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Molecular heterogeneity of beta-thalassaemia in Malaysia: a practical approach to diagnosis

Thong MK, Law HY, Ng IS

Ann Acad Med Singap, 1996 Jan;25(1):79-83.
PMID: 8779552

The beta-thalassaemia mutations in 20 Malaysian children with beta-thalassaemia major were characterised by using a multi-modal approach, consisting of a slot-blot hybridisation with selected allele-specific oligonucleotides (ASO), followed by reverse dot-blot assay (RDB), amplification refractory mutation system (ARMS) and genomic sequencing. This strategy yielded a 94.4% mutation detection rate. The 6 most common mutations were codons 41/42 (-TTCT), IVS II nt 654(C --> T), IVS I nt 5(G --> C), IVS I nt 1(G -->T), codon 35 (-C) and codon 19 (A --> G), which accounted for 83.3% of all mutations detected. A strategy of initial screening with the above 6 selected ASOs for slot-blot hybridisation followed by RDB assay for the less common Asian mutations would give a mutation identification of 91.7%. Another feasible approach would be to analyse alleles from a particular racial group, by a judicious selection of 4 ASOs common to that particular subpopulation and then supplement this with RDB assay. This could yield a 100% coverage for the Chinese subpopulation in Malaysia. With these strategies, a practical approach has been identified to overcome the pitfalls posed by the molecular heterogeneity of beta-thalassaemia to enable prenatal diagnosis and carrier screening to be carried out. Regional collaborative studies are to be encouraged as an indispensable tool in providing better health care services to our patients.

Matched MeSH terms: beta-Thalassemia/genetics*
Molecular study and genotype/phenotype correlation of β Thalassemia in Malaysia

Sivalingam M, Looi ML, Zakaria SZ, Hamidah NH, Alias H, Latiff ZA, et al.

Int J Lab Hematol, 2012 Aug;34(4):377-82.
PMID: 22335963 DOI: 10.1111/j.1751-553X.2012.01405.x

INTRODUCTION: To study the ß-gene mutations spectrum, the genotype/phenotype correlation, the modulatory effect of co-inherited factors such as α-gene mutations and of Xmn1 polymorphism in a large cohort of Malaysian patients.
METHODS: A total of 264 cases clinically diagnosed as Thalassemia major (TM) (111), Thalassemia intermedia (21), HbE-β Thalassemia (131), and 1 HbE homozygous were studied. The detection of α and ß gene mutations and characterization of Xmn1 polymorphism were performed by multiplex PCR, amplification refractory mutation system (ARMS), DNA sequencing, and restriction fragment length polymorphism (RFLP)-PCR.
RESULTS: A total of 19 ß Thalassemia mutations were characterized. CD26 and CD41/42 were the most common found in the Malay and Chinese population, respectively. The sensitivity of the clinical diagnosis for β TM, thalassemia intermedia, and HbE/β thalassemia was 94.0%, 15.2%, and 89.2%, respectively. Patients with Xmn1 heterozygosity [+/-] required less frequent transfusion compared with those without the polymorphism. Co-inheritance of α-thalassemia alleviates the severity of HbE-β thalassemia in our cohort.
CONCLUSION: Molecular analysis should be used for a better diagnosis and management of β thalassemia.

Matched MeSH terms: beta-Thalassemia/genetics*
Multiplexed genotyping of beta globin mutations with MALDI-TOF mass spectrometry

Looi ML, Sivalingam M, Husin ND, Radin FZ, Isa RM, Zakaria SZ, et al.

Clin Chim Acta, 2011 May 12;412(11-12):999-1002.
PMID: 21315703 DOI: 10.1016/j.cca.2011.02.006

BACKGROUND: Beta thalassemia represents a great heterogeneity as over 300 mutations have been identified and each population at-risk has its own spectrum of mutations. Molecular characterization with high accuracy, sensitivity and economics is required for population screening and genetic counseling.
METHODS: We used the MALDI-TOF mass spectrometry (MS) platform to develop novel multiplex assays for comprehensive detection of 27 mutations in beta-thalassemia patients. Six multiplex assays were designed to detect 13 common known ß-mutations, namely CD41/42, CD71/72, IVS1-5, IVS1-1, CD26, IVS2-654, CAP+1, CD19, -28, -29, IVS1-2, InCD (T-G) and CD17; and 14 rare ß-mutations, i.e. InCD (A-C), CD8/9, CD43, -86, CD15, Poly A, Poly T/C, IVS2-1, CD1, CD35/36, CD27/28, CD16, CD37, and 619bpDEL in 165 samples. We compared the efficiencies of genotyping by MS and Amplification Refractory Mutation System (ARMS). Discrepant results were confirmed by sequencing analysis.
RESULTS: A total of 88.7% (260/293 allele) of MS and ARMS results was in agreement. More than fifty percent of the discrepant result was due to the false interpretation of ARMS results. Failed CD19 assay by MS method might be due to the assay design. The MS method detected 5 rare ß-mutations (CD15, CD35/36, CD8/9, Poly A and Poly T/C) presented in 13 alleles, which were not included in the ARMS screening panel.
CONCLUSION: We revealed that the MS method is a sensitive, high-throughput, highly automated, flexible, and cost-effective alternative to conventional ß-thalassemia genotyping methods.

Matched MeSH terms: beta-Thalassemia/genetics
Non-Invasive DNA Sampling for Molecular Analysis of Beta-Thalassemia: Amiable Alternative Sampling Methods with Accurate Results for Pediatric Patients

Abd Rahim MR, Kho SL, Kuppusamy UR, Tan JA

Clin. Lab., 2015;61(9):1325-30.
PMID: 26554253

BACKGROUND: Beta-thalassemia is the most common genetic disorder in Malaysia. Confirmation of the β-globin gene mutations involved in thalassemia is usually carried out by molecular analysis of DNA extracted from leukocytes in whole blood. Molecular analysis is generally carried out when affected children are around 1 - 2 years as clinical symptoms are expressed during this period. Blood taking at this age can be distressing for the child. High yield and pure DNA extracted from non-invasive sampling methods can serve as alternative samples in molecular studies for genetic diseases especially in pediatric cases.
METHODS: In this study, mouthwash, saliva, and buccal cytobrush samples were collected from β-thalassemia major patients who had previously been characterized using DNA extracted from peripheral blood. DNA was extracted from mouthwash, saliva, and buccal cytobrush samples using the conventional inexpensive phenol-chloroform method and was measured by spectrophotometry for yield and purity. Molecular characterization of β-globin gene mutations was carried out using the amplification refractory mutation system (ARMS).
RESULTS: DNA extracted from mouthwash, saliva, and buccal cytobrush samples produced high concentration and pure DNA. The purified DNA was successfully amplified using ARMS. Results of the β-globin gene mutations using DNA from the three non-invasive samples were in 100% concordance with results from DNA extracted from peripheral blood.
CONCLUSIONS: The conventional in-house developed methods for non-invasive sample collection and DNA extraction from these samples are effective and negate the use of more expensive commercial kits. In conclusion, DNA extracted from mouthwash, saliva, and buccal cytobrush samples provided sufficiently high amounts of pure DNA suitable for molecular analysis of β-thalassemia.

Matched MeSH terms: beta-Thalassemia/genetics*
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.

Matched MeSH terms: beta-Thalassemia/genetics
Fulltext Predictive SNPs for β0-thalassemia/HbE disease severity

Munkongdee T, Tongsima S, Ngamphiw C, Wangkumhang P, Peerapittayamongkol C, Hashim HB, et al.

Sci Rep, 2021 05 14;11(1):10352.
PMID: 33990643 DOI: 10.1038/s41598-021-89641-2

β-Thalassemia/HbE disease has a wide spectrum of clinical phenotypes ranging from asymptomatic to dependent on regular blood transfusions. Ability to predict disease severity is helpful for clinical management and treatment decision making. A thalassemia severity score has been developed from Mediterranean β-thalassemia patients. However, different ethnic groups may have different allele frequency and linkage disequilibrium structures. Here, Thai β0-thalassemia/HbE disease genome-wild association studies (GWAS) data of 487 patients were analyzed by SNP interaction prioritization algorithm, interacting Loci (iLoci), to find predictive SNPs for disease severity. Three SNPs from two SNP interaction pairs associated with disease severity were identifies. The three-SNP disease severity risk score composed of rs766432 in BCL11A, rs9399137 in HBS1L-MYB and rs72872548 in HBE1 showed more than 85% specificity and 75% accuracy. The three-SNP predictive score was then validated in two independent cohorts of Thai and Malaysian β0-thalassemia/HbE patients with comparable specificity and accuracy. The SNP risk score could be used for prediction of clinical severity for Southeast Asia β0-thalassemia/HbE population.

Matched MeSH terms: beta-Thalassemia/genetics
Preimplantation genetic diagnosis for beta-thalassemia using single-cell DNA analysis for codons 17 and 26 of beta-globin gene

Nasri NW, Jamal AR, Abdullah NC, Razi ZR, Mokhtar NM

Arch Med Res, 2009 Jan;40(1):1-9.
PMID: 19064120 DOI: 10.1016/j.arcmed.2008.10.008

Preimplantation genetic diagnosis (PGD) of monogenic autosomal hereditary disorders following assisted conception usually involves the removal of one or two blastomeres from preimplantation embryos. However, the amount of DNA from a single blastomere is insufficient to amplify the region of interest. Hence, the whole genome amplification (WGA) method is performed prior to amplifying the genes of interest before analysis of DNA material through polymerase chain reaction (PCR).

Matched MeSH terms: beta-Thalassemia/genetics
Review: Beta-thalassemia and molecular chaperones

Sumera A, Radhakrishnan A, Baba AA, George E

Blood Cells Mol. Dis., 2015 Apr;54(4):348-52.
PMID: 25648458 DOI: 10.1016/j.bcmd.2015.01.008

Thalassemia is known as a diverse single gene disorder, which is prevalent worldwide. The molecular chaperones are set of proteins that help in two important processes while protein synthesis and degradation include folding or unfolding and assembly or disassembly, thereby helping in cell homeostasis. This review recaps current knowledge regarding the role of molecular chaperones in thalassemia, with a focus on beta thalassemia.

Matched MeSH terms: beta-Thalassemia/genetics
Fulltext Screening of concurrent alpha-thalassaemia 1 in beta-thalassaemia carriers

Chong YM, Tan JA, Zubaidah Z, Rahimah A, Kuldip K, George E

Med J Malaysia, 2006 Jun;61(2):217-20.
PMID: 16898315

Thalassaemia is an inherited blood disorder and is a significant public health problem in Malaysia, with many not knowing they carry the 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 concurrent 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 (- -SEA) deletion. The significant carrier rate for alpha thalassaemia 1 indicates the need for the implementation of DNA analysis to complement thalassaemia screening in high risk populations.

Matched MeSH terms: beta-Thalassemia/genetics
Fulltext Serum ferritin concentrations in transfusion dependent beta-thalassaemia

George E, Wong HB, George R, Ariffin WA

Singapore Med J, 1994 Feb;35(1):62-4.
PMID: 8009283

Patients on a moderate red cell transfusion programme have iron overload where the concentrations of the serum ferritin were inappropriate to increases in the transfusion load as a result of limitations of apoferritin synthesis and conversion of ferritin into haemosiderin. This study confirms the limitations for the use of estimations of the serum ferritin to evaluate the iron status in patients with expected high overload as would be seen in patients on many years of maintenance red cell transfusions in the absence of iron chelation therapy. Poor compliance, inadequate dosage of Desferal (deferoxamine), and the late initiation of iron chelation therapy were factors that were considered in the patients with failure of response to iron chelation.

Matched MeSH terms: beta-Thalassemia/genetics
Fulltext Specific and straightforward molecular investigation of β-thalassemia mutations in the Malaysian Malays and Chinese using direct TaqMan genotyping assays

Kho SL, Chua KH, George E, Tan JA

Genet. Mol. Res., 2013;12(3):2409-15.
PMID: 23479149 DOI: 10.4238/2013.February.28.4

Beta-thalassemia is a life-threatening inherited blood disorder. Rapid characterization of β-globin gene mutations is necessary because of the high frequency of Malaysian β-thalassemia carriers. A combination real-time polymerase chain reaction genotyping assay using TaqMan probes was developed to confirm β-globin gene mutations. In this study, primers and probes were designed to specifically identify 8 common β-thalassemia mutations in the Malaysian Malay and Chinese ethnic groups using the Primer Express software. "Blind tests" using DNA samples from healthy individuals and β-thalassemia patients with different genotypes were performed to determine the specificity and sensitivity of this newly designed assay. Our results showed 100% sensitivity and specificity for this novel assay. In conclusion, the TaqMan genotyping assay is a straightforward assay that allows detection of β-globin gene mutations in less than 40 min. The simplicity and reproducibility of the TaqMan genotyping assay permit its use in laboratories as a rapid and cost-effective diagnostic tool for confirmation of common β-thalassemia mutations in Malaysia.

Matched MeSH terms: beta-Thalassemia/genetics*
Fulltext Spectrum of beta-thalassaemia mutations in transfusion dependent thalassaemia patients: practical implications in prenatal diagnosis

George E, George R, Ariffin WA, Mokhtar AB, Azman ZA, Sivagengei K

Med J Malaysia, 1993 Sep;48(3):325-9.
PMID: 8183146

The study concerned the identification of the beta-thalassaemia mutations that were present in 24 patients with beta-thalassaemia major who were transfusion dependent. The application of a modified polymerase chain reaction, the amplification refractory system (ARMS) was found to be an effective and rapid method for the identification of the beta-thalassaemia mutations. Six different mutations were detected. Seventy five percent of the patients were Chinese-Malaysians and showed the commonly occurring anomalies: 1. frameshift codon 41 and 42 (-TCTT); 2. the C to T substitution at position 654 of intron 2 (IVS-2); 3. the mutation at position -28(A to G); and the nonsense mutation A to T at codon 17. In the Malays, the common mutations seen were: 1. the G to C mutation at position 5 of IVS-1; 2. the G to T mutation at position 1 of intron 1 (IVS-1); and the A to T at codon 17. The delineation of the specific mutations present will enable effective prenatal diagnosis for beta-thalassaemia to be instituted.

Matched MeSH terms: beta-Thalassemia/genetics*
Fulltext Thalassaemia and haemoglobinopathies in Brunei Darussalam

Ismail JB

Med J Malaysia, 1992 Jun;47(2):98-102.
PMID: 1494340

One thousand consecutive Brunei Darussalam patients referred with low Hb, and/or low MCV and MCH (Hb < 12.5g/dl, MCV < 76fl, MCH < 27pg) were studied in the laboratory for underlying haemoglobinopathies. 30.0% of such patients were proved to have either beta-thalassaemia trait, beta-thalassaemia major, Hb AE, Hb EE, Hb E beta-thalassaemia or Hb H disease. In some, the haemoglobin abnormality was not identified precisely. Alpha-thalassaemia was suspected in an additional 4.3% of cases but confirmation study by globin-chain synthesis was not available. Beta-thalassaemia trait which was the predominant disorder was equally distributed among the three major race groups of Brunei Darussalam. Hb E was found exclusive among the Malay population. Hb H disease appeared as more common among the Chinese or the Malays (p > 0.05). This study reveals that thalassaemia and haemoglobinopathies are prevalent in Brunei Darussalam.

Matched MeSH terms: beta-Thalassemia/genetics
Thalassemia intermedia in HbH-CS disease with compound heterozygosity for beta-thalassemia: challenges in hemoglobin analysis and clinical diagnosis

Tan JA, Kok JL, Tan KL, Wee YC, George E

Genes Genet Syst, 2009 Feb;84(1):67-71.
PMID: 19420802

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 alpha-thalassemia alleles (-alpha/alphaalpha --/alphaalpha, --/-alpha). The co-inheritance of beta-thalassemia with alpha-thalassemia with a single gene deletion (-alpha/alphaalpha) is usually associated with thalassemia major. In contrast, the co-inheritance of beta-thalassemia with two alpha-genes deleted in cis or trans (--/alphaalpha 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 alpha-gene deletion in cis (--SEA/alphaalpha) is generally not present in the Malays, who more commonly possess the two alpha-gene deletion in trans (-alpha/-alpha). 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.

Matched MeSH terms: beta-Thalassemia/genetics*
The clinical severity of beta-thalassemia mutations in West Malaysia

George E

Southeast Asian J. Trop. Med. Public Health, 1995;26 Suppl 1:225-8.
PMID: 8629111

Beta-thalassemia in West Malaysia is caused by 14 molecular defects with differing clinical severity. In Chinese patients from West Malaysia, the main beta-thalassemia mutations seen were (a) a 4 base pair-TCTT deletion in codon 41-42 [frameshift mutation (FSC 41-42)]; (b) a C to T substitution at the second intervening sequence (IVS2-654); (c) an A to G substitution in the TATA box [-28 (A to G)], and (d) an A to T substitution in codon 17[17 A to T]. In the Malays, the main mutations seen were (a) a G to C in nucleotide 5 at the intervening sequence I [IVS1-5 (G to C)]; (b) G to T substitution in nucleotide I at the intervening sequence I [IVS1-1 (G to T)]; (c) a A to T substitution in codon 17 (17 A to T); (d) removal of C from codon 35 [codon 35 (-C)], and (e) a 4 base pairs-TCTT deletion in codon 41-42 [frameshift mutation (FSC 41-42)]. A scoring system (Tha1 CS) has been formulated to predict clinical severity. It is the type of beta-thalassemia mutation present that decides on the clinical phenotype. The most severe beta-thalassemia mutation is assigned a score of 4. A score of 8 indicates severe thalassemia.

Matched MeSH terms: beta-Thalassemia/genetics*
Fulltext The spectrum of beta-globin gene mutations in children with beta-thalassaemia major from Kota Kinabalu, Sabah, Malaysia

Thong MK, Soo TL

Singapore Med J, 2005 Jul;46(7):340-3.
PMID: 15968446

Beta-thalassaemia major is one of the commonest genetic disorders in South East Asia. The strategy for the community control of beta-thalassaemia major requires the characterisation of the spectrum of beta-globin gene mutations in any multi-ethnic population. There is only a single report of mutation analyses of the beta-globin gene in an isolated Kadazandusun community in Kota Belud, Sabah, Malaysia, which showed the presence of a common 45 kb deletion.

Matched MeSH terms: beta-Thalassemia/genetics*
Fulltext The spectrum of beta-thalassemia mutations in Malays in Singapore and Kelantan

Abdullah WA, Jamaluddin NB, Kham SK, Tan JA

Southeast Asian J. Trop. Med. Public Health, 1996 Mar;27(1):164-8.
PMID: 9031421

The spectrum of beta-thalassemia mutations in Malays in Singapore and Kelantan (Northeast Malaysia) was studied. Allele specific priming was used to determine the mutations in beta-carriers at -28, Codon 17, IVSI #1, IVSI #5, Codon 41-42 and IVSII #654 along the beta-globin gene. The most common structural hemoglobin variant in Southeast Asia, Hb E, was detected by DNA amplification with restriction enzyme (Mnl1) analysis. Direct genomic sequencing was carried out to detect the beta-mutations uncharacterized by allele-specific priming. The most prevalent beta-mutations in Singaporean Malays were IVSI #5 (45.83%) followed by Hb E (20.83%), codon 15 (12.5%) and IVSI #1 and IVSII #654 at 4.17% each. In contrast, the distribution of the beta-mutations in Kelantan Malays differed, with Hb E as the most common mutation (39.29%) followed by IVSI #5 (17.86%), codon 41-42 (14.29%), codon 19 (10.71%) and codon 17 (3.57%). The beta-mutations in Kelantan Malays follow closely the distribution of beta-mutations in Thais and Malays of Southern Thailand and Malays of West Malaysia. The AAC-->AGC base substitution in codon 19 has been detected only in these populations. The spectrum of beta-mutations in the Singaporean Malays is more similar to those reported in Indonesia with the beta-mutation at codon 15 (TGG-->TAG) present in both populations. The characterization of beta-mutations in Singaporean and Kelantan Malays will facilitate the establishment of effective prenatal diagnosis programs for beta-thalassemia major in this ethnic group.

Matched MeSH terms: beta-Thalassemia/genetics*
The spectrum of beta-thalassemia mutations in southern Thailand

Nopparatana C, Panich V, Saechan V, Sriroongrueng V, Nopparatana C, Rungjeadpha J, et al.

Southeast Asian J. Trop. Med. Public Health, 1995;26 Suppl 1:229-34.
PMID: 8629112

Beta-thalassemia mutations in 282 alleles of 253 unrelated individuals originating from various provinces in the south of Thailand were characterized by dot blot hybridization, specific PCR-amplification and direct DNA sequencing. It was possible to characterize the mutations in 274 (97.2%) of alleles studied. Twelve different point mutations and two different large deletions of the beta-globin gene were identified. Seven common mutations, namely 4 bp deletion at codons 41/42. IVS1 position 5 (G-C), codon 19 (AAC-AGC), codon 17 (AAG-TAG), IVS1 position 1 (G-T), position -28 (A-G) and 3.5 kb deletion, accounted for about 91.5%. The mutations at mRNA cap site + 1 (A-C) and IVS1 position 1 (G-A), previously undescribed in Thailand, were found in 1 and 2 individuals, respectively. A novel mutation of 105 bp deletion at the 5' end of beta-globin gene was detected in a family originating from this area. The knowledge from this study should be useful for planning of genetic counseling and prenatal diagnosis programs for patients with beta-thalassemia in the south of Thailand.

Matched MeSH terms: beta-Thalassemia/genetics*
The use of Taqman genotyping assays for rapid confirmation of β-thalassaemia mutations in the Malays: accurate diagnosis with low DNA concentrations

Teh LK, Lee TY, Tan JA, Lai MI, George E

Int J Lab Hematol, 2015 Feb;37(1):79-89.
PMID: 24725998 DOI: 10.1111/ijlh.12240

In Malaysia, β-thalassaemia is a common inherited blood disorder in haemoglobin synthesis with a carrier rate of 4.5%. Currently, PCR-incorporating techniques such as amplification refractory mutation system (ARMS) or reverse dot blot hybridization (RDBH) are used in β-thalassaemia mutation detection. ARMS allows single-mutation identification using two reactions, one for wild type and another for mutant alleles. RDBH requires probe immobilization and optimization of hybridization and washing temperatures which is time consuming. The aim of our study was to investigate whether β-thalassaemia mutations can be identified in samples with low DNA concentrations.

Matched MeSH terms: beta-Thalassemia/genetics*
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.

Matched MeSH terms: beta-Thalassemia/genetics*

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