Origin and history of the IVS-I-110 and codon 39 beta-thalassemia mutations in the Lebanese population

Using restriction fragment length polymorphisms (RFLPs) and sequence haplotype analysis, we studied the chromosomal background of the beta-globin gene in 31 unrelated Lebanese IVS-I-110 or codon 39 (Cd39) subjects, and five normal betaAbeta/A individuals. Our results are compared with those from similar studies in other parts of the Mediterranean in an attempt to provide insights into historical patterns of selection and disease. The great majority of the Lebanese chromosomes with the IVS-I-110 mutation are associated with the RFLP haplotype I and sequence haplotype HT1, which is probably the ancestral structure on which the mutation first emerged. The remainder of the IVS-I-110 alleles are linked to the 5'-subhaplotype 12 RFLP haplotype and/or HTR sequence haplotype. In contrast, in Turkey, IVS-I-110 is associated with six distinct sequence haplotypes and four distinct RFLP haplotypes, suggesting that the mutation probably emerged there. The diversity of sequence haplotypes described in Turkey was probably generated through recombination or gene conversion events with the most frequent betaA autochthonous structures. Our data on Lebanese betaA chromosomes and Algerian betaA chromosomes, along with previously described Turkish betaA chromosomes, strengthen this hypothesis. Following its emergence in Turkey, the IVS-1-110 mutation was probably introduced to Lebanon later, by migration or settlements. Cd39 demonstrates a remarkable level of sequence and RFLP haplotype heterogeneity in Algeria, in contrast to its relative homogeneity in Turkish samples. However, its rarity in the Near East, and more specifically in Lebanon, does not allow us to draw any conclusions concerning its origin and gene flow.     

Molecular basis of beta-thalassemia in Morocco: possible origins of the molecular heterogeneity

We present the molecular spectrum of beta-thalassemia in the Moroccan population obtained by the identification of molecular defects responsible for this disease, and herewith we show that the Moroccan population is genetically heterogeneous; 18 different mutations have been found in the 158 beta-globin chromosomes studied. Eight mutations [codon 39 (C --> T), FSC-8 (-AA), IVS-II-745 (C --> G), -29 (A --> G), FSC-6 (-A), IVS-I-110 (G --> A), IVS-I-2 (T --> C), and IVS-I-1 (G --> A)] out of 18 beta-thalassemia mutations identified accounted for 76% of the Moroccan beta-thalassemia chromosomes. Restriction fragment length polymorphism (RFLP) haplotype analysis showed that the observed genetic diversity originated from both new mutational events and gene flow due to migration.     

Molecular analysis of alpha/beta-thalassemia in a southern Chinese population

Thalassemia is endemic to many regions in southern China. The screening of severe determinants of thalassemia is of critical importance in management and control of thalassemia. We designed a protocol based on microarray technology to screen for a spectrum of alpha/beta-globin gene mutations in the Chinese population. A total of 38 probes were capable of screening 98% of alpha/beta-globin gene mutations in the China population, including 16 mutations of beta-globin [beta(41-42)(-TCTT), IVSII-654(C-->T), beta17(A-->T), -28(A-->G), beta(71-72)(+A), beta(71-72)(+T), HbE26(G-->A), -29(A-->G), beta(27-28)(+C), IVSI-1(G-->T), IVSI-5(G-->C), beta(14-15)(+G), IVSII-5(G-->C), beta41(+T), 37(G-->A), and beta43(G-->T)] and five mutations of alpha/beta[three deletions of -alpha;(3.7), -alpha(4.2), and --(SEA); two nondeletions of alpha(Quong Sze) codon alpha125(T-->C) and alpha(Constant Spring) codon alpha142(T-->C)]. Multiplex PCR products were amplified from human genomic DNA and allowed to hybridize with the oligonucleotide array. alpha/beta-Globin genotypes were assigned by quantitative analysis of the hybridization results. The protocol, standardized by analysis of 100 thalassemia samples with known mutations and 13 recombinant plasmids, was 100% reliable in genotyping all mutant alleles. In subsequent screening of 2,030 Chinese with unknown mutations, the protocol was 100% accurate. This method provides unambiguous detection of complex combinations of heterozygous, compound heterozygous, and homozygous alpha/beta-thalassemia genotypes. The protocol was also flexible, detecting globin gene mutations from different population groups.     

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.     

Heterozygosity for the IVS-I-5 (G-->C) mutation with a G-->A change at codon 18 (Val-->Met; Hb Baden) in cis and a T-->G mutation at codon 126 (Val-->Gly; Hb Dhonburi) in trans resulting in a thalassemia intermedia.

We have analyzed the hemoglobins of a young German patient with beta-thalassemia intermedia and of his immediate family and included in these studies an evaluation of possible nucleotide changes in the beta-globin genes through sequencing of amplified DNA. One chromosome of the propositus and one of his father's carried the GTG-->GGG mutation at codon 126 leading to the synthesis of Hb Dhonburi or alpha 2 beta (2)126(H4)Val-->Gly; this variant is slightly unstable and is associated with mild thalassemic features. His second chromosome and one of his mother's had the common IVS-I-5 (G-->C) mutation that leads to a rather severe beta(+)-thalassemia and the GTG-->ATG mutation at codon 18, resulting in the replacement of a valine residue by a methionine residue. This newly discovered beta-chain variant, named Hb Baden, was present for only 2-3% in both the patient and his mother. This low amount results from a decreased splicing of RNA at the donor splice-site of the first intron that is nearly completely deactivated by the IVS-I-5 (G-->C) thalassemic mutation. The chromosome with the codon 18 (GTG-->ATG) and the IVS-I-5 (G-->C) mutations has thus far been found only in this German family; analysis of 51 chromosomes from patients with the IVS-I-5 (G-->C) mutation living in different countries failed to detect the codon 18 (GTG-->ATG) change.     

Beta-thalassemia genes in French-Canadians: haplotype and mutation analysis of Portneuf chromosomes.

beta-Thalassemia minor occurs at approximately 1% frequency in French-Canadians--in families residing in Portneuf County (population approximately 40,000) of Quebec province. We found eight different RFLP haplotypes at the beta-globin gene cluster in 37 normal persons and in 12 beta-thalassemia heterozygotes from six families. beta-Thalassemia genes in these families associated with two haplotypes only: Mediterranean I and Mediterranean II. There were two different beta-thalassemia mutations segregating in the Portneuf population: an RNA processing mutation (beta(+)IVS-1,nt110) on haplotype I (five families) and a point mutation leading to chain termination (beta(0) nonsense codon 39) on haplotype II (one family). The distribution of 5' haplotypes on normal beta A Portneuf chromosomes compared with other European populations was most similar to that in British subjects (data for French subjects have not yet been reported). Genealogical reconstructions traced the ancestry of carrier couples to settlers emigrating from several different regions of France to New France in the 17th century. These findings indicate genetic diversity of a greater degree among French-Canadians than recognized heretofore.     

Molecular characterization of seven beta-thalassemia mutations in Asian Indians

To characterize systematically the mutations which produce beta-thalassemia in Asian Indians, we first determined the DNA polymorphism haplotype in the beta-globin gene cluster of 44 beta-thalassemia chromosomes in the ethnic group. Nine different haplotypes were observed. Upon molecular cloning and partial DNA sequencing of one beta-gene from each of eight haplotypes and two from the ninth, seven different mutations were found. None of these have been identified in Mediterranean patients, even among the five haplotypes which appeared identical in the two groups. Asian Indian mutations included one nonsense and three frameshift mutations, one deletion affecting an acceptor splice site, and two mutations affecting a donor splice site. The correlation of a specific mutation with a specific haplotype was high but not invariant. Two mutations were associated with more than one haplotype but, in each instance, the mutation spread to a new haplotype could be explained most simply by recombination 5' to the beta-globin gene. In addition, four mutations, one reported here and three others previously reported, have been observed on two chromosome backgrounds that are identical except for the status of a polymorphic HinfI site 5' to the beta gene. This HinfI site does not show significant linkage disequilibrium with markers both 5' and 3' to it, suggesting that it lies within a region of relative sequence randomization.     

Reliable and high-throughput mutation screening for beta-thalassemia by a single-base extension/fluorescence polarization assay

beta-thalassemia is one of the most common inherited diseases with incidence varying between 3% and 10% in the high-prevalence regions of South China. The molecular defects are mostly due to single-nucleotide substitutions, minor insertions, and deletions in the beta-globin gene. Large-scale population genetic screening combined with prenatal diagnosis is necessary for the effective prevention of this disease. We present a single base extension (SBE) method based on homogenous fluorescence polarization (FP) for simultaneous detection of the eight most common causative mutations [CDs 41-42 (-TCTT), IVS-2-654 (C-->T), -28 (A-->G), CD17 (A-->T), CD 71/72 (+A), CD26 (G-->A), -29 (A-->G), and CD43 (G-->T)] in the beta-globin gene in a Chinese population. This assay has been validated by a blind experiment with 100 clinical samples previously characterized by reverse dot-blot and direct sequencing. The results demonstrate that this high-throughput method is simple, reliable, and cost effective. We expect this approach can be used in large-scale genetic screening for beta-thalassemia.     

Clinical expression of Leber hereditary optic neuropathy is affected by the mitochondrial DNA-haplogroup background

Leber hereditary optic neuropathy (LHON) is due primarily to one of three common point mutations of mitochondrial DNA (mtDNA), but the incomplete penetrance implicates additional genetic or environmental factors in the pathophysiology of the disorder. Both the 11778G-->A and 14484T-->C LHON mutations are preferentially found on a specific mtDNA genetic background, but 3460G-->A is not. However, there is no clear evidence that any background influences clinical penetrance in any of these mutations. By studying 3,613 subjects from 159 LHON-affected pedigrees, we show that the risk of visual failure is greater when the 11778G-->A or 14484T-->C mutations are present in specific subgroups of haplogroup J (J2 for 11778G-->A and J1 for 14484T-->C) and when the 3460G-->A mutation is present in haplogroup K. By contrast, the risk of visual failure is significantly less when 11778G-->A occurs in haplogroup H. Substitutions on MTCYB provide an explanation for these findings, which demonstrate that common genetic variants have a marked effect on the expression of an ostensibly monogenic mtDNA disorder.     

Red cell genetic abnormalities, beta-globin gene haplotypes, and APOB polymorphism in the Great Andamanese, a primitive Negrito tribe of Andaman and Nicobar Islands, India.

The Great Andamanese are a primitive Negrito tribe of the Andaman and Nicobar Islands, India, with a total population of 37. We studied 29 individuals from eight families from this population for abnormal hemoglobins, G6PD deficiency, DNA haplotypes, and apolipoprotein B (APOB, gene) polymorphism. Hb E was detected in five individuals, the prevalence of Hb E heterozygotes being 14.3%. One individual had beta-thalassemia trait. One female was G6PD deficient and showed the G6PD Orissa mutation. Haplotype analysis of the beta-globin gene cluster showed that the betaE chromosomes were linked to two haplotypes (- - - - - + + and + + - + + + +) representing the framework 1 gene, whereas the betaA chromosomes showed eight different haplotypic patterns corresponding to framework 1 and 3 genes. APOB polymorphism analysis showed that the 631-base-pair (bp) allele was the predominant one with a high homozygosity rate, which could be due to the higher rate of inbreeding in this isolated group. The presence of Hb E and our findings on haplotype analysis supports the hypothesis that the Great Andamanese are reasonably believed to be the surviving representatives of the Negrito race that once flourished in the entire Southeast Asian region in ancient times.     

beta-Thalassemia due to a deletion of the nucleotide which is substituted in the beta S-globin gene.

Sickle-cell anemia results from an A leads to T transversion in the second nucleotide of codon 6 of the beta-globin gene. We now report an uncommon beta-thalassemia gene that contains a deletion of this nucleotide. Thus, one mutation (GAG leads to GTG) produces sickle-cell anemia, while the other (GAG leads to GG) eliminates beta-globin production. These data establish that different alterations affecting one specific nucleotide can produce either an abnormal hemoglobin or beta-thalassemia. Moreover, the nucleotide sequence comprising codons 6-8 of the beta-globin gene appears to be particularly susceptible to mutations affecting nucleotide number.     

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.     

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.     

Molecular characterization of an atypical beta-thalassemia caused by a large deletion in the 5'' beta-globin gene region.

We describe a Canadian family of Czechoslovakian descent that came to our attention because of an HbA2 percentage approximately twice that of an average case of heterozygous beta-thalassemia. This unique phenotype suggested to us the possibility of a novel genetic mechanism being responsible for their beta-thalassemia. To investigate this possibility, we mapped, cloned, and sequenced the mutant beta-globin allele. This molecular analysis demonstrated the presence of a unique 4,237 base pair (bp) deletion extending from 3.3 kilobases (kb) 5' of the beta-globin mRNA cap site to approximately the middle of beta IVS-2. This truncated beta-globin gene further extends the heterogeneity of mutations known to cause beta-thalassemia and delineates new sequences involved in nonhomologous recombination events in the beta-globin gene region.     

Humanized beta-thalassemia mouse model containing the common IVSI-110 splicing mutation

Splicing mutations are common causes of beta-thalassemia. Some splicing mutations permit normal splicing as well as aberrant splicing, which can give a reduced level of normal beta-globin synthesis causing mild disease (thalassemia intermedia). For other mutations, normal splicing is reduced to low levels, and patients are transfusion-dependent when homozygous for the disease. The development of therapies for beta-thalassemia will require suitable mouse models for preclinical studies. In this study, we report the generation of a humanized mouse model carrying the common IVSI-110 splicing mutation on a BAC including the human beta-globin ((hu)beta-globin) locus. We examined heterozygous murine beta-globin knock-out mice ((mu)beta(th-3/+)) carrying either the IVSI-110 or the normal (hu)beta-globin locus. Our results show a 90% decrease in (hu)beta-globin chain synthesis in the IVSI-110 mouse model compared with the mouse model carrying the normal (hu)beta-globin locus. This notable difference is attributed to aberrant splicing. The humanized IVSI-110 mouse model accurately recapitulates the splicing defect found in comparable beta-thalassemia patients. This mouse model is available as a platform for testing strategies for the restoration of normal splicing.     

Only neutral polymorphisms found in the TIGR/myocilin gene of 45 Polish patients with primary open-angle glaucoma

The aim of the study was to identify mutations of the TIGR gene in Polish patients with primary open-angle glaucoma (POAG) and to define possible genotype-phenotype correlations. The study included 45 patients with a verified diagnosis of POAG. The PCR amplification of all three exons of the TIGR gene and screening for the sequence changes by CSGE analysis was done for every patient. The probes with identified heteroduplexes were sequenced. Altogether 315 PCR products were obtained. The CSGE analysis detected 60 possible changes of the sequence in 28 patients. 34 heteroduplexes were chosen for sequencing, including 29 unique changes and 5 changes representative of identical heteroduplexes. Direct sequencing enabled detection of only four different changes in the TIGR gene sequence. Three of them: 5'UTR -83G-->A (in 14 patients), +227 exon 1 G-->A, Arg76Lys (in 14 patients) and +311 exon 3 T-->C, Tyr347Tyr (in 4 patients) have already been described in the literature as neutral polymorphisms of the gene. Only one change in the promoter, 5'UTR -126T-->C (in 2 patients), has not been described in the literature to date. However, this change does not alter directly the sequence of amino acids in myocilin, so it is difficult to conclude on its pathogenetic role. Thus our study showed only neutral polymorphisms of the TIGR gene. This suggests that the patients probably have mutations in other genes, so other loci that predispose to POAG must be analyzed.     

Implications of the genetic epidemiology of globin haplotypes linked to the sickle cell gene in southern Iran

To determine the origin of sickle cell mutation in different ethnic groups living in southern Iran, we studied the haplotype background of the betaS and betaA genes in subjects from the provinces of Fars, Khuzestan, Bushehr, Hormozgan, and Kerman and from the islands of Khark and Qeshm. beta-globin gene cluster haplotypes were determined using the PCR-RFLP technique. Detection of -alpha 3.7 deletion and beta-thalassemia mutations were defined by PCR and reverse dot blot techniques, respectively. The framework of the beta-globin gene was determined using denaturing gradient gel electrophoresis. We found that the betaS mutation in southern Iran is associated with multiple mutational events. Most of the patients were from two ethnic groups: Farsi speakers (presumably Persian in origin) from Fars province and patients of Arab origin from Khuzestan province. In both ethnic groups the Arab-Indian haplotype was the most prevalent. The frequencies of the Arab-Indian and African haplotypes in sickle cell anemia patients from the provinces of Fars and Khuzestan were similar. Among betaA chromosomes the Bantu A2 haplotype was the most prevalent. The decrease in alpha-globin production in SS patients and AS individuals appeared to be related to the reduction in mean cell volume and mean cell hemoglobin. The Arab-Indian haplotype gene flow into this region of Iran can be traced to the Sassanian Empire. It is likely that the influx of betaS genes linked to the Benin and Bantu haplotypes, of African origin, must have occurred during the Arab slave trade.     

Detection of beta-globin gene mutations by polymerase chain reaction.

The polymerase chain reaction and oligonucleotide probe hybridization technique were applied to the detection of two common mutations of the beta-globin gene found in Chinese, namely the 4-base pair deletion at the 41-42 codons and the C to T substitution at nucleotide 654 of IVS-2. The accuracy of the method was established using beta-thalassemia cases with known mutations or haplotypes of the restriction fragment length polymorphism (RFLP). A further 11 cases of thalassemia intermediate and thalassemia minor were then analysed with the same approach. Our results showed that 5 of the 11 cases carried the TCTT-deletion at codons 41-42. Our method is economical both in terms of materials and time needed and in an alternative to the use of the molecular RFLP approach in the prenatal diagnosis of beta-thalassemia.     

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.