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.

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

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

Utilidade do RDW e da interpretaçåo eritrocitária no diagnóstico das anemias / RDW utility and interpretation of erythrocyte distribution curve for anemia diagnoses

Os contadores hematológicos de última geraçåo fornecem novos parâmetros que podem ser úteis na diferenciaçåo laboratorial das anemias. Analisamos o valor do RDW (Red cell distribuition width) fornecido pelo COBAS ARGOS 5 DIFF 9ROCHE) na distinçåo entre anemia ferropriva (n=43) e beta talassemia heterozigótica (n=31), compararando-o com outras funçöes discriminantes, baseadas nos índices hematimétricos. Observamos que entre todas as fórmulas matemáticas utilizadas, baseadas nos índices hematimétricos. Observamos que entre todas as fórmulas matemáticas utilizadas, o RDW apresentou o melhor valor preditivo para anemia ferropriva (91,6 por cento), enquanto que na beta talassemia heterozigótica o melhor resultado (88,6 por cento) foi observado quando da combinaçåo do RDW com valores de VCM e Hb. A interpretaçåo do valor do RDW associado à curva de distribuiçåo volumétrica das hemácias foi analisada em diversos tipos de anemia e pudemos observar uma correlaçåo entre as alteraçöes sugeridas pela análise realizada pelo aparelho e as obtidas através da observaçåo microscópica. Os dados por nós obtidos indicam que a interpretaçåo cuidadosa dos dados fornecidos pela automaçåo podem auxiliar no esclarecimento diagnóstico das anemias, completando as informaçöes obtidas através da análise microscópica da série eritrocitária

Diferenciaçäo entre talassemia beta-heterozigótica e anemia ferropriva / Differentiation between heterozygotic beta-thalassemia and iron deficiency anemia

Várias funcöes discriminantes baseadas nos parâmetros hematimétricos fornecidos pelos contadores eletrônicos de células têm sido desenvolvidas e utilizadas nas diferenciaçäo de várias formas de anemia. A eficiência de alguns desses critérios comumente empregados na distinçäo entre a talassemia beta-heterozigótica e a anemia ferropriva foi testada em 192 indivíduos talassêmicos e em 72 adultos com anemia ferropriva. Foram avaliados: 1) funçäo discriminante de England e Fraser: VCM - (5xHb) - H - 8,4;2) HCM/H; 3) relaçäo de Mentzer: VCM/H; 4) (VCM)² x HCM; e 5) número de hemácias. Os resultados obtidos demonstraram falha na distinçäo entre estas duas entidades, em percentagens variáveis de 9 a 90//, sendo que a equaçäo de England e Fraser demonstrou os resultados mais satisfatórios, enquanto a relaçäo de Mentzer apresentou maiores índices de erro no diagnóstico da anemia ferropriva. Assim, embora algumas dessas fármulas possam ser utilizadas em programa de triagem, em casos individuais a diferenciaçäo entre talassemia beta-heterozigótica e anemia ferropriva deve ser sempre realizada com dosagens de HbA2, ferro sérico, capacidade máxima de transporte de ferro e ferritina