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

In order to establish the spectrum of ß-thalassemia (ß-thal) mutations in the Venezuelan population for the first time, 127 unrelated subjects either with a suspicion of ß-thal trait or with a clinically recognized ß-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 ß-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 ß-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 δß-thal 0.7%. Overall, these data demonstrate that the major sources of ß-thal alleles in Venezuela, as expected, are of Mediterranean and African origins. This is the first large study defining the molecular spectrum of ß-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 ß-thal mutations can be explained by in situ recombination events in Venezuela.

Hemoglobin variants, hematological parameters and beta-globin gene cluster haplotypes in an isolated Amerindian group from the Orinoco River Delta.

BACKGROUND: Several previous studies reported that the Venezuelan Warao Indians presented unusual genetic characteristics. AIM: The present study checked previous reports of a high frequency of hereditary persistence of fetal hemoglobin (HPFH) and examined other hematological traits. SUBJECTS AND METHODS: Standard hematology, electrophoresis on cellulose acetate, fetal hemoglobin alkali denaturation, gamma-globin chain, DNA amplification and sequencing, and denaturing gradient gel electrophoresis determinations were performed in 269 individuals living in two localities of the Orinoco River Delta. RESULTS: Two beta(s) genes, in apparently non-related individuals, were found. HPFH, detected in this same population of Warao Indians 25 years ago, was present in heterozygous form in five individuals from a large kindred, with hemoglobin F levels ranging from 3.7% to 8%, and with a pancellular distribution. The HPFH mutation was of the deletional type. beta-globin gene haplotypes were determined by direct counting (through family studies) in 150 chromosomes; 26% of the 150 examined cluster presented haplotype 2, 22% haplotype 6, and 13% a new, Warao haplotype. Haplotype 3, of probable African origin, was also found with a frequency of 5%. CONCLUSIONS: The presence of the HPFH mutation was confirmed, and the new beta-globin gene haplotype together with the presence of other rare variants indicates that the Warao are very distinctive in relation to other Native Americans. Evidence was also found of a slight admixture from Africa-derived subjects (Layrisse et al. 1988).

Hemoglobinopatías en Venezuela / Hemoglobinopathies in Venezuela

Entre las hemoglobinopatías, grupo heterogéneo de alteraciones congénitas, destacan: las variantes de hemoglobina, las talasemias y la persistencia hereditaria de hemoglobina fetal (HPFH). Se determinó la distribución de estas patologías en Venezuela y se demostró que presentan una elevada frecuencia, constituyendo un problema de salud pública. Se analizaron 80400 individuos provenientes de estudios poblacionales de diferentes regiones del país y pacientes referidos por presentar anemia hemolítica fueron referidos a dos centros de investigación hematológica. Fueron estudiadas 76400 muestras mediante electroforesis en acetato de celulosa y en citrato agar y 4000 por cromatografía líquida de alta presión de intercambio catiónico (HPLC-CE). Se encontró que el 9 por ciento de los individuos presentaron hemoglobinopatías. La variante más frecuente fue la Hb S, seguida de las variantes C y D. Además, se observó la presencia de la beta talasemia y su asociación a las hemoglobinas S y C. La frecuencia de los haplotipos del gen bs estudiados en 272 de pacientes con síndrome drepanocítico (SCA) y heterocigotos para la Hb S fue de 50,8 por ciento Benin, 32,2 por ciento CAR, 14,2 por ciento Senegal y 2,3 por ciento Camerún. Se encontró que en los pacientes homocigotos para la Hb S estudiados, solo el 8 por ciento fue homocigoto para el haplotipo Ben, 82 por ciento fueron doble heterocigotos para los haplotipos Ben/CAR, 8,8 por ciento presentaron haplotipos Ben/Senegal, CAR/Senegal y Ben/Camerún y un caso fue homocigoto para el haplotipo CAR. La detección de estas patologías es de importancia para establecer un tratamiento precoz y consejo genético, logrando una mejor calidad de vida y gran ahorro para el sistema de salud.

[Phenotyping and genotyping studies in a family with the compound heterozygosity deltabeta Thalassemia/beta(IVSII-849) Thalassemia]. / Fenotipo y genotipo del doble heterocigoto para deltabeta Talasemia/beta(IVSII-849) Talasemia en una familia venezolana.

The propositus is a two year old child with a severe hemolytic anemia and increased level of Hb F. The Hbs A, A2 and F were eluted and quantitated by cation exchange high-performance liquid chromatography (HPLC-CE). DNA was isolated from peripheral blood leukocytes by a salting-out extraction procedure. The beta globin gone was amplified and the presence of the beta thalassemia mutation was determined by PCR followed of Reverse Dot Blot. Her hematological parameters were as follows: Hb: 7.0 g/dL, Hct: 24.8%, VCM: 87.4 fl, CHCM: 27.8 fl. The haemoglobin study showed an 97% increase of Hb F and Hb A2 normal. The molecular study suggested the presence of beta(IVSII-829) mutation in trans to deltabeta Thalassemia. The propositus inherited her mother's deltabeta-thalassemia gene mutation and her father's beta(IVSH-829) mutation. This is the first time the diagnosis has been performed in a Venezuelan family at-risk of compound heterozygotes for beta-thalassemia and delta beta-thalassemia.

Fenotipo y genotipo del doble heterocigoto para &ß talasemia/ßIVSII-849 talasemia en una familia venezolana / Phenotyping and genotyping studies in a family with the compound heterozygosity &ß thalassemia/ßIVSII-849 thalassemia

El propósito es una niña de 2 años de edad, con anemia hemolítica severa, transfusión dependiente, quien presenta un cuadro clínico sugestivo de ß talasemia mayor. Al examen físico se constata la presencia de esplenomegalia, malformaciones óseas y retardo en el crecimiento. La presencia de las distintas hemoglobinas: Hb A, Hb F, Hb A2 y su cuantificación fue determinada utilizando la técnica de cromatografía líquida de alta presión, de intercambio catiónico (HPLC-CE). El ADN genómico fue aislado a partir de leucocitos de sangre periférica mediante el método de salting-out. La detección de la mutación beta talasémica fue realizada por medio de las técnicas de reacción en cadena de la polimerasa (PCR) seguida de Reverse Dot Blot. Sus parámetros hematológicos fueron: Hb: 7,0 g/dL, Hcto: 24,8 por ciento, VCM: 87,4 fl, CHCM: 27,8 fl. Los resultados de la HPLC-CE mostraron un elevado incremento en los niveles de Hb Fetal en un 97 por ciento y niveles de Hb A2 dentro de los valores normales. El estudio molecular y familiar demostró la presencia de la mutación bIVSII-849 en trans con una deleción db talasemia. El propósito heredó de la madre la mutación db-talasemia y del padre la mutación bIVSII-849. Esta es la primera vez que se ha realizado este diagnostico en una familia Venezolana, con riesgo de un heterocigoto compuesto para ß-talasemia y &ß-talasemia

[Advantages in the use of high performance liquid chromatography technique for screening hemoglobinopathies in Venezuela]. / Ventajas de la técnica de cromatografía líquida de alta presión (HPLC-CE) en el estudio de hemoglobinopatías en Venezuela.

The hemoglobinopathies are a very heterogeneous group of congenital hemolytic anemias, which includes hemoglobin (Hb) variants, thalassemia and hereditary persistence of fetal hemoglobin (HPFH). The aim of this study was to determine the frequency of hemoglobinopathies using the High Performance Liquid Chromatography (HPLC-CE) technique with the beta-thalassemia Short Program of Variant* Bio Rad. Four thousand blood samples from anemic patients from the Laboratorio de Investigación de Hemoglobinas Anormales, Hospital Universitario de Caracas were studied. Twenty six percent of the anemia patients had hemoglobinopathies. The Hb S was the most frequent variant found, followed by the Hb C and Hb D. Also we observed the association of beta thalassemia with Hb S and Hb C. The quantification of the Hb A by HPLC-CE allowed us to classify the double heterozygote Hb S-Beta Thalassemia in Hb S-beta+ Tal Type 1, Hb S-beta+ Tal Type 2, Hb S-beta(0) Thalassemia. The double heterozygote patients with Hb C-Beta thalassemia were also classified. The HPLC-CE is a rapid, reproducible and precise technique. The reliability of HbA2 measurement by HPLC for the detection of beta thalassaemia without any false positive or false negative results is of great advantage. HPLC may be an appropriate method for rapid screening in population surveys for beta thalassemia and hemoglobin variants carriers. Due to the high incidence of cases, in our country this is very important for their clinical management and the genetic and anthropological impact of an early and precise diagnosis.