Spectrum of b thalassemia Mutation in Bahrain
Jassim N, M Phil*, Shaikha Al-Arrayed, PhD*
Al-Muharraq H, MD*, Merghoub T, PhD
Krishnamoorthy R, MD, PhD
Objective: to study the molecular characterization of b thalassemia mutations among Bahrainis.
Methods: we used a variety of polymerase chain reaction (PCR)- based procedures including reverse dot blot (RDB), denaturing gradient gel electrophoresis (DGGE) and DNA sequencing, to study the b-thal mutation in 87 Bahraini individuals from 51 unrelated Bahraini families.
Results: Thirteen different b-thala mutations were identified. Four mutations (Intervening Sequence I (IVSI)-3' end(-25 bas pairs (bp) deletion; Codon (Cd) 39 (C-T) and IVSI-5 (G-C), account for 80% of all b-thal alleles.
Conclusion: we conclude that IVSI-3' end (-25bp) deletion is the major b-thalassemia allele in Bahrain.
Recommendations: Based upon our findings, a preventive approach of b-thalassemia needs to be embloyed for the Bahraini people. This stludy can be used in implementing a cost effective strategy for screening and diagnosis of b-thal among Bahrainis.
Bahrain Med Bull 2000; 22(1): 8-12
The hemoglobinopathis are the most common among genetic disorders in Bahrain, represented mainly by sickle cell disease (SCD) and thalassemia1. among thalassemias both alpha and beta forms are identified. The b-thalassemias are group of inherited anemias characterized by a reduced (b+) or absent (b) production of the b-globin chain from the affected allele2. this leads to an imbalanced alpha/non-alpha globin chain production and subsequent accumolation of the alpha-chains which is the major pathophysiologic route in the b thalassemia syndromes. The vast majority of mutations causing b-thalassemia are non-deletional forms of which more than 180 point mutations have been characterized to date.
The incidence of b-thalassemia trait in Bahrain is low as 2%2 but it is the most severe among other haemoglobinopathies. It is manifested clinically in homozygous state, with a life-long blood transfusion dependency phenotype2. on the other hand most of b-thalassemia heterozygote state are clinically asymptomatic with a distinctive hematolofical phenotyper represented by hypo chromic, microcytic red blood cells and characteristically raised levels of Hb A2. exceptions to the latter include coinheritance of alpha thalassemia which will render HbA2. into a normal/borderline level. Moreover, the ingeritance of b-thalassemia might be masked by coinheritance of sickle cell gene. Thus, molecular diagnosis took an important place as a useful tool to overcome these diagnostic obstacles.
The techniques of denaturing gradient gel electrophoresis (DGGE) and reverse dot blot (RDB) technique as well as direct DNA sequencing were applied in this first study to uncover the molecular basis of b-thalassemia in Bahrain. Moreover, b-thalassemia (patterns of arrangements of the restriction fragment length polymorphisms (RFLPs) in the b-globin gene cluster0 were investigated for the uncovered mutations in order to identify possible origin(s) of these mutations.
Patients: eighty seven native Bahraini individuals representing 51 unrelated families were studied. The patients were divided as follow: 33 clinically homozygote b-thalassemia, 17 S-b-thal and 37 simple heterozygotes.
All of these individuals are attendees of the genetic and pediatric departments at Salmaniya Medical Complex, Bahrain. The genetic study was done in the molecular lavoratories in Robert Debre Hopital in Paris, France.
Some of the individuals studied were having b-thal major. They were blood transfusion dependent. Others were heterozygous (carrier) for b-thal with elevated HbA2 microcytosis and hypochromia.
Blood Analysis: the whole blood samples were collected in EDTA-anticoagulatd vacationers and analysis were performed according to established methodologies2,4.
DNA Extraction: Genomic DNA was isolated from leucocytes by the phenol-chloroform extraction method as described by Dracopli et al5.
PCR-DGGE and Sequencing: the specific amplification of the different b-globin gene fragments with subsequent DGGE analysis were performed according to previously published procedures6. sequencing protocol was performed according to the dideoxy termination method7,8 utilizing the Sequenase Verion 2.0 DNA sequencing kit (US Biochemical, Cleveland, USA). The same PCR products and primers of DGGE were used for sequencing.
Reverse Dot Blot: The reverse dot blot technique was used for further searching of explored mutations as described in previous studies9.10, except for untilising hybridization washing at temperature of 42 c instead of 45c.
Haplotype Analysis: it is defined by seven polymorphic sites in the b-globin gene cluster, was performed using a PCR-RFLP procedure as describe11-13. the polymorphic restricition sites studied were XmnI-5' Gy,HindIIIGy, HindIII-Ay, HincII-Ψβ, HincII-3' Ψβ, AvaII-b, and HinfI-3'b
A total of 70 βtha chromosomes were characterized in this study. The frequency of each mutation is presented in Table 1 along with previously published frequency data from three neighboring countries. The IVSI-3' end (-25bp) deletion allele represents the first major mutation in Bahrain with a frequency of 36%, followed by Cd 39 (c-T), a nonsense Mediterranean type mutation, that accounts for 26% of the mutations. The third major mutation is IVSI-5 (G-C). which was found at a frequency of 16%. Thus, four bthal alleles comprised~80% of all characterized β-thal mutations. The remaining 20% of the β-thal alleles were distributed among 10 different less frequent or rare mutations.
Haplotype analysis of the major and most common β-thalassemia mutations in Bahrain revealed each of them to be in linkage disequilibrium with specific haplotype(s). However, each single mutation has a common framework background (defined here by the presence or absence of the restriction sites for Ava II and HinfI).
In addition to the above findings the following technical features are noteworthy:
1. DGGE profile of homozygous state for codon 44 (-c) was behaving like normal pattern without any alteration in the melting profile. Consequently, this mutation was not possible to be discovered by ordinary DGGE analysis. however by premixing PCR produt of the patient with a homologous normal pcr product succeeded by instant denaturation and annealing steps prior to DGGE analysis, it was possible to characterize the abnormality in