --> GAC) and a deletion alpha+ (-alpha(3.7)) thalassaemia in which use of high performance liquid chromatography (HPLC) C-gram Hb subtype profile and DNA molecular analysis helped establish the diagnosis.

Abstract: Introduction: HbE is the commonest beta haemoglobin (Hb) variant in Southeast Asia. It causes a reduction in synthesis of the beta-globin E chain. Studies indicate HbE coinherited with a-thalassaemia leads to milder clinical phenotype. This study investigates the commitant inheritance of a-thalassaemia in Malays with HbE. Methods: Four hundred and fourteen (414) blood samples were screened for haemoglobinopathy using primarily the first three steps of the BHES [ (B) blood counts, blood film; (H), HPLC; (E) electrophoresis; (S), stability ] protocol. Complete blood cell analyser, Hb typing with cation exchange high-performance liquid chromatography (HPLC) and Hb electrophoresis at an alkaline pH (pH 8.5) Forty-five (10.9%) were identified as HbE trait and DNA analysis was done for deletional a-thalassaemia using a single-tube multiplex-PCR assay. Results: Among the 45 subjects with HbE trait, 4 (8.9%) were found to have alpha-thalassaemia -2 (a) (a-37 kb deletion) and 1 (2.2%) the alpha-thalassaemia-1 (a0) (–-SEA 20.5 kb deletion) defects respectively. Discussion: These findings show that 11.1% of Malays with HbE inherit alpha-thalassaemia concurrently. The most prevalent interaction found was a double heterozygote for HbE /a-thalassaemia 2, followed by HbE/a-thalassaemia 1. Conclusion: Molecular screening of deletional a-thalassaemia identified its concurrent inheritance in 11.1%o of Malays who were HbE carriers. This information will guide genetic counseling and the planning of treatment modalities in patients with HbE alpha-thalassaemia.

Abstract: Background/Aims: Individuals with double heterozygosity for alpha- and beta-thalassaemia and heterozygous beta-thalassaemia show a similar haematological picture. Co-inheritance of (alpha- and beta-thalassaemia in both partners may result in pregnancies with either Hb Bart’s hydrops foetalis or beta-thalassaemia major, or pregnancies with both disorders. Methods: The co-inheritance of a-thalassaemia in 322 P-thalassaemia carriers in Malaysia was studied. Results: The frequency of (alpha-thalassaemia in the beta-thalassaemia carriers was 12.7% (41/322), with a carrier frequency of 7.8% for the SEA deletion, 3.7% for the -alpha(3.7) deletion, 0.9% for Hb Constant Spring and 0.3% for the -alpha(4.2) deletion. Conclusion: Double heterozygosity for (alpha- and beta-thalassaemia was confirmed in 5 out of the 41 couples and the risk of the fatal condition Hb Bart’s hydrops foetalis was confirmed in two of these couples. Detection of the Southeast Asian (SEA) deletion in the Malaysian Malays in this study confirms that Hb Bart’s hydrops foetalis can occur in this ethnic group. Results of this study have provided new information on the frequency and different types of alpha-thalassaemia (–(SEA), -alpha(3.7) and -alpha(4-1) deletions, Hb Constant Spring) in Malaysian beta-thalassaemia carriers.Copyright (C) 2008 S. Karger AG, Basel.

Abstract: Thalassaemia is an inherited blood disorder and is a significant public health problem in Malaysia, with many not knowing they carry gene for thalassaemia. The two major forms are alpha and beta thalassaemia. An individual can co-inherit both the alpha and beta thalassaemia genes. This study determined the frequency of cincurrent carriers of alpha thalassaemia in 231 beta thalassaemia carriers. Gap-PCR was done on extracted DNA of the beta thalassaemia samples to check for alpha thalassaemia 1 molecular defect. Eight (3.5%) samples were found to have concurrently inherited the alpha thalassaemia 1 deletion. The significant carrier rare for alpha thalassaemia 1 indicates the need for the implementation of DNA analysis to complement thalassaemia screening in high risk populations.

Abstract: Aims: In Malaysia, about 4.5% of the Malay and Chinese populations are heterozygous carriers of beta-thalassaemia. The initial identification of rare beta-globin gene mutations by genomic sequencing will allow the development of simpler and cost-effective PCR-based techniques to complement the existing amplification refractory mutation system (ARMS) and gap-PCR used for the identification of beta-thalassaemia mutations. Methods: DNA from 173 beta-thalassaemia carriers and five beta-thalassaemia major patients from the Malay, Chinese and Indian ethnic groups were first analysed by ARMS and gap-PCR. Ninety-five per cent (174/183) of the 183 beta-globin genes studied were characterised using these two techiques. The remaining nine uncharacterised beta-globin genes (4.9%) were analysed using genomic sequencing of a 904 bp amplified PCR product consisting of the promoter region, exon 1, intervening sequence (IVS) 1, exon 2 and the 5’ IVS2 regions of the beta-globin gene. Results: The rare beta-globin mutations detected in the Chinese patients were CD27/28 (+C) and CD43 (GAG-TAG), and 288 (C-T) in an Indian patient. Beta-globin mutations at CD16 (-C), IVS1-1 (G-A), IVS2-1 (G-A), 286 (C-G) and Haemoglobin South Florida (CD1, GTG-ATG) were confirmed in the Malay patients. Conclusions: The seven rare beta-globin mutations and a rare haemoglobin variant confirmed in this study have been described in other populations but have not been previously described in Malaysian beta-thalassemia patients.

Abstract: Aim: Interactions between different determinants of alpha-thalassemia raises considerable problems, particularly during pregnancies where antenatal diagnosis is necessary. This study aims to determine the different types of deletional alpha-thalassemia and Hemoglobin Constant Spring (HbCS), and their frequency in Malays, Chinese and Indians in Malaysia. Methods: DNA from 650 pregnant women from the Antenatal Clinic of the University of Malaya Medical Center in Kuala Lumpur, Malaysia who showed mean cell volume <= 89 fL and/or mean cell hemoglobin <= 28 pg were analyzed for the double alpha-globin gene South-East Asian deletion (–(SEA)), the -alpha(3.7) and -alpha(4.2) single alpha-globin gene deletions and HbCS. Results: One hundred and three (15.8%) of the pregnant women were confirmed as alpha-thalassemia carriers: 25 (3.8%) were alpha-thalassemia-1 carriers with the –(SEA)/alpha alpha genotype, 64 (9.8%) were heterozygous for the -alpha(3.7) rightward deletion (-alpha(3.7)/alpha alpha), four (0.6%) were heterozygous for the -alpha(4.2) leftward deletion (-alpha(4.2)/alpha alpha), nine (1.4%) were heterozygous for HbCS (alpha(CS)alpha/alpha alpha) and one (0.2%) was compound heterozygous with the -alpha(3.7)/alpha(CS)alpha genotype. The double alpha-globin gene –(SEA) deletion was significantly higher in the Chinese (15%) compared to the Malays (2.5%) and not detected in the Indians studied. The -alpha(3.7) deletion was distributed equally in the three races. HbCS and -alpha(4.2) was observed only in the Malays. Conclusion: The data obtained gives a better understanding of the interactions of the different alpha-thalassemia determinants in the different ethnic groups, thus enabling more rapid and specific confirmation of alpha-thalassemia in affected pregnancies where antenatal diagnosis is necessary.

Abstract: beta-thalassaemia major, an autosomal recessive hemoglobinopathy, is one of the most common single gene disorders in multi-racial Malaysia. The control of beta-thalassaemia major requires a multi-disciplinary approach that includes population screening, genetic counselling, prenatal diagnosis and the option of termination of affected pregnancies. To achieve this objective, the molecular characterisation of the spectrum of beta-globin gene mutations in each of the affected ethnic groups is required. We studied 88 consecutive unrelated individuals and their respective families with beta-thalassaemia (74 beta-thalassaemia major, 12 HbE-beta-thalassaemia, 2 with HbE homozygotes) and four individuals with beta-thalassaemia trait that contributed a total 180 alleles for study. Using a 2-step molecular diagnostic strategy consisting of amplification refractory mutation system (ARMS) to identify the 8 most common mutations followed by other DNA-based diagnostic techniques, a total of 177 (98.3 per cent) of the 180 beta-thalassaemia alleles were characterised. One out of 91 (1 per cent) of the Chinese alleles, one out of 46 (2.2 per cent) Malay alleles and one out of two Indian alleles remained unknown. A 100 per cent success rate was achieved in studying the Kadazandusun community in this study. A strategy to identify beta-globin gene mutations in Malaysians with beta-thalassaemia is proposed based on this outcome.

Abstract: Tumour suppressor gene p53 is a common target in carcinogenesis, reported to be altered and functionally inactive in 70% of human cancers. Although p53 mutations are less commonly present in haematological malignancies when compared with other solid tumours, they have been reported in histological transformation of follicular lymphoma. We aimed to investigate the frequency of p53 gene alterations in paraffin-embedded tissue using commercially available PCR-SSCP, and to correlate the results with P53 protein expression by immunohistochemistry.

Abstract: p53 gene mutation is not a frequent event in the tumorigenesis of lymphomas and the expression of p53 protein is independent of p53 gene mutations. The present study aimed to investigate mutations in the p53 gene in a series of extranodal B-cell lymphomas, and its association with p53 protein expression. A total of 52 cases were graded histologically into Grade 1, Grade 2 and Grade 3 tumors and p53 protein expression was detected using immunohistochemistry. Mutations in the p53 gene were analyzed using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and mobility shifts were confirmed by direct sequencing. The tumors comprised 26 (50%) Grade 1, 9 (17%) Grade 2 and 15 (29%) Grade 3. A high proportion of Grade 2 (25%) tumors expressed p53 protein (P = 0.051) and carried p53 gene mutation (33%) (P = 0.218). However, p53 protein expression was not associated with p53 gene mutations (P = 0.057). Transversion mutations (88%) were more frequently detected than transition mutations (12%). The present study revealed that p53 gene mutations and p53 protein expression occurred in higher frequencies in Grade 2 tumors, which may be of pathogenetic importance. The high frequency of transversion mutations may reflect the influence of an etiological agent in the tumorigenesis of mucosa-associated lymphoid tissue (MALT lymphoma).

Abstract: Aims: Follicular lymphoma is frequently associated with t(14;18)(q32;q21) translocation. This study was undertaken to determine the pattern of Bcl-2, CD10 and Bcl-6 expression in relation to t(14;18) translocation in follicular lymphoma from a cohort of a multi-ethnic Asian population. Methods and results: Sixty-two cases of follicular lymphoma were retrieved for immunohistochemistry, and t(14;18) translocation analysis by polymerase chain reaction and fluorescent in-situ hybridization techniques. Bcl-2 expression was present in 74% of the cases. CD10 expression was also relatively low (61%), with decreasing frequency of expression in high-grade tumours. Bcl-6 protein was expressed in most of the tumours (88%) regardless of the tumour grade. The t(14;18) translocation was detected in 46 cases (74%) with an extremely high rate of t(14;18) translocation in ethnic Indian cases (100%). Conclusion: The frequency of t(14;18) translocation in this series of follicular lymphomas was higher when compared with previous Asian reports, but in accordance with European and North American findings. CD10 expression is strongly associated with a t(14;18) translocation event, but the overall CD10 expression was relatively low, possibly due to the high proportion of high-grade tumours in the series. t(14;18) translocation was not associated with Bcl-2 or Bcl-6 expression.

Abstract: beta- thalassemia is the most-common genetic disorder of hemoglobin synthesis in Malaysia, and about 4.5% of the population are heterozygous carriers of the disorder. Prenatal diagnosis was performed for 96 couples using the Amplification Refractory Mutation System and Gap-Polymerase Chain Reaction. We identified 17 beta-globin defects-initiation codon for translation (T-G), - 29 (A-G), 28 (A-G), CAP + 1 (A-C), CD 8/9 (+ G), CD 15 (G-A), CD 17 (A-T), CD 19 (A-G), Hb E (G-A), IVS1-1 (G-T), IVS1-5 (G-C), CD 41/42 (- CTTT), CD 71 - 72 (+ A), IVS2-654 (C-T), poly A(A-G), 100-kb (G)gamma((A)gammadeltabeta)degrees and 45-kb Filipino deletions. The 192 beta-alleles studied comprised Chinese (151 patients), Malay (21), Orang Asli from East Malaysia (15), Filipino ( 1), Indian ( 1), Indonesian Chinese ( 2), and Thai (1). In the Chinese, 2 beta-globin defects at CD 41/42 and IVS2-654 were responsible for 74% of beta-thalassemia. beta-mutations at CD 19, IVS1-1 (G- T), IVS1-5, poly A, and hemoglobin E caused 76% of the hemoglobin disorders in the Malays. The Filipino 45-kb deletion caused 73.3% of b-thalassemia in the Orang Asli. Using genomic sequencing, the rare Chinese beta-mutation at CD 43 (G-T) was confirmed in 2 Chinese, and the Mediterranean mutation IVS1-1 ( G- A) was observed in a Malay beta-thalassemia carrier. The beta-globin mutations confirmed in this prenatal diagnosis study were heterogenous and 65 (68%) couples showed a different globin defect from each other. The use of specific molecular protocols has allowed rapid and successful prenatal diagnosis of beta-thalassemia in Malaysia.

Abstract: t(11; 18)(q21; q21) Translocation and trisomy 3 are the most common chromosomal aberrations reported in low-grade mucosa-associated lymphoid tissue ( MALT) lymphoma. The current study aims to investigate the frequency of these chromosomal aberrations in a series of 52 extranodal B-cell lymphomas. The tumours were categorised into three histological grades: grade 1 (low-grade lymphoma of MALT type), grade 2 [ diffuse large B-cell lymphoma (DLBCL) with MALT component] and grade 3 ( DLBCL without MALT component). Fluorescence in situ hybridisation analyses on paraffin tissue sections were performed using a locus-specific probe for the 18q21 region and a centromeric probe for chromosome 3. The 18q21 rearrangement was detected in 9 of 40 (23%) cases, including 7 of 23 (30%) grade-1 and 2 of 11 (18%) grade-3 tumours. Amplification of the 18q21 region was detected in 10 of 40 (25%) cases, and trisomy 3 was detected in 9 of 34 (26%) cases. Amplification of the 18q21 region may be an important alternative pathogenetic pathway in MALT lymphoma and was found almost exclusively in tumours without 18q21 rearrangement. Our study showed that tumours with 18q21 rearrangement and 18q21 amplification develop along two distinct pathways, and the latter was more likely to transform into high-grade tumours upon acquisition of additional genetic alterations, such as trisomy 3. Trisomy 3 was more frequently found in coexistence with 18q21 abnormalities, suggesting that it was more likely to be a secondary aberration.

Abstract: The analysis of circulating nucleic acids has revealed applications in the noninvasive diagnosis, monitoring, and prognostication of many clinical conditions. Circulating fetal-specific sequences have been detected and constitute a fraction of the total DNA in maternal plasma. The diagnostic reliability of circulating DNA analysis depends on the fractional concentration of the targeted sequence, the analytical sensitivity, and the specificity. The robust discrimination of single-nucleotide differences between circulating DNA species is technically challenging and demands the adoption of highly sensitive and specific analytical systems. We have developed a method based on single-allele base extension reaction and MS, which allows for the reliable detection of fetal-specific alleles, including point mutations and single-nucleotide polymorphisms, in maternal plasma. The approach was applied to exclude the fetal inheritance of the four most common Southeast Asian beta-thalassemia mutations in at-risk pregnancies between weeks 7 and 21 of gestation. Fetal genotypes were correctly predicted in all cases studied. Fetal haplotype analysis based on a single-nucleotide polymorphism linked to the beta-globin locus, HBB, in maternal plasma also was achieved. Consequently, noninvasive prenatal diagnosis in a mother and father carrying identical beta-thalassemia mutations was accomplished. These advances will help in catalyzing the clinical applications of fetal nucleic acids in maternal plasma. This analytical approach also will have implications for many other applications of circulating nucleic acids in areas such as oncology and transplantation.

Abstract: Background: Apolipoprotein E (apoE) is encoded by a polymorphic gene located on chromosome 19. The three common apoE alleles are epsilon2, epsilon3 and epsilon4. We studied the frequencies of the apoE alleles and genotypes in the three ethnic groups-Malay, Chinese and Indian-in Malaysia using DNA amplification followed by agarose gel electrophoresis. Methods: EDTA blood was collected and DNA was extracted using proteinase K-SDS digestion and purified by phenol-chloroform extraction. The apoE gene sequence was amplified using the PCR and apoE genotyping was performed by restriction enzyme digestion with HhaI. Results: Genotyping of the apoE gene produces six genotypes-E2/E2, E2/E3, E3/E3, E2/E4, E3/E4 and E4/E4. The most common apoE genotype in the Malays, Chinese and Indians studied was ENE3, thus the most common apoE allele was epsilon3. The three common apoE genotypes were E3/E3 followed by E3/E4 and E2/E3, except in the Indians where E2/E3 was not detected. The three apoE alleles were confirmed in the Malays, Chinese and Indians except for the e2 allele which was absent in the Indians. Conclusion: The combined frequency of the apoE alleles in the Malays, Chinese and Indians was 0.058, 0.829 and 0.114 for epsilon2, epsilon3 and epsilon4, respectively. (C) 2003 Elsevier B.V. All rights reserved.

Abstract: 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.

Abstract: Aims: PCR has been the primary method used for the detection of t(14;18) translocation in formalin-fixed, paraffin-embedded tissues. This technique mainly targets the well-characterised breakpoint regions in chromosomes 14 and 18. FISH is now applicable on paraffin tissue sections and has been suggested to be capable of detecting essentially 100% of t(14;18) translocated cases. In this study, we described the application of both PCR and FISH for the detection of t(14;18) translocation. Methods: Fifty follicular lymphoma cases were retrieved from the files of the Department of Pathology, University of Malaya Medical Centre (UMMC). Nested PCR amplification of MBR/JH and mcr/JH was performed in these cases, and those cases that did not demonstrate the translocation were subjected to FISH analysis. Results: Thirty cases (60%) had t(14;18) translocation detected by PCR, 25 (50%) had breakpoint with MBR and five (10%) involved mcr. Twenty cases without detectable t(14;18) translocation by PCR were analysed by FISH. Eleven cases were successfully probed, and four of them showed positive translocation signal. Conclusions: The combination of PCR and FISH analysis on paraffin tissue sections for the detection of t(14;18) translocation increases the sensitivity of detection from 60 to 68%. Problems encountered in our FISH analysis on tissue sections impose certain limitations in using this technique for retrospective screening of large number of samples. Therefore, we suggested the application of PCR as the first screening tool on retrospective archival materials, followed by FISH on those PCR-negative cases.

Abstract: Thirty patients with early onset breast cancer or familial breast cancer from Malaysia were analysed for germline mutation in the early onset breast cancer I gene (BRCA1). Direct sequencing of the entire coding region of BRCA1 identified a frameshift mutation, c.5447-5448insC (insC5447) (codon 1776 of exon 21) in a patient aged 32 of the Malay ethnic origin, who had no family history of breast and/or ovarian cancer. Eight polymorphisms (2201C > T, 2430T > C, P871L, E1038G, K1183R, 4427T > C, S1613G and IVS8-57delT) were identified in the samples tested.

Abstract: beta -Thalassemia major patients have chronic anemia and are dependent on blood transfusions to sustain life. Molecular characterization and prenatal diagnosis of beta -thalassemia is essential in Malaysia because about 4.5% of the population are heterozygous carriers for beta -thalassemia. The high percentage of compound heterozygosity (47.62%) found in beta -thalassemia major patients in the Thalassaemia Registry, University of Malaya Medical Centre (UMMC), Malaysia, also supports a need for rapid, economical, and sensitive protocols for the detection of beta -thalassemia mutations. Molecular characterization of beta -thalassemia mutations in Malaysia is currently carried out using ARMS, which detects a single beta -thalassemia mutation per PCR reaction. We developed and evaluated Combine amplification refractory mutation system (C-ARMS) techniques for efficient molecular detection of two to three beta -thalassemia mutations in a single PCR reaction. Three C-ARMS protocols were evaluated and established for molecular characterization of common beta -thalassemia mutations in the Malay and Chinese ethnic groups in Malaysia. Two C-ARMS protocols (cd 41-42/IVSII #654 and -29/cd 71-72) detected the beta -thalassemia mutations in 74.98% of the Chinese patients studied. The C-ARMS for ed 41-42/IVSII #654 detected beta -thalassemia mutations in 72% of the Chinese families. C-ARMS for ed 41-42/IVSI #5/cd 17 allowed detection of beta -thalassemia mutations in 36.53% of beta -thalassemia in the Malay patients. C-ARMS for ed 41-42/IVSI #5/cd 17 detected beta -thalassemia in 45.54% of the Chinese patients. We conclude that C-ARMS with the ability to detect two to three mutations in a single reaction provides more rapid and cost-effective protocols for beta -thalassemia prenatal diagnosis and molecular analysis programs in Malaysia.

Abstract: We report a Chinese kindred with an atypical sex-linked form of isolated adrenal hypoplasia without hypogonadotropic hypogonadism, Evidence of sex linkage was supported by DNA analysis using three polymorphic markers from the X-chromosome: a restriction fragment length polymorphism 200 kb centromeric of the DAX-1 gene, a tetranucleotide repeat marker in the DAX-1 promoter (DAX-P), and a microsatellite in the Duchenne muscular dystrophy locus (3’-19), This pedigree therefore presents the novel phenotype of sex-linked hypoadrenalism without hypogonadotropic hypogonadism, with evidence of possible linkage to the DAX-1 gene. However, all three affected individuals were examined for mutations in the DAX-1 gene, and found to have no sequence anomalies in the coding region, splice sites or 5’ non-coding region, This presentation may be due to a defect in the DAX-1 gene outside its known coding region, possibly modulated by functional polymorphisms at other loci, and/or environmental effects, or to a defect in a novel gene on the X chromosome which selectively influences adrenal development.

Abstract: Advances in molecular diagnostic techniques have resulted in the characterisation of over 200 mutations causing beta-thalassaemia. In addition, each population appears to have its own unique set and frequency of beta-globin gene mutations. The strategy for prenatal diagnosis therefore involves screening at-risk couples for the most common mutations known to be present in the ethnic group or population. As a result of this selective approach, uncommon or novel mutations are not detected. We undertook to characterise beta-globin gene mutations which remain undetected following polymerase chain reaction (PCR) and amplification refractory mutation system (ARMS) analyses. In this study, we successfully identified 13 of’the 16 ’unknown’ alleles using chemical cleavage of mismatch (CCM) method and direct sequencing. As a result of this coupled strategy (ARMS and CCM), 98.5% of the beta-globin gene mutations in the Thalassaemia Registry, University Hospital Kuala Lumpur were characterised.

Abstract: Engraftment following allogenic BMT in four beta-thalassaemia major patients was evaluated using two DNA amplification protocols - DNA amplification with restriction enzyme (RE) analysis and the Amplification Refractory Mutation System (ARMS). Seven common mutations - -28, Codon 17, Codon 26 (Hb E), IVSI #1, IVSI #5, Codon 41-42 and IVSII #654 - responsible for beta-thalassaemias in the Chinese and Malays in Malaysia were studied. In each case, the recipient was a beta-major patient and the donor was a normal or beta-carrier sibling. Using ARMS and DNA amplification with RE analysis, one patient (Hb E/IVSI #5) was found to have only normal beta-sequences after BMT. Using ARMS alone, normal beta-sequences were also detected in two patients (previously with beta-mutations at IVSII #654/Codon 41-42 and IVSII #654/Codon 17) while the fourth patient (Codon 41-42/-28) was found to be a beta-carrier (beta-mutation at Codon 41-42). Confirmation of engraftment was carried out by characterization of patient’s haemoglobin and measurement of their alpha/beta chain synthetic ratios after BMT. We conclude that DNA amplification with RE analysis and ARMS provide a direct and rapid assessment of engraftment after BMT in beta-thalassaemia.

Abstract: Restriction fragment length polymorphism (RFLP) of the gene encoding the beta chain of the human T cell receptor (TcR) was studied in three ethnic groups in Singapore by Southern blotting. Polymorphism in the beta chain gene was identified in BglII-digested DNA samples using a 770-bp TcR beta cDNA clone containing the joining and constant region segments. The TcR beta/BglII polymorphism was studied in 136 Chinese, 93 Indian and 88 Malay samples. The frequency of the less frequent allele (TcR beta*2) in all the ethnic groups was significantly lower (0.15-0.29, p < 0.01) than that in the Caucasians (0.46). Indians had a significantly lower frequency of this allele (0.15) than the Chinese (0.29) and Malays (0.26).

Abstract:

Abstract: A population genetic study was undertaken to provide gene frequency data on the additional blood genetic markers in the Semai and to estimate the genetic relations between the Semai and their neighboring and linguistically related populations by genetic distance and principal components analyses. Altogether 10 poly morphic and 7 monomorphic blood genetic markers (plasma proteins and red cell enzymes) were studied in a group of 349 Senoi Semai from 11. aboriginal settlements (villages) in the Pahang State of western Malaysia, Both the red cell glucosed-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (PGD) loci reveal the presence of polymorphic frequencies of a nondeficient slow allele at the G6PD locus and a fast allele at the PGD locus. The Semai are characterized by high prevalences of ahaptoglobinemia and G6PD deficiency, high frequencies of HP*1, HB*E, RH*R1, ACP*C, GLO1*1, PGM1*2+, and GC*1F and corresponding low frequencies of ABO*A, Hb(CoSp), HB*B0, TF*D, CHI, and GC*2. Genetic distance analyses by both cluster and principal components models were performed between the Semai and 14 other populations (Malay; Javanese; Khmer; Veddah; Tamils of Malaysia, Sri Lanka, and India; Sinhalese; Oraon; Toda and Irula of India; Chinese; Japanese; Koreans) on the basis of 30 alleles at 7 polymorphic loci. A more detailed analysis using 53 alleles at 13 polymorphic loci with 10 populations was carried out. Both analyses give genetic evidence of a dose relationship between the Semai and the Khmer of Cambodia. Furthermore, the Semai are more closely related to the Javanese than to their close neighbors-the Malay, Chinese, and Tamil Indians. There is no evidence for close genetic relationship between the Semai and the Veddah or other Indian tribes. The evidence fits well with the linguistic relationship of the Semai with the Mon-Khmer branch of the Austro-Asiatic language family.

Abstract: DNA amplification of three Mycobacterium tuberculosis-specific DNA sequences by the polymerase chain reaction (PCR) were evaluated as a means for rapid diagnosis of tuberculous meningitis (TBM). The DNA sequences amplified were a 123 bp region of the IS6110 insertion elements which occur in multiple copies in the mycobacterial genome, a 240 bp region (nts 460-700) from the MPB 64 protein coding gene, and the 383 bp region of the 65 kDa heat shock protein (HSP) antigen. Twenty-seven cerebrospinal fluid (CSF) specimens were studied. Six were obtained from patients with TBM diagnosed by culture (4/6) or by the patients’ response to anti-tuberculous therapy (2/6). The remaining 21 specimens were obtained from patients with febrile seizures (3/21), aseptic meningitis (3/21), septic meningitis (14/21), and cryptoccocal meningitis (1/21), and these served as negative controls. Our results indicate that although the protocols involving the 3 DNA sequences were able to detect TB DNA in the 6 TBM specimens, the main drawback was their extreme sensitivity, thus giving rise to false positive results. In particular, the repeat copy sequence, IS6110, and the 65 kDa HSP gave unacceptably large numbers of false positive results (62% and 33%, respectively).

Abstract: beta-Thalassaemia major patients have chronic anaemia and since 3-4 per cent of Singaporeans carry the beta-gene, prenatal diagnosis is essential. We evaluated the amplification refractory mutation system (ARMS) technique as a routine test for prenatal diagnosis of beta-major. Six mutations along the beta-gene were studied-41-42 (-TCTT), IVSII #654 (C-T), 17 beta (A-T), -28 TATA (A-G), IVSI #5 (G-C), and IVSI #1 (G-T). Our results indicate that prenatal diagnosis using these mutations can be offered to 90 per cent (35/39) of our Chinese couples and 54.6 per cent (12/22) of our Malay couples at risk. Confirmation of ARMS results was carried out using allele-specific oligonucleotide hybridization. Prenatal diagnosis using ARMS was successfully carried out in nine cases which included a set of triplets and twins. The triplets were diagnosed with the beta-trait carrying the 41-42 mutation. The couple with twins possessed the #654 mutation and one twin was diagnosed with the beta-trait and the other with #654 homozygosity. Genomic sequencing of the undefined mutations in the Chinese couples revealed rarer mutations at -29 and an ATG-AGG base substitution at the initiation codon for translation. In the Malay couples, genomic sequencing detected mutations at codon 15 (TGG-TAG) and codon 26 (GAG-AAG). We conclude that ARMS with its direct detection of amplified products by gel electrophoresis provides an accurate, rapid, and simpler method for our beta-thalassaemia prenatal diagnosis programme in Singapore.

Abstract: Major histocompatibility complex (MHC) antigen expression on cells is a prerequisite for immune interaction with activated T-cells. This study examined the ability of sera from patients with systemic lupus erythematosus (SLE) to modulate MHC expression on vascular endothelial cells. SLE sera were able to selectively upregulate MHC class I antigen expression on cultured human umbilical venous endothelial (HUVE) cells, without concomitant induction of MHC class II antigen. The stimulation index (SI) for MHC class I expression produced by SLE sera (1.21 ± 0.23) was significantly higher than those for normal controls (1.01 ± 0.10) (P < 0.0001) and non-SLE patients (1.12 ± 0.14) (P < 0.05). Additionally, active SLE patients had higher mean SI than inactive patients (P < 0.001). Preincubation of SLE sera with Protein A-Sepharose beads conjugated with antibodies against tumor necrosis factor-α and interferon-α was able to significantly reduce their ability to upregulate class I MHC expression by HUVE cells, indicating that these cytokines were responsible for the modulatory effect. This could be an important mechanism for the immune-mediated vascular injury seen in SLE.

Abstract: Sixty-five beta-thalassaemia genes from 14 unrelated Chinese beta-thalassaemia major patients and 37 Chinese beta-carriers were analysed by allele-specific oligonucleotide (ASO) hybridization after DNA amplification by the polymerase chain reaction (PCR). Six mutations were studied and are represented by 49.2% of codon 41-42, 30.8% of IVSII #654, 6.2% of 17 beta, 3.1% of IVSI #5 (G–>C) and 1.5% of - 28 TATA box. The complete mutations responsible for beta-thalassaemia major in 13 of our 14 affected families were identified. For these families prenatal diagnosis at 10 weeks gestation using DNA amplification and ASO hybridization will replace the globin chain biosynthesis technique at 19 weeks gestation. Using ASO analysis, our results indicate that 5 oligo-probes (41-42, II-#654, 17 beta, IVSI-#5 and - 28) allow determination of beta-thalassaemia mutations in 59/65 (90.8%) of the Singaporean Chinese studied.

Abstract: The distribution of restriction fragment length polymorphism (RFLP) at the BamHl site of the beta-globin gene was investigated in the Chinese, Indian, and Malay race in Singapore. The sample comprised of 183 normal individuals and 35 beta-thalassemia carriers in which 13 were couples with at least one beta-major child. The results from this study indicate that BamH1 polymorphism will be informative in 22% of pregnancies at risk for beta-thalassemia major in Chinese, 19% in Malays and 7% in Indians. In prenatal diagnosis using BamH1 polymorphism for one beta-major affected family, the fetus was diagnosed to be normal or beta-carrier. The validity of BamH1 polymorphism in the exclusion of beta-thalassemia major was subsequently confirmed at birth by globin chain biosynthesis.

Abstract: The effect of a DNA polymorphism (MspI) of the apolipoprotein (apo) A-II gene on serum lipid and apo levels was studied in a group of 125 healthy Chinese of both sexes. The frequency of the 3.7-kb rarer allele (M2) was found to be significantly higher in the Chinese (0.30) than in Caucasians (0.16; p < 0.025). The distribution of apo A-II genotypes was in Hardy-Weinberg equilibrium in the Chinese population. The presence of a polymorphic site (MspI) within an Alu sequence at the 3’ end of the gene, the 3.0 kb (M1) allele, was associated with significantly higher levels of serum apo A-I and A-II (p < 0.05 and < 0.01, respectively). Serum high-density Lipoprotein cholesterol levels were also correspondingly higher in individuals with Ml, but did not reach a significant level. Male heterozygotes of the apo A-II polymorphism had significantly higher levels of serum triglycerides compared to homozygotes (p < 0.05). Thus the MspI polymorphism of the apo A-II gene appears to be associated with altered levels of lipids and apos in the Chinese population.

Abstract: The frequency of deletional alpha-thalassaemia in a Javanese population sample (n = 103) was investigated at three restriction sites of the alpha-globin gene (BamHI, BglII and RsaI). The overall gene frequency of alpha+ deletional thalassaemia was found to be very low (0.03). Leftward (-alpha4.2) and rightward (-alpha3.7) deletions and triplicated genes were present in equal frequency (0.015 and 0.005, respectively).