GENERAL INFORMATION

1. Programme/Project Title : Study of morbidity pattern in sickle cell disease in Western Orissa and its correlation with fetal hemoglobin concentration and different epistatic factors like malaria.

2. Category of the Programme/Project  :

• Programme Support

• R&D

• Establishment of Centre of Excellence

3. Priority Area :  Human Genetics

4. Duration: Three Years

5. Total Programme/Project Cost:   Rs. 99.4 Lakhs

6. Is the project Single Institution or Multiple-Institutional (S/M)? :  M

7.Name of Programme/Project Coordinator :    Dr. Dilip Kumar Patel

Affiliation :   Assistant Professor, Department of Medicine

V.S.S. Medical college, Burla

Address :       3R/27, Doctors’ Colony, At/Po: Burla, Orissa, PIN – 768 017,

Ph. No. 2430273 (0663),     Mobile : 9437062273

TECHNICAL DETAILS OF PROGRAMME/PROJECT 

Project Summary

Sickle Cell Disease caused by point mutation in the b globin gene is widespread and has protean manifestations affecting various organ system in the body. Of the various globin gene cluster haplotype, Arab-Indian type is thought to be milder. The strong association of Xmn–1 site with the Arab Indian haplotype is thought to be associated with high fetal hemoglobin concentration and confer a benign course of the disease. However the clinical presentation of sickle cell disease in different regions in our country is highly variable. In this part of Western Orissa the severity of sickle cell disease is found to be more compared to that of Gujarat (Mohanty et al). The genetic factor like a-thalassemia, b-globin gene and fetal hemoglobin haplotype are some of the factors responsible for these variations. Environmental factors like falciparum malalaria is also thought to play a significant role.

This proposed study area is also highly endemic for malaria. It was Haldane (1948) who believed that natural selection had been responsible in alleviating and maintaining the gene frequencies of all haemoglobinopathies in malaria endemic area. Although results of an epidemiological study in Africa and several in-vitro and in-vivo studies (Luzzato et.al. 1970, Carlson et.al. 1994, Aluoch, 1997, Ntoumi et.al.1997) supported this theory that sickle cell haemoglobin  protects against malaria infection. Olumes et.al. 1997 found that those with sickle cell trait (HbAS) and persons with normal haemoglobin (HbAA) are equally susceptible to severe falciparum malaria. Recently, observation in a small number of cases in Ispat General Hospital, Rourkela, it was found that sickle cell traits are equally vulnerable to severe falciparum malaria like that in normal population. A similar observation in a limited number of patients at V.S.S. Medical College, Burla by Dr. Patel et al found that patients of sickle cell disease are susceptible to severe fa1ciparum malarila infection and have a worse outcome compared to persons without sickle cell disease. ­

            The present study is designed to ,find out different clinical presentations of sickle cell disease in two study populations at Burla and Rourkela centers and compare there morbidity pattern. In these patients fetal hemoglobin concentrations, sickle cell genotype and Xmn polymorphism will be studied and correlated with clinical severity. The incidence and severity of P. fjalciparum infection in sickle cell disease and sickle cell trait will be studied and compared this with age and sex matched controls without sickle cell hemoglobinopathy. A family study for assessment of hemoglobinopathy, status in different; family members will be done for providing counseling. Patients attending the hospitals in the two study sites will be screened for presence of sickle cell hemoglobin. The clinical data will be collected and blood will be examined by PCR analysis for Xmn polymorphism and sickle cell genotype at V.S.S. Medical College, Burla. Hemoglobin variants will be analyzed at Ispat General Hospital, Rourkela by HPLC. In selected cases DNA sequencing will be done at ILS, DBT, Bhubaneswar. The patients having concomitant malaria will be confirmed by parasite genotyping at Ispat General Hospital, Rourkela. The impact of malaria on the morbidity and mortality pattern of sickle cell hemoglobin will be compared with age and sex matched control. Following identification of sickle cell disease and trait cases genetic counseling and follow up of this target group will be down.

            This project is expected to generate data for risk stratification of sickle cell disease patients and identify high risk groups with increased morbidity and mortality, so that health care resource can be successfully targeted at them. Further, this study will create awareness and educate the patients and the family for adoption of simple remedial measures. The expertise and equipment can be utilized further in future for establishment of a prenatal diagnostic clinic for pregnant sickle cell disease mothers.

REFERENCES:

1.       ALLISON AC. PROTECTION AFFORDED BY SICKLE CELL TRAIT AGAINST SUB TERTIAN MALARIAL INFECTION.  BRITISH MEDICAL JOURNAL 1: 290, 1954.

2.       AMBE JP, FATUNDE JO, SODEINDE O. ASSOCIATED MORBIDITIES IN CHILDREN WITH SICKLE CELL ANAEMIA PRESENTING WITH SEVERE ANAEMIA IN MALARIOUS AREA. TROPICAL DOCUMENT  31(1): 26-27, JAN 2001.

3.       DONALDSON A ET AL., HBF IN HOMOZYGOUS SICKLE CELL DISEASE: A STUDY OF PATIENTS WITH LOW HBF LEVEL. ACTA HEMATOLOGY, 105(1): 27-31, 2001.

4.       GILMAN JG, HUISMAN TH. DNA SEQUENCE VARIATION ASSOCIATED WITH ELEVATED FETAL HEMOGLOBIN PRODUCTION. BLOOD 66: 783-787, 1985.

5.       HALDANE   JBS. THE RATE OF MUTATION OF HUMAN GENES. PROCEEDINGS OF THE VIII INTERNATIONAL CONGRESS  ON GENETICS AND HEREDITY 35SUPP.: 369, 1949.

6.       KAR BC, SICKLE CELL DISEASE IN INDIA, J. ASSO. PHYSICIAN IND. 39: 954-64, 1991.

7.       KOTILA TR ET AL, HBF AND CLINICAL SEVERITY OF OF SICKLE CELL ANAEMIA IN NIGERIAN ADULTS, AM. J. MED. SCI. 308(5): 259-65, NOV’1994.

8.       LEHMAN, CUTBUSH M. SICKLE CELL TRAIT IN SAUTH INDIA BRITISH MED. J. 1: 404-06, 1952.

9.       LUKENS JN, HEMOGLOBINOPATHIES – S, C, D, E & O AND ASSOCIATED DISEASE. IN LEE R ET AL(EDS). WINTROBES CLINICAL HEMATOLOGY, PHILADELPHIA USA.1:1061-1101, 1993.

10.   LUZZATTO L., NWACHUKU J., REDDY S. INCREASED SICKLING OF PARASITIZED ERYTHROCYTES IN MECHANISM OF RESISTANCE AGAINST  MALARIA IN SICKLE CELL TRAIT. LANCET 1:319-322, 1970.

11.   MUKHERJEE MB, LU CY, DUCROCQ R, GANGAKHADKAR  RR, COLAH RB, KADAM MD, MOHANTY D, NAGEL RL, KRISHNAMOORTY. EFFECT OF ALPHA THALASSEMIA ON SICKLE CELL LINKED TO THE ARABIAN INDIAN HAPLOTYPE IN INDIA, AM. J. HEMATOLOGY 55:104-109, 1997.

12.   NAGEL RL, HEMATOLOGICALLY AND GENETICALLY DISTINCT FORMS OF SICKLE CELL ANAEMIA IN AFRICA. THE SENEGAL TYPE AND BENIN TYPE. N. ENGL. J. MED., 317:850-54, 1984.

13.   NAGEL RL, STEINBERG MH. GENETICS OF OF THE ‘S’ GENE: ORIGINS, GENETICS, EPIDEMIOLOGY AND EPISTASIS IN SICKLE CELL ANEMIA. IN STEINBERG MH, FORGET BG, HIGGS DR ET AL (EDS). DISORDERS OF HEMOGLOBIN: GENETICS, PATHOPHYSIOLOGY, CLINICAL MANAGEMENT, CAMBRIGE, UK: CAMBRIDGE UNIVERSITY PRESS, 2001.

14.   NANDA BK ET AL. HEMAGLOBIN S IN AGHARIA COMMUNITY IN ORISSA, JIMA 48: 150-52, 1967.

15.   OLUMES PE ET AL, THE CLINICAL MANIFESTATIONS OF CEREBRAL MALARIA AMONG NIGERIAN CHILDREN WITH THE SICKLE CELL TRAIT. ANNALS OF TROPICAL PEDIATRICS 17(2): 141-45, JUN 1997.

16.   PATEL DK ET AL, CLINICAL PROFILE OF P. FALCIPARUM MALARIA AND GEOGRAPHICAL AREA OF WESTERN ORISSA ASSOCIATED WITH HIGH INCIDENCE OF SICKLE CELL HEMOGLOBINOPATHY. PROCEEDINGS OF EIGHTY SEVENTH SESSION OF INDIAN SCIENCE CONGRESS, PUNE, 2000.

17.   SERGEANT GR DISTRIBUTION OF SICKLE CELL DISEASE. OXFORD UNIVERSITY PRESS 3^RD EDN.: 18-30.

18.   SERJEANT GR, BASIC CONCEPTS IN SICKLE CELL DISEASE, 3^RD EDITION 2001 OXFORD UNIVERSITY PRESS: 3-15.

13. INTRODUCTION :

            Sickle Cell Diseases caused by point mutation in the b globin gene is widespread and has protean manifestations affecting various organ system in the body.⁽¹⁾  Of the various globin gene cluster haplotype, Arab Indian type is thought to be milder.  The strong association of Xmn-I site with the Arab Indian haplotype of Sickle Cell disease is thought to be associated with high fetal hemoglobin concentration and confer a benign course of the disease.⁽²⁾  However the clinical presentation of Sickle Cell disease in various Indian patients is variable.  In this part of Western Orissa the morbidity of Sickle Cell Disease is found to be more compared to that of Gujrat (Mohanty et al. current opinion in hematology).  In our institution yearly around three hundred patients of Sickle Cell disease are admitted with various complications.  The majority (70%) present with painful crisis.  The next common presentation is chronic severe anemia (48%).  These patients have chronic anemia, icterus and various organ dysfunction like chronic renal failure, hepatic failure & heart failure.  Avascular necrosis of femoral head is found in 8% of cases.  Pregnant women with sickle cell disease suffer from preterm labor, urinary tract infections and fetal intrauterine growth retardation.  Approximately 10% of the admitted patients die in the hospital from various complications.

            The various epistatic factors which modify the clinical course and severity of sickle cell disease could be genetic, like mutations linked to fetal hemoglobin and µ thalassaemia.  Environmental epistatic factors like plasmodium falciparum malaria also plays an important role in the morbidity of sickle cell disease (Patel et al, Mohanty et al)

            In view of the common incidence P.falciparum infection in this region it would be interesting to study the effect of these epistatic factors of Sickle Cell disease.

13.1:  Aim :

            Study of morbidity pattern in sickle cell disease in Western Orissa and its correlation with fetal hemoglobin concentration and different epistatic factors like malaria.

           Objectives :

1.      To findout different clinical presentations of Sickle Cell Disease in two study populations at Burla and Rourkela centres and compare them.

2.      To estimate fetal hemoglobin concentration and genotype in these patients by studying Xmn polymorphism and correlate this with the severity of clinical manifestations.

3.      To study the incidence and severity of P.falciparum infection on sickle cell disease.  Sickle cell trait and compare this with age and sex matched controls without sickle cell hemoglobinopathy.

4.      To have a family study for assessment of hemoglobinopathy status in different family members and provide counselling.

5.      To followup these cases at periodic intervals over 3 years and give simple intervention measures.

13.2 :  Specific research project(s) that will be initiation in the first year of the programme support

(i)                Procurement of equipments and standardization of laboratory.

(ii)             Training

(iii)           Pilot study.

14.       Review of current status of research and development in subject.

14.1:   International Status :

            Sickle Cell disease is wide spread in various parts of the world with high frequency in Africa, USA, Caribbean islands, Soudi Arabia and India (Luken’s, 1993).  The most common HbS haplotype is the Benin type found in Benin & Central West Africa.  The second, Senegal haplotype is prevalent in Senegal and African West Coast.  The third haplotype i.e. Banty haplotype is found in central African Republic.  The Asian haplotype probably represent an independent HbS mutation and is found in Eastern Soudi Arabia and India (Nagal RL et al).  There is also a Cameroon haplotype.  Of these various genotypes the Arab-Indian variety is thought to be milder probably because of high HbF concentration.  But various studies in this aspect is far from conclusive.  Kotila et al in a study in Nigerian Sickle Cell disease patients found that there is no statistically significant difference of HbF level in patients with milder and severe presentation Donaldson A et al in a study of 50 Jamican patients found that low HbF level in sickle cell disease did not appear to increase the clinical severity of the disease and may be protective against leg ulceration.

14.2. National Status :

            HbS gene was first recognized in India by Lehman and Cutbush in 1952 in veddoids in South India.  Subsequent studies revealed the frequency of HbS gene to be around thirty-two percent in tribal population of Kerala, in Abhug Maria in Madhya Pradesh and twenty percent in schedule tribes in Orissa.  Kar et al in an extensive epidemiological study revealed that the HbS gene in India is not confined to tribals but is widely prevalent permeating through the different caste and communities of the country.  He drew a Sickle map of India depicting the largest volume of cases in Western Orissa.

            Nanda et al reported the first case of Sickle Cell disease in 1967 from this Institution in Orissa.  From various clinical studies performed in this institution since then it is found that the clinical manifestations of the disease are extremely variable ranging from completely asymptomatic patients surviving upto sixth decade to crippling or life threatening complication and death in early childhood.  Moreover, as reported by Mohanty Deepika et al the severity of the disease in Orissa is more than that found in Gujurat.  Various genetic factors like mutations linked to fetal hemoglobin and             µ-thalassemia and environmental epistatic factors like malaria are thought to be responsible for this varied phenotypic expression of the disease.

            In view of the large number of complicated P.falciparum cases being admitted to our institution we conducted a preliminary study on interaction of P.falciparum malaria and sickle cell disease and found that cases of Sickle cell trait are also susceptible to falciparum malaria and this infection in sickle cell disease patients results in multiorgan failure and high mortality (Patel et al).

14.3.                Importance of the proposed programme/project in the context of current status.

            The prevalence of sickle cell disease in Western Orissa is very high.  Their clinical presentation is highly variable in this group of population and some what more severe compared to other parts of the country.  If all the Indian sickle disease patients belong to a single Arab-Indian haplotype it is difficult to explain extremely variable phenotypic expression in these patient.  So it will be pertinent to study various genetic and environmental factors which alter the clinical manifestations of this disease.  In this way patients of sickle cell disease with frequent complication and high risk of death can be segregated.  In the back drop of large sickle cell disease population and scarcity of health resources it will be prudent to focus our attention to these high risk people and institute simple and cheap interventional measures for prevention and amelioration of complications e.g. if falciparum malaria could be a cause of excess death in these patients then simple chloroquine prophylaxis would save many valuable lives.

14.4. Description of Institutions and recent research interest.

(I)  V.S.S. Medical College, Burla, Orissa.

            V.S.S. Medical College, Burla is a twelve hundred bedded tertiary care hospital situated in Western Orissa under Ministry of Health & Family Welfare, Government of Orissa.  In addition to basic science departments like Anatomy, Physiology, Biochemistry, Pathology, Pharmacology etc and various clinical specialties like Internal Medicine, Surgery, Obstetrics & Gynaecology, Orthopaedics and Pediatrics are available. This apart, superspeciality departments like Cardiology, Neurology, Urology, Neurosurgery are also available.  In addition to imparting specialized care for referral patients of Western Orissa, it is an under graduate & postgraduate teaching Hospital.

            The Department of Internal Medicine has a specialized unit i.e. Sickle Cell clinic which was initially started with financial support of Indian Council of Medical Research (ICMR) way back in 1986.  After completion of ICMR project in 1992 the clinic is run by the department of medicine under the guidance of Dr. D.K. Patel, Principal Investigator.

            In addition to special interest in research of Sickle Cell disease, we are also studying other diseases like P.falciparum malaria, hypokalemic psiodic pralysis, multiple myeloma and HIV infection.

(II)  I.G.H. Rourkela, Orissa :      

(III)     Institution of Life Science, Bhubaneswar, Orissa (under Department of Biotechnology)

            This is an institution under Department of Biotechnology, Government of India, located in Bhubaneswar, the capital city of Orissa.  This is a centre for research in advanced molecular biology with the state of the art facilities.

14.5. Expertise Available :

(I)        V.S.S. Medical College, Burla, Orissa :

Ø     Clinical and research experience :  In 1992 with help of a Council of Scientific & Industrial Research (CSIR) assisted grant we studied the prevalence of Hepatitis-B virus infection in Sickle Cell disease and found very high incidence of the Hepatitis B infection in comparison to the controls.  In addition, the various aspects of the sickle cell disease that we have studied are :

1.      Plasma viscosity and its role in vaso-oclusive crisis in sickle cell disease.

2.      Renal changes, pancreatic pathology, the pregnancy outcome (maternal and fetal morbidity and mortality) in sickle cell disease.

Besides this research experience we have got a fifteen year clinical experience in management of sickle cell disease and complications.

Ø     Investigative expertise available :

·        Sickling test

·         Hb electrophoresis by agar gel and cellulose acetate.

·        Department of Pathology and hematology.

·        Department of Radiology with CT Scan and Ultrasonography facilities.

(II)             I.G.H. Rourkela, Orissa                       

(III)         Institute of Life Sciences, Bhubaneswar, Orissa.

Ø     P.C.R.

Ø     D.N.A. Sequencing facilities

14.6.                Anticipated products and processes of Potential/Technological Applications/Socioeconomic relevance expected to be evolved by pursuing the programme/project.

1.                  This research will generate data for risk stratification of sickle cell disease patients and identify high risk groups with increased morbidity and mortality.  So that health care resource can be targeted to the population for saving valuable life.

2.                  Subsequent steps can be taken for prenatal diagnosis of sickle cell disease with the P.C.R. facilities of this project.

14.7.                No new drug trial will be included in the present study.

15.                       WORK PLAN :

15.1.       Methodology :

(A)       V.S.S. Medical College, Burla, Orissa.

(a)               Clearance by Institutional ethical committee.

(b)              Patients attending to the Department of Medicine Surgery, Orthopaedics, obstetrics and gynecology and sickle cell clinic.

Inclusion Criteria :

            Cases of sickle cell disease and trait diagnosed by hemoglobin electrophoresis will be taken after obtaining informed consent.

Exclusion Criteria :

            Patient with connective tissue disorder and gout will be excluded from the study. Clinical data will be collected in a specially designed format.

(c) A.   At   V.S.S. Medical College, Burla

            Blood will be collected for routine tests and the genetic studies.

            5ml heparinised blood will be collected from the patients before institution of any therapy.  About 1ml of whole blood will be rremoved for estimtion of haemoglobin and other red cell indices by cell counter.  The remaining portion of blood will be centrifuged at 1500 rpm for 10 minutes and plasma separated.  The plasma will be used for biochemical tests such as glucose, urea, Creatinine, bilirubin and alanine amino transferase by auto-analyzer.  The erythrocytes would used for preparation of haemolysate.  Haemoglobin electrophoresis of the haemolysate will be done using both agar gel and cellulose acetate membrane.  Electrophorresis will be run with normal as well as sickle cell haemoglobin controls.

Some haemolysate will be transported to IGH, Rourkela for HPLC.

DNA samples will be sent to Institute of Life Science for DNA sequencing.

Other investigations as may be needed in individual cases like X-rays, CT Scan, USG and other will done in our institution.

(B)      AT I.G.H., Rourkela -         

(C)      At institute of Life Science, Bhubaneswar DNA sequencing will be done statistical analysis will be done at the end of the study.

15.2.       Proprietary/Patented items, if any, expected to be used for this programme or project – Nil.

15.3.       Organization of work elements including training : 

(A)      At V.S.S medical college, Burla

Principal Investigator & Programme Coordinator :

            He will coordinate the research activity in the three institutions.  He will also supervise and coordinate the various activities of co-investigators.  He will be associated with the clinical study of these patients.

Co-investigator – 1 : In addition to assisting principal investigator, will take part extensively in the clinical study and statistical analysis.

Co-investigator – 2 : He will be trained in P.C.R. and look after the P.C.R. analysis of genetic materials and study of biochemical parameters.

Co-investigator – 3 : He will after the Pathological and hematological aspects of the patient.

Co-investigator – 4 : She will study the obstetrics and Gynaecological aspect of the Sickle Cell disease.

Training  - Co-investor & (Dr. U.K. Das) will be sent to Institute of Immunohematology

KEM Hospital, Mumbai for PCR

(B) AT IGH, Rourkela, Orissa    -           Dr. Pati

(C) At Institute of Life Sciences, Bhubaneswar, Orissa.  Co-investigator will take part in sequencing of DNA.

15.4.       Suggested plan of action for utilization of research outcome expected from the programme/project.

The research out come of this project will be utilized in the following manner :

(a)    Patient care : for managing high risk sickle cell disease patients in a better way Molecular diagnosis of sickle cell disease will help in genetic counselin

The information can be utilized in future for development of novel  treatment strategies like drugs to increase the fetal Hb concentration and gene therapy.

(b)   Education :

Molecular diagnostic facility established in this project can be utilized by undergraduate and post graduate  students and encourage them for research in molecular biology.

(c)    Research :

The research outcome can be conveyed to the medical community by publications in journals so as to help further research in this field.

15.5 Time schedule of activities giving milestones :

15.6  Programme/Project implementing Agency/Agencies:

16. Linkaes with other Institutions :

            Institute of Immunohematology  13^th floor, New Building, KEM Hospital Parel, Mumbai.