Combine-ARMS: a rapid and cost-effective protocol for molecular characterization of 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.     

The molecular basis of beta-thalassemia in Thailand: application to prenatal diagnosis.

To enable the prenatal diagnosis of beta-thalassemia by direct detection of the mutant beta-globin genes, we have determined the spectrum of mutations causing this disease in Thailand. The techniques employed included a combination of synthetic oligonucleotide probe hybridization, direct sequencing of genomic DNA enzymatically amplified by the polymerase chain reaction, and cloning and sequencing of the beta-globin genes. A total of 116 beta-thalassemia genes from 78 Hb E/beta-thalassemia patients and from 19 homozygous beta-thalassemia patients were analyzed, and the mutation was characterized in 112/116 (97%) of them. Eleven mutations were found, of which four (-CTTT in codon 41/42, AAG----TAG in codon 17, C----T in position 654 of the IVS-2 region, and A----G in position -28 upstream of the beta-globin gene) accounted for 83%; two previously undescribed mutations have been identified. The spectrum of beta-thalassemia mutations is similar to that reported among the Chinese. However, within the Thai population itself, patients with homozygous beta-thalassemia show a wider spread of mutations in comparison with the Hb E/beta-thalassemia group, in whom the frameshift 41/42 mutation predominates at a frequency of 62%. This difference in distribution may reflect the difference in ethnic origin of the two groups. Characterization of these mutations should aid the planning of a prenatal diagnosis program for beta-thalassemia in Thailand.     

Distribution of beta-thalassemia mutations in south China and their association with haplotypes.

DNA from 93 Chinese beta-thalassemia chromosomes were hybridized to eight different mutant oligomers to determine their specific mutation. Four mutations accounted for 87% of the chromosomes; in descending frequencies, these mutations were codon 41/42, IVS-2 nt654, codon 17, and -28. Since codon 41/42 mutation can be associated with multiple beta-thalassemia haplotypes, codon 41/42 is probably a hot spot for the 4-bp deletion. The distributions of these mutations were mapped to various regions in south China. These data are useful for the planning of prenatal diagnosis programs in other Chinese communities worldwide.     

Molecular characterization of beta-thalassemia in the Sardinian population.

This study reports the molecular characterization of beta-thalassemia in the Sardinian population. Three thousand beta-thalassemia chromosomes from prospective parents presenting at the genetic service were initially analyzed by dot blot analysis with oligonucleotide probes complementary to the most common beta-thalassemia mutations in the Mediterranean at-risk populations. the mutations which remained uncharacterized by this approach were defined by denaturing gradient gel electrophoresis (DGGE) followed by direct sequence analysis on amplified DNA. We reconfirmed that the predominant mutation in the Sardinian population is the codon 39 nonsense mutation, which accounts for 95.7% of the beta-thalassemia chromosomes. The other two relatively common mutations are frameshifts at codon 6 (2.1%) and at codon 76 (0.7%), relatively uncommon in other Mediterranean-origin populations. In this study we have detected a novel beta-thalassemia mutation, i.e., a frameshift at codon 1, in three beta-thalassemia chromosomes. The DGGE procedure followed by direct sequencing on amplified DNA is a powerful approach for the characterization of unknown mutations in this genetic system. The results herein presented allowed an expansion of the applicability of prenatal diagnosis by DNA analysis, to all couples at risk for beta-thalassemia in our population.     

Determination of the spectrum of beta-thalassemia genes in Spain by use of dot-blot analysis of amplified beta-globin DNA.

We have delineated the molecular lesions causing beta-thalassemia in Spain, a country that has witnessed the passage of different Mediterranean populations over the centuries, in order to evaluate the extent of heterogeneity of these mutations and to make possible simplified prenatal diagnosis of the disorder in that country. The use of the polymerase chain-reaction (PCR) technique to preferentially amplify beta-globin DNA sequences that contain the most frequent beta-thalassemia mutations in Mediterraneans enabled us to rapidly analyze 58 beta-thalassemia alleles in a dot-blot format either by hybridization with allele-specific radiolabeled oligonucleotide probes or by direct sequence analysis of the amplification product. The Spanish population carries seven different beta-thalassemia mutations; the nonsense codon 39 is predominant (64%), whereas the IVS1 position 110 mutation, the most common cause of beta-thalassemia in the eastern part of the Mediterranean basin, is underrepresented (8.5%). The IVS1 mutation at position 6 accounts for 15% of the defects and leads to a more severe form of beta+-thalassemia than originally described in most of the patients we studied. In this study, we demonstrate further the usefulness of the dot-blot hybridization of PCR-amplified genomic DNA in both rapid population surveys and prenatal diagnosis of beta-thalassemia.     

Identification of a beta-thalassemia mutation associated with a novel haplotype of RFLPs.

The study of the molecular defects that result in beta-thalassemia in Mediterraneans has uncovered a large number of unique mutations. This information is already being utilized for prenatal diagnosis of pregnancies at risk. Here, we report the definitive identification, by molecular cloning, of the beta-thalassemia mutation associated with a Mediterranean chromosome bearing a novel haplotype of restriction fragment length polymorphisms (RFLPs) in the beta gene cluster that has been previously designated as haplotype X. The thalassemia mutation was identified as a T----C base substitution at IVS-1 position 6, a mutation previously described in association with haplotype VI. We describe the use of the restriction enzyme SfaNI for the detection of this mutation and point out a possible pitfall that should be avoided if such an approach is used for the detection of this mutation, which appears to be a common cause of mild beta+-thalassemia in some populations.     

Characterization of beta-thalassemia mutations in patients from the state of Rio Grande do Norte, Brazil

35 unrelated individuals were studied for characterization as either heterozygous or homozygous for beta-thalassemia. Molecular analysis was done by PCR/RFLP to detect the mutations most commonly associated with beta-thalassemia (β(0)IVS-I-1, β(+)IVS-I-6, and β(0)39). In the patients who showed none of these mutations, the beta-globin genes were sequenced. Of the 31 heterozygous patients, 13 (41.9%) had the β(+)IVS-I-6 mutation, 15 (48.4%) the β(0)IVS-I-1 mutation, 2 (6.5%) the β(+)IVS-I-110 mutation and 1 (3.2%) the β(+)IVS-I-5 mutation. IVS-I-6 was detected in the four homozygotes. The mutation in codon 39, often found in previous studies in Brazil, was not detected in the present case. This is the first study aiming at identifying mutations that determine beta-thalassemia in the state of Rio Grande do Norte.     

Molecular nature of beta-thalassemia in Tajikistan: a four base pair deletion in codons 41-42 of the beta-globin gene]

Thirty tajiks, whose relatives had beta-thalassemia traits (revealed in previous investigations by determination of the HbA-2 and HbF levels) were selected to screen beta-thalassemia mutations. DNA samples from each individual were subjected to the PCR (polymerase chain reaction) to amplify the 635 bp beta-globin gene fragment. One additional band was detected in three samples after the amplified fragment underwent electrophoresis in 2% agarose gel and the EtBr was stained, and two additional ones were revealed by 6% PAAGE and staining of the EtBr. All additional bands migrated more slowly than appropriate 635 bp fragment. It is supposed that additional bands are heteroduplexes formed from the wild type chains and mutated chains carrying a deletion or insertion. The 4 bp deletion of the 41-42 (-tctt) was detected after the direct sequencing of the amplified fragments. This mutation is common among Chinese but it was not revealed in the Middle Asia populations. The mutation can be easily screened using the PCR and electrophoresis in 2% agarose gel or PAAG of the amplified beta-globin gene fragments.     

Characterization of a spontaneous mutation to a beta-thalassemia allele.

We have studied a nuclear family containing a single child with severe beta-thalassemia intermedia, a Greek-Cypriot mother with hematological findings of beta-thalassemia trait, and a Polish father who is hematologically normal. Since both the child and her father were heterozygous for a DNA polymorphism within the beta-globin gene, it was possible to clone and sequence the beta-globin gene identical by descent from both the child and her father. A nonsense mutation in codon 121 (GAA----TAA) was found in the beta-globin gene of the child, while the same gene from her father lacked this mutation and was normal. This mutation has not been previously observed among over 200 beta-thalassemia genes characterized in Caucasians. Since the mutation eliminates an EcoRI site in the beta-globin gene, we could show that the mutation is not present in genomic DNA of the father. To rule out germinal mosaicism, sperm DNA of the father was also digested with EcoRI, and the mutant EcoRI fragment was not observed under conditions that would detect the mutation if it were present in at least 2% of sperm cells. Routine HLA and blood group testing supported stated paternity. In addition, studies with 17 DNA probes that detect multiple allele polymorphisms increased the probability of stated paternity to at least 10(8):1. These data provide evidence that the G----T change in codon 121 of the beta-globin gene in the child is the result of a spontaneous mutation that occurred during spermatogenesis in a paternal germ cell.     

Identification of five rare mutations including a novel frameshift mutation causing beta zero-thalassemia in Thai patients with beta zero-thalassemia/hemoglobin E disease.

6 out of 14 uncharacterized beta-thalassemia alleles from 187 Thai beta-thalassemia/HbE patients were identified by direct sequencing of DNA amplified by polymerase chain reaction. A novel mutation occurring from an insertion of adenosine in codon 95, which results in a shift of the reading frame with terminator at the new codon 101, was detected in one patient. In addition, two frameshift mutations not previously reported among the Thai population were also detected in 3 patients: one with a deletion of thymidine in codon 15 and two with an insertion of cytidine in codons 27/28. A frameshift mutation that occurred from a cytidine deletion in codon 41 was also found in one patient in this study. The remaining case was an amber mutation, GAG-TAG, in codon 43 in exon 2 of the beta-globin gene. These mutations bring the number of mutations known to be present in the Thai population to a total of 20, 15 of which were detected in beta-thalassemia/HbE patients.     

Testing of G----A mutation in position 110 of a minor intron of beta-globin genes in patients with thalassemia in Azerbaijan

A point mutation G-A in the 110 position of the beta-globin gene small intron has been revealed by cloning and sequencing from the material of a homozygote beta-thalassemia patient in Azerbaijan. In the present study two allele-specific oligonucleotide probes for testing the mutation have been synthesized. Assessment frequency of the mutation among the beta-thalassemia patients in Azerbaijan has been performed with the use of the amplified beta-globin gene fragments obtained by using the thermostable DNA-polymerase from T. thermophilus with the subsequent dot-hybridization in gel of the amplified material with the oligonucleotide probes. The possibility to test the mutation by hybridization of the oligonucleotide probes with the donors and beta-thalassemia patients restricted genomic DNA has been analyzed. Only one of 50 thalassemia alleles of beta-globin genes under study has been shown to possess the mutation mentioned.     

Scanning of beta-globin gene for identification of beta-thalassemia mutation in Romanian population

Beta-thalassemia is uncommon (0.5%) in the Romanian population, but it must be considered in the differential diagnosis of hypochromic anemia. The molecular characterization of beta-thalassemia is absolutely necessary for molecular diagnosis, as well as any genetic epidemiological study in this region. Molecular analyses consist of mutation detection by molecular scanning of beta-globin gene. This gene has 3 exons and 2 introns, involved in beta-thalassemic pathogenesis. Clinical application of DNA analysis on beta-thalassemic chromosomes allowed characterization of 29 persons with different beta-thalassemia mutations among 58 patients with anemia. The experimental strategy was based on sequential PCR amplification of most of the beta-globin gene and running on denaturing gradient gel electrophoresis of amplification products. Definitive characterization of mutations in samples identified with shifted DGGE patterns was performed ARMS-PCR and/or PCR-restriction enzyme analysis methods. Eight different beta-thalassemia alleles were identified, the most common being IVS I-110 (G-A) and cd 39 (C-T). Comparison of overall frequency of mutations in the neighboring countries, shows that these results are in the frame of overall distribution of these mutations in Mediterranean area, especially in Greece and in Bulgaria. Molecular diagnosis is useful for differentiating mild from severe alleles, for genetic counseling, as well as for mutation definition in carriers, identified by hematological analysis necessary for prenatal testing and genetic counseling.     

A beta-thalassemia lesion abolishes the same Mst II site as the sickle mutation.

Digestion of DNA from a patient with homozygous beta zero thalassemia from Calabria, Italy with the restriction endonuclease Mst II produced a pattern similar to the one obtained with sickle cell trait DNA in that the Mst II site at the beta 6 position on one chromosome was abolished. We cloned the DNA from this beta-thalassemia chromosome and performed sequence analysis. The deletion of a single nucleotide (A) at the GAG codon of the beta 6 position results in a frame shift and early beta-globin chain termination. This mutation occurs on a chromosome with a haplotype similar to two other Mediterranean beta-thalassemia lesions. The Mst II enzyme is useful for prenatal diagnosis of beta thalassemia in this population.     

Prenatal diagnosis of inherited hemoglobinopathies

In this paper we reviewed the different methods presently available for prenatal diagnosis of hemoglobin disorders and the impact of this technology in the control of beta-thalassemia in several Mediterranean populations. The vast majority of the inherited hemoglobinopathies can now be detected in the fetus by amniocyte or trophoblast DNA analysis. alpha-thalassemias, delta beta-thalassemias and gamma delta beta-thalassemias, which are usually caused by a gross structural rearrangement of the DNA, may be directly detected by Southern blot analysis. Only a few beta-thalassemia lesions are caused by gene deletion or affect a restriction recognition site and thus may be directly identified by this method. The major part of beta-thalassemia are due to single nucleotide substitution, small deletion or addition which do not alter a restriction recognition site. These mutations may be directly detected by complementary oligonucleotide probes. Alternatively, when normal or affected children are available, fetal diagnosis may be accomplished by linkage analysis with polymorphic restriction sites. Fetal blood analysis is used at present time for those cases presenting too late in the pregnancy for characterization of the molecular defect and in prospective parents in whom the defect is not known. Introduction of prenatal diagnosis in combination with carrier screening in several mediterranean populations led to a consistent reduction in the incidence of homozygous beta-thalassemia.     

Identification of the Chinese IVS-II-654 (C-->T) beta-thalassemia mutation in an immigrant Turkish family: recurrence or migration?

In this study we describe the Chinese IVS-II-654 (C-->T) beta-thalassemia mutation for the first time in an immigrant Turkish family living in Istanbul and originating from Xanthe, Greece. Four members of the family, representing 3 generations, are heterozygous for this mutation. A detailed family history demonstrated a Greek origin for members of 5 generations with no records of migration or consanguineous marriages. Analysis of polymorphic nucleotides located at the 5' end of the beta-globin chromosomes bearing the IVS-II-654 mutation in the family described carried the (AT)9(T)5 type of microsatellite sequence and the ACATCCCCA haplotype. These 2 haplotype components favor a non-Eastern Asian origin for this chromosome, hence suggesting an independent origin for the IVS-II-654 mutation described in this family.     

A humanized mouse model for a common beta0-thalassemia mutation

Accurate animal models that recapitulate the phenotype and genotype of patients with beta-thalassemia would enable the development of a range of possible therapeutic approaches. Here we report the generation of a mouse model carrying the codons 41-42 (-TTCT) beta-thalassemia mutation in the intact human beta-globin locus. This mutation accounts for approximately 40% of beta-thalassemia mutations in southern China and Thailand. We demonstrate a low level of production of gamma-globins from the mutant locus in day 18 embryos, as well as production of mutant human beta-globin mRNA. However, in contrast to transgenic mice carrying the normal human beta-globin locus, 4-bp deletion mice fail to show any phenotypic complementation of the knockout mutation of both murine beta-globin genes. Our studies suggest that this is a valuable model for gene correction in hemopoietic stem cells and for studying the effects of HbF inducers in vivo in a "humanized" thalassemic environment.     

Prenatal diagnosis of hemoglobinopathies: evaluation of techniques for analysing globin-chain synthesis in blood samples obtained by fetoscopy.

Three techniques for analysing hemoglobin synthesis in blood samples obtained by fetoscopy were evaluated. Of the fetuses studied, 12 were not at risk of genetic disorders, 10 were at risk of beta-thalassemia, 2 were at risk of sickle cell anemia and 1 was at risk of both diseases. The conventional method of prenatal diagnosis of hemoglobinopathies, involving the separation of globin chains labelled with a radioactive isotope on carboxymethyl cellulose (CMC) columns, was compared with a method involving globin-chain separation by high-pressure liquid chromatography (HPLC) and with direct analysis of labelled hemoglobin tetramers obtained from cell lysates by chromatography on ion-exchange columns. The last method is technically the simplest and can be used for diagnosing beta-thalassemia and sickle cell anemia. However, it gives spuriously high levels of adult hemoglobin in samples containing nonlabelled adult hemoglobin. HPLC is the fastest method for prenatal diagnosis of beta-thalassemia and may prove as reliable as the CMC method. Of the 13 fetuses at risk for hemoglobinopathies, 1 was predicted to be affected, and the diagnosis was confirmed in the abortus. Of 12 predicted to be unaffected, 1 was aborted spontaneously and was unavailable for confirmatory studies, as were 3 of the infants; however, the diagnosis was confirmed in seven cases and is awaiting confirmation when the infant in 6 months old in one case. Couples at risk of bearing a child with a hemoglobinopathy should be referred for genetic counselling before pregnancy or, at the latest, by the 12th week of gestation so that prenatal diagnosis can be attempted by amniocentesis, safer procedure, with restriction endonuclease analysis of the amniotic fluid cells.     

Molecular genetic testing of beta-thalassemia patients of Indian origin and a novel 8-bp deletion mutation at codons 36/37/38/39

Hemoglobinopathies are the most commonly inherited genetic disorders in India. Certain communities in India have a high predisposition to beta-thalassemia. To offer prenatal diagnosis and to prevent the birth of an affected child, mutation testing in clinically diagnosed beta-thalassemia patients/carriers is a prerequisite. Over a period of 4 years, we have conducted DNA analysis in 385 carriers for 15 beta-thalassemia mutations, HbD, HbE, and HbS. Using reverse dot blot (RDB) and amplification refractory mutation system (ARMS), we have been able to identify mutations in 381 of 385 thalassemia chromosomes. The study included the analysis of five common mutations found in Asian Indians, namely IVS1-5 (G-C), 619-bp deletion, IVS1-1 (G-T), and the frameshifts at CD8/9(+G) and CD41/42(-CTTT). The occurrence of these five mutations was seen in 299 (91.2%) carriers referred to us, the percentage of mutations varying between 4.0 and 68.9%. We also found Cd16 (-C) in 2.1%, CD30 (G-C) in 1.5%, and CD 15(G-A) in 0.6%; these are considered common mutations in the Indian population, as well. The beta-thalassemia anomaly in 4 (0.6%) carriers remained uncharacterized by RDB and ARMS analysis. During delineation of the mutations in uncharacterized carriers by single-stranded conformational polymorphism (SSCP) and sequencing analysis, we have also been able to identify two unusual mutations, one involving an initiation codon and the second involving a novel 8-bp deletion, in Indian families of Uttar Pradesh origin.     

Erroneous diagnosis of iron deficiency anemia

Haematological data on children with mild iron deficiency-anaemia are compared with those of patients with heterozygous beta-thalassemia. The differential diagnosis of beta-thalassemia minor may suspected on the grounds of the blood smear. Confirmation of the diagnosis is based on the MCV, HbA2 and the graphic determination of EVR50 as well as by family survey. With these simple methods beta-thalassemia minor may be diagnosed with reasonable certainty even in the absence of a special laboratory for the determination of the beta-chain deficiency of hemoglobin. The importance of the correct differential diagnosis is stressed because of the danger of unnecessary iron therapy in thalassemia.     

A new codon 31 (-C) mutant resulting in beta zero-thalassemia.

A new beta zero-thalassemia mutation, a frameshift mutation with deletion of a single cytosine nucleotide in codon 31, is described. The propositus, which is compound heterozygous for this mutation and the 17 beta A-T beta zero-thalassemia mutation, has the phenotype of severe beta-thalassemia major.