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.
A total of 103 beta thalassemia genes from 78 children (45 with Hb E/beta thalassemia, 8 with beta thalassemia heterozygotes, and 25 with homozygous beta thalassemia) were analyzed using dot-blot hybridization of the polymerase chain reaction-amplified DNA and direct DNA sequencing. Nine mutations were characterized in 98/103 (95%) of beta thalassemia alleles, of which six (a 4 bp deletion in codons 41-42, a G-C transition at position 5 of IVS-1, A-G transition at codon 19, an A-T transition at codon 17, an A-G transition at position -28 upstream of the beta globin gene, a G-T transition at position 1 of IVS-1), accounted for 92%. The spectrum of beta thalassemia mutations in Chinese Thai is similar to that reported among the Chinese from other parts of the world. The distribution of beta thalassemia mutations in Muslim Thai is similar to that reported among Malaysians. The most common beta thalassemia mutation in Thai and Chinese Thai patients is the frameshift mutation at codons 41-42, in comparison with the Muslim Thai in whom the G-C transition at position 5 of the IVS-1 mutation predominates. The heterogeneity of molecular defects causing beta thalassemia should aid in the planning of a prenatal diagnosis program for beta thalassemia in the South of Thailand.
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.
The distribution of restriction fragment length polymorphism (RFLP) at the BamH1 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.
Patients with the Hb beta + [IVS 1-5 (G-->C)] clinically presented as beta-thalassaemia intermedia and remained asymptomatic in the absence of blood transfusions. With or without blood transfusions the patients were short and had moderate to marked thalassaemia facies. Children who received blood transfusions showed progressive iron loading with age. The serum ferritin and serum alanine transaminase levels were significantly raised in the patients who were given blood transfusions. In the presence of blood transfusions, and absence of adequate iron chelation therapy, splenectomy became an inevitable event at some stage of the disease because of increasing transfusing requirements.
The clinical severity of the mutations causing beta-thalassaemia in West Malaysia is presented. Thalassaemia clinical scores (Thal CS), a scoring system, has been formulated to predict clinical severity. It is the type of beta-thalassaemia mutation present that decides on the clinical phenotype. The most severe beta-thalassaemia mutation is assigned a score of 4. A score of 8 indicates a severe thalassaemia phenotype. Alpha-thalassaemia, increased synthesis of Hb F, and glucose-6-phosphate deficiency may ameliorate the clinical condition at phenotype level, and the co-inheritance of hereditary ovalocytosis aggravates it.
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.
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.
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.
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.
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.
We have systematically analyzed beta-thalassemia genes using polymerase chain reaction-related techniques, dot-blot hybridization with oligonucleotide probes, allele specific-polymerase chain reaction, and sequencing of amplified DNA fragments from 41 unrelated patients, including 37 beta-thalassemia homozygotes, three with beta-thalassemia/Hb E, and one with beta-thalassemia/Hb S. Four different beta-thalassemia mutations were detected in 78 alleles. These are the IVS-I-5 (G-->C), codon 30 (AGG-->ACG) [also indicated as IVS-I (-1)], IVS-I-1 (G-->A), and codons 41/42 (-TTCT) mutations. The distribution of the beta-thalassemia mutations in the Maldives is 58 alleles (74.3%) with the IVS-I-5 (G-->C) mutation, 12 (15.4%) with the codon 30 (AGG-->ACG) mutation, seven (9%) with the IVS-I-1 (G-->A) mutation, and one with the codons 41/42 (-TTCT) mutation. The first three mutations account for 98.7% of the total number of beta-thalassemia chromosomes studied. These mutations are clustered in the region spanning 6 bp around the junction of exon 1 and the first intervening sequence of the beta-globin gene. These observations have significant implications for setting up a thalassemia prevention and control program in the Maldives. Analysis of haplotypes and frameworks of chromosomes bearing each beta-thalassemia mutation suggested that the origin and spread of these mutations were reflected by the historical record.
Beta-thalassemia major is one of the commonest genetic disorders in South-East Asia. The spectrum of beta-thalassemia mutations in the various ethnic sub-populations on the island of Borneo is unknown. We studied 20 Dusun children from the East Malaysian state of Sabah (North Borneo) with a severe beta-thalassemia major phenotype, using a combination of Southern analysis, polymerase chain reaction analysis and direct sequencing. We found the children to be homozygous for a large deletion, which has a 5' breakpoint at position -4279 from the cap site of the beta-globin gene (HBB) with the 3' breakpoint located in a L1 family of repetitive sequences at an unknown distance from the beta-globin gene. This was similar to a recent finding of a large deletion causing beta-thalassemia first described in unrelated beta-thalassemia heterozygotes of Filipino descent. This report describes the first 20 families with homozygosity of the deletion causing a severe phenotype. It provides the first information on the molecular epidemiology of beta-thalassemia in Sabah. This finding has implications for the population genetics and preventative strategies for beta-thalassemia major for nearly 300 million individuals in South-East Asia.
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.
Beta-thalassemia major patients have chronic anemia and are dependent on blood transfusions to sustain life. Molecular characterization and prenatal diagnosis of beta3-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 CARMS for cd 41-42/IVSII #654 detected beta-thalassemia mutations in 72% of the Chinese families. C-ARMS for cd 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 cd 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.
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.
DNA technology provides a new avenue to perform neonatal screening tests for single-gene diseases in populations of high frequency. Thalassemia is one of the high-frequency single-gene disorders affecting Singapore and many countries in the malaria belt. The authors explored the feasibility of using PCR-based diagnostic screening on 1,116 unselected sequential cord blood samples for neonatal screening. The cord blood samples were screened for the most common reported alpha- and beta-thalassemia mutations in each ethnic group (Chinese, Malays, and Indians) in a multiracial population. The carrier frequency for alpha-thalassemia mutations was about 6.4% in the Chinese (alpha deletions = 3.9%, alpha deletions = 2.5%), 4.8% in Malays, and 5.2% in Indians. Only alpha deletions were observed in the Chinese. The carrier frequency for beta-thalassemia mutations was 2.7% in the Chinese, 6.3% in Malays, and 0.7% in Indians. Extrapolating to the population distribution of Singapore, the authors found a higher overall expected carrier frequency for alpha- and beta-thalassemia mutations of 9% compared with a previous population study of 6% by phenotype. The highly accurate results make this molecular epidemiologic screening an ideal method to screen for and prevent severe thalassemia in high-risk populations.
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 (CT), poly A(A-G), 100-kb Ggamma(Agammadeltabeta) 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 bthalassemia 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.