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ISSN - 0972-0847

Columban J. Life Sci.

Vol. 11

No. 1 & 2

41 - 50

2010

SICKLE-CELL DISEASE WITH ORTHOPEDIC COMPLICATIONS IS NOT ASSOCIATED WITH BMP- 6 GENE 567C/G MUTATION BUT STRONGLYASSOCIATED WITH INCREASED LDH AND URIC ACID LEVEL IN INDIAN PATIENTS FROM CHHATTISGARH AND JHARKHAND STATES
Kumar Abhisheka, d, Mohammad Sohailb *, Ritesh Kumarc, Vishwaprakash Roye, Avinash Sonif, Kumar Vivekg, Narendra Kumarh, P. K. Patraa, Mohammad Raziuddini and S. B. Choudharyd aDepartment of Biochemistry, Pandit Jawaharlal Nehru Memorial Medical College, Raipur, Chhattisgarh, India bDepartment of Medical Parasitology, NYU, Old Public Health Building, Rm 107B 341 East 25th Street New York, NY 10010 cDepartment of Biotechnology, Hamdard University, New Delhi, India
defghi-

Department of Botany, Vinoba Bhave University, Hazaribag, Jharkhand, India Faculty of Life Science, MATS University, Raipur, Chhattisgarh, India School of Life Sciences, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India Vinoba Bhave University, Hazaribag, Jharkhand, India Department of Dermatology, RIMS, Ranchi, Jharkhand, India Department of Zoology, Vinoba Bhave University, Hazaribag, Jharkhand, India

ABSTRACT
Sickle cell disease has numerous consequences; one of the most characteristic is orthopedic complications. Bone morphogenetic proteins (BMPs) are involved in the various orthopedic complications and play important role in bone physiology influencing bone growth, turnover, bone formation and cartilage induction. We investigate a possible association of sickle cell disease with orthopedic disorders through BMP6 gene polymorphism. Among the population studied in Chhattisgarh and Jharkhand states (a total of 200 cases and 172 control groups), the association was examined between SNP 567C/G of BMP6 and orthopedic complications in sickling patients by employing PCR-RFLP and biochemical analysis. 567C/G SNP has not been implicated in disease and doesnt increase the risk (OR=1.27, OR=0.85). We observed no significant association between the 567C/G polymorphism and case group in the studied population (P=0.64, P=0.91 respectively). However, significantly elevated Uric acid (UA) level(P=0.0001, P=0.0001, P=0.0001 and P=0.0001, P=0.0001, P=0.0001 respectively) and Lactate dehydrogenase (LDH) level (P=0.0001, P=0.0001, P=0.0001 and P=0.0001, P=0.0001, P=0.0001 respectively) in GG, CG and CC in case group as compared to control group among the studied population. 567C/G polymorphism in BMP6 gene is not associated with case group and in view of present observation, we suggest that evaluation of LDH and UA level and its association with polymorphisms in the BMP6 may be considered as a reliable molecular and biochemical markers possesses promising rational for diagnostic potential in clinical cases.

Key words: SCD, BMP6, Orthopedic Complication, Chhattisgarh, Jharkhand, India.


INTRODUCTION Sickle cell anaemia is an autosomal recessive disease caused by a point mutation in the hemoglobin beta gene (HBB) found on chromosome 11p15.4, caused by homozygosity for a mutation in the haemoglobin gene (HBB; glu6val) has multiple complications due to sickle vasculopathy and hemolytic anaemia. Sickle cell disease (SCD) clinically counts as a multi-system disorder. The disorder of orthopedic complication during SCD is complex and involves interaction of hormonal, nutritional and multiple environmental factors. BMPs are known for their ability to induce the growth of bone, cartilage, repairs of fractures, and are multifunctional growth factors. Several classes of BMPs have been identified, BMP-2, 4 and 7 have been
* Corresponding author- Dr. Mohammad Sohail, Department of Medical Parasitology, NYU Old Public Health Building, Rm 107B 341 East 25th Street New York, NY 10010. E-mail- soh.khan@gmail.com , abhi.biotech@gmail.com Received 14 September 2009; Accepted 21 May 2010 aa

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SICKLE-CELL DISEASE WITH ORTHOPEDIC COMPLICATIONS demonstrated to be potent osteotropic factors 567C/G polymorphisms in BMP6 gene. In this study, promoting bone formation in vivo and in vitro we investigated whether this polymorphism in the (Yamaguchi et al, 1996; Jane et al, 2002; Cook and region of BMP6 gene is associated to SCD with Rueger, 1996). BMPs are proteins secreted by cells, orthopedic complications, LDH, UA and Calcium which act as ligands for receptors present on the level in different clinical groups and their possible plasma membrane of different types of cells correlations in Indian population. (autocrine and paracrine effects), thus establishing cell and tissue organization. BMPs were originally MATERIAL AND METHODS extracted from bone and identified by their ability to Study population form normal, endochondral bone when implanted into The consent were taken from the participants and nonskeletal sites in rodents (Urist, 1965; Reddi, 1972; human blood samples were taken as per protocols Reddi, 1981; Urist, 1982). This de novo bone forming approved by the Ethical Review Board of the Pt. activity was found to contain several structurally J.N.M. Medical College, Raipur, Chhattisgarh, human related proteins from the transforming growth factorethical guideline as reflected in the guidelines of the . superfamily (Wozney et al, 1988). BMP6 is a medical ethics committee, Ministry of Health, multifunctional growth factor belonging to the TGFGovernment of India. Subjects were taken from superfamily and located on chromosome Chhattisgarh and Jharkhand state for this study, 6p12.1which is known to play an important role in which consists of 244 and 128 subjects respectively. skeletal development, joint integrity and bone The group was divided in two categories: case and formation (Hogan, 1996; Bellusci et al, 1996). BMP6 control. In case group, we have taken sickle cell is the key regulator of hepcidin, the small peptide disease (SS) and orthopedic complication with the secreted by the liver which is the major regulator of sickle cell disease (Ortho+SS) subjects, whereas, in iron metabolism in mammals (Mungall, 2003). In control group, we have taken sickle cell trait (AS) addition to their ability to induce bone formation, these and healthy (AA) subjects. The selection criterion proteins show diverse expression patterns throughout for patients was that they had Sickle cell disease embryonic development and are thought to regulate with orthopedic complications (Ortho+SS) and no proliferation, apoptosis, differentiation, cell-fate other malformations. An individual with any other determination, patterning, and morphogenesis in abnomalies, other than the above said has not multiple organ systems (Lyons, 1990; Hogan, 1996). included in study. To be classified as a case, all the patients were diagnosed through clinical examinations and their diseased status was checked by However, our knowledge of the BMP6 polymorphism electrophoresis. The outdoor patients and inpatients and its relation to SCD, in patients with orthopedic with no such complications were considered as complications, is very limited. Thus, to further study control group. The selection criteria for controls had the interaction between SCD patients and orthopedic no evidence of any other serious illness, particularly complications with BMP6. We investigated the impact of the content of LDH, Calcium, UA and for other hereditary diseases. The control group was matched to the case group by age, weight, height, Haemoglobin in plasma on SCD and orthopedic complications in adults and childrens, who visited haemoglobin, calcium, UA and LDH. Pandit Jawaharlal Nehru Memorial Medical College located at Raipur, Chhattisgarh, and Rajendra Blood samples Institute of Medical Sciences, Ranchi, Jharkhand. Peripheral venous blood samples were collected from LDH, Calcium and UA were determined because all the subjects (2 ml. in each tube) aseptically by of their biological relevance in SCD and orthopedic dripping from the syringe without anticoagulant in complications responses in order to obtain a global sterile microfuge tube for assessment of plasma measure of the patients actual reactivity towards LDH activity, Haemoglobin assay, UA and Calcium SCD disorder. activity and with anticoagulant (heparin or EDTA) for DNA isolation. Age, weight and height were Apart from this, the second objective of investigation obtained at the time of obtaining blood sample form was to explore the polymorphisms in BMP6 gene, every subject. Plasma was obtained by centrifugation which is important for regulating gene expression. of the samples for biochemical studies and used for However, there has been no published data for the estimation of total calcium, UA, LDH level. Blood examining the association between SCD and the
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was allowed to coagulate at 4 C for 4 to 6 hour prior to processing via centrifugation. Plasma was preserved in 3 to 5 aliquots at -70 0 C until measurements were performed. Blood samples were drawn from all the patients and control subjects. Biochemical Parameters Approximately 0.5ml of venous blood was collected with a heparinized syringe and plasma was obtained by centrifugation. Calcium and UA concentration were determined in 0.05ml plasma aliquot using a calcium estimation kit (QuantiChromTM Calcium Assay Kit (DICA-500)) and uric acid estimation kit (Biochain Uric acid Assay Kit). Haemoglobin and LDH concentrations were determined in 0.05ml plasma aliquot using a haemoglobin (Biochain Haemoglobin Assay Kit) and LDH estimation kit (BioAssay Systems QuantiChromTM LDH Assay Kit separately. The coefficients of variations for inter and intra-batch assessments of all biochemical parameters were < 5%. DNA extraction and BMP6 genotyping DNA was extracted from blood spots dried on filter paper using a DNA isolation kit (QIAmp Blood Kit: Qiagen, Krefeld, Germany). The BMP6 567C/G genotype was determined by using a PCR-RFLP assay and DNA sequencing analysis. The PCR primers were designed based on the GenBank reference sequence (AL135778). Primer sequences for PCR reactions were 5ATGAAACCCTGTCCCAAAAGA-3 (forward) and 5- TGAGCAGTATGGCACAGTG-3 (reverse). PCR reactions were performed in a total volume of 25 L containing 100 ng genomic DNA, 20pM of each primer, 0.2mM dNTPs, 20mM TrisHCl (pH 8.8), 10mM KCl, 10mM (NH4)2SO4, 2mM MgCl 2, 0.1% Triton X-100, and 1 unit of Taq polymerase (New England BioLabs, Ipswich, MA). PCR cycle conditions consisted of an initial denaturation step at 940C for 12 min followed by 35 cycles of 1 min at 940C, 1 min at 540C, 1 min at 720C, and a final elongation at 720C for 7 min. PCR products were digested at 370C for 6 hour with 2.5U of BsaJI (New England BioLabs, Cambridge,UK) restriction enzyme in 10 L of PCR products in total reaction mixture of 20 L. PCR products were electrophoresed through 1.5% agarose gel and the BMP6 polymorphism was screened by the Restriction Fragment Length Polymorphism and the digested PCR fragments were separated on 2%
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COLUMBAN J. LIFE SCI. VOL. 11 (1&2), 2010 agarose gel in the presence of ethidium bromide and visualized by fluorescence in UV light. Statistical Analysis Statistical analyses were performed using Graph Pad Prism 5.0 software. A goodness of fit test was used for testing the HardyWeinberg equilibrium and a chi 2 test compared the genotype and allele frequencies of BMP6 567C/G polymorphism between the two groups. All biochemical data were expressed as MeanSE.The mean of the parameters for case group and control group was compared by using Students t-test. The differences were considered significant when p < 0.05. RESULTS Biochemical Parameters and haemoglobin assay, calcium activity, UA activity and LDH levels in cases and controls In the above study 244 subjects (61 (Ortho+SS), 68 (SS), 76 (AS) and 39 (AA)) from Chhattisgarh state and 128 subjects (35 (Ortho+SS), 36 (SS), 35 (AS) and 22 (AA)) from Jharkhand state were considered for analysis. Table-I and Table-II shows the clinical, demographic, haemoglobin, calcium, UA and LDH levels in case and control group from both the state. We observed overall significant elevation of UA and LDH (p=0.0001, p=0.0001 respectively) and significant depletion of haemoglobin and calcium (p=0.0001, p=0.0001 respectively) as compared to control group, in the population of Chhattisgarh state as shown in figure-1A-D. On the other hand, we observed overall significant elevation of UA and LDH (p=0.0001, p=0.0001 respectively) and significant depletion of haemoglobin and calcium (p=0.0001, p=0.0001 respectively) as compared to control group, in the population of Jharkhand state as shown in figure-2A-D.In addition to this, we observed significantly higher UA and LDH level (p=0.0001, p=0.0001 respectively) as compared to haemoglobin and calcium (p=0.0001, p=0.0001 respectively), in population of both states. Polymorphism at BMP6 567C/G gene Amplification of 261 base pair fragment for variants of codon 567C/G of BMP6 gene was carried out in the blood samples collected from case group and control group. To confirm the desired amplicon and RFLP findings the representative amplified PCR products were gel-purified using a gel extraction kit (Qiagen), ligated into the pGEM T-Easy vector

SICKLE-CELL DISEASE WITH ORTHOPEDIC COMPLICATIONS


Table-I Physical and Clinical characteristics of Sickle Cell patients and healthy subjects from Chhattisgarh State
Parameters CASE Sickling patients with Sickling patients without orthopedic complications orthopedic complications (SS) (Ortho+SS) (Mean SE) (Mean SE) 61 68 31.571.0 9.430.8 55.020.8 20.631.5 5.50.03 3.690.1 8.160.1 7.870.2 2.480.05 2.720.06 5.460.08 5.210.09 360.18.84 370.46.59 CONTROL Sickling carrier Healthy subjects (AS) (Mean SE) 76 23.230.9 50.401.2 5.290.05 14.520.1 3.700.05 4.240.11 272.25.93 (AA) (Mean SE) 39 32.231.21 61.281.14 5.550.05 15.760.21 4.220.05 4.130.16 197.50.21 P

Sample size (N) Age (years) Weight (kg) Height (ft. & inches) Haemoglobin (gm %) Calcium (mmol/L) Uric acid (mg/dl) LDH (U/I)

0.005 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001

Table-II Physical and Clinical characteristics of Sickle Cell patients and healthy subjects from Jharkhand State
CASE Sickling patients without Sickling patients with orthopedic complications orthopedic complications (SS) (Ortho+SS) (Mean SE) (Mean SE) 35 36 30.46+1.18 12.60+1.13 55.57+0.99 27.02+2.45 5.59+0.04 4.16+0.14 10.08+1.91 8.46+0.25 2.47+0.07 2.68+011 5.42+0.10 5.21+0.12 361.1+9.09 371.9+8.71 CONTROL Healthy subjects Sickling carrier (AS) (Mean SE) 35 26.11+1.71 51.34+1.96 5.23+0.10 14.35+0.25 3.00+0.07 4.22+0.12 271+5.15 (AA) (MeanSE) 22 18.09+1.88 40.14+4.11 4.76+017 15.79+0.31 4.21+0.11 4.01+0.25 203+7.13

Parameters

Sample size (N) Age (years) Weight (kg) Height (ft. & inches) Haemoglobin (gm %) Calcium (mmol/L) Uric acid (mg/dl) LDH (U/I)

NS NS NS 0.0001 0.0001 0.0001 0.0001

Table-III Genotypic distribution of BMP6 gene 567C/G polymorphism in case group as compared to control group from Chhattisgarh

Genotype /Alleles

GG CG CC C G
OR, odd ratio; CI, confidence interval. GG-CG vs. CC Table-IV Genotypic distribution of BMP6 gene 567C/G polymorphism in case group as compared to control group from Jharkhand

OR, odd ratio; CI, confidence interval. GG-CG vs. CC

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COLUMBAN J. LIFE SCI. VOL. 11 (1&2), 2010


Table-V Association of BMP6 polymorphism with Calcium, Uric acid, Haemoglobin and LDH in case group as compared to control from Chhattisgarh
Paramet ers Sickling patients with orthopedic complications (Ortho+SS) (Mean SE) GG CG CC 2.350.13 2.550.13 2.510.01 CASE Sickling patients without orthopedic complications (SS) (Mean SE) GG CG CC 2.800.15 2.680.08 2.670.0 5 5.380.1 0 374.09. 33 7.980.2 7 Sickling carrier (AS) (Mean SE) CG 3.150.09 CONTROL Healthy subjects (AA) (Mean SE) CG 4.280.14

Genotyp e Calcium (mmol/L ) Uric acid (mg/dl) LDH (U/l) Hb (gm %)

GG 3.150.0 9 4.300.2 2 259.29. 51 14.650. 27

CC 2.970.0 7 4.330.1 6 278.88. 53 14.780. 26

GG 4.290.09

CC 4.140.09

5.410.18

5.160.22

5.560.09

5.180.13

4.870.32

3.910.25

3.950.33

4.220.23

4.240.22

369.218. 73 8.030.18

346.821. 35 7.910.15

360.611. 46 8.310.18

364.513. 62 7.600.45

371.410. 19 8.100.26

280.515. 22 14.130.4 1

212.814. 69 15.680.4 0

187.917. 69 16.610.3 6

189.311. 44 15.490.2 8

Table-VI Association of BMP6 polymorphism with Calcium, Uric acid, Haemoglobin and LDH in case group as compared to control from Jharkhand
Parameters Sickling patients with orthopedic complications (Ortho+SS) (Mean SE) GG CG CC 2.430.09 5.380.19 356.016.52 12.184.19 2.420.28 5.320.26 2.530.10 5.510.13 CASE Sickling patients without orthopedic complications (SS) (Mean SE) GG CG CC 2.600.14 5.320.20 2.550.10 4.920.31 2.820.26 5.280.14 Sickling carrier (AS) (Mean SE) CG 2.850.17 4.000.31 CONTROL Healthy subjects (AA) (Mean SE) CG 4.200.14 3.320.65

Genotype Calcium (mmol/L) Uric acid (mg/dl) LDH (U/l) Hb (gm %)

GG 3.090.15 3.970.19

CC 3.000.10 4.590.19

GG 4.360.25 4.220.46

CC 4.100.16 4.130.33

340.015.66 374.511.20 373.413.67 8.860.54 8.110.26 8.370.56

384.313.33 363.416.50 265.29.65 289.33.87 8.610.28 8.470.33 14.920.41 14.140.58

267.97.80 199.015.84 206.016.59 205.18.15 13.890.35 15.480.58 16.300.55 15.830.49

Figure-1 (A) Comparison of Calcium level between case and control groups in Chhattisgarh population. (B) Comparison of Uric acid level between case and control groups in Chhattisgarh population. (C) Comparison of LDH level between case and control groups in Chhattisgarh population. (D) Comparison of Haemoglobin level between case and control groups in Chhattisgarh population.

Figure-2 (A) Comparison of Calcium level between case and control groups in Jharkhand population. (B) Comparison of Uric acid level between case and control groups in Jharkhand population. (C) Comparison of LDH level between case and control groups in Jharkhand population. (D) Comparison of Haemoglobin level between case and control groups in Jharkhand population.

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SICKLE-CELL DISEASE WITH ORTHOPEDIC COMPLICATIONS

Figure-3 (A) Comparison of Calcium level between case and control groups based of 567C/G polymorphism in BMP6 gene of Chhattisgarh state population. (B) Comparison of Uric acid level between case and control groups based of 567C/G polymorphism in BMP6 gene of Chhattisgarh state population. (C) Comparison of LDH level between case and control groups based of 567C/G polymorphism in BMP6 gene of Chhattisgarh state population. (D) Comparison of Haemoglobin level between case and control groups based of 567C/G polymorphism in BMP6 gene of Chhattisgarh state population.

Figure-4 (A) Comparison of Calcium level between case and control groups based of 567C/G polymorphism in BMP6 gene of Jharkhand state population. (B) Comparison of Uric acid level between case and control groups based of 567C/G polymorphism in BMP6 gene of Jharkhand state population. (C) Comparison of LDH level between case and control groups based of 567C/G polymorphism in BMP6 gene of Jharkhand state population. (D) Comparison of Haemoglobin level between case and control groups based of 567C/G polymorphism in BMP6 gene of Jharkhand state population.

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(Promega, Madison, WI), and transformed into DH5 E.coli. Sequencing reactions were conducted with a BigDye Terminator Cycle Sequencing Ready Reaction Kit in an ABI 3730xl automatic DNA sequencer (Applied Biosystem, Foster City, CA) and cloned sequence was submitted to GenBank at the NCBI [GenBank: GQ863579, GQ863580] Restricted PCR products with BsaJI restriction enzyme resulted in complete digestion. The gel analysis demonstrated that fragments of 206 and 54 basepair for the homowild (GG), 137 and 69, 54 basepair for the homomutant (CC) and 206,137, 69, 54 basepairs for the heteromutant (CG) genotype. Distribution of BMP6 567C/G genotype in orthopedic complication in sickle cell anaemia patients The genotype distributions for BMP6 at position 567C/G were compared between case and control group. A total of 244 patients, 129 from case and 115 from control group of Chhattisgarh state and a total of 128 patients, 71 from case and 57 from control group of Jharkhand state were genotyped for BMP6 567C/G mutation as shown in Table-III and Table-IV respectively. We observed that the BMP6 genotype was not associated with an orthopedic complications (OR 1.27, 95% CI 0.742.18) as compared to control group of Chhattisgarh state and similar finding was observed (OR 0.85, 95% CI 0.42-1.74) as compared to control group of Jharkhand state. However, genotype frequency data of Chhattisgarh and Jharkhand patients were analyzed and interestingly no difference was found statistically significant (p=0.64 and p=0.91 respectively), as shown in Table-III and Table-IV respectively. The odds ratio (OD) and 95% confidence interval provide a measure for the strength of association, e.g., the indicating of the decrease in the odds of a given case group as compared to control group in both the studied population demonstrating lower risk category with this mutation. The model used for risk assessment was the logistic regression adjusted for gender and age. All tests were conducted at P<0.05 level of significance. Association of BMP6 567C/G mutation and clinical parameter in SCD patients We observed significant depletion of Calcium and Haemoglobin level in all the three genotypes in case group as compared to control group shown in figure3A, 3D and figure- 4A, 4D respectively, in both the
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COLUMBAN J. LIFE SCI. VOL. 11 (1&2), 2010 studied population. Further, we also observed significantly elevated UA and LDH level in all the three genotype, i.e., GG, CG and CC in case group as compared to control group as depicted in figure3B, 3C and figure- 4B, 4C respectively, in both the studied population from Chhattisgarh and Jharkhand. In addition to this, we analyzed the effect of Calcium, UA, LDH and Haemoglobin on these genotype in Sickling patients with and without orthopedic complications and interestingly we observed the elevated calcium and LDH level in sickling patients without orthopedic complication as compared to patients with orthopedic complications, whereas UA and haemoglobin was observed to be higher in patients with orthopedic complication in population of Chhattisgarh as shown in Table-V. In case of population of Jharkhand, we observe elevated uric acid and LDH level and decreased calcium level in patients without orthopedic complication, whereas haemoglobin was higher in patients with orthopedic complication as shown in Table-VI. DISCUSSION SCD is primarily a haematological disorder, which leads to multiple diseases such as vascular function, bone metabolism, oxidant stress and inflammation showed an association with osteonecrosis in patients with sickle cell anaemia. The skeleton bears the brunt of the complications due to marrow hyperplasia initially, and later due to vascular occlusion in up to 50% of the patients (Diggs, 1967; Middlemiss, 1966). In this study, we have shown that the BMP6 567C/ G polymorphism was not associated with the risk of Sickling with orthopedic complications. There has been no published study demonstrating the association of BMP6 between sickle cell disease with and without orthopedic complication. However, our investigation is the first report attempting to evaluate the association of 567C/G of BMP6 gene in sickle cell patient with and without orthopedic complication in Indian population. It is unlikely that single genetic polymorphism alone will have a major effect. However, our study suggests that the BMP6 567C/G polymorphism could be used as genetic marker of the sickle cell patient with and without orthopedic complication in association with biochemical markers. Till date, the possible effects of this polymorphism on structure and processing of the BMP6 propeptide are unknown. However, our findings regarding the non-association of BMP6 567C/G polymorphism with orthopedic complications

SICKLE-CELL DISEASE WITH ORTHOPEDIC COMPLICATIONS in sickling patients can be substantiated by the findings observed by Hongwei Li et al. (2006) (Hongwei et al, 2006) in which they found that recombinant human BMP4 and BMP6 second generation adenoviral vectors had not shown any improvement in the induction of ectopic bone formation in immunocompetent rats and thus, not associated. The reason for findings indicates that the osteogenic potentials of BMP4 or BMP6 are significantly reduced in immunocompetent rats and probably similar kind of the mechanisms are likely to be operated in the human. This limitation may be caused by the innate host immune response, rather than by the adaptive immune response (Gahery et al, 1997). Interestingly, we observe higher percentage of rare homozygous (CC) genotypes in sickling patients with orthopedic complications than that in sickling patients without orthopedic complications in both the studied population, although the difference is not significantly associated with the disease. Furthermore, we observed increased proportion of mutant allele (C) in case subject as compared to control subject in population of Chhattisgarh whereas similar proportion of mutant allele (C) was observed in the population of Jharkhand. Heterozygous (CG) genotype distribution is slightly higher in case group than that of control group in Chhattisgarh population, whereas higher proportion of heterozygous (CG) genotype in control group than that in case group was observed in population of Jharkhand. The reason for genetic disparities and varied degree of association with disease susceptibly can be attributed to highly polymorphic chromosomal location, biological effect and extensive linkage disequilibrium to the locus (Kaijzel, 2001) such differences are likely to exist. In addition, the different ages, ethnicity and geographical differences in the studied population can also contribute genetic association and disease outcome (Ramasawmy et al, 2007). The decreased frequency of 567C position implies that the C allele could provide protection from disease progression. LDH has long been considered a useful clinical marker that has been associated with low haemoglobin concentration, high levels of LDH during sickle cell disease and elevated in patients as also observed in our findings which are in accordance with the study carried out by the Kato et al., 2006a (Kato et al, 2006). Our observation regarding low haemoglobin during disease state can be attributed
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to the LDH elevation, which is associated with low transcutaneous oxygen saturation also. This is consistent with publications from two other groups, who have found in patients with sickle cell disease the association of desaturation with anaemia and reticulocytosis, suggesting the same link between hemolysis and hypoxemia (Setty et al, 2003; Quinn and Ahmad, 2005). Further, genotype is an important determinant of serum LDH levels, also significantly higher in case group as compared to control in our experiment. Similar to our observation some previously published studies (Kato et al, 2006a; Ataga et al, 2006), looked at LDH as a biomarker in adults with SCD and showed significant correlation between LDH and other disease. Findings from above studies suggest that the increase in endogenous LDH and UA level early in the course of sickling contributes significantly to onset of orthopedic complications, perhaps by losing some of the counterbalance to the increased TGF- activity and decreased calcium and haemoglobin level in diseased state. BMP6 is involved in inflammatory processes (Rosendahl et al, 2002) and is important for bone formation and in association with parathyroid hormone (PTH) and vitamin D, appears to be involved in inducing bone development by human bone marrow derived mesenchymal stem cells (Sammons et al, 2004) . Our finding of decreased calcium level in diseased state may be due to shape transition from discocyte to spherocyte, shedding of microvesicles into the extracellular fluid, and enhanced susceptibility to the hydrolytic action of secretory phospholipase A2.These responses to decreased intracellular calcium were all blunted in erythrocytes containing haemoglobin S.The reduction of both the shape transition and the shedding of microvesicles were greater than the impairment of phospholipase susceptibility, and both of them correlated strongly with the intracellular content of haemoglobin S and calcium level. Normally, in healthy human erythrocytes display several responses to elevated intracellular calcium levels. The effect was more prominent among samples from patients heterozygous for haemoglobin S than that in sample from homozygous individuals. These results reveal additional abnormalities in the membranes of sickle cell erythrocytes beyond those described previously and demonstrate that red blood cells from both heterozygous and homozygous are affected. Furthermore, they suggest a possible means by which sickle cell disease and trait patients may

display enhanced vulnerability to inflammatory stimuli (Hongwei et al, 2006). UA has long been considered as simply a waste product of human metabolism. However, and perhaps significantly, Ames et al. (1981) have suggested that in various physiological situations, some of the reductive activity of ascorbic acid may be replaced by the same activity of UA, which is comparable to ascorbic acid in trapping certain free radicals in vitro; thus, in body fluids, UA may act as a free-radical scavenger. Furthermore, concentrations of UA in blood have shown to be increased after a severe oxidative stress. Therefore, we can speculate that compared with normal individuals sickle-cell patients may be able to provoke an increase in UA in body fluids to offset a substantial loss of cellular antioxidants as observed in our case. The reason for elevated cellular antioxidant within human erythrocytes is due to the perturbation in the balance between oxidant and antioxidant, as evidenced by the disastrous consequences when this balance is disrupted in pathological oxidation. The perturbed balance in sickle-cell disease is suggested by the demonstration that sickled erythrocytes spontaneously generate about double the normal amount of oxygen radicals. Thus, oxidant stress and cellular inadequacies in dealing with that stress might be important in producing the membrane damage that leads to the formation of irreversibly sickled cells. Evidence now indicates that the erythrocytes in most sickle-cell patients have subnormal resistance to oxidant stress because of low cellular concentrations of glutathione (Ames et al, 1981). In addition, the data of this study suggest that BMP6 567C/G polymorphism is not involved in the etiology of orthopedic complications in sickling patients, and it may serve as a valuable molecular marker, possesses promising rational for diagnostic potential in assessing the disease state. Although the evidence of a correlation between 567C/G alleles and LDH was found in the studied population yet the relationship was not strong. The contribution of this study was to further characterize the adult SCD population regarding the pathophysiology of orthopedic complications in sickling patients. It explored the possibility of further research regarding BMP6 and its polymorphism. These data also provide a foundation of future genotype-phenotype association studies involving both disease risk and variation in drug response. As the findings are novel
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COLUMBAN J. LIFE SCI. VOL. 11 (1&2), 2010 and well aware of the small number of subjects, they deserve to be investigated in a large study group. In conclusion, this data provide link between the clinically acquired immunity and polymorphism through complex interplay of molecular cascade in population. However, the reliability of BMP6 and their polymorphisms, as a sensitive, genetic and diagnostic marker in clinical usefulness await further well conducted clinical investigations as to permit interventions in order to access sickle cell disease. ACKNOWLEDGEMENTS We would like to thank Professor G.B.N.Chainy, Utkal University, Orissa, India and Dr. Mohd. Asim, Department of Dermatology, University of Wisconsin Madison, US for critical comments on parts of the manuscript and analytical help in experimental planning.

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