MOLECULAR BASIS OF 6-THALASSEMIA IN TURKEY AND ITS PRENATAL DIAGNOSIS BY DNA ANALYSIS

Abstract

B-Thalassemia is an autosomal recessive disorder caused by the absence or reduced synthesis of the B-chains of hemoglobin. As in many Mediterranean and Southeast Asian countries B-thalassemia is a major health concern in Turkey . B-Thalassemia is generally caused by point mutations and so far more than 130 of these have been described worldwide (Baysal and Carver, 1995). Since no satisfactory treatment is available for this inherited disorder, carrier detection and prenatal diagnosis in the couples at risk are the main approaches to control the disease. Each population tends to have its own group of mutations causing B-thalassemia, therefore, determination of prevalence and distribution of these mutations in different populations is extremely important for establishing a comprehensive prenatal diagnosis program based on DNA analysis. Due to its geographical location as a cross road between Asia and Europe, Turkey has a heterogeneous population. The characterization of mutations causing B-thalassemia and their frequencies in this population is an important prerequisite to the implementation of a DNA based prenatal diagnostic program. The present study was mainly conducted to determine the distribution pattern of B-thalassemia mutations in the Turkish population in order to design a prenatal diagnosis strategy for this genetic syndrome and later on to develop a similar program for the Pakistani population. In this study a total of 235 B-thalassemia alleles consisting of 93 transfusion dependent thalassemic homozygotes (major/intermedia) and 49 heterozygotes (carriers/trait) randomly selected from all over the Turkey were screened for mutations causing B-thalassemia. Polymerase chain reaction (PCR) based techniques used in this study for the carrier detection and prenatal diagnosis of B-thalassemia include radioactive and nonradioactive allele specific oligonucleotide (ASO) hybridization, amplification refractory mutation system (ARMS), restriction endonuclease analysis and direct sequencing of genomic DNA by dideoxy chain termination method. Our results indicate that great molecular heterogeneity exists among the Turkish population with 15 different mutations causing B-thalassemia including one rare [lVS-II-848 (C-A)] previously undescribed in this population. The first ten mutations identified represent 86% of the chromosomes studied and include IVS-I- 110 (G-A: 32.34%), IVS-I-6 (T-C: 13.19%), IVS-II-745 (C-G: 9.36%), IVS-II- l (G-A: 8.93%), IVS-I-l (G-A: 8.51 %), -30 (T-A: 4.68%), FSC-8 (-AA: 3.40%), IVS-I-116 (T-G: 2.12%), Codon 39 (C-T: 1.7%) and Codon 44 (-C: 1. 7 %). The other five mutations include FSC-8/9 (+ G: 0.85%), FSC-5 (-CT: 0.85%), IVS-II-848 (C-A: 0 .85 %) , IVS-I-5 (G-C: 0.85%) and -87 (C-G: 0.42%) which account for only 4% of the alleles analyzed. Two hemoglobin variants i.e. HbS (A-T) and HbD (G-C) were also detected in five and one chromosomes respectively. Inspite of the high genotypic variability, majority of the homozygotes were found to be true homozygotes (58.06%) for a single mutation (e.g. IVS-I-IlO and IVS-I-6 etc.). In addition, nine prenatal diagnosis using first trimester chorionic villus sampling (CVS) were carried out. Using the PCR based techniques the prenatal diagnosis was always completed within one to three days if the parental mutations were known to us before hand. Fortunately, all the fetuses diagnosed in this study proved to be either completely negative for the parental Bthalassemia mutations or carrier for only one mutant allele, therefore, all the pregnancies were continued. The PCR based techniques employed in this study have greatly facilitated the rapid carrier detection and prenatal diagnosis of B-thalassemia. Moreover, the first trimester chorionic villus sampling (CVS) instead of fetal blood or amniotic fluid has made prenatal diagnosis possible at an early stage of pregnancy. The present study indicates that existence of high genotypic variability in the Turkish population complicates the carrier detection and establishment of prenatal diagnosis for B-thalassemia and would not be as simple as in many other Mediterranean countries, however, possible in all the affected families