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On Sep 2018




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"Journal of Clinical and Diagnostic Research is at present a well-known Indian originated scientific journal which started with a humble beginning. I have been associated with this journal since many years. I appreciate the Editor, Dr. Hemant Jain, for his constant effort in bringing up this journal to the present status right from the scratch. The journal is multidisciplinary. It encourages in publishing the scientific articles from postgraduates and also the beginners who start their career. At the same time the journal also caters for the high quality articles from specialty and super-specialty researchers. Hence it provides a platform for the scientist and researchers to publish. The other aspect of it is, the readers get the information regarding the most recent developments in science which can be used for teaching, research, treating patients and to some extent take preventive measures against certain diseases. The journal is contributing immensely to the society at national and international level."



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On Aug 2018




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Dr. Arundhathi. S
MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
Bengaluru.
On Aug 2018




Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
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Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help one’s reviewing skills.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".



Dr. Mamta Gupta
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018




Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.


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Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
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Indexation and online availability: Indexation transforms the journal in some sense from its local ownership to the worldwide professional community and to the public.JCDR is indexed with Embase & EMbiology, Google Scholar, Index Copernicus, Chemical Abstracts Service, Journal seek Database, Indian Science Abstracts, to name few of them. Manuscriptspublished in JCDR are available on major search engines ie; google, yahoo, msn.
In the era of fast growing newer technologies, and in computer and internet friendly environment the manuscripts preparation, submission, review, revision, etc and all can be done and checked with a click from all corer of the world, at any time. Of course there is always a scope for improvement in every field and none is perfect. To progress, one needs to identify the areas of one's weakness and to strengthen them.
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Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."



Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
E-mail: drrajendrak1@rediffmail.com
On May 11,2011




Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."



Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
E-mail: ravi.dr.shankar@gmail.com
On April 2011
Anuradha

Dear team JCDR, I would like to thank you for the very professional and polite service provided by everyone at JCDR. While i have been in the field of writing and editing for sometime, this has been my first attempt in publishing a scientific paper.Thank you for hand-holding me through the process.


Dr. Anuradha
E-mail: anuradha2nittur@gmail.com
On Jan 2020

Important Notice

Original article / research
Year : 2023 | Month : February | Volume : 17 | Issue : 2 | Page : BC05 - BC09 Full Version

Association of β-Globin Gene Haplotypes with Haematological Parameters and Foetal Haemoglobin among Patients with Sickle Cell Disorder in Raipur, Chhattisgarh, India


Published: February 1, 2023 | DOI: https://doi.org/10.7860/JCDR/2023/58342.17445
Sanjana Bhagat, Amar Singh Thakur

1. Assistant Professor, Department of Biotechnology, Government Nagarjuna PG College of Science, Raipur, Chhattisgarh, India. 2. Professor, Department of Biochemistry, Government Medical College, Jagdalpur, Chhattisgarh, India.

Correspondence Address :
Dr. Sanjana Bhagat,
Assistant Professor, Department of Biotechnology, Government Nagarjuna PG College of Science, GE Road, Raipur-492010, Chhattisgarh, India.
E-mail: sanjana_bhagat@rediffmail.com

Abstract

Introduction: Sickle cell disease is caused by a single nucleotide substitution in the β-globin gene. The variations in Foetal Haemoglobin (HbF) levels, β-globin gene cluster haplotype have been used as predictors of disease severity in sickle cell disease patients.

Aim: To determine the frequency of β-globin gene haplotypes in sickle cell disease patients and also to establish their association with haematological parameters and HbF.

Materials and Methods: The present cross-sectional study was conducted in Department of Biochemistry, Government Medical College, Jagdalpur, Chhattisgarh, India, in collaboration with Department of Biotechnology, Government Nagarjuna PG College of Science, Raipur, Chhattisgarh, India, from April 2021 to May 2022. A total of 100 patients with Sickle Cell Disease (SCD) and 50 with sickle cell traits were included in the study. Haplotypes were identified by Restriction Fragment Length Analysis (RFLP) method for seven polymorphic sites in β-globin gene cluster. Haematological parameters such as Hb, Haematocrit (HCT), Mean Cell Volume (MCV), Mean Cell Haemoglobin (MCH) and HbF levels were estimated. Data was analysed using various statistical tests such as Shapiro-Wilk test, Levene’s test, student t-test, Mann-Whitney test and Kruskal-Wallis tests as per analysis requirement.

Results: In the present study, 51 (51%) males and 49 (49%) females were in sickle cell disease group (SS), while 25 (50%) males and 25 (50%) females were in sickle cell trait group (AS). The mean age of the sickle cell disease patients was 23.84±8.38 years and for sickle cell trait group was 26.3±7.37 years. There was a significant difference (p-value <0.0001) in HbF levels among haplotypes. Additionally, higher HbF concentration was found in Arab-Indian haplotypes in SCD patients. No significant association was observed between the haplotypes and haematological parameters.

Conclusion: The findings suggested that haematological parameters were not significantly associated with β-globin gene haplotypes. The β-globin gene haplotypes influence the HbF levels in sickle cell patients.

Keywords

Chromatography, Disease severity, Restriction fragment length analysis, Sickle cell traits

Sickle Cell Disease (SCD) is caused by a single nucleotide substitution in the β-globin gene. However, despite being a monogenic disease, sickle cell disease has been associated with diverse range of disease severity. The severity of sickle cell disease has correlation with various clinical phenotypes between different individuals (1). The clinical severity is influenced by variations in Foetal Haemoglobin (HbF) levels, the β-globin gene haplotype and presence of α-thalassaemia (2).

Foetal Haemoglobin is a well-known major genetic modulator of the clinical heterogeneity observed in sickle cell disease patient (3),(4),(5). Higher HbF levels protect against many of the haematologic and clinical complications of sickle cell anaemia. Recently, it is reported that higher expression of HbF in adulthood ameliorates morbidity and mortality in sickle cell disease (6),(7),(8),(9). In this context, much work has been done for analysis of genetic determinants in the β gene cluster that might affect globin gene expression and thus relate to the clinical diversity of sickle cell disease (10),(11),(12). This observation stimulated research to identify genetic determinants associated with disease severity such as β-haplotypes based on Restriction Fragment Length Analysis (RFLP) analysis in Chhattisgarh, India.

The β-globin gene haplotype is characterised by the non random association of cleavage sites recognised by restriction endonucleases enzymes (13). Five major β-haplotypes designated as Benin, Bantu, Cameroon, Senegal and Arab-Indian haplotypes are named according to the geographic region or ethnic group in which they were most commonly found (14),(15),(16),(17),(18). Other less common haplotypes, known as atypical haplotypes, are generated by a number of genetic mechanisms (19),(20),(21).

Till date, the relationship between βS haplotypes and HbF level with haematological parameters has not been defined for SCD patients in Chhattisgarh. A better understanding of β-globin gene haplotypes is of particular relevance to population genetic studies and may also be useful for understanding the varying clinical outcomes seen among individuals with sickle cell disease. Hence; present study was conducted to determine the frequency of βS haplotypes and their association with HbF level, haematological parameter in SCD patients of Chhattisgarh, India.

Material and Methods

This cross-sectional study was conducted in the Department of Biochemistry, Government Medical College Jagdalpur, India, in collaboration with Department of Biotechnology, Government Nagarjuna PG College of Science, Raipur, Chhattisgarh, India, from April 2021 to May 2022. The study was approved by the Institutional Ethical Committee (reference no.3572/GMCJ/Estt/22). Written informed consent was taken from all the subjects before commencement of the study.

Inclusion criteria: Patients with positive result of solubility test, cellulose acetate electrophoresis and Sickle Haemoglobin (HbS) mutation {confirmed using Polymerase Chain Reaction (PCR)-RFLP method of Dde I restriction enzymes}, were included in the study.

Exclusion criteria: Patients with systemic disease like Human Immunodeficiency Virus (HIV) and renal failure, taking any kind of treatment (other than folic acid supplementation) and blood transfusions during the four months prior to sample collection were excluded from the study. Pregnant women and patients with sickle cell disease and sickle cell trait belonging to same family were also excluded from the study.

Sample size calculation: A total of 100 sickle cell disease patients (SS) and 50 sickle cell traits (AS) were enrolled in the present study by convenience sampling. Samples were collected by random sampling method.

Demographic data such as age, gender were collected from all the participants. Venous blood samples (5 mL) were collected under aseptic precautions, using Ethylenediamine Tetraacetic Acid (EDTA) as an anticoagulant.

Molecular Diagnosis of Patients (PCR-RFLP Method)

Genomic Deoxyribonucleic Acid (DNA) was extracted using Himedia DNA kit and amplified by PCR (Bio-Rad thermal cyclers, model no. T100) using forward 5’-ACC TCA CCC TGT GGA GCC AC-3’ and reverse 5’-GAG TGG ACA GAT CCA AAG GAC TCA AAG A-3’ primers followed by restriction digestion and agarose gel inspection. The presence of an A or T nucleotide in the 6th codon of β-globin was confirmed for all DNA samples using the restriction enzyme Dde I analysis (fermentas) (22).

Polymerase chain reaction mix were prepared by adding the following:

• 2.5 μL of 10x buffer,
• 1.5 μL (25 mM) MgCl ,
• 1.56 μL (2 Mm) deoxynucleoside triphosphate dNTP’s,
• 1 μL of forward and reverse primer (10 picomoles),
• 0.5 μL of taq polymerase and nuclease free water 15.9 μL, and
• 1 μL of genomic DNA (5 ng/μL).

The reactions consisted of an initial step of DNA denaturation at 94°C for three minutes, followed by 35 cycles of one minutes at 94°C for denaturation, one minutes at 56°C for annealing, one minutes at 72°C for polymerisation and a final step of seven minutes at 72°C.

Haematological Study

Complete blood counts were carried out using an automated cell counter (BC-3000 Plus Auto Haematology Analyser). Haematological parameters such as Hb level, Haemotocrit (HCT), Mean Corpuscular Value (MCV), Mean Corpuscular Haemoglobin (MCH) were studied by haematological analysis (23).

HbF Level Determination

The HbF% was quantified by ion-exchange high-performance liquid chromatography. High Performance Liquid Chromatography (HPLC) was carried out using VARIANTTM β-thalassaemia Short Program, Bio-Red L 70018803 model instrument equipped with a dual-wavelength filter photometer (415 and 690 nm) (24).

Haplotype Analysis by RFLP

Polymerase chain reaction based restriction enzyme digestion method was used for haplotype analysis with following polymorphic restriction site; Hinc II ε, Hind III Gγ, Hind III Aγ, Hinc II 5’ψβ, Hinc II 3’ψβ, Ava II β, Hinf I 3’-β in the beta globin gene cluster. The PCR amplified products containing each of these polymorphic sites were analysed by digested with the appropriate restriction enzyme and visualised by agarose gel analysis (25),(26). In the present study 10 haplotypes were compared numbered as: 1) Arab-Indian haplotype; 2) Atypical Arab-Indian; 3) Atypical Benin haplotype; 4) Atypical Bantu haplotype; 5) Atypical Cameroon haplotype; 6) Benin haplotype; 7) Atypical Cameroon/Benin haplotype; 8) Cameroon haplotype; 9) Rare 1 and 10) Rare 2.

Statistical Analysis

Statistical analysis was analysed by using the Statistical Package for the Social Sciences (SPSS) software version 16.0. Firstly, data was analysed for normal distribution and homogeneity of variances assumption according to Shapiro-Wilk test and Levene’s test, respectively, when the data showed the normal distribution and met the assumptions for parametric test. Further variable was compared by using the student t-test for two groups. Those groups that did not met the parametric assumptions were further compared by non parametric test by using Mann-Whitney U test and Kruskal-Wallis test. Results were presented as mean±Standard Deviation (SD). A p-value of ≤0.05 was considered statistically significant.

Results

In present study, 51 (51%) males and 49 (49%) females were in sickle cell disease group (SS), while 25 (50%) males and 25 (50%) females were in sickle cell trait group (AS). The mean age of the sickle cell disease patients was 23.84±8.38 years and for sickle cell trait was 26.3±7.37 years.

The maximum HbF level of haplotypes was found in Arab-Indian haplotype (22.71±5.27), followed by atypical Arab-Indian (20.85±3.16), rare 2 (14.70±0.14) haplotype, rare 1 (13.70±1.81) and atypical Cameroon (12.84±0.75) (Table/Fig 1).

A haematological comparison between different haplotypes showed insignificant difference for Hb, HCT, MCV and MCH parameters among SS patients. There was a significant difference (p-value <0.0001) only for HbF concentration between all haplotypes in SS patients (Table/Fig 2).

Mean HbF levels in sickle cell trait were 1.96±0.43 for Arab-Indian haplotype, 0.94±0.19 for atypical Arab-Indian, 0.14±0.05 for atypical Benin, 0.42±0.13 for atypical Cameroon, 0.11±0.01 for rare 1 and 0.2 for rare 2 haplotype (Table/Fig 3).

No significant differences in the mean levels of Hb, HCT, MCV and MCH were observed between haplotypes among sickle cell trait (AS). However, the significant difference (p-value <0.001) was observed only for HbF level between haplotype in AS individuals (Table/Fig 4).

A significant difference was observed for HbF level, Hb and MCV values for Arab-Indian and Atypical Arab-Indian haplotype, while in Atypical Cameroon and Atypical Benin haplotype show significant difference only for Hb and HbF level between AS and SS patient. The result with Arab-Indian haplotype was found significant difference for HbF (p-value <0.001), Hb (p-value <0.002) and MCV (p-value <0.001), Atypical Arab-Indian haplotype for HbF (p-value <0.001), Hb (p-value <0.006) and MCV (p-value <0.023), Atypical Benin haplotype for HbF (p-value <0.017), Atypical Cameroon haplotype for HbF level (p-value <0.008) and Hb (p-value <0.032), respectively. Additionally, no significant difference was observed for rare 1 and rare 2 atypical haplotype between the SS and AS groups (Table/Fig 5).

Discussion

In this study, the haematological differences between β-globin gene haplotypes among sickle cell disease and sickle cell trait patients were examined. The Arab-Indian haplotype was the most common haplotype encountered, followed by atypical Arab-Indian. Due to small number of other atypical haplotype found in this study the haematological parameters were compared between most frequent found haplotypes and other haplotypes. This is in accordance with the recent studies on Benin haplotype was the most predominant in Nigerian sickle cell patients (13). In the current study, no significant differences were observed between haematological parameter and β-globin gene haplotypes except for HbF levels.

Foetal haemoglobin is related to the haplotype and correlate with clinical course of Sickle Cell Anaemia (SCA) (27). Senegal and Arab-Indian haplotypes, by producing the highest levels of HbF in the blood, are associated with less severe clinical evolution of sickle cell anaemia, with a lower occurrence of organic damage (12),(28). As to the Benin and Cameroon haplotypes, the clinical picture is off intermediate severity. The Bantu or Central African Republic (CAR) haplotype is associated with greater clinical severity (29).

Several studies confirmed the association of β-globin gene haplotypes as modulators for clinical presentation of sickle cell disease (27),(28). The Arab-Indian haplotypes influence the disease severity in SCA patients (30),(31),(32),(33). The Benin haplotype is generally associated with lower HbF levels and a severe disease presentation. Elevated HbF levels clearly play a role in decreasing clinical severity, possibly through interfering with HbS sickling process (2),(34),(35),(36).

Despite the fact some haematological parameters are associated with disease severity of SCD patients (5),(24). In the present study, Hb, HCT, MCV and MCH values were not significantly distributed between haplotypes both in AS and SS patients, probably due to the fact that the studied patients presented a variable clinical course like painful episode, vaso-occlusive crises, jaundice, splenomegaly, anaemia and infection. This is in accordance with the previous studies (27),(29),(34).

The present findings are in agreement with others studies, which reported similar results for the association of haematological data among the most frequent haplotypes, only MCV had statistical significance in CAR/Benin haplotypes (13),(22). The sickle cell patients showing the severe normocytic normochromic anaemia were associated with reticulocytosis and leucocytosis, thus the haematological parameters like MCV, MCH and HCT were higher in SS patients (37). Recent study also shows that no significant association was established between the haematological parameters and the various haplotypes; it was observed that patients with the BEN/SEN (Senegal) haplotype displayed a three-fold increased association with improved Hb levels when compared with the BEN/BEN haplotype (13).

In addition, highest HbF level were found in Arab-Indian haplotypes followed by in Atypical Arab-Indian haplotype. These observations suggested that clinical course affect the haematological parameter beside the fact that HbF level is still elevated in SS patients in this study. Beta globin gene haplotypes has been used as a marker for phenotypic heterogeneity of sickle cell disease because of its association with variable HbF levels. However, influence of the Haemoglobin Beta Globin (HBB) gene locus and the XmnI-HBG2 site on HbF levels in SCA has been validated by many studies in several populations (33),(35),(36).

In SCA patients with Bantu haplotype often have low HbF levels, while those with the Cameroon and Benin haplotypes show intermediary HbF levels. However, the present results agree with some previous published data on HbF levels and βS-globin haplotype (18). In this study, the presence of intermediate HbF levels for the Cameroon haplotype could be due to sequence variations in regulatory regions, such as the 5’HS2 and flanking region of the γ gene (38),(39).

Limitation(s)

The limitations of this study were the other genetic polymorphisms that might associate with disease severity were not studied, which can be included in the further studies to determine the correlation with disease severity.

Conclusion

In the present study, HbF level was significantly associated with haematological parameters and β-globin haplotypes. High HbF level and β-globin gene cluster haplotypes act as modulators for disease severity of sickle cell patients of Chhattisgarh, India. More studies as modulators for sickle cell disease are essential to find the disease severity that can be helpful for treatment and management of sickle cell disease.

References

1.
Steinberg MH. Predicting clinical severity in sickle cell anaemia. Br J Haematol. 2005;129:465-81. [crossref] [PubMed]
2.
Akinsheye I, Alsultan A, Solovieff N, Ngo D, Baldwin CT, Sebastiani P, et al. Fetal haemoglobin in sickle cell anaemia. Blood. 2011;118:19-27. [crossref] [PubMed]
3.
Steinberg MH, Sebastani P. Genetic modifiers of sickle cell disease. Am J Hematol. 2012;87:795-03. [crossref] [PubMed]
4.
Thein SL, Menzel S, Lathrop M, Garner C. Control of fetal hemoglobin: New insight emerging from genomics and clinical implications. Hum Mol Genet. 2009;18:R216-R23. [crossref] [PubMed]
5.
Steinberg MH. Genetic etiologies for phenotypic diversity in sickle cell anaemia. Sci World J. 2009;9:46-67.[crossref] [PubMed]
6.
Lettre G, Sankaran VG, Bezerra MA, Araujo AS, Uda M, Sanna S, et al. DNA polymorphisms at the BCL11A, HBS1L-MYB, and β-globin loci associate with fetal hemoglobin levels and pain crises in sickle cell disease. Proc Natl Acad Sci USA. 2008;105:11869-74. [crossref] [PubMed]
7.
Platt OS, Brambilla DJ, Rosse WF, Milner PF, Castro O, Steinberg MH, et al. Mortality in sickle cell disease. Life expectancy and risk factors for early death. N Engl J Med. 1994;330:1639-44. [crossref] [PubMed]
8.
Rees DC, Williams TN, Gladwin MT. Sickle-cell disease. Lancet. 2010;376:2018-31. [crossref] [PubMed]
9.
Harp KO, Botchway F, Dei-Adomakoh Y, Wilson MD, Mubasher M, Adjei AA, et al. Analysis of clinical presentation, hematological factors, self-reported bed net usage, and malaria burden in sickle cell disease patients. Eclinicalmedicine. 2021;39:101045. [crossref] [PubMed]
10.
Vathipadiekal V, Farrell JJ, Wang S, Edward HL, Shappell H, Al-Rubaish AM, et al. A candidate transacting modulator of fetal hemoglobin gene expression in the Arab-Indian haplotype of sickle cell anemia. Am J Hematol. 2016;91(11):1118-22. [crossref] [PubMed]
11.
Sebastiani P, Solovieff N, Hartley SW, Milton JN, Riva A, Dworkis DA, et al. Genetic modifiers of the severity of sickle cell anaemia identified through a genome-wide association study. Am J Hematol. 2010;85:29-35. [crossref] [PubMed]
12.
Cabral CH, Serafim ES, de Medeiros WR, de Medeiros Fernandes TA, Kimura EM, Costa FF, et al. Determination of βS haplotypes in patients with sickle cell anaemia in the state of Rio Grande do Norte, Brazil. Genet Mol Biol. 2011;34:421-24. [crossref] [PubMed]
13.
Adabale A, Lobo Makanjuola SB, Akinbami A, Dosunmu A, Akanmu A, Javid FA, et al. Frequency of beta S globin gene haplotypes among sickle cell patients in Nigeria. J Int Med Res. 2021;49:01-11. [crossref] [PubMed]
14.
Al-Ali AK, Alsulaiman A, Alzahrani AJ, Obeid OT, Vatte CB, Cyrus C, et al. Prevalence and diversity of haplotypes of sickle cell disease in the eastern province of Saudi Arabia. Hemoglobin. 2020;44:78-81. [crossref] [PubMed]
15.
Esoh K, Wonkam A. Evolutionary history of sickle-cell mutation: Implications for global genetic medicine. Hum Mol Genet. 2021;30:R119-28. [crossref] [PubMed]
16.
Choudhury A, Aron S, Sengupta D, Hazelhurst S, Ramsay M. African genetic diversity provides novel insights into evolutionary history and local adaptations. Hum Mol Genet. 2018;27:R209-18. [crossref] [PubMed]
17.
Lindenau JD, Wagner SC, Castro SM, Hutz MH. The effects of old and recent migration waves in the distribution of HBB*S globin gene haplotypes. Genet Mol Biol. 2016;39:515-23. [crossref] [PubMed]
18.
Zago MA, Silva WA Jr, Dalle B, Gualandro S, Hutz MH, Lapoumeroulie C, et al. Atypical βS haplotypes are generated by diverse genetic mechanisms. Am J Hematol. 2000;63:79-84. 3.0.CO;2-D>[crossref]
19.
Liu L, Muralidhar S, Singh M, Sylvan C, Kalra IS, Quinn CT, et al. High-density SNP genotyping to define β-globin locus haplotypes. Blood Cells Mol Dis. 2009;42:16-24. [crossref] [PubMed]
20.
Rahimi Z, Karimi M, Haghshenass M, Merat A. β-globin gene cluster haplotypes in sickle cell patients from Southwest Iran. Am J Hematol. 2003;74:156-60. [crossref] [PubMed]
21.
Clark BE, Thein SL. Molecular diagnosis of haemoglobin disorders. Clin Lab Haematol. 2004;26:159-76. [crossref] [PubMed]
22.
Camilo-Araujo RF, Amancio OM, Figueiredo MS, Cabanas-Pedro AC, Braga JA. Molecular analysis and association with clinical and laboratory manifestation in children with sickle cell anaemia. Rev Bras Hematol Hemoter. 2014;36:334-39. [crossref] [PubMed]
23.
Ngo D, Bae H, Steinberg MH, Sebastiani P, Solovieff N, Baldwin C, et al. Fetal hemoglobin in sickle cell anaemia: Genetic studies of the Arab-Indian haplotype. Blood Cells Mol Dis. 2013;51:22-26. [crossref] [PubMed]
24.
Silva MAL, Friedrisch JR, Bittar CM, Urnau M, Merzoni J, Valim VS, et al. β-globin gene cluster haplotypes and clinical severity in sickle cell anaemia patients in Southern Brazil. Open Journal of Blood Disease. 2014;4:16-23. [crossref]
25.
Sutton M, Bouhassira EE, Nagel RL. Polymerase chain reaction amplification applied to the determination of β-like globin gene cluster haplotypes. Am J Hematol. 1989;32:66-69. [crossref] [PubMed]
26.
Mukherjee MB, Surve RR, Gangakhedkar RR, Ghosh K, Colah RB, Mohanty D. Beta-globin gene cluster haplotypes linked to the βS gene in western India. Hemoglobin. 2004;2:157-61. [crossref] [PubMed]
27.
Loggetto SR. Sickle cell anaemia: Clinical diversity and beta S-globin haplotypes. Rev Bras Hematol Hemoter. 2013;35:155-57. [crossref]
28.
Ogedegbe HO. β-Globin gene cluster haplotype analysis as a predictor of sickle cell disease severity. Lab Medicine. 2007;38:563-68. [crossref]
29.
Adorno EV, Zanette A, Lyra I, Seixas MO, Reis MG, Goncalves MS. Clinical and molecular characteristics of sickle cell anaemia in the northeast of Brazil. Genet Mol Biol. 2008;31:621-25. [crossref]
30.
Habara AH, Shaikho EM, Steinberg MH. Fetal hemoglobin in sickle cell anaemia: The Arab-Indian haplotype and new therapeutic agents. Am J Hematol. 2017;92:1233-42. [crossref] [PubMed]
31.
Fong C, Lizarralde-Iragorri MA, Rojas-Gallardo D, Barreto G. Frequency and origin of haplotypes associated with the beta-globin gene cluster in individuals with trait and sickle cell anaemia in the Atlantic and Pacific coastal regions of Colombia. Genet Mol Biol. 2013;36:494-97. [crossref] [PubMed]
32.
Yaseen NT, Al-Mamoori HS, Hassan MK. Sickle ß-globin haplotypes among patients with sickle cell anaemia in Basra, Iraq: Across-sectional study. Iraqi J Hematol. 2020;9:23-29. [crossref]
33.
Bhagat S, Patra PK, Thakur AS. Fetal haemoglobin and b-globin gene cluster haplotypes among sickle cell patients in Chhattisgarh. J Clin Diagn Res. 2013;7:269-72. [crossref] [PubMed]
34.
Donaldson A, Thomas P, Serjeant BE, Serjeant GR. Foetal haemoglobin in homozygous sickle cell disease: A study of patients with low HBF levels. Clinical and Laboratory Haematology. 2001;23:285-89. [crossref] [PubMed]
35.
El-Hazmi MAF. Heterogeneity and variation of clinical and haematological expression of haemoglobin S in Saudi Arabs. Acta Haematol. 1992;88:67-71. [crossref] [PubMed]
36.
Adekile A. The genetic and clinical significance of fetal hemoglobin expression in Sickle Cell Disease. Med Princ Pract. 2021;30:201-11. [crossref] [PubMed]
37.
Carneiro JS, de Souza Goncalves M, Albuquerque SRL, Fraiji NA, de Moura Neto NP. Beta-Globin Haplotypes and Alpha-Thalassaemia 3.7 kb deletion in sickle cell disease patients from the occidental brazilian amazon. J Hematol. 2016;5:I23-28. [crossref]
38.
Goncalves MS, Bomfim GC, Maciel E, Cerqueira I, Lyra I, Zanette A, et al. βS haplotypes in sickle cell anaemia patients from Salvador, Bahia Northeasthern Brazil. Braz J Med Biol Res. 2003;36:1283-88. [crossref] [PubMed]
39.
Lanclos KD, Oner C, Dimovski AJ, Gu YC, Huisman TH. Sequence variations in the 5’ flanking and IVS-II regions of the G gamma-and A gamma-globin genes of beta S chromosomes with five different haplotypes. Blood. 1991;77:2488-96.[crossref] [PubMed]

DOI and Others

DOI: 10.7860/JCDR/2023/58342.17445

Date of Submission: Jun 08, 2022
Date of Peer Review: Jul 22, 2022
Date of Acceptance: Nov 08, 2022
Date of Publishing: Feb 01, 2023

AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. Yes

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