Beta-thalassemia in the Korean population

Beta-Thalassemia is uncommon in the Korean population, however, 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. We analyzed the molecular basis of beta-thalassemia in 38 Korean families. Using direct sequencing of genomic DNA amplified through polymerase chain reaction and haplotype analysis, 35 beta-thalassemic genes were characterized, all of which were heterozygous. Twelve different mutations were identified. The most common mutations noted included the initiation codon ATG-->AGG (beta0) (28.9%), codon 17 (beta0) (A-->T) (18.4%), and IVS-II-1 (beta0) (G-->A) (10.5%). Interestingly, mutations causing dominantly inherited beta-thalassemia were also common (15.7%). All four cases with the IVS-II-1 (G-->A) mutation were linked to the silent mutation of codon 91 (C-->T) of the beta-globin gene. The initiation codon A7G-->AGG and IVS-II-1 (G-->A) with codon 91 (C-->T) mutations were found in the Far East only, and may be inherited from a common origin for each mutation, at least in Koreans. The codon 17 (A-->T) and codons 41/42 (beta0) (-TTCT) were suggested to be introduced by gene-flow from southern China. Otherwise, only Hb Korea [codons 33/34 (beta0) (-GTG)] and a novel beta-thalassemic mutation, codons 89/90 (beta0) (-GT), were identified in Koreans. This mutation spectrum is characteristic of the low prevalence area of beta-thalassemia in Korea, it is, however, quite different from the adjacent countries, Japan and China.     

Compound heterozygosity of Hb D(Iran) (beta(22) Glu-->Gln) and beta(0)-thalassemia (619 bp-deletion) in India

The present report describes the hematologic and molecular study of the second case of Hb D(Iran) associated with beta(0)-thalassemia (619 bp-deletion) found in India and the first case in which the mutations have been identified at molecular level. The patient showed hypochromic, microcytic red cell picture with reduced red cell indices. The characterization of the hemoglobinopathy was made by electrophoretic and chromatographic techniques and confirmed by sequencing of the beta-globin gene. Both the propositus and her father were found to be carriers of the gene for beta(0)-thalassemia owing to the 619 bp-deletion mutation as seen by the polymerase chain reaction (PCR). Single base substitution GAA > CAA (indicative of Hb D(Iran)) in the heterozygous form was seen in the propositus as well as the mother by sequencing.     

Spectrum of beta-globin gene mutations among thalassemia patients in the West Bank region of Palestine

beta-Thalassemia (thal) is an autosomal recessive disorder that results in hypochromic hemolytic anemia in affected patients. In the West Bank area of Palestine, the prevalence of beta-thal trait is approximately 3.5% among the population, with an estimated 120,000 carriers. Seventeen beta-globin gene mutations could be identified in 148 patients using polymerase chain reaction (PCR), amplification refractory mutation system (ARMS)-PCR and direct sequencing. The predominant mutations included: IVS-I-6 (T --> C) (28.7%), IVS-I-110 (G --> A) (17.6%), codon 37 (G --> A) (10.4%), IVS-I-1 (G --> A) (9%), codons 106/107 (+ G) (6.8%) and codon 39 (C --> T) (4.6%). Other less frequent and rare mutations included: IVS-II-1 (G --> A), codon 5 (-CT), IVS-II-848 (C --> A), -30 (T --> A), codons 8/9 (+ G), IVS-I-5 (G --> C), -28 (A --> C), IVS-II-745 (C --> G), codon 6(-A), codon 27 (G --> T) and codon 30 (AGG --> ACG). Most patients (62.2%) were homozygous for one type of mutation, while the rest (27.3%) were compound heterozygotes. Some patients were heterozygous for beta-thal and sickle cell anemia traits. No mutations could be detected in both alleles of eight patients, while in seven patients only one mutant allele could be detected. Further investigations are needed to resolve the corresponding genotypes of these patients. This study represents a comprehensive investigation of the type, frequency, and distribution of thalassemia mutations among the Palestinian population in the West Bank region of Palestine. A degree of similarity and significant variations was evident in the type and frequency of mutations when the present mutations profile was compared with similar ones among various Arab and non Arab populations. The association between the identified mutations and the corresponding genotypes of our patients with specific polymorphism frameworks in the beta-globin gene was performed and the results revealed linkage disequilibrium.     

A novel beta-Thalassemic allele due to a two nucleotide deletion: beta76 (-GC)

We have identified and characterized a novel beta-thalassemic mutation in a North African adult. The molecular defect consists of a two nucleotide (nt) deletion in the beta-globin gene at codon 76 [beta76 (-GC), c.229-230delGC]. This frameshift mutation generates a TGA stop codon at position 89. The carrier presented with mild microcytic anemia (Hb 12.8 g/dL, MCV 60 fL), no detectable Hb F, an elevated Hb A2 level (5.5%) with no other mutation in the beta-globin gene and none of the more common known deletions in the alpha-globin cluster. No abnormal hemoglobin (Hb) was present in routine electrophoresis or in high performance liquid chromatography (HPLC) analyses. Pathologic inclusions were absent in both mature red cells and in reticulocytes. This observation reinforces the hypothesis that nonsense and frameshift mutations that result in a premature stop codon in exon 1 or exon 2 inherited in the heterozygous state do not generate dominant beta-thalassemia (thal). This is the first example of a premature stop codon at position 89.     

Hb Amsterdam [alpha32(B13)Met--Ile (alpha2)]: a new unstable variant associated with an alpha-thalassemia phenotype and a new African polymorphism

We have characterized a new abnormal hemoglobin (Hb) at position 32 of the alpha-globin chain. The proband, a 38-year-old woman of Surinamese Black ancestry, was referred to the Academic Hospital in Amsterdam, The Netherlands, after 3 years of Prednisone treatment in Surinam. Kidney failure was diagnosed at the Nephrology Department, Free University Medical Center, Amsterdam, The Netherlands; the cortisone treatment was interrupted and dialysis was started. At this stage, a microcytic hypochromic anemia was observed with high reticulocyte (40%) and ferritin (500 microg/L) levels, and hemoglobinopathy was suspected. No abnormal bands were visible on alkaline electrophoresis and high performance liquid chromatography (HPLC). The Hb A2 level was normal (2.7%) and the erythrocyte count was low (3.59 x 10(12)/L) with a normal haptoglobin level (68 mg/100 mL). None of the common alpha-thalassemia (thal) deletion defects were present. The beta-globin gene sequence was normal but the alpha2-globin gene sequence revealed an ATG-->ATA transition at codon 32, changing the methionine into an isoleucine residue. The mutation, called Hb Amsterdam, was observed in the mother of the proband, who was also heterozygous for the--alpha3.7-thal deletion and affected by a moderate microcytic hypochromic anemia. Both Hb Amsterdam and the--alpha(-3.7) allele were found in association with a new polymorphism, IVS-I-39 (C-->T), previously observed in our laboratory in seven patients of African origin, on both the alpha1 and alpha2 genes. In addition, Hb Amsterdam was also associated with the common African alpha2 polymorphism (G-->CTCGGCCC at position 7238 and T-->G at position 7174). Hb Amsterdam is the first mutation ever described at codon alpha32, a position involved in alpha1/beta1 interaction. The possibility of a contribution of this mutation to the nephropatic state of the proband is discussed.     

The IVS-II-1 (G-->a) beta0-thalassemia mutation in cis with HbA2-Troodos [delta116(G18)Arg-->Cys (CGC-->TGC)] causes a complex prenatal diagnosis in an Iranian family

The beta-thalassemia (thal) minor phenotypes with normal Hb A2 levels and decreased MCV and MCH values are relatively rare beta-thal traits. Here, we describe a family with normal Hb A2 and decreased MCV and MCH levels. Amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) revealed the IVS-II-1 (G-->A) mutation in the beta-globin gene of the proband and her father. Direct sequencing of the gamma-globin gene of the proband and her father also revealed a previously reported variant called Hb A2-Troodos [gamma116(G18)Arg-->Cys] [in cis with the IVS-II-1 (G-->A) beta0-thal mutation]. This is the first case report of Hb A2-Troodos in association with the beta0 IVS-II-1 mutation. Reduced Hb A2 expression by a concomitant Hb A2 beta-thal in cis or trans, may cause problems in carrier diagnostics, and eventually in genetic counseling and prenatal diagnosis when insufficient molecular analyses are performed.     

Compound heterozygosity for Hb S [beta6(A3)GluVal, GAG-->GTG] and a new thalassemic mutation [beta132(H10)Lys-->term, AAA-->TAA] detected in a family from West Africa

We describe a Hb S/beta-thalassemia (beta-thal) mutation involving an AT transition at codon 132 of the beta-globin gene. The mutation, in the heterozygous state, unlike several other mutations in exon 3, shows no signs of dominant thalassemia but those of a typical beta(0) carrier. Compound heterozygosity with Hb S [beta6(A3)GluVal, GAGGTG] showed a severe clinical picture.     

Hb Suan-Dok [alpha109(G16)Leu-->Arg; CTG-->CGG (alpha2)] described in a patient of African ancestry

A 58-year-old Black female from Cura?ao (West Indies) was recently referred to our Laboratory for a persistent microcytic hypochromic anemia. An analysis 13 years earlier had shown no abnormal hemoglobin (Hb) fractions and a balanced beta/alpha synthetic ratio. The hematological indices were again compatible with thalassemia and no abnormal fractions were observed on electrophoresis or high-performance liquid chromatography (HPLC). None of the seven common alpha-thalassemia (thal) deletion defects were present. Direct sequencing of the alpha2 gene revealed a CTG-->CGG single base substitution at codon 109. This mutation was previously described in a Thai patient (Hb Suan-Dok), inducing Hb H disease in association with a - -(SEA) allele. In contrast with earlier reports we were unable to identify any native Hb fraction. The balanced beta/alpha ratio indicated that alpha2-Suan-Dok is formed but does not form tetramer formation unless alpha-thal is present.     

Distribution of beta-thalassemia mutations in the northern provinces of Iran

Beta-Thalassemia (thal) is one of the most common autosomal recessive disorders in Iran. There are more than two million carriers of beta-thal and over 15,000 people affected with beta-thal major who live in Iran. Prevalent mutations were identified by examining genomic DNAs isolated from 392 blood samples of beta-thal carriers from three northern provinces of Iran. Furthermore, 172 pregnant women were analyzed from the 196 couples who requested pregnant diagnosis for beta-thal. Allele identification was carried out using routine reverse dot-blot, amplification refractory mutation system (ARMS), and genomic sequencing. The most common mutation, IVS-II-1 (GA), is followed, in order of frequency, by codon 30 (GC), frameshift codons (FSC) 8,9 (+G), FSC 22/23/24 (-AAGTTGG), IVS-I-110 (GA), IVS-I-5 (GC), IVS-II-745 (CG), IVS-I-2 (TC), FSC 8 (-AA), IVS-I,3'-end (-25 bp), IVS-I-1 (GA), FSC 36/37 (-T), IVS-I-6 (TC), FSC 5 (-CT), -28 (AC), codon 37 (GA), IVS-II-2,3 (+11/-2), -30 (TA), and -88 (CA). We have also revealed the existence of five new mutations from northern Iran, one of which (codon 37) is the first reported for Iran. Furthermore, the rate of unknown mutations is significantly reduced in our study (about 6%). These results could help with establishing a center for prenatal diagnosis, prevention, and control of thalassemia in the northern provinces of Iran.     

Dominantly Inherited beta-Thalassemia

Dominantly inherited beta-thalassemia (thal) or "inclusion body beta-thalassemias" are heterogeneous at the molecular level and are due to mutations at or near the beta-globin gene locus. Many of these involve mutations of exon 3 of the beta-globin gene. They include frameshifts, premature chain termination (nonsense) mutations, and complex rearrangements that lead to the synthesis of truncated or elongated and highly unstable beta-globin gene products. The resulting beta chain variants are very unstable, and in many cases, the products of the dominantly inherited beta-thal are not detectable. Hematological and clinical observations made in several families with comparable forms of beta-thal and with certain highly unstable hemoglobin (Hb) variants, have indicated a striking overlap; many subjects with detectable unstable Hb variants and with a dominant type of beta-thal without a detectable abnormal Hb, have similar phenotypes. Here, a review of dominantly inherited beta-thal is given, and new examples of hyperunstable Hbs (Hb Stara Zagora and Hb Jambol) are presented. The first example is a hyperunstable variant named Hb Stara Zagora that was found in a 2-year-old Bulgarian boy. The abnormal Hb is associated with severe hemolytic anemia as a consequence of its hyper instability. The anemia was noticed at the age of 2 months and since then he has been on a regular monthly blood transfusion regimen. Hemoglobin analysis revealed no abnormalities, except the presence of inclusion bodies. Sequencing of the beta-globin gene revealed a heterozygosity for a 6 bp deletion (-TGGCTA) at codons 137 (the second and third bp), 138 and 139 (the first bp), thus forming a new codon at position 139 (GAT). This event eliminates three amino acids (Val-Ala-Asn) and introduces a new residue (Asp). It creates a new restriction site for HphI. The parents and his twin brother had no history of hemolysis. The paternity of the child was confirmed by DNA analysis. The second example is a new hyperunstable variant named Hb Jambol, found as a de novo mutation in a 2-year-old Bulgarian girl with severe hemolytic anemia. The mutation was detected through RNA/DNA analysis. It represents a complex genomic rearrangement involving an insertion of 23 nucleotides (nts) after IVS-II-535, a deletion of 310 nts extending from IVS-II-550 to the first nt of codon 108, and an insertion of 28 nts at the deletion junctions (derived from inverted sequence between nts +3707 and +3734 3' to the beta-globin gene termination codon). At the protein level, this mutation leads to a deletion of four amino acid residues (Leu-Leu-Glu-Asn) at positions 105, 106, 107 and 108, and an insertion of nine residues (Val-Pro-Ser-Val-Thr-Leu-Phe-Phe-Asp) at the same location, creating an abnormal elongated beta chain of 151 amino acid residues. The parents had no history of hemolysis. The paternity of the child was confirmed by DNA analysis.     

Globin chain synthesis is a useful complementary tool in the differential diagnosis of thalassemias

The present study aimed at differentiating rare types of heterozygous beta-thalassemia (thal) with normal Hb A(2) values from alpha-thal in Iranian carriers by globin chain synthesis in addition to other hematological parameters. Our study groups consisted of 51 normal subjects, 24 heterozygous beta- thalassemic subjects with high Hb A(2), 62 alpha-thal-2 subjects, 34 alpha-thal-1 subjects, six Hb H disease thalassemic subjects, 14 silent beta-thal subjects with normal Hb A(2) values, five deltabeta-thal subjects and two subjects with an association of alpha- and deltabeta-thal (total = 198). Analysis of globin chains was performed by high performance liquid chromatography (HPLC). The results showed that the alpha/beta ratio averages were close to the ones in the published literature, but with a greater standard deviation and a wider range. Globin chain synthesis (GCS) could be valuable in differentiating between microcytosis produced by silent beta-thal (heterozygous beta-thal with a normal Hb A(2) level) and that caused by alpha-thal. Since the complex genotype/phenotype relationship can lead to diagnostic difficulties, GCS cannot be used as the only diagnostic tool for thalassemia carrier detection. Therefore, a combination of different tests for each patient is required.     

Diagnosis of concurrent hemoglobin H disease and heterozygous beta-thalassemia

Definitive diagnosis of concurrent hemoglobin (Hb) H disease and heterozygous beta-thalassemia cannot be made from Hb analysis alone, but necessitates genotype analysis and family study. Interactions between alpha- and beta-thalassemia must be considered when investigating moderate to severe hypochromic microcytic anemia of uncertain cause in adult patients from areas with a high prevalence of globin gene mutations.     

Beta-thalassemia in the Korean population

Beta-thalassemia is uncommon in the Korean population, however 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. We analyzed the molecular basis of beta-thalassemia in 47 Korean families. Using direct sequencing of genomic DNA amplified through PCR and haplotype analysis, 44 beta-thalassemia genes were characterized, all of which were heterozygous. Fourteen different mutations were identified. The common mutations noted included the initiation codon (CD) ATG-->AGG (23.4%), CD 17 A-->T (21.2%), and IVS-II-1 G-->A (12.7%). Interestingly, mutations causing dominantly inherited beta-thalassemia were common (17.0%). All cases of IVS-II-1 G-->A mutations were linked to the silent mutation of CD 91 C-->T of the -globin gene. The initiation CD ATG-->AGG and IVS-II-1 G-->A with CD 91 C-->T were found in the Far East only, and may be inherited from a common origin for each mutation, at least in Koreans. CD17 A-->T and CDs 41/42-TTCT were suggested to be introduced by gene-flow from southern China. Otherwise, Hb Korea, CDs 89/90 -GT and a novel beta-thalassemia mutation, CD 131 CAG-->TAG, were only identified in Koreans. This mutation spectrum is characteristic of the low prevalent area of beta-thalassemia, however it is quite different even from the adjacent countries, Japan or China.     

Molecular spectrum of beta-thalassemia mutations in Northwestern Iran

Beta-thalassemia (beta-thal) is a hereditary autosomal disorder with decreased or absent beta-globin chain synthesis. This study was designed to identify the common and rare beta-thal mutations in the Azerbaijan provinces, Northwestern Iran, and to set up a prenatal diagnostic laboratory. One hundred unrelated patients with known beta-thal major and intermedia, registered with the thalassemia clinics in the provincial capitals of Tabriz and Ardebil, were included. Mutations were studied in 200 chromosomes, by polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) and direct sequencing methods. We found 17 beta-thal mutations in this region of Iran. The results showed that IVS-II-1 (-->GA) was the most frequent mutation, comprising 21% of all mutations. Other common mutations were IVS-I-110 (-->GA) 18%, frameshift codons (FSC) 8/9 (+G) 14.5%, FSC 8 (-AA) 8% and IVS-I-1 (GA) 7.5%. This is the first comprehensive study in this region and could be useful for developing a beta-thal molecular screening in Azerbaijan-Iran.     

The rare Hb Showa-Yakushiji [beta110(G12)Leu-->Pro, CTG-->CCG] in combination with an alpha gene triplication found in a Dutch patient during her first pregnancy examination

We report a semi dominant beta-thalassemia (thal) phenotype caused by the rare Hb Showa-Yakushiji [beta110(G12)Leu-->Pro, CTG-->CCG] mutation in combination with an alpha gene triplication. This combination of two rare mutations was observed during hemoglobinopathy carrier diagnostics in a 26-year-old Dutch female at 9 weeks gestation, at the first pregnancy examination in the midwives practice. The partner was promptly examined and no abnormalities were found. The beta-thal trait was diagnosed by a standard high performance liquid chromatography (HPLC) procedure showing a normal separation but an elevated Hb A(2) level of 5.9% in the presence of pronounced hypochromic microcytic parameters and mild chronic hemolysis. Direct sequencing of the beta-globin genes was subsequently performed revealing a CTG-->CCG transition at codon 110. This rare mutation was previously described as two independent events in a few Japanese and Indian individuals. The mutation induces a Leu-->Pro substitution and the gene product is highly unstable. Gap-polymerase chain reaction (gap-PCR) revealed a heterozygosity for the alpha gene triplication as well. The excess of alpha-globin chains contributed only marginally to the hematological abnormalities of the patient and did not aggravate the phenotype to an intermediate level.     

Alpha-thalassemia phenotype induced by the new IVS-II-2 (T --> A) splice donor site mutation on the alpha2-globin gene

We present a family of North European extraction referred for a refractory non iron depleted microcytic anemia. The proband, a 36 year-old male, presented with chronic borderline anemia and microcytic hypochromic parameters. No abnormal hemoglobin (Hb) fractions were observed on high performance liquid chromatography (HPLC) or on alkaline electrophoresis. Gap-polymerase chain reaction (gap-PCR) excluded the seven common alpha-thalassemia (thal) deletion defects. However, the beta/alpha-globin chain synthesis ratio measured in vitro was unbalanced, indicating a reduced expression of the alpha-globin genes. Direct sequencing of the alpha-globin genes revealed heterozygosity for a T --> A transversion at the IVS-II-2 position of the alpha2 gene. This is the first IVS-II splice donor site mutation described on the alpha2-globin gene.     

Adult onset of a Thalassemia intermedia genotype in association with a -alpha-3.7 homozygosity. Hb G-Accra [beta73(e17)Asp-->Asn] in combination with beta- and alpha-thalassemia in the same family

We present the case of a 39-year-old male of mixed Black and Chinese Surinamese origin referred because of abdominal pain and extreme tiredness. The patient reported that he had received a single blood transfusion in his youth and presented at intake with a severe microcytic hypochromic anemia. A chest X-ray and computer tomography (CT)-scan revealed bilateral mediastinal lymphadenopathy and interstitial infiltrates. Elevated Hb F (80%) and an unbalanced synthesis ratio (beta/alpha = 0.18) were compatible with severe beta-thalassemia (thal) intermedia. DNA analysis revealed a double heterozygoty for the -88 (C-->T) and the IVS-II- 654 (C-->T) mutations in the presence of a homozygosity for the -alpha3.7 deletion. The two daughters of the proband were both heterozygous for the IVS-II-654 (C-->T) mutation and the -alpha3.7 deletion. The youngest daughter also carried the Hb G-Accra [beta73(E17)Asp-->Asn] mutation, inherited from the mother. Hb G-Accra, a mutant of presumed Ghanaian origin, described as non pathological in the carrier, is reported for the first time in combination with a severe fbeta(+)thal. The molecular background, haplotype of the mutations and a new A--> polymorphism at -309, 5' to the G(gamma) romoter, are reported.     

Beta-thalassemia due to a novel nonsense mutation at codon 37 (TGG-->TAG) found in an Afghanistani family

We have identified and characterized a novel beta-thalassemic mutation in an Afghanistanifamily. The molecular pathology consists of a single base substitution (TGG-->TAG) at codon 37 of the beta-globin gene, giving rise to a stop codon (TAG). Premature stop of translation results in a truncated protein and usually the phenotype of beta-thalassemia (thal) major in homozygous individuals. However, this was not the case in our proband, who was homozygous for the codon 37 mutation. He presented with the phenotype of thalassemia intermedia with a hemoglobin (Hb) level of 8.1 g/dL and no previous history of blood transfusions. High performance liquid chromatography (HPLC) analysis showed exclusively Hb F except for a Hb A2 level within normal limits. Subsequent analysis demonstrated homozygosity for the XmnI Ggamma polymorphism and heterozygosity for a deletional alpha-thal (alphaalpha/-alpha(-3.7)). These findings might, at least partly, explain the beta-thal intermedia phenotype observed in the proband.     

α-Thalassemia Phenotype Induced by the New IVS-II-2 (T→A) Splice Donor Site Mutation on the α2-Globin Gene

We present a family of North European extraction referred for a refractory non iron depleted microcytic anemia. The proband, a 36-year-old male, presented with chronic borderline anemia and microcytic hypochromic parameters. No abnormal hemoglobin (Hb) fractions were observed on high performance liquid chromatography (HPLC) or on alkaline electrophoresis. Gap-polymerase chain reaction (gap-PCR) excluded the seven common α-thalassemia (thal) deletion defects. However, the β/α-globin chain synthesis ratio measured in vitro was unbalanced, indicating a reduced expression of the α-globin genes. Direct sequencing of the α-globin genes revealed heterozygosity for a T→A transversion at the IVS-II-2 position of the α2 gene. This is the first IVS-II splice donor site mutation described on the α2-globin gene.