1. ABSTRACT
Beta-thalassemia is of the most prevalent single gene disorder in the world. Beta-thalassemia is caused by the reduced (beta+) or absent
(beta0) synthesis of the beta globin chains of the haemoglobin tetramer in blood. Three clinical and haematological conditions of
increasing severity are recognised, i.e. beta thalassemia carrier state, thalassemia intermedia, and thalassemia major. In the present study,
the prevalence of beta-thalassemia was studied in Indian population. A total of 118 patient samples were collected; all were beta-
thalassemia major patients from the Thalassemia and Sickle Cell Society, Hyderabad. Screening for mutations were carried out in 19
samples. Screening was carried out by genomic DNA isolation from patient blood samples and then amplifying a region of HBB gene, a
mutation hotspot (319bp, from 62187 to 62506). The amplified region was then sequenced to identify the probable mutations present. The
IVS I-5 (G-C) or HBB:c. 92+5 (G>C) was found in homozygous condition in 7 patients and heterozygous condition in 8 patients, was the
most common mutation identified. HBB:c. 47 G>A was observed in two patients in homozygous condition. Heterozygous condition of
HBB:c. 126 C>G and HBB:c. 127 T>A was observed in one patient. HBB:c. 92 G>C or Hb Monroe, identified in one of the patient
sample, has been reported to be a rare mutation in Indian population by previous studies. The work on the remaining samples is still being
carried out. Such a comprehensive mutation screening is essential for prenatal diagnosis beta-thalassemia and control of this highly
prevalent monogenic disorder in Indian population.
INTRODUCTION
Beta thalassemia is one of the most common autosomal recessive
disorder worldwide. High prevalence is present in population in the
Mediterranean, Middle-East, Transcaucasus, Central Asia, Indian
subcontinent and Far East. It is also relatively common in populations of
African descent. The highest incidences are reported in Cyprus (14%),
Sardinia (12%), and South East Asia. It is also a common
haemoglobinopathy in India as per WHO records.
The beta globin (HBB) gene maps in the short arm of chromosome 11 in a
region also containing the delta globin gene, the embryonic epsilon gene,
the fetal A-gamma and G-gamma genes, and a pseudogene (ψB1). The five
functional globin genes are arranged in order of their developmental
expression.
Beta thalassemia is inherited in autosomal recessive fashion.
OBJECTIVES
In silico retrieval and analysis of sequences and primer design to amplify
the mutation hotspot.
Genomic DNA isolation from patient samples.
PCR amplification of a region in hbb gene.
Sequencing of the amplified region.
Mutation analysis and carrier/diseased state identification.
Genetic counseling of the patients.
METHODOLOGY
Molecular Analysis of Beta-thalassemia Variants
Deepanjan Ghosh, Dr. M. Parani
Department of Genetic Engineering, School of Bioengineering, SRM University
REFERENCES
1. Verma IC, Saxena R, Thomas E, Jain PK (1997) Regional distribution of β- thalassemia mutations in India. Human Genetics 100: 109-113.
2. Agarwal S, Hattori Y, Agarwal SS (2000) Rare- beta- thalassemia mutations in Asian Indians. American Journal of Hematology 65: 322- 323.
3. Flint J, Harding R, Boyce AJ, Clegg JB (1993) The population genetics of the haemoglobinopathies. Baillière's Clinical Haematology 6: 215-262.
Major beta-thalassemia variants in Indian
population are:
IVS -1-5 (G-C) codon 41/42 (-TTCT) codon 42 (C-G)
codon 8/9 (+G) codon 15 (G-A)
codon 43 (T-A) codon 31 (G-C)
RESULTS AND DISCUSSION
MUTATIONS SCREENED
IVS I-5(G>C) (-/-) IVS I-5(G>C) (+/-)
HBB:c. 47 G>A (-/-) HBB:c. 92 (G>C) (+/-)
HBB:c. 126(C>G) (+/-) HBB:c. 127 (T>A) (+/-)
This study revealed that IVS I-5 (G>C) or HBB:c. 92+5(G>C), both heterozygous
and homozygous state is the most common mutation found in the sample cases.
The mutation HBB:c. 9 G>C was also found in many patients in the group studied.
A mutation HBB:c. 92 G>C or Hb Monroe, which is located in the last nucleotide
of Exon 1 was identified and this mutation has been termed as a rare mutation by
previous studies
This study has been successfully extended to provide genetic counselling to the
affected families in terms of carrier detection, antenatal screening, and prenatal
diagnosis by the Thalassemia and Sickle Cell Society, Hyderabad.
gDNA isolation from blood by Modified Miller’s Method
PCR amplifiacation of a 319 bp region (mutation hotspot) in
HBB gene
PCR product purification (EZ
10 spin column)
Automated DNA sequencing
Mutation Analysis and Reporting
Figure 1: Genomic DNA from blood samples Figure 2: PCR amplified region of HBB gene