TO MONITOR TREATMENT OF A LARGE COHORT OF PATIENTS WITH β-THALASSAEMIA MAJOR, INTERMEDIA & SICKLE CELL ANAEMIA IN ORDER TO ESTABLISH THE RESPONSE TO TRANS-RESVERATROL AND THE ASSOCIATED ELEMENTS

Anirban Roy Chowdhury 1 , Sudipa Chakravarty 2 and Amit Chakravarty 2 . 1. Department of Genetics, Institute of Genetic Medicine and Genomic Science. 30A Thakurhat Road. Kolkata700128, West Bengal, India. 2. Department of Genetics, Institute of Genetic Engineering. 30 Thakurhat Road. Kolkata700128, West Bengal, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Though several genetic, non-genetic and pharmacological factors reported to influence the Trans-Resveratrol response in different early studies, the response to Trans-Resveratrol is significantly different among good, moderate and non-responders irrespective of the IVS I-5 (GC), the common beta mutation here and even among other β0 or β+thalassaemia mutations. Here we study whether HbF level has any relation to beta variants responding to Trans-Resveratrol therapy, it has been shown that even among good responders in some cases (8.39 %) patients are not showing high HbF values ( < 20% HbF values are taken). In our study (among 220 patients), Trans-Resveratrol therapy completely replace blood transfusion in as expected in this Eastern part of India, the predominant β-thalassemia defect is the IVSI-5(GC), the most frequent β0-thal mutation in the area. This mutation is found in 87.66% of the CR patients and in 86.66% of the PR / NR patients. The next represented defects are the β+-thalassemia mutations like Cod 8/9, Fr. 41/42, Cod 15, Cod 30 etc. The common IVSI-5 (GC) mutation in beta thalassemia patients (either in homozygous or heterozygous form) is present in 31.5% among the good responders (CR) while it is present in 66.66% among PR / NR (the moderate responders / non-responders) and in HbE-beta patients present in 73.28% among good responders (CR) while in 33.33% among moderate / non responders (PR / NR).
Hence, we would like to study the nucleotide variations in the -globin gene cluster and their association with the Trans-Resveratrol response so that we could explore the genetic basis of the clinical diversity of the Trans-Resveratrol therapy in beta-thalassaemic and sickle cell anaemia patients. Again, we may assume that a large proportion of the thalassaemia patients in Eastern India have a molecular background favorable to Trans-Resveratrol response, because, during the one year of treatment (followed by Thalassaemia Foundation) we did not observe any significant problems regarding drug compliance and no myelogenic or clinically adverse events occurred. This in agreement with other long-term clinical trials which reported no significant increases in secondary malignancy following Trans-Resveratrol therapy. The good response to Trans-Resveratrol treatment that a significant number of Eastern Indian patients with β-thalassemia had seems to correlate not with the particular haplotypes, but with some other molecular factors, which we have to screen further.

Materials and Methods:-
Study groups:-Patients with HPLC-screened documented Sickle cell anaemia, S-beta thalassemia, beta thalassaemia, HbE thalassaemia, HbE-beta thalassaemia, HPFH genotypes have been considered in this primary analysis.
Collection of Sample:-Sample was collected from OPD of Thalassaemia Foundation, Kolkata. Total 220 patients were evaluated. Among which 140 patients with Hb-E-beta and 69 patients with Beta and HPFH and 11 patients with other hemoglobinopathies were observed.

Molecular Analysis:-
Samples for DNA isolation have been collected. DNA has been extracted and genotyping has been done by ARMS-PCR for the common beta mutations of this region, like IVSI-5(GC), Cod 8/9, Fr.41/42, Cod15, Cod26 and also for Cod6 (Sickle cell anaemia).
Fetal hemoglobin studies:-Hb variants' (HbA / HbA2 / HbF & others) levels was estimated by HPLC (High Performance Liquid Chromatography) (Bio-Rad, USA). Estimation of HbF was also done by using HPLC method.

Result:-
Clinical profile:-We were able to classify three categories of response: a Complete Response (52.2%) in patients who can able to maintain at an average Hb level of 6-9 gm/dL without blood transfusion, in this group 12.3% patients are without any previous H/O blood transfusion, others shifted from monthly blood transfusion dependency to a stable transfusionfree condition; a Partial Response (18.2%) in patients who remained transfusion dependent but at longer intervals (2-1192 3 months or more), and Non response (15.9%) in patients who, after more than one year of treatment, remained at the same level of transfusion dependency. [ Table 1]   Table 2].

IVSI-5 (GC) / cd 6 --------
The common IVSI-5 (GC) mutation in beta thalassemia patients (either in homozygous or heterozygous form) is present in 31.5% among the good responders (CR) while it is present in 66.66% among PR / NR (the moderate responders / non-responders) and in HbE-beta patients present in 73.28% among good responders (CR) while in 33.33% among moderate / non responders (PR / NR). [ Table 3].  The response to Trans-Resveratrol is equal in males and females and the age distribution is not significantly different in the three response categories. Similarly no significant difference in response was found between splenectomized and non-splenectomized patients. 1193

Discussion:-
It has been shown that low-dose resveratrol induces early maturation of normal erythroid precursors by activation of the FoxO3 a transcriptional factor, inhibition of Akt and upregulation of antioxidant response genes such as catalase. The effects of resveratrol on cell maturation are highly dependent on resveratrol concentration and on cell types (1,(5)(6)(7)(8). The findings from previous studies are not directly applicable to the studies due to either the use of erythroid leukemia cell lines or study of primary erythroid cells without detailed characterization of their stage of cell differentiation (5)(6)(7). CFU-E cells are the most susceptible erythroid cell population to the effects of low dose resveratrol. Since resveratrol has no effect on the expression of erythropoietin receptors during erythroid differentiation (8), hence it can be proposed that resveratrol might hamper cell proliferation and may induce cell maturation as supported by the early expression of GPA and band 3 in resveratrol-treated cells and by similar observations in other cell models (7,17). We recently showed that resveratrol targets FoxO3 a key transcriptional factor in erythropoiesis involved in up-regulation of scavenging enzymes (4,12). We explored the possibility of resveratrol playing a pivotal role as an exogenous anti-oxidant agent and as a modulator of endogenous anti-oxidant systems.
In our present study we observed that whether HbF level has any relation to beta variants responding to Trans-Resveratrol therapy, it has been shown that even among good responders in some cases (8. According to Bianchi et al, when erythroid precursor cells from normal subjects were treated with increasing concentrations of resveratrol and analysis of accumulation of globin mRNA sequences was performed by quantitative RT-PCR, a clear increase in accumulation of γ-globin mRNA content was found. Increase in accumulation of α-globin and β-globin mRNA was much lower. Taken together these data strongly indicate resveratrol as a strong inducer of HbF and a selective stimulator of the expression in γ-globin genes. (17) Conclusion:-

1.
Though several genetic, non-genetic and pharmacological factors reported to influence the Trans-Resveratrol response in different early studies, the response to Trans-Resveratrol is significantly different among good, moderate and non-responders irrespective of the IVS I-5 (GC), the common beta mutation here and even among other β 0 or β + thalassaemia mutations.

2.
To study whether HbF level has any relation to beta variants responding to Trans-Resveratrol therapy, it has been shown that even among good responders in some cases (8.39 %) patients are not showing high HbF values ( < 20% HbF values are taken). 3.

Funding statement:-
This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. All the research work done by the affiliated institution funding.