Blocking of Gardos channel and KCL Co- Transport system chloride channel may be the other therapeutic strategy to prevent sickling complication in Sickle Cell Anaemia

Authors
 *Professor Pranab Kumar Bhattacharya MD(cal), FIC Path(Ind), Now Professor and Head, Dept. of Pathology, Convener In-charge DCP course WBUHS; *Miss Upasana Bhattacharya - Only daughter of Prof. P.K. Bhattacharya, Mahamaytala, Garia kol-84 ; **Rupak BhattacharyaBSc(cal),MSc(JU);**Ritwik Bhattacharya B.com(cal); **Soumyak Bhattacharya BHM(cal) MSc Student PUSHA, New Delhi ;** Rupsa Bhattacharya-All of residence 7/51 Purbapalli, Sodepur, Kol-110 West Bengal ; India; *** Mrs Dalia Mukherjee BA(hons) cal, ***Miss Oindrila Mukherjee*** Mr Debasis Mukherjee BSC(cal),all of residence Swamijinagar, South Habra, North 24 Parganas ;West Bengal India **** Surajit Sarkar Bsc(cal) ,DMLT(cal) ,Msc Lab Tech
profpkb@yahoo.co.in*

*Dept of Pathology  Calcutta School of Tropical Medicine, C.R avenue Calcutta-73, W.B , India** 7/51 Purbapalli; Sodepur; 24 Parganas(north) Kol-110 W.B, India *** Swamijinagar, South Habra, North 24 Parganas ;West Bengal India; B**** Dept of pathology, IPGME&R, Kolkata-20 W.B, India
Molecular Structure of HbF  composed ofα₂γ₂ of globin chain.  RBC containing HBF have higher affinity for oxygen than  adult RBC. This property of HbF is for amino acid differences in amino acid terminus in gamma-globin chains that impair binding of 2,3DPG. HbF also have alkaline Bhor effect.  RBC containing HbF is called F cells / AF cells.  Gamma- globin chains are encoded by 2 nearly identical genes (HbG2 and HbG1) within  beta-globin gene-like cluster on chromosome 11p that differ by a glycine or alanine residue at amino acid position γ136. Cy- and Ay- globins have similar effects on HbS polymerization with rapid decrease in numbers of circulating fetal erythrocytes, the ratio of Gy to Ay- globin falls from 0.7 at birth to 0.4 at age 5 months. This is accompanied by progressive decline in number of erythrocytes with measurable HbF, called F cells^[2]. In adults, HbF is < 1% of total Hb, is distributed unevenly among erythrocytes. In certain thalassemia and HPFH , number of F cells are increased, HbS in sickle cell results from mutation of thymine in adenine in  6th position codon of Beta gene, there by encoding valine instead of glutamine. Study of Beta gene cluster polymorphisms from different population suggest that mutation arise at least five times in  context of different ancient haplotypes. In Africa, there major sickle haplotype is Senegal (Atlantic Senegalese has high proportion of γ G HbF. A 5^th  haplotype , found in Orissa, West Bengal and Poona, India, and patients from these regions have rather milder from of disease, as they have elevated level of HbF^[1] .HbF levels in SCA range between 5% and 8%. Sickle cell trait carriers have a mean HbF of 1.4% and 14.1% ? 7.5% F cells. In SCA, F cells survive longer than non-F cells, and this depends on  amount of HbF/F cells^.[2] A high correlation (R2 = 0.967) is present between  number of F cells and  percentage of HbF.
Polymerization occur with de-oxygenation and increase of 2,3 DPG decrease affinity of HbS for oxygen and enhance gelation. Gelation occurs when concentration of deoxy HBS is raised above 20.8 g/dl (MHC of RBC is normally >30 g/dl) HbF have inhibitory effect on gelation by dilutional effect. HbS gelation studies shows that HbF did not interact with HbS compound heterozygotes for SCA is remarkable variable in its clinical setting expression based on HbF in  members of the same family even. When many individuals may have recurrent ,severe complications, others enjoy relatively benign course and remain virtually disease free life. The modulators of such clinical and hematological severity or benign profile are HbF, presence of  thalassemia , gender and Beta globulin chain cluster haplotype like Indian. The cause of mild disease in SCA homogygotes in Orissa, West Bengal of India is associated with HbF level> 30%. Hb level<20% confers minimum benefit towards disease complications. Another factor is F cells, level of MCV and MCH and More HB A2. sickle cell trait and hereditary persistence of HbF are clinically so normal ,despite having a very high HbS concentration (reviewed in Rodgers and Steinberg)^[2]. Higher HbF levels were associated with a reduced rate of acute painful episodes, fewer leg ulcers, less osteonecrosis, less frequent acute chest syndromes, and reduced disease severity. However, HbF level had a weak or no clear association with priapism, urine albumin excretion, stroke and silent cerebral infarction, systemic blood pressure, and perhaps sickle vasculopathy as estimated by tricuspid regurgitant velocity (reviewed in Steinberg et al. The Saudi-Indian and Senegal haplotypes of the HbB-like globin gene complex, discussed in "Haplotypes of the HbB-like cluster," are associated with high HbF levels, and carriers of these haplotypes can have milder disease in Southwestern patients, splenomegaly is common and stroke and leg ulcers are rare^[2]. The rarity of stroke might be a result of the higher hemoglobin concentration and a high incidence of  alfa- thalassemia; little information on the incidence of pulmonary vasculopathy is available. Sickle cell trait carriers or persons with HbA and the Saudi -Indian haplotype did not have high HbF, but the cultured erythroblasts of sickle cell trait patients with the Saudi-Indian haplotype made increased amounts of HbF^[2] The drug Hydroxyurea-  has no known effect on DNA methylation but increases HbF production. It may have a direct effect on globin synthesis by early erythroid progenitors, as it increases F reticulocytes numbers. Hydroxy urea therapy is associated with intravascular and intra erythrocytic generation of NO which may have a role in improvement of fetal Hb level. and therapeutic induction of HbF affects and that lessen common complications, such as priapism, leg ulcers, pulmonary vasculopathy, ACS, mortality, reduces transfusion, hospitalization, and stroke. Hydroxy urea treatment increases fetal Hb, Hb concentration, MCV and decreases WBC, platelet reticulocyte. Hydroxy urea has been approved by FDA in adults with SCA. The Ideal candidate is an SS patient older than 18 years with a high rate of pain, ACS or both. Hydroxy urea require monitoring every two weeks of absolute neutrophil count <2000/ul, absolute reticulocyte count 80,000/ul, platelet count<80,000, LFT, urea, nitrogen creatinine every month .But  main problem in SCA lies with sickling phenomenon . And for this, blocking of ca ²⁺ activated K+ channel( Gardos channel) is required. Clotrimazole-an actnimycosite can prevent sickle cell hydration by inhibiting Gardos path way. It also increases HbF KCL Co transport system, is probably the most important dehydrating transport pathway in Sickle RBC and does not require deoxygenation, polymerization or sickling for its activation. KCL Co transport pathway actively result in a net loss of water and that pathway is chloride channel dependent and is stimulated by Acid pH(<7.4) cell swelling and Sulphhydril modification. Blocking of this transport channel may be one of the therapeutic strategies. West of Africa) Benin(central West of Africa) and Bantu (central Africa Republic).
Reference
1] Kalozik AE, Wainscoat JS, Serjeant GR etal Geographical survey of BetaS globin chain gene haplotypes: evident for independent Assian origin of Sickcle cell mutation" letter published in American J Hum Gen 1986:39;239- 244
2] Idowu Akinsheye, Abdulrahman Alsultan, Nadia Solovieff, Duyen Ngo, Clinton T. Baldwin, Paola Sebastiani ,David H. K. Chui, and Martin H. Steinber Fetal hemoglobin in sickle cell anemia blood-2011-03-325258v1 118/1/19

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Conflict of Interest:None declared   word count 961