Characterization of alpha thalassemic genotypes by multiplex ligation-dependent probe amplification in the Brazilian population

Alpha-thalassemia is the most common inherited disorder of hemoglobin synthesis. Genomic deletions involving the alpha-globin gene cluster on chromosome 16p13.3 are the most frequent molecular causes of the disease. Although common deletions can be detected by a single multiplex gap-PCR, the rare and novel deletions depend on more laborious techniques for their identification. The multiplex ligation-dependent probe amplification (MLPA) technique has recently been used for this purpose and was successfully used in the present study to detect the molecular alterations responsible for the alpha-thalassemic phenotypes in 8 unrelated individuals (3 males and 5 females; age, 4 months to 30 years) in whom the molecular basis of the disease could not be determined by conventional methods. A total of 44 probe pairs were used for MLPA, covering approximately 800 kb from the telomere to the MSLN gene in the 16p13.3 region. Eight deletions were detected. Four of these varied in size from 240 to 720 kb and affected a large region including the entire alpha-globin gene cluster and its upstream regulatory element (alpha-MRE), while the other four varied in size from 0.4 to 100 kb and were limited to a region containing this element. This study is the first in Brazil to use the MLPA method to determine the molecular basis of alpha-thalassemia. The variety of rearrangements identified highlights the need to investigate all cases presenting microcytosis and hypochromia, but without iron deficiency or elevated hemoglobin A2 levels and suggests that these rearrangements may be more frequent in our population than previously estimated.

Influence of the polymorphisms of the alpha-major regulatory element HS-40 on in vitro gene expression

The α-MRE is the major regulatory element responsible for the expression of human α-like globin genes. It is genetically polymorphic, and six different haplotypes, named A to F, have been identified in some population groups from Europe, Africa and Asia and in native Indians from two Brazilian Indian tribes. Most of the mutations that constitute the α-MRE haplotypes are located in flanking sequences of binding sites for nuclear factors. To our knowledge, there are no experimental studies evaluating whether such variability may influence the α-MRE enhancer activity. We analyzed and compared the expression of luciferase of nine constructs containing different α-MRE elements as enhancers. Genomic DNA samples from controls with A (wild-type α-MRE) and B haplotypes were used to generate C-F haplotypes by site-directed mutagenesis. In addition, three other elements containing only the G→A polymorphism at positions +130, +199, and +209, separately, were also tested. The different α-MRE elements were amplified and cloned into a plasmid containing the luciferase reporter gene and the SV40 promoter and used to transiently transfect K562 cells. A noticeable reduction in luciferase expression was observed with all constructs compared with the A haplotype. The greatest reductions occurred with the F haplotype (+96, C→A) and the isolated polymorphism +209, both located near the SP1 protein-binding sites believed not to be active in vivo. These are the first analyses of α-MRE polymorphisms on gene expression and demonstrate that these single nucleotide polymorphisms, although outside the binding sites for nuclear factors, are able to influence in vitro gene expression.

Screening for mutations in human alpha-globin genes by nonradioactive single-strand conformation polymorphism

Point mutations and small insertions or deletions in the human alpha-globin genes may produce alpha-chain structural variants and alpha-thalassemia. Mutations can be detected either by direct DNA sequencing or by screening methods, which select the mutated exon for sequencing. Although small (about 1 kb, 3 exons and 2 introns), the alpha-globin genes are duplicate (alpha2 and alpha1) and highy G-C rich, which makes them difficult to denature, reducing sequencing efficiency and causing frequent artifacts. We modified some conditions for PCR and electrophoresis in order to detect mutations in these genes employing nonradioactive single-strand conformation polymorphism (SSCP). Primers previously described by other authors for radioactive SSCP and phast-SSCP plus denaturing gradient gel electrophoresis were here combined and the resultant fragments (6 new besides 6 original per alpha-gene) submitted to silver staining SSCP. Nine structural and one thalassemic mutations were tested, under different conditions including two electrophoretic apparatus (PhastSystemÖ and GenePhorÖ, Amersham Biosciences), different polyacrylamide gel concentrations, run temperatures and denaturing agents, and entire and restriction enzyme cut fragments. One hundred percent of sensitivity was achieved with four of the new fragments formed, using the PhastSystemÖ and 20 percent gels at 15ºC, without the need of restriction enzymes. This nonradioactive PCR-SSCP approach showed to be simple, rapid and sensitive, reducing the costs involved in frequent sequencing repetitions and increasing the reliability of the results. It can be especially useful for laboratories which do not have an automated sequencer.

Thalassemia intermedia as a result of heterozygosis for beta 0 thalassemia and aaa3.7 /aa genotype in a Brazilian patient

We report a case in which the interaction of heterozygosis for both the beta0-IVS-II-1 (G->A) mutation and the aaa anti-3.7 allele was the probable cause for the clinical occurrence of thalassemia intermedia. The propositus, a 6-year-old Caucasian Brazilian boy of Portuguese descent, showed a moderately severe chronic anemia in spite of having the beta-thalassemia trait. Investigation of the alpha-globin gene status revealed heterozygosis for alpha-gene triplication (aaa /aa). The patient's father, also presenting mild microcytic and hypochromic anemia, had the same alpha and beta genotypes as his son, while the mother, not related to the father and hematologically normal, was also a carrier of the aaa anti-3.7 allele. The present case emphasizes the need for considering the possibility of alpha-gene triplication in beta-thalassemia heterozygotes who display an unexpected severe phenotype. The beta-thalassemia mutation found here is being described for the first time in Brazil

Molecular identification of Sicilian (deltaß)º-thalassemia associated with ß-thalassemia and hemoglobin S in Brazil

We describe the clinical and molecular characteristics of two unrelated Brazilian families with an association of the Sicilian form of (deltaß)º-thalassemia with hemoglobin S and ß-thalassemia. Direct sequencing of the ß-globin gene showed only the hemoglobin S mutation in patient 1 and the ß-thalassemia IVS1-110 in patient 2. The other allele was deleted in both patients and PCR of DNA samples of the breakpoint region of both patients showed a band of approximately 1,150 bp, expected to be observed in the DNA of carriers of Sicilian (deltaß)º-thalassemia. The nucleotide sequence of this fragment confirmed the Sicilian deletion. There are few reports concerning the Hb S/(deltaß)º-thalassemia association and patient 2 is the first reported case of Sicilian type of (deltaß)º-thalassemia in association with ß-thalassemia documented at the molecular level

High prevalence of alpha-thalassemia among individuals with microcytosis and hypochromia without anemia

In order to determine the contribution of alpha-thalassemia to microcytosis and hypochromia, 339 adult outpatients seen at Unicamp University Hospital (with the exception of the Clinical Hematology outpatient clinics), who showed normal hemoglobin (Hb) levels and reduced mean corpuscular volume and mean corpuscular hemoglobin, were analyzed. Ninety-eight were Blacks (28.9 percent) and 241 were Caucasians (71.1 percent). In all cases, Hb A2 and F levels were either normal or low. The most common deletional and nondeletional forms of alpha-thalassemia [-alpha3.7, -alpha4.2, --MED, -(alpha)20.5, alphaHphIalpha, alphaNcoIalpha, aaNcoI and alphaTSAUDI] were investigated by PCR and restriction enzyme analyses. A total of 169 individuals (49.9 percent) presented alpha-thalassemia: 145 (42.8 percent) were heterozygous for the -alpha3.7 deletion (-alpha3.7/aa) and 18 (5.3 percent) homozygous (-alpha3.7/-alpha3.7), 5 (1.5 percent) were heterozygous for the nondeletional form alphaHphIalpha (alphaHphIalpha/aa), and 1 (0.3 percent) was a --MED carrier (--MED/aa). Among the Blacks, 56 (57.1 percent) showed the -alpha3.7/aa genotype, whereas 12 (12.2 percent) were -alpha3.7/-alpha3.7 and 1 (1.0 percent) was an alphaHphIalpha carrier; among the Caucasians, 89 (36.9 percent) were -alpha3.7/aa, 6 (2.5 percent) had the -alpha3.7/-alpha3.7 genotype, 4 (1.7 percent) presented the nondeletional form (alphaHphIalpha/aa), and 1 (0.4 percent) was a --MED carrier. These results demonstrate that alpha-thalassemia, mainly through the -alpha3.7 deletion, is an important cause of microcytosis and hypochromia in individuals without anemia. These data are of clinical relevance since these hematological alterations are often interpreted as indicators of iron deficiency

Alpha-Globin genes: thalassemic and structural alterations in a Brazilian population

Seven unrelated patients with hemoglobin (Hb) H disease and 27 individuals with alpha-chain structural alterations were studied to identify the alpha-globin gene mutations present in the population of Southeast Brazil. The -alpha3.7, --MED and -(alpha)20.5 deletions were investigated by PCR, whereas non-deletional alpha-thalassemia (alphaHphalpha, alphaNcoIalpha, aaNcoI, alphaIcalpha and alphaTSaudialpha) was screened with restriction enzymes and by nested PCR. Structural alterations were identified by direct DNA sequencing. Of the seven patients with Hb H disease, all of Italian descent, two had the -(alpha)20.5/-alpha3.7 genotype, one had the --MED/-alpha3.7 genotype, one had the --MED/alphaHphalpha genotype and three showed interaction of the -alpha3.7 deletion with an unusual, unidentified form of non-deletional alpha-thalassemia [-alpha3.7/(aa)T]. Among the 27 patients with structural alterations, 15 (of Italian descent) had Hb Hasharon (alpha47Asp->His) associated with the -alpha3.7 deletion, 4 (of Italian descent) were heterozygous for Hb J-Rovigo (alpha53Ala->Asp), 4 (3 Blacks and 1 Caucasian) were heterozygous for Hb Stanleyville-II (alpha78Asn->Lys) associated with the alpha+-thalassemia, 1 (Black) was heterozygous for Hb G-Pest (alpha74Asp->Asn), 1 (Caucasian) was heterozygous for Hb Kurosaki (alpha7Lys->Glu), 1 (Caucasian) was heterozygous for Hb Westmead (alpha122His->Gln), and 1 (Caucasian) was the carrier of a novel silent variant (Hb Campinas, alpha26Ala->Val). Most of the mutations found reflected the Mediterranean and African origins of the population. Hbs G-Pest and Kurosaki, very rare, and Hb Westmead, common in southern China, were initially described in individuals of ethnic origin differing from those of the carriers reported in the present study and are the first cases to be reported in the Brazilian population

Hematological phenotype and the type of ß thalassemia mutation in Brazil

O heterozigoto para a talassemia ß apresenta um típico padräo hematológico. A severidade desses parâmetros varia consideravelmente de valores claramente anormais até próximos à normalidade. Postula-se que essa variaçäo seria influenciada pelo tipo de mutaçäo de talassemia beta encontrada e que portanto seria possível suspeitar-se do tipo de alteraçäo pelos índices hematológicos. Nossos resultados demonstram que mutaçöes brandas (ß IVSI-nt 6) mostram maiores níveis de hemoglobina corpuscular média (MCH) do que as formas mais severas (ߺ39 or ß IVSI-nt 1) e que os níveis de hemoglobina A2 säo menores em mutaçöes ߧ do que em formas brandas (ß IVSI-nt 6). Entretanto, contrariando estudos anteriores, näo pudemos indicar o MCH como um discriminador entre as mutaçöes ߺ ou ß+.

Rapid identification of beta-globin structural mutations by sequencing the mRNA from peripheral blood reticulocytes

A simple and rapid method for the molecular detection of beta-globin structural mutations is described using a reverse transcription-polymerase chain reaction of reticulocyte mRNA and direct sequencing of the product. The amplified segment (employing a sense primer 5'-ATTTGCTTCTGACACAACTGT-3', located at position + 1 with respect to the Cap site and an antisense primer 5'-TCCAGATGCTCAAGGCCCTTC-3', located at position + 1772 with respect to the Cap site) encompasses the cDNA sequence including the three globin exons. Employing this method we were able to characterize two hemoglobin structural variants: Hb S (beta 6 (A3) Glu-Val: GAG-GTG) and Hb Porto Alegre (beta 9 (A6) Ser-Cys: TCT-TGT). The approach described in this paper should be very useful to detect hemoglobin structural variants because the RNA extraction is simple, rapid and does not require cesium chloride, guanidinium and proteinase K. In addition, the direct sequencing of the RT-PCR product permits the screening of the entire globin genes with only two reactions

Hemoglobin Bart's in a Brazilian black population

Hemoglobin Bart's was measured spectrophotometrically after electrophoreis on cellulose acetate strips in cord blood from 320 full-term randomly selected black new borns from the University Hospital of Campinas, State of Säo Paulo, in the southeast of Brazil. Hb Bart's was detected in 38 of the 320 newborns (11.0%), with the levels presenting the following bimodal distribution: 10.3% of the infants in the 1-3.5% range and 1.6% in the 5-10% range. The data suggest that the frequency of alfa-talassemia gene in the Brazilian black population is individuals with Bart's Hb in the 5-10% range are usually alfa+ -talassemia homozygotes