Molecular epidemiology of β-thalassemia in Pakistan: Far reaching implications.

Background: β -Thalassaemia, an autosomal recessive hemoglobinopathy, is one of the commonest genetically transmitted disorders throughout the world. Collective measures including carrier identification, genetic counseling and prenatal diagnosis are required for preventing β-thalassemia. Aim: To achieve this objective, Identification of the spectrum of genetic mutations, especially for various ethnic backgrounds in Pakistan. Therefore, we designed a cross sectional prospective study to identify the frequency of various gene mutations in different ethnic groups of Pakistan. Materials and Methods: Over a 5-year period, DNA from 648 blood samples {including specimens of chorionic villus sampling (CVS)} were analyzed for the twelve most common β-thalassemia mutations found in the Pakistani population by a Multiplex amplification refractory mutation system (ARMS). Each sample was analyzed for the mutation as well as the normal gene, appropriate with negative and positive controls, and reagent blanks. Results: Out of 648 samples mutations were identified in 640 (98.75%) samples by multiplex ARMS. 8 common β-thalassemia mutations were identified in 8 different ethnic groups accounting for 93.9% of the β-thalasemia alleles. Conclusions: Based on the outcome of this study a cost effective proposal is formulated for detection of β-thalassemia mutations.

Resultados de un programa de pesquisa prenatal de la talasemia grave / Results of an antenatal screening programme for beta-thalessemia

Objetivos: Detectar el rasgo beta-talasémico durante el control prenatal de mujeres en la primera etapa del embarazo; reducir el nacimiento de niños homocigotos con beta-talasemia. Métodos: Se incluyeron embarazadas de hasta 18 semanas de gestación con antecedentes de no más de 3 embarazos, que asistían a una clínica de control prenatal. Se realizó una pesquisa mediante la estimación del volumen corpuscular medio, la hemoglobina corpuscular media y la prueba NESTROF. Se controló también a los esposos de las mujeres con pruebas positivas. Si ambos padres eran positivos, se confirmó el diagnóstico por medio de cromatografía líquida de alto rendimiento. Para la detección fetal de las parejas positivas se efectuó biopsia de vellosidades coriónicas o amniocentesis. Se ofreció la interrupción del embarazo a las mujeres con fetos con talasemia grave. Resultados: Se efectuó la pesquisa a 17339 madres en el período comprendido entre octubre de 1999 y marzo de 2010. Las pruebas de cribado fueron positivas para el volumen corpuscular medio, la hemoglobina corpuscular media y la pruebas de NESTROF en el 11.02%, 18.76% y 12.62% de las embarazadas, respectivamente. El 1.98% de las mujeres eran portadoras confirmadas. Un total de 54 parejas requirieron pruebas diagnósticas prenatales fetales. Se detectó talasemia grave en 19 fetos; todos esos embarazos fueron interrumpidos. Se encontró rasgo talasémico en 16 fetos. Conclusión: La pesquisa prenatal de la beta-talasemia es un abordaje rentable para evitar el nacimiento de niños afectados, en especial en países de alta prevalencia.

[ frequency of SspI polymorphism in intron II of beta globin gene in Mazandaran province]

Due to the high annual birth rate of thalassemia major in our country, its prevention by prenatal diagnosis is of important priority. Gene mutation remains unknown in 10-20% of thalassemia trait people in Iran. In these cases, linkage analysis using polymorphic sites which are located near or within the gene is necessary to follow the mutant or the normal chromosome. SspI polymorphic site which is studied for the first time in Iran is located in the second intron of beta globin gene. The aim of this study was to determine the polymorphism frequency of this site in Mazandaran province. Peripheral blood of 211 thalassemia trait patients living in Mazandaran province was collected. After DNA extraction and amplification of the beta globin gene region containing the SspI polymorphic site, the effect of SspI restriction enzyme was evaluated on agarose gel. In 422 analyzed chromosomes, 20.6% were negative for SspI polymorphic site. Negative sites were almost equally associated with normal and mutant alleles [11.9% and 14.3% respectively]. SspI site analysis can be applied to follow the normal or mutant alleles of beta globin gene

'NESTROFT'--an effective screening test for beta thalassemia trait.

OBJECTIVE: To evaluate the efficacy of NESTROFT (Naked Eye Single Tube Red Cell Osmotic Fragility Test) as a screening tool for detection of beta thalassemia trait. DESIGN: Prospective study. SETTING: Field camps in various parts of Gujarat and Maharashtra States. METHODS: A total of 2525 subjects were screened. NESTROFT, complete hemogram including red cell indices and calculation of Mentzer's Fraction (MF) and discriminant functions (DF1-4) were done in all subjects. HbA2 was performed in 830 initial subjects to compute sensitivity, specificity and predictive values for various parameters. RESULTS: NESTROFT (sensitivity 94.4%), as a single screening parameter was superior to any of the other evaluated parameters individually, besides being cost effective. Mean corpuscular volume (MCV) < 80 fl followed NESTROFT closely (sensitivity 93.7; p > 0.05). MCV < 75 fl had a significantly (p < 0.001) lower sensitivity (87.3%) in comparison to both of these parameters. In contrast, MF, DF1, DF2, DF3 and DF4 did not meet the requirements of a good screening test with sensitivity values of 66.2%, 54.9%, 47.2%, 64.1% and 55.6%, respectively. NESTROFT in combination with MCV < 80 fl proved 100% sensitive. However, the combination was not cost effective. CONCLUSION: NESTROFT is a sensitive, cost effective, rapid and reliable screening test for detection of beta thalassemia trait in a population.