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

Efficacy of a single dose treatment of Wuchereria bancrofti microfilaria carriers with diethylcarbamazine in Matara, Sri Lanka.

OBJECTIVE: To test the efficacy of diethylcarbamazine DEC single dose regimen of 6 mg/kg body weight (bw) on a sample of Wuchereria bancrofti microfilaria (mf) carriers in Matara. DESIGN: 6 mg/kg bw DEC dose in 50 mg tablets given under direct observation to the subjects at 23.00 hours after pre-treatment blood collection for mf counts. Post-treatment mf counts were at 1, 2 weeks and 1, 3, 6, 12 months. SUBJECTS: 31 asymptomatic mf carriers, 14 males, age range 6 to 62 years. RESULTS: Treatment resulted in 89 to 97% success rate, 19 to 28% cure rates and 74 to 80% reduction in mf density. There was no difference statistically in the success rate and cure rate at 6 and 12 months. The effect of DEC treatment at 6 and 12 months compared by sex, age group and pre-treatment mf level showed no difference. 64.5% of the carriers treated had at least one mild adverse reaction. Rates of the common reactions were 41.9% fever, 22.6% headache and 16.1% joint pains. CONCLUSION: A new mass treatment program has been initiated by the national Antifilariasis Campaign using the single dose DEC 6 mg/kg bw regimen. The satisfactory reduction in mf density at 6 and 12 months following DEC single dose treatment we observed provides support for this program.

Microfilarial periodicity of Wuchereria bancrofti and man landing periodicity of the vector Culex quinquefasciatus say in Matara, Sri Lanka.

OBJECTIVE: To compare the microfilarial periodicity of Wuchereria bancrofti, with the man landing periodicity of the vector Culex quinquefasciatus in Matara, Sri Lanka. DESIGN: Periodicity was estimated using a statistical method. 60 microliters finger prick (FP) blood was smeared from a single subject every 2 hours for 24 hours of the day to make 12 samples. Smears were stained with Giemsa and the microfilariae (mff) counted. Man landing catches of mosquitoes were made inside a bedroom of a house in the same area on a sleeping volunteer during the night, between 18.00 and 06.00 hours. Each hourly catch was placed in separate paper cups. Hourly C. quinquefasciatus taken were counted. SUBJECTS: 10 asymptomatic microfilaria (mf) carriers. RESULTS: The individual mf peaks in the 10 carriers varied from 22.00 to 04.00 hours. Using the statistical method the parameter k showing the mf peak hour was 1.19 estimating the peak mf density at 01.11 hours. The influence of different times of blood collection on false negatives among the very low density carriers was estimated by the periodicity curve. It would be desirable to collect blood during the estimated time interval when the mf count was 80% of the peak count, between 21.55 and 04.27 hours in Matara. The results of 25 all-night mosquito landing catches gave a peak activity hours of k as 7.78, corresponding to 01.47 hours. CONCLUSION: The close agreement in the peak hours of mf density and vector activity suggests a perfect adaptation between parasite and vector for optimum transmission.

Fetal hemoglobin in patients with chronic renal disease

To investigate whether hemoglobin (Hb) synthesis is affected by different treatment protocols used for end-stage renal disease, we analyzed the electrophoretic pattern of hemoglobin in 136 adult patients with chronic renal failure. Forty-seven patients were not in a dialysis program (ND), 29 individuals were on continuous ambulatory peritoneal dialysis (CAPD), 33 patients were on hemodialysis(HD), and 27 subjects had received a kidney transplant (KT). We found 3.6 per cent hemoglobin C, 1.4 per cent hemoglobin S and 3.6 per cent beta-thalassemia minor as reported in other studies of Brazilian patients. In addition, we found increased fetal hemoglobin (Hb F) levels in 7.4 per cent of the patients which contrasts with the reported 0.01 per cent prevalence rate of hereditary persistence of Hb F in Brazil. Seven out of ten patients with elevated Hb F belonged to either the CAPD or the KT group. We postulate that stress erythropoiesis is probably the mechanism responsible for the Hb F increase in these patients. However, properly designed clinical studies are still necessary to clarify these questions.

Avaliaçäo do estado de ferro em indivíduos beta-talassêmicos heterozigotos / Evaluation of iron status in individuals with heterozygotic beta-thalassemia

A sobrecarga de ferro (Fe) é problema cosmopolita, que tem a hemocromatose idiopática e a talassemia como as doenças genéticas a que maisfreqüentemente está associada. Enquanto a sobrecarga de ferro em beta-talassêmicos em hipertransfusäo é situaçäo bem definida, existindo esquemas terapêuticos que visam minorá-la, o mesmo näo acontece com a talassemia menor, na qual estoques de ferro aumentados têm sido encontrados, de forma näo consensual. A ferritinasérica é considerada o melhor índice para avaliaçäo do estoque do ferro, uma vezque existe correlaçäo entre os seus valores e a concentraçäo de ferro hepático. OBJETIVO. O presente trabalho tem por objetivo verificar o risco de traços talassêmicos desenvolverem sobrecarga de Fe e a correlaçäo entre o grau de anemia e oestado do ferro. MÉTODOS. Foram avaliados o Fe sérico, TIBC, saturaçäo de transferrina, ferritina e homeglobina de 35 talassêmicos menores assintomáticos e 35 indivíduos normais, pareados por sexo e idade (20 mulheres e 15 homens, entre 20 e 54 anos de idade). RESULTADOS. Näo houve diferença estatisticamente significante para nenhuma variável estudada nos grupos do sexo feminimo, diferentemente dosexo masculino, em que a ferritina sérica nos traços talassêmicos apresentou média de 253,69ng/mL, e no controle normal de 107,79ng/mL (U calc

Padronizaçäo da técnica de imunofixaçäo na detecçäo e identificaçäo de paraproteínas séricas / Standartization of immunofixation technique for detection and identification of seric paraproteins

O diagnóstico e seguimento das paraproteinemias requer a identificaçäo e tipagem de paraproteínas (PP). A imunoeletroforese (IEF) é o método comumente usado embora demorado e pouco sensível. A técnica de imunofixaçäo (IF) é superior por ser mais sensível, rápida e de fácil interpretaçäo, particularmente no reconhecimento de PP presentes em baixa concentraçäo no soro e/ou urina. Consiste de fase eletroforética, seguida de fixaçäo, quando o anti-soro é colocado sobre o gel, precipitando a proteína. Objetivo. Este estudo objetiva padronizar a técnica de IF e compará-la à IEF. Métodos. Foram estudados os soros de 28 pacientes, sendo 25 portadores de mieloma múltiplo e 3 com hipergamaglobulinemia policlonal, comparados com 6 indivíduos normais. Todos foram submetidos à eletroforese (EF) em gel de agarose, à IEF e à IF. Resultados. O principal problema na padronizaçäo da IF foi a determinaçäo da diluiçäo que estabelecesse proporçäo ideal entre antígeno e anticorpo. A concentraçäo sérica ideal da PP, neste estudo, variou de 28 a 35 g/dL. A PP foi detectada e caracterizada por ambas as técnicas em 21 (84 por cento) dos indivíduos e näo detectada por nenhuma delas em 2 (8 por cento). Em outros 2, somente a IF conseguiu identificar a PP. Näo houve banda monoclonal à EF e à IEF que näo fosse identificada pela IF. Conclusäo. Nossos resultados permitem concluir que a IF é mais sensível que a IEF e deve ser incorporada à rotina de diagnóstico

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

Diethylcarbamazine: inhibitory effect on acetylcholinesterase of Dirofilaria immitis and Brugia pahangi.

Anti-acetylcholinesterase activity of diethylcarbamazine (DEC) was studied. Acethylcholinesterase (AchE) of adult worms of Dirofilaria immitis, those of the 4th-stage larvae, early 5th-stage larvae and adults worms of Brugia pahangi, and that of hamster brain tissue were all inhibited by DEC. Michaelis constant (Km) of D. immitis and B. pahangi adult worm AchE were 1.47 x 10(-4) and 1.81 x 10(-4) M respectively. DEC was a competitive inhibitor of the filarial AchE. Inhibition constant (Ki) for AchE of D. immitis and B. pahangi adult worms were 2.56 x 10(-4) and 6.39 x 10(-4) M, respectively. DEC is a less potent anticholinesterase inhibitor, because Ki of DEC is 10(4) times higher than that of eserine, a potent anti-cholinesterase agent.

Antitumor effects of Rhodium (II) citrate in mice bearing Chrlich tumors

Rhodium II citrate was tested in mice for acute toxicity, antitumoral activity against Ehrlich ascites carcinoma and inhibition of DNA synthesis by Ehrlich tumor, malignant adrenocortical cells (Y-1) and normal adrenocortical cells (AR-1)_. At ip doses up to 260 mg/Kg, the compound had no toxic effects for up to 14 days. The same total dose given over 4 days significantly increased the survial rat of mice bearing Ehrlich ascites cells. Thymidine incorporation by Ehrlkich tumor, Y-1 cells in vitro was inhibited 50% by a.1 to 0.2 mM concentrations of the compound. We conclude that the increase survival of the tumor-bearing mice was due at least in part to the inhibition of DNA synthesis with a consequet reduction of cell division and tumor growth

Comparison of methods for the detection of microfilariae of Wuchereria bancrofti in Western Samoa.

The 1 ml nuclepore filtration technique (NP method), 60 c.mm finger-prick or venous blood smear method and the counting chamber method were compared for their effectiveness in detecting microfilariae. The loss of microfilariae by the NP method was small enough to be disregarded. The 60 c.mm blood smear method was shown to be as efficient as the 60 c.mm NP method, when using finger-prick blood. The finger-prick blood contained 32% more microfilariae than venous blood when compared by the 60 c.mm blood smear method. Probably reflecting this difference of mf count between finger-prick and venous blood, 1 ml NP method using venous blood detected only 12.4 times more microfilariae than 60 c.mm NP method using finger-prick blood, despite the fact that the former requires 16.7 times more blood than the latter. No difference of mf count was shown between the 60 c.mm blood smear and 60 c.mm counting chamber methods, when using finger-prick blood.

Plasma level of diethylcarbamazine in jirds and hamsters.

The relation between the dosage and plasma level of diethylcarbamazine (DEC) in jirds and hamsters was examined by gas-liquid chromatography. When the drug was given intraperitoneally to jirds at 100 mg/kg body weight, the plasma level rose rapidly and reached the maximum level (20-25 micrograms/ml) at 10 minutes and afterwards fell quickly to undetectable level at 4 hours. Even if larger dosage (300 mg/kg) was given, DEC was eliminated completely from the blood circulation within 8 hours. When the same dosages of DEC were given intraperitoneally, the DEC levels remained 2-4 hours longer in the blood of hamsters than in the blood of jirds. DEC given by stomach intubation at 100 mg/kg remained detectable much longer in hamsters (8 hrs) than in jirds (2 hrs). A repeated doses schedule did not show a tendency for the drug to accumulate. DEC was excreted in the urine and feces, but the total amount of drug excreted without metabolic changes was only about 8% of the given dosage. The majority of unmetabolized DEC was excreted in the urine within 0-12 hours.

Studies on Brugia pahangi in inbred hamsters. 1. Susceptibility of inbred GN and APG hamsters.

The susceptibility of inbred GN and APG hamsters to Brugia pahangi infection was studied. Male GN hamsters infected subcutaneously with 100 infective larvae showed 100% patency with the average prepatent period of 69 days. The average microfilarial density was 148/40 c.mm at 26 weeks and reached 1,000/40 c.mm at days 280. The rate of adult recovery was as high as 36%. Most worms were recovered from the heart and lungs (46%) and testes (40%). The male GN hamster-B. pahangi model could be a good substitution for the jird-B. pahangi model. Female GN and male APG hamsters seemed to be less susceptible than male GN hamsters. However, they could still be a better alternative to outbred hamsters due to their high rates of patency (92 and 100%), relatively high microfilarial densities (31 and 39/40 c.mm at 26 weeks) and reasonable rates of adult recovery (23.5 and 17.2%).

Isolation and characterization of variant clones resistant to adrenocorticotropin from Y-1 mouse adrenocortical carcinoma cell line.

A acao de ACTH foi estudada por um enfoque genetico usando a linhagem corticoadrenal functional Y-1. Alem de estimular esteroidogenese, ACTH causa alteracoes morfologicas e bloqueia totalmente o crescimento da populacao. Estudos cineticos indicam que somente celulas do comeco de G1 sao afetadas pelo hormonio. Uma abordagem genetica levou ao isolamento de variantes resistentes a ACTH (variantes AR) a partir da linhagem parental Y-1. Embora estes variantes sejam ainda capazes de metabolizar [14C]-colesterol, a funcao de secretar esteroides nao foi mantida. Os efeitos da sintese de DNA por soro ou fator de crescimento extraido de pituitaria(PF) mostraram que: 1) alguns clones variantes AR sao totalmente resistentes a ACTH enquanto outros so parcialmente (50%); 2) todos os clones variantes mostraram-se sensiveis ao bloqueio por cAMP quando estimulados com soro mas resistentes a cAMP quando estimulados com PF. Os variantes AR sao morfologicamente diferentes das celulas parentais Y-1 e excluem uma as outras em culturas mistas. A composicao da superficie celular e as caracteristicas de crescimento permitem propor que o variante AR-1 e um revertente normal da linhagem maligna Y-1