Geographical distribution of β-globin gene mutations in Syria

ABSTRACT

Objectives: β-Thalassemia disease is caused by mutations in the β-globin gene. This is considered as one of the common genetic disorders in Syria. The aim of this study was to identify the geographical distribution of the β-thalassemia mutations in Syria.

Methods: β-Globin gene mutations were characterized in 636 affected patients and 94 unrelated carriers using the amplification refractory mutations system-polymerase chain reaction technique and DNA sequencing.

Results: The study has revealed the presence of 38 β-globin gene mutations responsible for β-thalassemia in Syria. Important differences in regional distribution were observed. IVS-I.110 [G?>?A] (22.2%), IVS-I.1 [G?>?A] (17.8%), Cd 39 [C?>?T] (8.2%), IVS-II.1 [G?>?A] (7.6%), IVS-I.6 [T?>?C] (7.1%), Cd 8 [?AA] (6%), Cd 5 [?CT] (5.6%) and IVS-I.5 [G?>?C] (4.1%) were the eight predominant mutations found in our study. The coastal region had higher relative frequencies (37.9 and 22%) than other regions. A clear drift in the distribution of the third common Cd 39 [C?>?T] mutation in the northeast region (34.8%) to the northwest region (2.5%) was noted, while the IVS-I.5 [G?>?C] mutation has the highest prevalence in north regions. The IVS-I.6 [T?>?C] mutation had a distinct frequency in the middle region. Ten mutations ?86 [C?>?G], ?31 [A?>?G], ?29 [A?>?G], 5′UTR; +22 [G?>?A], CAP?+?1 [A?>?C], Codon 5/6 [?TG], IVS-I (?3) or codon 29 [C?>?T], IVS-I.2 [T?>?A], IVS-I.128 [T?>?G] and IVS-II.705 [T?>?G] were found in Syria for the first time.

Conclusions: These data will significantly facilitate the population screening, genetic counseling and prenatal diagnosis in Syrian population.     

HbA2-Partinico or δ(A2)Pro→Thr, a new genetic variation in the δ-globin gene in cis to the β+ thal IVS-I-110 G>A, and the heterogeneity of δ-globin alleles in double heterozygotes for β- and δ-globin gene defects

The study of the alleles of the δ-globin gene is relevant to the prevention of β-thalassemia homozygosis; in fact, the increase of the HbA2 is an invaluable hematological marker of the β-thalassemia heterozygosis and the double heterozygosis for alleles of δ- and β-globin genes can cause the decrease of the HbA2 up to normal or borderline values. We carried out the characterization of alleles of the δ- and β-globin genes, restriction fragment length polymorphism (RFLP) haplotype background, and hematologic phenotype in 23 double heterozygotes belonging to 18 unrelated families. A wide heterogeneity of the δ-globin alleles was detected; seven known alleles in trans to the β-globin gene defects were revealed in 17 out of 18 families, while a new allele in cis to a β-thalassemia allele was detected in one family. Moreover, the relative frequency of the δ-mutants was quite different from that found among heterozygotes. The new allele δ-cod 5 CCT>ACT, in cis to the allele β⁺ thal IVS-I-110 G>A, was found in five carriers of a Sicilian family. The new variant δ5(A2)Pro→Thr, named HbA2-Partinico upon the origin of the family, was detected with high-performance liquid chromatography; it overlapped the HbA2 peak which was partially split. The double in cis heterozygotes had increased percentage of normal and variant HbA2 of comparable size. The variant originated most likely from a new mutational event because it was associated with RFLP haplotype I, commonly found with the β⁺ thal IVS-I-110 G>A, even if crossing over or gene conversion cannot be excluded.     

Identification of a novel δ-globin gene mutation in an Iranian family

δ-Thalassemia (δ-thal) has no clinical symptoms, but its coinheritance with β-thal may cause misdiagnosis, especially in countries with a high prevalence of β-thal where prevention programs have been implemented. The molecular basis of most β-thal syndromes have been defined, while the spectrum of mutations causing δ-thal have not been well characterized. A couple was referred to us for thalassemia molecular screening. Since she had rather low values of Hb A? and normal Hb F, her δ-globin gene was amplified and directly sequenced. We found two different mutations on her δ-globin genes: HBD: c.92+5G>T/HBD:c.428C>A. The c.92+5G>T mutation has not been previously reported. Two different mutations in trans may explain the reduced Hb A? level.     

Novel therapeutic candidates, identified by molecular modeling, induce γ-globin gene expression in vivo

The β-hemoglobinopathies and thalassemias are serious genetic blood disorders affecting the β-globin chain of hemoglobin A (α(2)β(Α)(2)). Their clinical severity can be reduced by enhancing expression of fetal hemoglobin (γ-globin), producing HbF (α(2)γ(2,)). In studies reported here, γ-globin induction by 23 novel, structurally-unrelated compounds, which had been predicted through molecular modeling and in silico screening of a 13,000 chemical library, was evaluated in vitro in erythroid progenitors cultured from normal subjects and β-thalassemia patients, and in vivo in transgenic mice or anemic baboons. Four predicted candidates were found to have high potency, with 4- to 8-fold induction of HbF. Two of these compounds have pharmacokinetic profiles favorable for clinical application. These studies thus effectively identified high potency γ-globin inducing candidate therapeutics and validated the utility of in silico molecular modeling.     

MBD2 contributes to developmental silencing of the human ε-globin gene

During erythroid development, the embryonic ε-globin gene becomes silenced as erythropoiesis shifts from the yolk sac to the fetal liver where γ-globin gene expression predominates. Previous studies have shown that the ε-globin gene is autonomously silenced through promoter proximal cis-acting sequences in adult erythroid cells. We have shown a role for the methylcytosine binding domain protein 2 (MBD2) in the developmental silencing of the avian embryonic ρ-globin and human fetal γ-globin genes. To determine the roles of MBD2 and DNA methylation in human ε-globin gene silencing, transgenic mice containing all sequences extending from the 5' hypersensitive site 5 (HS5) of the β-globin locus LCR to the human γ-globin gene promoter were generated. These mice show correct developmental expression and autonomous silencing of the transgene. Either the absence of MBD2 or treatment with the DNA methyltransferase inhibitor 5-azacytidine increases ε-globin transgene expression by 15-20 fold in adult mice. Adult mice containing the entire human β-globin locus also show an increase in expression of both the ε-globin gene transgene and endogenous ε(Y) and β(H1) genes in the absence of MBD2. These results indicate that the human ε-globin gene is subject to multilayered silencing mediated in part by MBD2.     

The genetic heterogeneity of β-globin gene defects in Sicily reflects the historic population migrations of the island

The aim of this study is to update the incidence and the distribution of the globin gene defects causing β-thalassemia and abnormal hemoglobins in Sicily. The data derived from a total of 8875 beta-thalassemia alleles and 1330 variant hemoglobin chromosomes studied in Sicily from 1990 during a hemoglobinopathy control program. Fifty-four beta-globin gene defects were characterized, involving 30 different beta-thalassemia mutations and 24 variant hemoglobins. Eight of 30 β-thalassemia defects accounted for 95.11% of examined alleles while other beta-globin gene defects were found at lower frequencies (<1%). A consistent number (24) of variant hemoglobins were identified of whom Hb S was the most represented (72.1%). Our data underline the heterogeneity of the beta-globin gene defects in the Sicily. The enormous progress in the technique for β-globin gene analysis permitted to characterize 99.93% of mutated alleles and it has made a first trimester prenatal diagnosis program possible in our region in all cases with a great improvement in thalassemia management. The origin of the large spectrum of mutations is discussed taking in consideration the history of the island.     

Hemoglobin Köln: Analysis of linkage relationships between the mutant gene and polymorphic restriction sites in the β-globin gene cluster

Summary Nuclear DNA has been analyzed by means of restriction endonuclease mapping procedure to identify chromosomes that carry mutant Hb Köln -globin genes in a family with individuals heterozygous for this disease. Inherited DNA polymorphisms within the -globin gene cluster yielded a direct linkage of the Hb Köln mutation to haplotype constellations that are diagnostic for further off-spring.Supported by the Deutsche Forschungsgemeinschaft     

Molecular characterisation of β-globin gene mutations in Penang and Kedah,Malaysia

IntroductionBeta-thalassaemia is an autosomal recessive disorder and it is a public health problem in the Malaysian Malays and Chinese. This disorder mainly results from point mutations, small insertion or deletions in the β-globin gene complex. Beta-thalassaemia major patients require life-long monthly blood transfusions and iron-chelation therapies to sustain their lives. Mutation characterisation is necessary for affected couples at risk of having a β-thalassaemia major child.Objective1. To develop the TaqMan genotyping platform as a time- and cost-effective approach for characterisation of β-globin gene mutations. 2. To characterise the mutations using the developed assays in transfusion-dependent patients in Penang and Kedah.MethodsTen sets of primers and TaqMan probes were designed to identify the common mutations in Malaysian Malays and Chinese: −28 (A→G), CD17 (A→T), CD19 (A→G), HbE (G→A), IVS1-1 (G→T), IVS1-5 (G→C), CD 41/42 (-CTTT), CD71/72 (⁺A), IVS2-654 (C→T) and Poly A (AATAAAHAATAGA). Another 7 sets of TaqMan genotyping assays were designed to identify the rare mutations in Malays and Chinese: −29 (A→G), Cap (⁺1) (A→C), CD8/9 (⁺G), CD16 (-C), CD27/28 (⁺C), IVS1-1 (G→A) and CD43 (G→T). The developed assays were used to screen 54 and 62 transfusion-dependent patients in Penang and Kedah respectively.Results & DiscussionThe developed assays detected 92.9% of mutations in the β-thalassaemia major patients. The remaining mutations were detected by ARMS, gap-PCR and DNA sequencing. The most common mutation in β-thalassaemia major patients in Penang is CD41/42 with a frequency of 20.9%. The most common mutation in β-thalassaemia major patients in Kedah is HbE with a frequency of 30.8%.ConclusionThe simplicity and reproducibility of the TaqMan genotyping assays enable rapid and cost-effective analysis of the β-globin gene mutations in Malaysia.     

Molecular analysis of the β-globin gene cluster haplotypes in a Sudanese population with sickle cell anaemia

Introduction: Sudan has a multiethnic population with a high frequency of Hb S, but little is known about the β^S haplotypes in this population. Methods: Blood samples from Sudanese Hb SS individuals were taken at two locations. Family history, age, ethnicity and clinical symptoms were recorded for each subject. Hb S was investigated using cellulose acetate electrophoresis (CAE) and cation exchange–high performance liquid chromatography. Dried blood samples from 93 individuals were used for β^S haplotype identification based on restriction fragment length polymorphism analysis for seven restriction sites. Results: Haplotypes could be assigned unequivocally to 143 chromosomes. Four of the five typical β^S‐globin haplotypes were identified. The most frequent was the Cameroon (35.0%), followed by the Benin (29.4%), the Senegal (18.2%) and the Bantu (2.8%). The Indian‐Arab haplotype was not observed. Three atypical haplotypes were identified in 17 patients, occurring at a combined frequency of 14.6%. One of these, found at the high frequency of 11.8%, possibly represented a new Sudan haplotype. Conclusion: β^S Haplotyes were demonstrated successfully from dried blood samples. A new haplotype is apparent in Sudan, in addition to the four African haplotypes.     

Molecular spectrum of β-thalassemia mutations in the admixed Venezuelan population, and their linkage to β-globin gene haplotypes

In order to establish the spectrum of β-thalassemia (β-thal) mutations in the Venezuelan population for the first time, 127 unrelated subjects either with a suspicion of β-thal trait or with a clinically recognized β-thal syndrome of different degrees of severity, were studied. DNA from these subjects was analyzed by a polymerase chain reaction (PCR)-based reverse dot-blot method or amplification refractory mutation system (ARMS). Prototype β-globin gene sequencing of relevant DNA was performed to confirm the mutations. Fifteen different mutations were identified accounting for 92.0% of the mutant alleles explored, revealing a significant genetic heterogeneity at the β-globin gene locus in this population. The most frequent mutations were codon 39 (C >T) 34.1%, IVS-I-1 (G >A) 11.1%, IVS-I-6 (T > C) 6.6%, IVS-I-110 (G >A) 6.6%, IVS-II-849 (A >G) 6.6%, -88 (C >T) 6.0%, -29 (A >G) 5.2%, followed by the less common IVS-I-5 (G >A) 3.7%, the 1,393 bp deletion 3.0%, IVS-II-1 (G >A) 3.0%, -86 (C >G) 2.2%, IVS-II-1 (G >T) 1.5%, codons 41/42 (-TCTT) 1.5%, IVS-II-745 (C >G) 0.7% and deletional δβ-thal 0.7%. Overall, these data demonstrate that the major sources of β-thal alleles in Venezuela, as expected, are of Mediterranean and African origins. This is the first large study defining the molecular spectrum of β-thal in the highly admixed population of Venezuela and lays the foundation for genetic counseling as well as implementing comprehensive clinical care programs. Diversity of haplotypes associated with some of the β-thal mutations can be explained by in situ recombination events in Venezuela.     

In vivo monitoring of intracellular Ca2+ dynamics in the pancreatic β-cells of zebrafish embryos

ABSTRACT

Assessing the response of pancreatic islet cells to glucose stimulation is important for understanding β-cell function. Zebrafish are a promising model for studies of metabolism in general, including stimulus-secretion coupling in the pancreas. We used transgenic zebrafish embryos expressing a genetically-encoded Ca²⁺ sensor in pancreatic β-cells to monitor a key step in glucose induced insulin secretion; the elevations of intracellular [Ca²⁺]_i. In vivo and ex vivo analyses of [Ca²⁺]_i demonstrate that β-cell responsiveness to glucose is well established in late embryogenesis and that embryonic β-cells also respond to free fatty acid and amino acid challenges. In vivo imaging of whole embryos further shows that indirect glucose administration, for example by yolk injection, results in a slow and asynchronous induction of β-cell [Ca²⁺]_i responses, while intravenous glucose injections cause immediate and islet-wide synchronized [Ca²⁺]_i fluctuations. Finally, we demonstrate that embryos with disrupted mutation of the Ca_V1.2 channel gene cacna1c are hyperglycemic and that this phenotype is associated with glucose-independent [Ca²⁺]_i fluctuation in β-cells. The data reveal a novel central role of cacna1c in β-cell specific stimulus-secretion coupling in zebrafish and demonstrate that the novel approach we propose – to monitor the [Ca²⁺]_i dynamics in embryonic β-cells in vivo – will help to expand the understanding of β-cell physiological functions in healthy and diseased states.     

Analysis of linkage disequilibrium between the 5′ and 3′ haplotypes of the β-globin gene cluster in Mexican afromestizos

Analysis of the 5′ and 3′ haplotypes (Hps) of the β-globin gene cluster was performed in 110 β^A chromosomes from unrelated Mexican afromestizo individuals in order to determine Hardy-Weinberg equilibrium, allelic frequencies, linkage disequilibrium (LD) and association between the 5′ and 3′ haplotypes. All sites were found to be in Hardy–Weinberg equilibrium (p > 0.05). In the whole β-cluster, only 12.87% of the pairs of loci exhibited significant LD (22/171) (r² > 0.33). Within the 5′Hp, three pairs of loci were associated (?HincII/^GγHindIII, ?HincII/3′ψβHincII and ^GγHindIII/3′ψβHincII). In the 3′Hp, 19 pairs of loci showed significant LD and were distributed mostly among the ? 551, ? 340, Exon1nt6 and IVS2nt16 polymorphisms. The absence of pairs of loci significantly linked between both 5′ and 3′ Hps is noteworthy. The allelic combinations of the 40 studied polymorphisms (5 sites in the 5′ Hp and 35 sites in the 3′ Hp) displayed 69 distinct haplotypes, 22 of them belonging to group A, 27 to B, 18 to C and 2 to D, which denoted the great heterogeneity of our population. Further, 1a7A1, 1a7B1 and 1a1C1 were the most common sequences with 8, 9 and 9 chromosomes each. Association analysis between both 5′ and 3′ Hps revealed strong coherence with the proposed evolutionary histories for the β-globin gene polymorphisms. 5′Hp1 (+––––), which is considered to be an ancestral haplotype, was the most frequent haplotype found in our population and was linked to 24 different sequences in the 3′Hp, demonstrating great heterogeneity. A similar result was found in the 3′ Hps, where older alleles (a17A1 and a7B1) were linked to a higher number of 5′Hps.This is the first time that an analysis of association among the 5′ and 3′ haplotypes and the LD has been performed with 40 polymorphisms distributed in the β-globin gene cluster in the Mexican afromestizo population. The poor LD observed between and within the 5′ and 3′ Hps show that this region is very prone to recombination events.     

Thymosin β4 and angiogenesis: modes of action and therapeutic potential

Here we review the mechanisms by which Thymosin β4 (Tβ4) regulates angiogenesis, its role in processes, such as wound healing and tumour progression and we discuss in more detail the role of Tβ4 in the cardiovascular system and significant recent findings implicating Tβ4 as a potential therapeutic agent for ischaemic heart disease.