Beta-thalassemia intermedia from southern Iran: IVS-II-1 (G-->A) is the prevalent thalassemia intermedia allele

The preliminary results of a pilot study are reported, intended as an initiation of a research plan, focused on the prevention of beta-thalassemia in Iran. The aims of this study are: (i) to improve the knowledge of the molecular background of beta-thalassemia intermedia in Southern Iran; (ii) to verify the role of the -158 (G)gamma (C-->T) (Xmn I) polymorphism as a modulating factor in thalassemia intermedia; (iii) to test the validity of the multiplex and single mutation specific amplification refractory mutation system in analyzing the molecular defects causing beta-thalassemia in multiethnic populations; and (iv) to develop suitable strategies for the application of prevention protocols in Iran. To accomplish the task we have selected 87 beta-thalassemia intermedia patients and adapted the DNA methodology to detect the following 11 frequent mutations in Iran: codon 5 (-CT); frameshift codons (FSC) 8/9 (+G); codon 30 (G-->C); IVS-I-1 (G-->A); IVS-I-5 (G-->C); IVS-I-6 (T-->C); IVS-I-110 (G-->A); codons 36/37 (-T); codon 44 (-C); IVS-II-1 (G-->A); IVS-II-745 (C-->G). Because of the multiethnicity of the population we have also included the Indian IVS-I (25 bp deletion) and the Mediterranean IVS-I-130 (G-->C) and codon 39 (C-->T) mutations. Forty-eight patients were randomly studied for the Xmn I polymorphism together with 50 healthy volunteers as a control group. The molecular analysis conducted in Iran, identified only 31% of the alleles that were presumed to be thalassemic, revealing either a strategic or a technical insufficiency of the chosen method. However, the mutations with the highest prevalence in the country (IVS-II-1, IVS-I-110, IVS-I-1 and FSC 8/9) were found. As expected the IVS-II-1 defect, being the most frequent in south Iran, was present at the highest rate (24%). The Xmn I polymorphism was found in association with this prevalent mutation and was detected in the homozygous state in 87.5% of the patients homozygous for the IVS-II-1 (G-->A) mutation. The overall positivity for Xmn I was found in 40.6% of the thalassemic alleles vs. 14% in the non-thalassemic, confirming the hypothesis of an older event, antecedent to the IVS-II-1 mutation. In trying to assess a more suitable molecular detection method we intend to continue this study in collaboration with the European centers involved, applying more effective technologies and better defining the molecular spectrum of beta-thalassemia in the sub-populations. We also intend to verify the effect of alpha-thalassemia in the genotype/phenotype correlation of beta-thalassemia intermedia.     

Molecular basis of thalassemia syndromes in Serbia and Montenegro

This study reports the molecular characterization of thalassemia syndromes in Serbian and Montenegrin populations. We identified eight beta-thalassemia mutations [codon 39 (C-->T), IVS-I-110 (G-->A), IVS-II-745 (C-->G), codon 44 (-C), -87 (C-->G), IVS-II-1 (G-->A), IVS-I-6 (T-->C), IVS I-1 (G-->A)] in 70 members of 29 families using polymerase chain reaction, reverse dot blot, amplification refractory mutation system and direct sequencing analysis. Hemoglobin (Hb) Lepore was found to be the most common cause of the thalassemia phenotype. Hb Sabine and alpha-thalassemia were detected as well. We also studied beta-globin gene cluster haplotypes and their association with the most common mutations. A novel haplotype associated with the Hb Lepore gene was identified. The results presented herein allowed the implementation of a prenatal diagnosis program in Serbia and Montenegro.     

Molecular basis of beta-thalassemia in the population of Tunisia

The present study attempts to delineate the spectrum of beta-thalassemia (thal) mutations in Tunisia by studying a large population from different parts of the country. A total of 285 unrelated subjects, 190 of whom had beta-thal major, 72 with Hb S/beta-thal, one with Hb C/beta-thal, one with Hb O-Arab/beta-thal and 21 beta-thal carriers, were studied. The molecular defects were detected in 97.7% of the beta-thalassemic chromosomes (n=475). Nineteen different beta-thalassemic alleles were identified. Two mutations, namely codon 39 (C-->T) and IVS-I-110 (G-->A) accounted for 70.0% of the studied chromosomes, followed by IVS-I-1 (G-->A) (4.5%). Five other mutations, frameshift codon (FSC) 44 (-C), codon 30 (G-->C), IVS-I-2 (T-->G), IVS-II-745 (C-->G), and FSC 6 (-A), are not uncommon in this population, while the remaining 11 mutations, IVS-I-5 (G-->A), -30 (T-->A), codons 25/26 (+T), IVS-I-6 (T-->C), FSC 5 (-CT), IVS-II-848 (C-->A), FSC 8 (-AA), -87 (C-->G), IVS-I-5 (G-->C), IVS-II-1 (G-->A) and IVS-II-849 (A-->C) are quite rare; four of these have not been previously reported in the Tunisian population. Potential origin and spread of these mutations to Tunisia are also discussed.     

The β-thalassaemia mutations in the population of Cyprus

We have identified the beta-thalassaemia alleles in nearly all known Turkish Cypriot beta-thalassaemia homozygotes and in over 700 Greek Cypriot beta-thalassaemia heterozygotes living on the island of Cyprus. The data confirmed earlier observations that the IVS-I-100 (G-->A) mutation is present for about 74-80%, while three other alleles [IVS-II-745 (C-->G), IVS-I-6 (T-->C), IVS-I-1 (G-->A)] occur at frequencies of 5-8%. Nearly identical percentages were observed for the two Cypriot groups, quite different from those for beta-thalassaemia patients from Greece and Turkey. This suggests close contacts between the two Cypriot communities during many centuries without a major recent influence from Greek or Turkish beta-thalassaemia carriers.     

Clinical, hematological, and molecular features in Sicilians with Hb S-beta-thalassemia.

The clinical, hematological, and molecular features of 81 patients with Hb S-beta-thalassemia and relatives from 76 unrelated families are reported. We analyzed the beta-thalassemia mutations and the beta S haplotypes in all patients and detected 6 different beta-thalassemia alleles: codon 39 (C-->T) (39 cases), IVS-I-1 (G-->A) (12 cases), IVS-II-1 (G-->A) (4 cases), IVS-I-6 (T-->C) (6 cases), IVS-I-110 (G-->A) (14 cases), and IVS-II-745 (G-->C) (6 cases). Eighty patients had haplotype #19 or the Benin type and one had haplotype #17 or the Cameroon type. The type of beta-thalassemia allele had the greatest influence on the phenotypic expression; this was observed for patients with Hb S-beta-thalassemia and for simple beta-thalassemia heterozygotes. The mild IVS-I-6 (T-->C) mutation produced borderline abnormal erythrocytic indices and Hb A2 levels in heterozygotes. Overall, there was a milder expression in beta(S) beta(+) patients (only 7.7% presented severe disease) than in those with the beta(S)beta(0) condition (22.6% had the severe form of the disease).     

Contribution to the description of the beta-thalassemia spectrum in Tunisia and the origin of mutation diversity

We determined the spectrum of beta-thalassemia (thal) mutations in 118 affected unrelated patients with different forms of beta-thal. Using a combination of reverse dot-blot analysis, denaturing gradient gel electrophoresis (DGGE), polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) and direct nucleotide sequencing, we identified the largest spectrum of beta-thal mutations so far reported in Tunisia, and to the best of our knowledge, within the Mediterranean Basin. A total of 18 distinct alleles were detected at different frequencies, with two alleles [codon 39 (C-->T) and IVS-I-110 (G-->A)] predominating all others. Seven other alleles [frameshift at codon (FSC) 6 (-A), FSC 8 (-AA), codon 30 (G-->C), IVS-I-1 (G-->A), IVS-I-2 (T-->G), IVS-I-6 (T-->C), FSC 44 (-C)] were rare, and nine alleles [-29 (A-->G), IVS-I-2 (T-->C), IVS-I-5 (G-->C), IVS-I-5 (G-->T), IVS-I-116 (T-->G), codon 37 (G-->A), IVS-II-1 (G-->A), IVS-II-745 (G-->C) and IVS-II-849 (A-->C)], albeit described elsewhere, are reported here in Tunisia for the first time. The codon 39 and IVS-I-110 mutations were the two predominant alleles occurring at frequencies of 43.8% and 10.8%, respectively. They are presumably the earliest mutations introduced into this country. The codon 39 allele could have been introduced in Tunisia during the Roman occupation. Similarly, the IVS-I-110 mutation might have been introduced by the Turkish and Phoenician influence. Both gene flow and private mutations may account for the diversity of alleles observed in Tunisia. These data provide the background for implementing prevention programs based on genetic counseling and prenatal diagnosis.     

β-THALASSEMIA INTERMEDIA FROM SOUTHERN IRAN: IVS-II-1 (G→A) IS THE PREVALENT THALASSEMIA INTERMEDIA ALLELE

The preliminary results of a pilot study are reported, intended as an initiation of a research plan, focused on the prevention of β-thalassemia in Iran. The aims of this study are: (i) to improve the knowledge of the molecular background of β-thalassemia intermedia in Southern Iran; (ii) to verify the role of the ?158 ^Gγ (C→T) (Xmn I) polymorphism as a modulating factor in thalassemia intermedia; (iii) to test the validity of the multiplex and single mutation specific amplification refractory mutation system in analyzing the molecular defects causing β-thalassemia in multiethnic populations; and (iv) to develop suitable strategies for the application of prevention protocols in Iran. To accomplish the task we have selected 87 β-thalassemia intermedia patients and adapted the DNA methodology to detect the following 11 frequent mutations in Iran: codon 5 (?CT); frameshift codons (FSC) 8/9 (+G); codon 30 (G→C); IVS-I-1 (G→A); IVS-I-5 (G→C); IVS-I-6 (T→C); IVS-I-110 (G→A); codons 36/37 (?T); codon 44 (?C); IVS-II-1 (G→A); IVS-II-745 (C→G). Because of the multiethnicity of the population we have also included the Indian IVS-I (25?bp deletion) and the Mediterranean IVS-I-130 (G→C) and codon 39 (C→T) mutations. Forty-eight patients were randomly studied for the Xmn I polymorphism together with 50 healthy volunteers as a control group. The molecular analysis conducted in Iran, identified only 31% of the alleles that were presumed to be thalassemic, revealing either a strategic or a technical insufficiency of the chosen method. However, the mutations with the highest prevalence in the country (IVS-II-1, IVS-I-110, IVS-I-1 and FSC 8/9) were found. As expected the IVS-II-1 defect, being the most frequent in south Iran, was present at the highest rate (24%). The Xmn I polymorphism was found in association with this prevalent mutation and was detected in the homozygous state in 87.5% of the patients homozygous for the IVS-II-1 (G→A) mutation. The overall positivity for Xmn I was found in 40.6% of the thalassemic alleles vs. 14% in the non-thalassemic, confirming the hypothesis of an older event, antecedent to the IVS-II-1 mutation. In trying to assess a more suitable molecular detection method we intend to continue this study in collaboration with the European centers involved, applying more effective technologies and better defining the molecular spectrum of β-thalassemia in the sub-populations. We also intend to verify the effect of α-thalassemia in the genotype/phenotype correlation of β-thalassemia intermedia.     

Genetic heterogeneity of beta-thalassemia at Cukurova in southern Turkey

Beta-thalassemia is the most common genetic abnormality causing health problems worldwide. Cukurova, in the southern part of Turkey, being on the Mediterranean, is in the thalassemic belt. Since there is no cure for the disease at present, the frequency of the mutation types of beta-thalassemia must first be identified to aid in clinical follow-up and prenatal diagnosis. Carriers identified during a screening survey and patients referred to our laboratory were studied for this purpose. After routine hematological analysis molecular screening was performed by the amplification refractory mutation system and DNA sequencing. The frequency of the common mutations were: IVS-I-110 (G-->A) 57.3%, IVS-I-1 (G-->A) 8.3%, codon 39 (C-->T) 6.4%, IVS-I-6 (T-->C) 5.7%, frameshift codon 8 (-AA) 5.7%, -30 (T-->A) 4.7%, IVS-II-1 (G-->A) 3.4%, IVS-II-745 (G-->C) 2.8%, and frameshift codon 5 (-CT) 1.1%. Some rare mutations (1%) such as frameshift codon 44 (-C) 0.7%, frameshift codons 74/75 (-C) 0.7%, IVS-1-5 (G-->C) 0.7%, frameshift codons 8/9 (+G) 0.4%, frameshift codons 36/37 (-T) 0.4%, frameshift codons 22/23/24 (-AAGTTGG) 0.4%, IVS-1-130 (G-->C) 0.4%, IVS-1-5 (G-->T) 0.2%, -28 (A-->C) 0.2%, codon 15 (TGG-->TGA) 0.2%, and frameshift codons 82/83 (-G) 0.2%, were detected by sequence analysis. The codon 15 (TGG-->TGA) and frameshift codons 82/83 (-G) mutations were seen in Turkey for the first time.     

The beta+-IVS-I-6 (T-->C) mutation accounts for half of the thalassemia chromosomes in the Palestinian populations of the mountain regions

A study of the spectrum of beta-thalassemia mutations in the southern part of the West Bank of the Palestinian Authority revealed the presence of 10 different beta-globin mutations. The study included 41 patients and 54 carriers of beta-thalassemia and sickle cell anemia. The spectrum of mutations observed was typically Mediterranean. However, their relative frequencies was unique. The predominant allele was IVS-I-6 (T-->C), with an exceptionally high frequency of 48.5% for this mutation. The homozygous IVS-I-6 patients had widely variable clinical presentations, from typical transfusion-dependent thalassemia major to non-transfusion-dependent thalassemia intermedia phenotype. Since it is so widespread in these West Bank populations, the IVS-I-6 mutation may date back to ancient times. The nonsense mutation at codon 37 (G-->A) was found at a relatively high frequency of 11.3%, supporting the hypothesis that it originated in this region. The other mutations, at decreasing frequencies ranging from 9.5-1.5%, were: IVS-I-110 (G-->A), frameshift codon 5 (- CT), IVS-I-1 (G-->A), IVS-II-1 (G-->A), Hb S [beta6(A3)Glu-->Val], frameshift codons 8/9 (+G), codon 39 (C-->T), and -30 (T-->A). Our findings will improve health care for the Palestinian population, and also has implications for the study of the origin and spread of thalassemia in the Middle East.     

Study of beta-Thalassemia mutations using the polymerase chain reaction-amplification refractory mutation system and direct DNA sequencing techniques in a group of Egyptian Thalassemia patients

The aim of this study was the molecular characterization of beta-thalassemia (thal) mutations in a group of 95 Egyptian thalassemic patients from Fayoum in Upper Egypt, Cairo, Alexandria and Tanta in Lower Egypt and the Nile Delta. To identify these anomalies, the polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) technique was used, complemented by direct DNA sequencing for uncharacterized cases. In 80 of the 95 patients, the beta-thal mutation was detected by PCR-ARMS. The most common allele encountered in our study was IVS-I-6 (T-->C) (36.3%); the second most common mutation was IVS-I-110 (G-->A) (25.8%). In addition, we report three homozygous cases for the promoter region -87 (C-->G) allele with a frequency of 3.2%. DNA sequencing of uncharacterized cases (14 cases, 15 alleles) revealed six cases (six alleles) of codon 27 (G-->T), and three cases (three alleles) of the IVS-II-848 (C-->A) mutation. Codon 37 (G-->A) in the homozygous state was found in one patient with positive consanguinity. The frameshift codon 5 (-CT) mutation was detected in two of our uncharacterized cases. The codon 15 (TGG-->TGA) mutations was detected in one patient (one allele, 0.5%). All studied cases were fully characterized by this strategy. Screening for beta-thalassemic mutations using ARMS-PCR for the seven most frequent alleles in Egypt succeeded in determining the beta-globin genotype in 84.2% of our patients (91.6% of the expected alleles). To improve the efficiency of routine screening, the PCR-ARMS mutation panel should be updated to include the reported rare alleles. Direct DNA sequencing is an additional way to allow a full characterization of beta-thal patients in the Egyptian population.     

Genotypic correlation between six common beta-thalassemia mutations and the XmnI polymorphism in the Moroccan population

beta-Thalassemia (thal) is the most common recessive inherited disorder in Mediterranean populations. It is estimated that the frequency of this disease in the Moroccan population is between 1.5 and 3.0%. Severe forms of homozygous thalassemia cases require expensive and technically demanding curative (bone marrow transplantation) or palliative (chronic transfusion/chelation) therapies. The -158 (C-->T) polymorphism of the (G)gamma-globin gene (XmnI polymorphism) is known to ameliorate the severity of the disease because of it strong association with an increased production of fetal hemoglobin (Hb F). Among the many known mutations in Morocco, six are common [codon 39 (C-->T), frameshift codon (FSC) 8 (-AA), IVS-II-745 (CG), FSC 6 (-A), -29 (A-->G) and IVS-I-1 (G-->A)]. In this study, we have investigated, in 82 Moroccan beta-thalassemic chromosomes, the correlation between the six common mutations and the XmnI polymorphism using the Fisher exact test. The XmnI polymorphism was divided into two categories, (XmnI [+] and XmnI [-]) and the six common Moroccan mutations into two groups (group I with FSC 8 and group II without FSC 8). Correlation was carried out between the XmnI [+] category and the six common mutations individually that showed that 68% of chromosomes in the XmnI [+] category had the FSC 8 (-AA) mutation. The results reported here show that there is a positive correlation between the XmnI polymorphism and FSC 8 mutation in linkage with haplotype IV [- + - + + - +] (p <10(-5)). In conclusion, molecular determination of genetic markers in early childhood will help to identify candidates for pharmacological Hb F switching by hydroxyurea (HU). In the Moroccan population, a good response to HU treatment should be suspected in cases with the -158 (C-->T) polymorphism in linkage with haplotype IV and internal beta-globin gene framework 3.     

Distribution of β-Thalassemia Mutations in the Northern Provinces of Iran

β-Thalassemia (thal) is one of the most common autosomal recessive disorders in Iran. There are more than two million carriers of β-thal and over 15,000 people affected with β-thal major who live in Iran. Prevalent mutations were identified by examining genomic DNAs isolated from 392 blood samples of β-thal carriers from three northern provinces of Iran. Furthermore, 172 pregnant women were analyzed from the 196 couples who requested pregnant diagnosis for β-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 (G→A), is followed, in order of frequency, by codon 30 (G→C), frameshift codons (FSC) 8,9 (+G), FSC 22/23/24 (?AAGTTGG), IVS-I-110 (G→A), IVS-I-5 (G→C), IVS-II-745 (C→G), IVS-I-2 (T→C), FSC 8 (?AA), IVS-I,3′-end (?25 bp), IVS-I-1 (G→A), FSC 36/37 (?T), IVS-I-6 (T→C), FSC 5 (?CT), ?28 (A→C), codon 37 (G→A), IVS-II-2,3 (+11/?2), ?30 (T→A), and ?88 (C→A). 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.     

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.     

Identification of three rare beta-thalassemia mutations in the Pakistani population

Three rare beta-thalassemia mutations, not reported previously in Asian Indians or the Pakistani population, were identified by single strand conformation polymorphism analysis followed by direct sequencing. Two mutations, IVS-II-848 (C-->A) and initiation codon (ATG-->ACG), were found in the homozygous condition in patients belonging to Balochi and Sindhi ethnic groups of Pakistan, together with heterozygous and homozygous alpha(-3.7) deletions, respectively. A frameshift mutation at codon 44 (-C) was identified in a patient belonging to the Gujrati ethnic group together with IVS-I-1 (G-->T) and a normal complement of four a-globin genes. Haplotype analysis was performed to identify the chromosomal background associated with these mutations, and for tracing the origin and spread of these 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 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.     

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 basis of beta-thalassemia in Alexandria

Thalassaemia is a group of inherited haemoglobin disorders characterized by reduced synthesis of one or more of the globin chains leading to imbalanced a /non-a globin synthesis which is the major factor in determining the severity of the disease in the thalassaemia syndromes. In Egypt, beta-thalassemia is the commonest cause of chronic haemolytic anaemia and it represents a major genetic disease and a public health problem. This study included 50 transfusion dependent beta-thalassaemic cases. They were subjected to detailed history taking, physical examination to assess the size of liver and spleen and laboratory investigations including complete haemogram, bone marrow (BM) aspiration, haemoglobin electrophoresis and serum ferritin. Genetic analysis for detection of point mutations was done by PCR amplification refractory mutation system (ARMS) which is a PCR method based on allele specific priming. Using this technology, it was possible to characterize mutations in 73% of beta-thalassaemia cases, while 27% remained uncharacterized. The five common mutations used were: IVS-1-110 (G-->A), IVS-1-6 (T-->C) IVS-I-1 (G-->T), IVS-II-1 (G-->A) and codon 39 (C-->T). The commonest was IVS-I-110 (62%) followed by IVS-1-6 (7%), then IVS-I-1 (4%). On the other hand mutations such as IVS-II-1 and Cd- 39 were not found in any of our patients. No significant difference was found between different genotypes regarding the frequency of blood transfusion needed, degree of anaemia and microcytosis, HbF% or serum ferritin levels. This may be due to the small sample size of some of the genotypes (IVS-I-110/IVS-I-1 & IVS-I-110/IVS-I-6) or due to repeated blood transfusions which mask the patient original CBC and Hb electrophoresis pattern or due to co-inheritance of other genetic modifying factors that alter the typical phenotype.     

Comparative in vivo expression of beta(+)-thalassemia alleles.

Double heterozygotes who inherit one abnormal though stable beta-globin variant in association with a molecularly identified beta(+)-thalassaemia allele provide unique opportunities to quantify the in vivo expression of particular beta(+)-thalassemia alleles. The globin products of the two alleles can be separated, quantified and the output of the beta(+)-thalassaemia allele expressed as the MCH-beta(A) in pg beta(A)-globin/beta(+)-thalassemia allele/RBC = 0.5 MCH x Hb A%. In this communication we provide new quantitative data on the expression of five mutations as follows: the beta(+)-87 (C-->G) = 3.8 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 1); the beta(+) IVS-I-1 (G-->A) = 0.2 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 1); the beta(+) IVS-I-6 (T-->C) = 2.9 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 7); the beta(+) IVS-I-110 (G-->A) = 1.1 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 13), and the beta(+) IVS-II-745 (C-->G) = 1.74 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 2). The values obtained are compared with those of other beta(+)-thalassemia alleles from the literature. It can be seen that the MCH-beta(A) value may be a correct index of thalassemia severity useful for the correlation of genotype with phenotype, and for understanding the effects of mutations in beta-globin genes on pathophysiologically meaningful beta-globin gene expression.     

The molecular analysis of beta-thalassemia mutations in Lorestan Province, Iran

Beta-Thalassemia (thal) is one of the most common genetic disorders in Iran and other countries. Getting information on the distribution of mutations in different ethnic groups of Iran is of fundamental importance for the purpose of health planning and prenatal diagnosis programs. One hundred and thirty chromosomes from 65 unrelated homozygous beta-thal patients were investigated for beta-globin gene mutations by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). The most common mutations of the Mediterranean region were examined in this study. Our results showed that the frameshift codons (FSC) 36/37 (-T) mutation, with a frequency of 33.8%, is the most common mutation in Lorestan Province. The other most frequent mutations were of the Mediterranean type and consisted of IVS-II-1 (G -->A), IVS-I-110 (G -->A), FSC 8/9 (+G) and IVS-I-5 (G -->C) with frequencies of 27.7, 11.5, 10.8 and 4.5%, respectively. The less frequent alleles, IVS-II-745 (C -->G), FSC 5 (-CT), IVS-I (25 bp deletion) and FSC 44 (-C) accounted for only 3.9% of the mutations. The unknown alleles comprised 7.7% of the mutations. These data showed that the spectrum of mutations found in Lorestan Province was different from those reported from other thalassemic regions of Iran and also of some neighboring countries.     

Molecular characterization of beta-thalassemia in the Dohuk region of Iraq

beta-Thalassemia (thal) is an important health problem in the Dohuk region of northern Iraq because of its high carrier rate and the frequency of consanguineous marriages. Thus, the need to establish an effective preventative program is paramount. As part of this effort, we initiated this study to determine the molecular basis of this disorder in the region. For the latter purpose, either parent of 104 registered beta-thal major/intermedia patients had their full blood counts, hemoglobin (Hb) electrophoresis, Hb A2 and Hb F quantitation performed. Their DNA was extracted, amplified and reverse hybridized to specific oligonucleotide probes to detect 20 beta-thal mutations. The testing detected 12 beta-thalassemic mutations. The eight most frequent were: IVS-II-1 (G-->A), codon 44 (-C), codon 5 (-CT), IVS-I-1 (G-->A), codon 39 (C-->T), IVS-I-6 (T-->C), codons 8/9 (+G) and IVS-I-5 (G-->C). These mutations accounted for 81.7% of the thalassemic defects in the studied individuals. The less frequent mutations were: codon 8 (-AA), IVS-I-110 (G-->A), codon 30 (G-->C) and codon 22 (-7 bp), and the beta-thalassemic defects remained uncharacterized in 11.5% of cases. This is the first study of beta-thal mutations from Iraq, and shows a frequency of thalassemic defects different from those reported in surrounding countries. It provides a foundation for prenatal genetic testing that will be part of a thalassemia prevention program in the Dohuk region.