Beta-thalassemia in China: a systematic molecular characterization of beta-thalassemia mutations

In order to initiate a program of prenatal diagnosis for the prevention of beta-thalassemia in China, we have begun systematic studies of the beta-thalassemia mutations among the Chinese. DNA polymorphisms in the beta-globin gene cluster were examined in 46 beta-thalassemia chromosomes. Six different haplotypes were observed. One beta-thalassemia gene associated with a new haplotype was cloned and sequenced. The mutation was a single base substitution (A----G) at position -29 within the highly conserved proximal promoter element (the "TATA" box). This mutation was not observed previously in the Chinese. The beta-thalassemia genes were further screened with oligonucleotide probes specific for all known mutations in the Chinese. Five mutations were identified and accounted for 35 beta-thalassemia alleles.     

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 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.     

Characterization of beta-thalassemia mutations among the Japanese

Characterization of beta-thalassemia mutations were attempted for 29 Japanese families clinically diagnosed as having beta-thalassemia. Following the identification of a mutation by cloning and sequencing, all families were screened for this particular mutation, using biotinylated allele-specific oligonucleotide probes. Seven different mutations were detected in 17 families: Six families had the frameshift mutation at codons 41/42, resulting from a 4 nucleotide deletion (TTCTTT----TT); four had the deletion at codons 127/128 (CAGGCT----CCT); and three had the TATA box mutation at nucleotide -31 (A----G). Four additional families had mutations at codon 24 (GGT----GGA), codon 26 (GAG----AAG), IVS-II-654 (C----T) and codon 110 (GTG----CCG), respectively. The newly discovered deletion mutation at codons 127/128, and mutations at nucleotide -31, and at codon 110 are peculiar to Japanese, and have not been found in any other ethnic group. The haplotypes of the beta-globin gene cluster were also determined. Some of the haplotypes and beta-thalassemia mutations are identical to those reported in the Chinese population. However, it is noteworthy that nearly half of the beta-thalassemia mutations were unique to Japanese.     

The molecular heterogeneity of beta-thalassemia in Greece

β-thalassemia is the most predominant genetic defect in Greece. In this study, we investigated the heterogeneity and the frequency of β-thalassemia mutations among 3796 heterozygotes detected in the course of DNA based diagnoses. The diagnostic strategy included Denaturing Gradient Gel Electrophoresis (DGGE), Allele Specific Oligonucleotide Hybridization (ASO), GAP PCR, Restriction Enzyme (RE) analysis and direct sequencing and led to 100% identification of the underlying molecular lesion. Six out of 33 different β-globin defects identified accounted for more than 91.4% of the total β-thalassemia chromosomes in Greece. The β-globin gene mutations cd29 C→T, IVS-I-2 T→C, IVS-I-5 G→T, cd37 G→A and poly A Kurdish AATAAA→AATAAG are for the first time reported in Greece, whereas cd7 GAG→TAG is a new β⁰-thalassemia mutation detected in an adult man from Albania residing in Greece. Three DNA single nucleotide polymorphisms (IVS-I-85 T→C, IVS-I-91 C→T and IVS-I-108 T→C) were also revealed; among these, IVS-I-85 T→C and IVS-I-91 C→T are new and described for the first time worldwide.     

Epidemiology of visceral leishmaniasis in northeast Brazil.

Epidemiologic aspects of the relationship between infection with Leishmania chagasi and development of clinical visceral leishmaniasis (VL) were studied in all children < 11 years old in a defined, endemic, rural area of the state of Ceará in northeast Brazil. Antileishmanial antibodies were measured in the same subjects by ELISA on six occasions between May 1987 and August 1989. Seroconversion was documented during this period in 108 children, with a cumulative annual incidence of 4.6%. Twelve (11.1%) of these children developed VL. Age < 4 years, hematocrit < 33%, and living in the mountains predicted the development of clinically apparent VL after seroconversion. Despite a high percentage of dogs serologically positive in the region (38%), there was no increased risk of infection for children living in the same household with dogs. Since children in households with a prior case of VL had a threefold increased risk of infection, human-sandfly-human transmission might have been important.     

Familial aggregation of mucosal leishmaniasis in northeast Brazil

To evaluate whether familial clustering occurs in mucosal leishmaniasis (ML), patients with ML (index cases) were randomly selected from medical records at a health post in an endemic area for Leishmania braziliensis infection. Control individuals (index controls) matched by age, gender, and place of residence to index cases were selected. Family members of index cases and controls were compared with respect to environmental factors and the incidence of cutaneous leishmaniasis (CL) and ML. Delayed type hypersensitivity test (DTH) to Leishmania antigen was tested in selected families. Among 289 members of 46 families enrolled, significant differences were found in the frequencies of CL (37% versus 20%) and ML (5% versus 0) in case versus control families, respectively. Families with 2 cases of ML had a higher frequency (29.6%) of DTH-positive individuals than control families (9.4%). These data demonstrate familial clustering of CL, ML, and positive DTH skin tests in an area endemic for L. braziliensis infection.     

Observations on the levels of Hb A2 in patients with different beta-thalassemia mutations and a delta chain variant.

Hb A2 and its variant B2 (alpha 2 delta 2(16)(A13)Gly----Arg) were quantitated in the blood of subjects with three different types of beta-thalassemia and with the delta-B2 anomaly in cis or in trans to the beta-thalassemia determinant. In one family, the delta-B2 mutation was in cis to a newly discovered codon 47 (+A) frameshift. The levels of Hbs A2 and B2 were nearly the same and approximately 70% higher than those in simple Hb B2 heterozygotes. In two additional families, the delta-B2 variant was in trans to either a deletional beta-thalassemia (1,393 bp) involving part of the beta-globin gene and part of the beta-globin gene promoter, or to the -88 C----T promoter mutation. In both instances, the Hb B2 level was increased by approximately 80%, but the Hb A2 level was increased by approximately 270% and 200%, respectively. These data indicate two mechanisms that will cause an increase in delta chain production. One is consistent with a general mechanism concerning the relative excess of alpha chains in beta chain deficiencies which will combine with delta chains to form variable levels of Hb A2 dependent on the severity of the beta chain deficiency. The second concerns the loss of beta-globin gene promoter activity, perhaps by an absence of (or decreased) binding of specific protein(s) to this segment of DNA and a concomitant increase in delta-globin gene promoter activity in cis.     

Plasma lipids and lipoproteins pattern in beta-thalassemia major

We studied serum lipids and lipoproteins in 20 patients with beta-thalassemia major, under high transfusion programme and regular chelation therapy, and in 20 control subjects. Total cholesterol, HDL-cholesterol, HDL2-and HDL3-cholesterol, apolipoprotein A and B levels were significantly lower in patients with Cooley's anemia, whereas free cholesterol, triglycerides and the HDL-/HDL3-cholesterol ratio did not differ in the two groups. We think that liver damage plays an important role in determining the altered lipoprotein pattern in beta-thalassemia major. However, other factors may contribute to cause such lipid changes.     

An emerging peri-urban pattern of infection with Leishmania chagasi, the protozoan causing visceral leishmaniasis in northeast Brazil

Peri-urban visceral leishmaniasis (VL) caused by Leishmania chagasi is emerging in a new epidemiologic pattern in Brazilian cities. We studied peri-urban VL in endemic neighborhoods surrounding Natal, Brazil, identified through hospitalized individuals with VL. Clinical and environmental information obtained for 1106 members of 216 families living in endemic neighborhoods enabled us to identify 4 groups: VL: individuals with current or prior symptomatic visceral leishmaniasis (n = 135); DTH+: individuals with positive delayed-type hypersensitivity response with no history of VL (n = 390); Ab +: individuals with negative DTH response and seropositive (n = 21); DTH -: individuals with negative DTH and seronegative (n = 560). The mean +/-SD age of VL was 9.3+/-12.3 y. The gender distribution was nearly equal below age 5, but skewed toward males at higher ages. Acutely infected VL subjects had significantly lower hematocrits, neutrophils, and eosinophils than other categories. AB+ subjects also had lower eosinophil counts than others, a possible immune marker of early infection. VL was not associated with ownership of dogs or other animals, raising the question whether the reservoir differs in peri-urban settings. This new pattern of L. chagasi infection enables us to identify epidemiological and host factors underlying this emerging infectious disease.     

Reverse dot-blot detection of the African-American beta-thalassemia mutations

DNA-based diagnosis of the beta thalassemias provides accuracy to newborn screening genetic counseling, and prenatal diagnosis. However, the use of polymerase chain reaction (PCR)-based methods is challenged by the great number of different-beta-thalassemia mutations that exist even within defined ethnic groups. In this regard, the reverse dot-blot method offers a means of screening for several mutations with a single hybridization reaction. We have applied the reverse dot-blot method to the detection of the beta-thalassemia mutations of African-Americans. We used two biotin-labeled primer pairs in a duplex reaction to amplify and label two beta-globin target DNA fragments that encompass all known African-American beta-thalassemia mutations. The PCR products were denatured and hybridized to polyT-tailed, membrane-fixed, allele- specific probe pairs for the hemoglobin (Hb) S, Hb C, and 14 beta- thalassemia mutations and their corresponding wild-type sequences. Seven common mutations plus Hb S and Hb C were included on one diagnostic strip, and seven less common beta-thalassemia mutations were included on another strip. Carefully controlled, high stringency hybridization allowed accurate distinction of these alleles. Reverse dot-blot diagnosis of the less common beta-thalassemia mutations precludes the need for alternative, more technically challenging methods. This method provides a rapid, accurate method for diagnosis of beta thalassemia among African-Americans and other ethnic groups in which beta thalassemia occurs.     

The molecular basis of beta-thalassemia in Argentina. Influence of the pattern of immigration from the Mediterranean Basin

In order to determine the molecular heterogeneity of the beta-thalassemia gene and to analyze the influence of immigration from the Mediterranean Basin, a total of 254 families (475 subjects) from Argentinean beta-thalassemia patients were investigated using molecular biology techniques. This allowed us to provide a simplified diagnosis and genetic counselling of this disorder in Argentina.     

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.     

Molecular characterization of beta-thalassemia intermedia in patients of Italian descent and identification of three novel beta-thalassemia mutations

In this study, we have defined by dot-blot analysis with allelic specific oligonucleotide probes or direct sequencing on amplified DNA the beta-thalassemia mutations in a large group of patients (23) of Italian descent with thalassemia intermedia. These patients had one parent with either the silent beta-thalassemia carrier phenotype or borderline-normal hemoglobin A2 (HbA2) levels (2.5% to 3.5%). Nearly all were genetic compounds for a severe beta-thalassemia mutation and a beta-thalassemia mutation associated with high residual output of beta-globin chains (beta + intervening sequence [IVS]-I-nt6, beta -87, beta -101), indicating that inheritance of a mild beta-thalassemia allele, even in a single dose, is the most common molecular mechanism producing thalassemia intermedia in the Italian population. In three cases, in whom we failed to define by dot-blot analysis the mutations, we sequenced the beta + globin gene and found three novel beta-thalassemia mutations, which are certainly very rare because they have been hitherto detected solely in a single patient. These mutations consist of: (1) a T-A substitution at position 2 of IVS-I, in a patient compound heterozygote for this mutation and the -87 promoter mutation; (2) a G-C substitution at position 844 of IVS-II, in a patient heterozygous for this mutation who showed normal sequences at the in trans beta-globin gene (The reason for the presence of clinical manifestations in a beta-thalassemia heterozygote has not been defined.); and (3) a deletion of one nucleotide (-T) at codon 126, resulting in a frameshift and readthrough of the 5' untranslated region and most likely producing an elongated Hb molecule of 156 amino acid residues, in a patient heterozygous for this mutation with normal beta-globin gene sequences at the other locus.     

Detection of new mutations and molecular pathology of mild and moderate haemophilia A patients from southern Brazil

A total of 76 unrelated male patients with mild (n = 55) or moderate (n = 21) haemophilia A living in the southern Brazilian state of Rio Grande do Sul were studied by direct sequencing of all F8 26 exons, the 5′ UTR and 3′ UTR, intron–exon junctions and the promoter region. When no mutation was found, a multiplex ligation‐dependent probe amplification analysis was performed. We identified the disease‐causing mutations in 69 patients, who showed 33 different mutations: 27 missense, one small deletion, two small duplications and three splice site mutations. Seven missense and two splice site mutations were not previously reported in HAMSTeRS and were not identified in any current literature search. Nine recurrent mutations were found, one of them never described before (p.Tyr1786Phe). Haplotype analysis indicated that this mutation had originated in the Brazilian population as a single event in a common ancestor. The possible influence of these mutations in the determination of the disease was carefully considered, including bioinformatic tools. These data add to the general knowledge of the disease and can also be useful for HA diagnosis and detection of carriers in the southern Brazilian population.