Abstract
β-Thalassaemia, the most common monogenic disorder, is characterized by genetic heterogeneity at the molecular level. More than 300 mutations of the β globin gene have been characterized all over the world, however, few common mutations account for majority of the cases in various populations. The present study aimed to screen known cases of β-thalassaemia in the Western part of Rajasthan state for five common mutations. The study included 144 known cases of β-thalassaemia of all clinical phenotypes. Cases were diagnosed based on clinical features, haematology investigations including haemogram and Hb-HPLC. Blood samples from cases were taken for mutation analysis. After DNA extraction, mutations were characterized by the polymerase chain reaction method employing allele specific priming technique (AMRS) to study the five mutations including IVS-I-5 (G → C), IVS-I-1 (G → T), CD41/42 (–TCTT), CD 8/9 (+G) and 619 bp deletion from the 3′ end of the β-globin gene using a total of seven different primers. Of all 144 cases, 74 (51.38% of all) cases were of β-thalassaemia major, five (3.4% of all) cases were of β-thalassaemia intermedia and 65 (45.14% of all) cases were of β-thalassaemia minor. Mutation analysis revealed that five common mutations were present in 130 (90.27% of all) cases. Among identified mutations, highest frequency of mutation was of IVS-I-5 (G → C) identified in 73 cases (50.7% of all cases). In 11 (7.63% of all) cases, more than one mutation was identified. β-Thalassaemias are common in Western Rajasthan; however, there is dearth of literature from this part of the country. We observed that five common mutations are common in this part of the country also. These observations are helping us in forming the basis for comprehensive diagnostic database that would not only be useful for genetic counselling but also for prenatal diagnosis.
Keywords: Thalassaemia, Haemoglobinopathy, Mutations, Rajasthan
Introduction
The inherited disorders of haemoglobin are the commonest human monogenic diseases with an autosomal recessive inheritance affecting at least seven percent of the world’s population. Among these, the beta-thalassaemias is, by far, the most important public health problem globally.
Beta-thalassaemias are characterized by defective beta-globin chain synthesis leading to imbalance in globin chain production and an excess of alpha-chains. This excess of alpha chains aggregates in bone marrow erythroid precursors causing their premature destruction.
In India, the average prevalence of beta-thalassemia carriers is 3–4% translating to more than 40 million carriers in our multi-ethnic, culturally and linguistically diverse population. Our population also includes around eight percent of tribal groups according to the Census of India 2011 and several ethnic groups have a much higher prevalence (4–17%) [1–3].
Thalassemia syndromes require long and specialized treatment causing severe distress and financial burden to the family as well as affecting nation’s health resources. Government of India has taken a major step to bring out a national policy for the diagnosis, management and prevention of thalassemias. With the launch of National Programme, it is expected that the care of thalassemia and haemoglobinopathies will improve significantly [4].
β-Thalassaemias are heterogeneous not only clinically but also at the molecular level. More than 350 beta-thalassaemia mutations have been reported so far in the IthaGenes database and majority of mutations are single nucleotide substitutions, deletions or insertions of oligonucleotides leading to frameshift; rarely does it result from gross gene deletion [5].
Further, each population or sub population has its own unique spectrum of mutations for beta-thalassaemia. Around 80 mutations have now been characterized in India and approximately 92% of β-thalassemia allele in India is accounted by common mutations such as IVS 1-5, IVS 1-1, 619 bp deletion, codon 8/9, and codon 41/42 [6].
These mutations for beta-thalassaemia are not uniform in their distribution across India and have geographic specificity with some common mutations in each ethnic group. There is also regional variation in prevalence across northern, eastern and southern parts of India. IVS 1-5 is the most common mutation across India as well as in Pakistan and Sri Lanka. IVS 1-5 (43.5%) and codon 8/9 (38.5%) are two most common mutations in North India whereas in South India codon 41/42 is more frequent and mutation HBE (codon 26 G−A) (c. 79G > A) is frequently found in Eastern India [7, 8].
Prevention of birth of beta thalassaemia major babies can be achieved by either carrier screening or by prenatal testing. However, in India, access to prenatal diagnosis is not convenient as the facility is available primarily in the major metropolitan cities. Paucity of literature exclusively from Rajasthan prompted us to undertake this study with the aim to evaluate cases of beta-thalassemia for common mutations in Western Rajasthan.
Material and Methods
The present study was conducted at a tertiary care centre of Western Rajasthan between October 2017 to December 2018. Patients were enrolled after obtaining approval from Institute’s Ethics Committees. Informed and written consent was taken from the patients or parents (in case of minors). Diagnosed cases of beta-thalassaemia including all clinical phenotypes, viz, thalassaemia major, thalassaemia minor and thalassaemia intermedia were included in the study. Cases of other haemoglobinopathies and cases wherein the diagnosis had not been established with certainty were excluded from the study.
Sample Collection
Samples of diagnosed cases of all clinical phenotypes of beta-thalassaemia were collected for mutation analysis. Cases of beta-thalassaemia trait were the ones encountered during routine haemoglobin high performance liquid chromatography (on Bio-Rad D-10 Hb-HPLC system) testing in department of Pathology. Cases of beta thalassaemia major and intermedia included both newly diagnosed as well as previously diagnosed cases who were taking regular transfusions in department of Immunohaematology and Blood Transfusion of the collaborating Institute. Blood samples were collected in EDTA vials and were transported immediately to the laboratory for DNA extraction.
DNA Extraction and Storage
DNA extraction was done using HiPurA blood genomic DNA Miniprep Purification Kit and the process principally comprised of three phases viz. adsorption of DNA to the membrane, removal of residual contaminants and elution of pure genomic DNA. Peripheral blood collected in EDTA vials was processed as per manufacturer’s instructions. DNA, extracted in the form of eluate, was transferred to a fresh-capped collection tube, numbered and stored at −80 °C.
Mutation Analysis
Mutation analysis was carried out using Himedia beta-thalassaemia detection kit which is a qualitative conventional polymerase chain reaction (PCR) kit containing seven different primers detecting five mutations, namely IVS-I-5 (G → C), IVS-I-1(G → T), CD 41/42 (–TCTT), CD 8/9 (+G) and Δ 619 bp deletion from the 3′ end of the β-globin gene.
Preparation of PCR reaction mixture was done as per manufacturer’s instructions. Template DNA was added to the PCR reaction mixture and amplified as per manufacturer’s instructions using recommended programme on Bio-Rad CFX96 real-time PCR system. Amplicon was loaded on a 1.5% agarose gel along with 6X DNA loading dye. 3 μL of 100 bp DNA ladder was loaded in separate well. Ethidium bromide was incorporated in the agarose gel. The presence of amplification product indicated mutation and absence of amplified product of expected size indicated absence of mutation.
Results
In the present study, a total of 144 cases of beta-thalassaemia were enrolled. Out of these 144 cases, 74 cases (51.38%) were of beta-thalassaemia major, 5 cases (3.4%) were of beta thalassaemia intermedia and 65 cases (45.14%) were of beta-thalassaemia minor.
Of all cases, one or more mutation was identified in 130 cases (90.27%) whereas 14 cases (9.73%) did not reveal any mutation. In 11 cases (7.63% of all cases), more than one mutation was identified. Among identified mutations, highest frequency of mutation was of IVS-1-5(G → C) which was identified in 73 cases (50.7% of all cases) as shown in Table 1.
Table 1.
Frequency of beta-thalassaemia mutation detected
| Mutations | Number of patients detected | Frequency (%) |
|---|---|---|
| No mutation | 14 | 9.72 |
| IVS-1-5 (G → C) | 73 | 50.69 |
| CD8/9 (+G) | 28 | 19.44 |
| IVS-1-1 (G → T) | 22 | 15.27 |
| CD 41/42 (–TCTT) | 13 | 9.02 |
| 619 bp deletion | 05 | 3.74 |
Cases of beta-thalassaemia major were those who had already received multiple transfusions and ranged from one year of age to 21 years of age. Among 74 cases, 27 were females and 47 were males. On mutation analysis, eight cases did not show any mutation and 66 cases revealed presence of one of the five mutations as shown in the Table 2.
Table 2.
Mutation frequency in various clinical phenotypes
| Beta-thalassaemia major | Beta-thalassaemia intermedia | Beta-thalassaemia minor | |
|---|---|---|---|
| Total cases | 74 | 5 | 65 |
| IVS-1-5 (G → C) | 37 | 2 | 34 |
| CD8/9 (+G) | 14 | 0 | 14 |
| IVS-1-1 (G → T) | 13 | 1 | 8 |
| CD 41/42 (−TCTT) | 7 | 0 | 6 |
| 619 bp deletion | 2 | 1 | 2 |
There were five cases of beta thalassaemia intermedia in the study. On Hb-HPLC study, one case was diagnosed as heterozygous beta-thalassaemia and rest of the cases were homozygous beta-thalassaemia. Two of the cases did not reveal any mutation whereas three cases revealed presence of one or more mutation shown in Table2.
Of all cases, 65 cases were of beta thalassaemia minor. Majority of these cases were clinically asymptomatic and were diagnosed on Hb-HPLC study. These cases had revealed microcytic hypochromic red cell indices on complete haemogram. On mutation analysis, four cases did not show any mutation and 61 cases revealed presence of one of the five mutations as shown in the Table 2.
Discussion
Prenatal genetic diagnosis may prevent births of beta-thalassaemia major babies as well as emotional, psychological and financial trauma of the family and molecular profiling of beta thalassaemia cases regionally can facilitate this. However, there is dearth of literature on molecular spectrum of beta-thalassaemia from Rajasthan.
The present study was conducted to fill the gap, to elucidate the mutation pattern in this part of country, and to compare this with the national data.
About 400 mutations have been identified in the beta-globin gene which can cause reduced or absent Hb production. In India, 64 β-globin mutations have been reported as documented in the Thailand database until date [9]. Various studies have been conducted on the Indian population before, and six mutations, codon 8-9 (+G), Codon 15 (G−A), codon 41/42 (–TCTT), IVS I-1 (G−T), IVS I-5 (G−C) and 619 bp deletion at 3′ end of β-globin gene, account for about 90% of β-thalassaemia mutation in Indian population.
Although India has multiple geographical, ethnic, religious, and language divisions yet each population seems to harbour only a few of all reported mutations. The regional difference in in frequency of mutation has been pointed out by various studies, e.g., 619 bp deletion which has a high prevalence rate in Western Indian states of Gujarat and Maharashtra, whereas its prevalence is quite low in the southern states. Other community-based studies have also shown the community based dominance of certain mutations [9–11].
The present study screened beta-thalassaemia cases for five common mutations viz. IVS-1-5 (G → C), IVS-1-1 (G → T), CD41/42 (–TCTT), CD8/9 (+G) and 619 bp deletion and revealed that these mutations are common in the studied population also. We report the dominance of IVS1-5 (G−C) mutation as the most common mutation attributing to half of all the cases studied which is in concordance with earlier studies [7, 8]. Second most common mutation observed was CD8/9 (+G) mutation. This finding was in concordance with a study from Uttar Pradesh whereas the observation is in contrast with the previous report showing it to be the fourth most common mutation in India [10, 12]. Overall, the five mutations comprised 90.27% of all the loci which is similar to mutational spectrum known for Northern India. Our study had limitation of not identifying other mutations which were present in remaining population.
Although the present study was not a community specific mutation study, our cohort included patients primarily from Western Rajasthan and gives an overall view which may be investigated further at community level.
Further, we tried to establish genotype–phenotype correlation but the results (presence or absence of mutations) cannot predict clinical phenotype as patients of beta-thalassaemia major and minor have been found to harbour similar mutations when screened for five common mutations. Further studies would be needed to look in to presence of multiple mutations as well as for study of disease modifiers [13].
Information of distribution and frequency of different beta-thalassemia mutations is vital for establishing programme for carrier screening, genetic counselling, prenatal diagnosis and for physicians to effectively manage patients at large. This information is being applied in setting up of centre for prenatal diagnosis in our Institute.
Acknowledgements
None.
Funding
Intramural research grant from All India Institute of Medical Sciences, Jodhpur.
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
Footnotes
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