Anita Nadkarni

Matrix metalloproteinase and its drug targets therapy in solid and hematological malignancies: An overview

Matrix metalloproteinase (MMP) comprises a family of zinc-dependent endopeptidases that degrade various components of the extracellular matrix (ECM) and basement membrane. MMPs are involved in solid and hematological malignancy through modification of cell growth, activation of cancer cells and modulation of immune functions. Several polymorphisms of different MMPs such as MMP-1 (-1607 1G/2G), MMP-2 (-1306 C/T), MMP-3 (-1171 5A/6A) & MMP-9 (-1562 C/T) and their expression levels have been well documented in different types of solid cancer. These polymorphic variations were found to be associated with angiogenesis, cancer progression, invasion and metastasis. There is paucity of data available in the field of hematological malignancies. Hence the field of matrix biology of hematological malignancies is an area of active exploration. A number of MMP inhibitors (MMPIs) have been developed for the cancer treatment. The most extensively studied classes of MMP inhibitors include Batimastat, Marismastat, Salimatat, Prinomastat and Tanomastat. However, their efficacy and action have not been confirmed and more data is required. The application of one or more selective targeted MMPIs in combination with conventional anti-leukemic treatment may represent a positive approach in combat against hematopoietic malignancies. Balance of MMPs and TIMPs is altered in different malignancies and biochemical pathways. These alternations will add another dimension in the matrix biology of both solid tumor and leukemia. MMP and TIMP singly and in combination are increasingly being recognized as an important player in basic cellular biology. Exploration and exploitation of MMP and TIMP balance in various malignant and nonmalignant lesions is going to be one of the most interesting facets of future use of this system for human health care.

Global burden, distribution and prevention of β-thalassemias and hemoglobin E disorders

The β-thalassemias, including the hemoglobin E disorders, are not only common in the Mediterranean region, South-East Asia, the Indian subcontinent and the Middle East but have now become a global problem, spreading to much of Europe, the Americas and Australia owing to migration of people from these regions. Approximately 1.5% of the global population are heterozygotes or carriers of the β-thalassemias. While the overall frequencies of carriers of these disorders are known in most countries, there have been few attempts at micromapping and wherever this has been done, significant variations are seen even within small geographic regions. Thus, the figures for the estimated numbers of births each year of homozygous β-thalassemia and the severe compound states involving other hemoglobin disorders may be an underestimate. Screening strategies have varied from premarital to antenatal in different countries depending on socio–cultural and religious customs in different populations. Prenatal diagnosis programs are ongoing in many countries and the knowledge of the distribution of mutations has facilitated the establishment of successful control programs. Many of these were through North–South partnerships and networking. Yet, there are many countries in Asia where they are lacking, and South–South partnerships are now being developed in South-East Asia and the Indian subcontinent to link centers with expertise to centers where expertise needs to be developed. Although the carrier frequencies will remain unaltered, this will eventually help to bring down the burden of the birth of affected children with β-thalassemias and hemoglobin E disorders in Asia.

Response to hydroxyurea in β thalassemia major and intermedia: Experience in western India

BACKGROUND The clinical and hematological response to hydroxyurea was evaluated in beta thalassemia patients in western India with variable clinical severity and correlated with genetic factors. MATERIALS AND METHODS Seventy-nine patients-[38-beta thalassemia intermedia-(group I), 41-beta thalassemia major-(group II)] on hydroxyurea therapy were followed-up for 20-24months. RESULTS Among the frequently transfused patients in group I, 58% became transfusion independent and 16% showed a 50% reduction in transfusions after therapy which correlated with a higher mean fold increase in HbF and gamma mRNA expression levels. Forty-one percent of patients in group I had associated alpha-thalassemia and 72.7% were XmnI (+/+). beta thalassemia chromosomes among the responders of group I (41%) were linked to haplotype (- + + - + + - - +) as against haplotype (+ - - - - - - - +) being more common among the non-responders. Response was not linked to the beta thalassemia mutations. Thirty-two percent of group II patients showed a 50% reduction in their transfusion requirements after therapy which also correlated with a higher mean fold increase in HbF and gamma mRNA expression levels. A significant decrease in serum ferritin was seen in both groups. 19% of patients could not tolerate the drug. CONCLUSIONS In group I, clinical response to hydroxyurea was better in patients with alpha-thalassemia, XmnI (+/+) and a higher mean fold increase in gamma mRNA expression. In group II, only one-third of patients showed a partial response.

Hydroxyurea in sickle cell disease--a study of clinico-pharmacological efficacy in the Indian haplotype.

There is clinical variability in the presentation of sickle cell disease among Indians. Vaso-occlusive crisis is common among non-tribal patients. Hydroxyurea, induces fetal hemoglobin (HbF) synthesis and reduces the clinical severity of sickle cell disease but individual patients have a variable response. This study was undertaken to investigate the efficacy and safety of hydroxyurea in Indians with severe manifestations where the beta(s) gene is linked to the Arab-Indian haplotype and is associated with higher HbF levels. Seventy-seven patients (29 adult sickle homozygous, 25 pediatric sickle homozygous, 23 adult sickle beta-thalassemia) selected for hydroxyurea therapy were evaluated for clinical, hematological, biochemical and genetic parameters and were followed for 24 months. Ninety-eight point seven percent of the sickle chromosomes were linked to the Arab-Indian haplotype, 27% of patients had associated alpha thalassemia and 65% were Xmn I +/+. Seventy-eight percent of the patients had no further crises after starting hydroxyurea. This effect was accompanied by a significant increase in HbF (p<0.001), but this increase was variable in individual cases. There was also an increase in gamma gene mRNA expression in the few cases so studied. Hemoglobin levels increased significantly (p<0.001) resulting in the cessation of blood transfusions. Leucopoenia was observed in one patient. Hydroxyurea was effective in reducing the clinical severity in Indian patients who initially had higher HbF levels and the presence of ameliorating factors, such as alpha-thalassemia and the Xmn I polymorphism. Hydroxyurea therapy with careful monitoring can thus change the quality of life of Indians with sickle cell disease.

Regional heterogeneity of β-thalassemia mutations in the multi ethnic Indian population

To determine the frequencies of beta-thalassemia mutations in different states of India and to compare this with the available data in Asian Indians for a comprehensive catalogue of molecular defects in the Indian population. beta-thalassemia mutations were characterized in 2456 heterozygotes using reverse dot blot hybridization, ARMS and DNA sequencing. 36 beta-thalassemia mutations were characterized from 18 different states in India. Seven mutations were common, accounting for 95.8% of mutated alleles. Marked regional diversity was seen in different parts of the country. Among the tribal populations, only 2 mutations (IVS I-5 (G-->C) and CD15 (G-->A) accounted for over 90% of mutant alleles. A compilation of all the studies in Asian Indians reported so far showed the presence of 63 mutations in the Indian population. This large study adds to the existing data to give a detailed account of the molecular basis of beta-thalassemia in India. This information is important for establishing prenatal diagnosis programmes in different states in India as well as other countries in which there is a major influx of Indian immigrants.

Epidemiology of β‐thalassaemia in Western India: mapping the frequencies and mutations in sub‐regions of Maharashtra and Gujarat

Although the average frequency of β‐thalassaemia carriers in India is 3–4% and the prevalent mutations have been studied, no micromapping has been done. This is the first attempt to provide an accurate estimate of the frequencies of β‐thalassaemia and the expected annual births of homozygous children in different districts of Maharashtra and Gujarat in Western India as well as to determine the molecular heterogeneity in different sub‐regions in these states. A total of 18 651 individuals were screened for haemo‐globinopathies and mutations were characterized in 1334 β‐thalassaemia heterozygotes. There was an uneven distribution of the frequencies of β‐thalassaemia, varying from 1·0% to 6·0% and 0% to 9·5% in different districts of Maharasthra and Gujarat. The rate of homozygosity per 1000 births annually was 0·28 in Maharashtra and 0·39 in Gujarat. The three most prevalent β‐thalassaemia mutations in Maharashtra were IVS 1‐5(G→C), Codon 15(G→A) and Codon 30(G→C) (87·9%) while in Gujarat they were IVS 1‐5(G→C), 619 bp deletion and Codon 5(‐CT) (68·5%). These data will help to develop adequate programmes for control and care where they are most needed. They also emphasize the importance of subgroup micromapping for determining the health burden of a common genetic disease.

Effect of Proinflammatory Cytokines (IL-6, TNF-α, and IL-1β) on Clinical Manifestations in Indian SLE Patients

Systemic lupus erythematosus (SLE) is an inflammatory rheumatic disease characterized by production of autoantibodies and organ damage. Elevated levels of cytokines have been reported in SLE patients. In this study we have investigated the effect of proinflammatory cytokines (IL-6, TNF-α, and IL-1β) on clinical manifestations in 145 Indian SLE patients. One hundred and forty-five healthy controls of the same ethnicity served as a control group. Clinical disease activity was scored according to SLEDAI score. Accordingly, 110 patients had active disease and 35 patients had inactive disease. Mean levels of IL-6, TNF-α, and IL-1β were found to be significantly higher in SLE patients than healthy controls (P < 0.001). Mean level of IL-6 for patients with active disease (70.45±68.32 pg/mL) was significantly higher (P = 0.0430) than those of inactive disease patients (43.85±63.36 pg/mL). Mean level of TNF-α was 44.76±68.32 pg/mL for patients with active disease while it was 25.97±22.03 pg/mL for those with inactive disease and this difference was statistically significant (P = 0.0161). Similar results were obtained for IL-1β (P = 0.0002). Correlation between IL-6, TNF-α, and IL-1β serum levels and SLEDAI score was observed (r = 0.20, r = 0.27, and r = 0.38, resp.). This study supports the role of these proinflammatory cytokines as inflammatory mediators in active stage of disease.

Prevalence and Molecular Characterization of α-Thalassemia Syndromes among Indians

OBJECTIVE This study was undertaken to determine the prevalence and molecular basis of alpha-thalassemia in the Indian population and its implications in genetic counseling and prenatal diagnosis. METHODS 1253 random samples were screened for hemoglobinopathies. Red cell indices were measured on the Sysmex K 1000 cell counter; HbA2 and HbF levels were quantitated using high performance liquid chromotography (HPLC). Cellulose acetate electrophoresis and isoelectric focusing (IEF) was done to detect the presence of Hb Barts in cord blood samples. alpha-Globin gene mapping was done using Southern blot hybridization of BamHI and BglII digests. RESULTS Of the 1253 subjects, 132 had a single alpha-gene deletion (10.5%) while 29 had two alpha-gene deletions (2.31%). Fifteen cases showed the presence of alpha-gene triplication (1.1%). A single case showed the presence of one alpha-gene deletion as well as alpha-gene triplication (-alpha/alphaalphaalpha). Overall, the prevalence of alpha-thalassemia was 12.9%. Region-wise and caste-wise analysis showed the highest prevalence of alpha-thalassemia among the Punjabi population originating from the northern region of India. CONCLUSION alpha-Thalassemia is by far the commonest hemoglobinopathy in India, but it is not a cause of serious genetic risk is the milder form (-alpha/alphaalpha) of alpha-thalassemia, which is predominant. Knowing the alpha-genotype is useful for genetic counseling for prenatal diagnosis in couples where one of the parents may have reduced indices coupled with a raised RBC count and normal HbA2 levels.

Effect of hydroxyurea on the transfusion requirements in patients with severe HbE-β-thalassaemia: a genotypic and phenotypic study

Background Haemoglobin E (HbE)-β-thalassaemia has a very variable clinical presentation. The management of severe cases that are often transfusion dependent is similar to that of cases of β-thalassaemia major; however, this is often not possible in India because of its high cost and the lack of availability of safe blood at many places. Thus there was a need for a drug such as hydroxyurea, which is known to reduce the transfusion requirements of patients with thalassaemia intermedia. This study was undertaken to evaluate the response of Indian patients with HbE-β-thalassaemia to hydroxyurea. Materials and methods 11 patients with HbE-β-thalassaemia receiving regular transfusion plus two less frequently transfused patients were selected for hydroxyurea therapy. Clinical and haematological evaluation was performed before and after treatment for 2 years. Molecular studies included β-globin genotype, β-globin gene haplotype, Xmn I polymorphism and α-genotyping. Results Four clinically severe patients became transfusion independent (responders) after hydroxyurea therapy, four patients showed a reduction in their transfusion requirements (partial responders), and three patients were non-responders. Responders showed a statistically significant increase in Hb, mean corpuscular volume, mean cell Hb, fetal Hb and F cells with a reduction in their transfusion requirements. A reduction in serum ferritin concentration was also seen in responders and non-responders. Conclusions Genetic markers such as Xmn I polymorphism and α-gene deletions were not always beneficial for the response to hydroxyurea therapy. Thus many more markers and a larger cohort need to be studied to predict the response in these patients.

Clinical, hematologic and molecular variability of sickle cell-β thalassemia in western India

BACKGROUND: Sickle cell-β thalassemia (HbS-β thalassemia) is a sickling disorder of varying severity, which results from compound heterozygosity for sickle cell trait and β thalassemia trait. The present study was undertaken to determine the genetic factors responsible for the clinical variability of HbS-β thalassemia patients from western India. MATERIALS AND METHODS: Twenty-one HbS-β thalassemia cases with variable clinical manifestations were investigated. The α and β globin gene clusters were studied by molecular analysis. RESULTS: Thirteen patients showed milder clinical presentation as against eight patients who had severe clinical manifestations. Four β thalassemia mutations were identified: IVS 1-5 (G→C), codon 15 (G→A), codon 30 (G→C) and codon 8/9 (+G). α thalassemia and XmnI polymorphism in homozygous condition (+/+) were found to be common among the milder cases. The βS chromosomes were linked to the typical Arab-Indian haplotype (#31). Framework (FW) linkage studies showed that four β thalassemia mutations were associated with different β globin gene frameworks. Linkage of codon 15 (G→A) mutation to FW2 is being observed for the first time. CONCLUSION: The phenotypic expression of HbS-β thalassemia is not uniformly mild and α thalassemia and XmnI polymorphism in homozygous condition (+/+) are additional genetic factors modulating the severity of the disease in the Indian subcontinent.