The Changing Trends in Prenatal Diagnosis of Hemoglobinopathies in India: The Quest of a Single Center to Reduce the Burden of Disease over Three Decades.

The ß-thalassemias and sickle cell disorders pose a considerable health burden in India. Of the more than 10,000 annual births of children with a severe hemoglobinopathy, only around 10.0% are managed optimally. Thus, genetic counseling and prenatal diagnosis (PND) is a valid option for a large and diverse country. Our center was one of the first to initiate PND and we present our experience over 30 years to evaluate the impact of awareness in changing the trends of PND of hemoglobinopathies. Both second and first-trimester diagnoses were undertaken by fetoscopy/cordocentesis and globin biosynthesis/high-performance liquid chromatography (HPLC) analysis of fetal blood and chorionic villus sampling (CVS) and DNA analysis. Over 30 years, 3478 couples (first trimester: 2475; second trimester: 1003) from all over India were offered PND. The number of couples coming in the first trimester increased significantly over each decade and couples coming prospectively increased from 2.5 to 18.4%. A cost-effective stepwise approach was used for molecular analysis. Eight hundred and one fetuses (23.0%) were affected and all except three couples opted for termination of these pregnancies. Genetic counseling and PND is the only way to reduce the burden of disease. With awareness, there was a shift from second trimester to first trimester PND over each decade, with an increasing number of couples coming during the first pregnancy. There are only 15 to 20 centers in India offering PND. We have compared our study with other reports on PND from different regions in India.

Rare ß- and δ-Globin Gene Mutations in the Pathare Prabhus: Original Inhabitants of Mumbai, India.

Genetic structure of the Indian population is influenced by waves of several immigrants from West Eurasia. Therefore, genetic information of various ethnic groups is valuable to understand their origins, the pattern of migration as well as the genetic relationship between them. No genetic data is available on Pathare Prabhu, which is a small indigenous Hindu community from Mumbai, Maharashtra State, India. The aim of this study was to screen the Pathare Prabhus for hemoglobinopathies, which is a major public health problem in India. Two hundred and fifty-seven unrelated Pathare Prabhus subjects were screened for various hemoglobinopathies. Complete blood counts (CBC) were done on an automated hematology counter. High performance liquid chromatography (HPLC) was used to identify ß-thalassemia (ß-thal) carriers. Molecular characterization of the ß gene defects was done by reverse dot-blot hybridization, amplification refractory mutation system (ARMS) and DNA sequencing. Deletional α-thalassemia (α-thal) was detected by multiplex polymerase chain reaction (PCR). Hb A2-Saurashtra (HBD: c.301C>T) was identified by DNA sequencing; its modeling was also done. The prevalence of ß-thal was 3.89%, while deletional α-thal was 5.4%. The initiation codon (ATG>ACG) (HBB: c.2T>C) was seen in eight individuals (80.0%), Hb D-Punjab (HBB: c.364G>C) and Hb A2-Saurashtra, was found in two and one individual, respectively. A community-specific ß-thal mutation was found in Pathare Prabhus in significant proportions. This information is useful in developing an algorithm for a prenatal diagnosis (PND) program.

A functional SNP MCP-1 (-2518A/G) predispose to renal disorder in Indian Systemic Lupus Erythematosus patients.

Systemic Lupus Erythematosus (SLE) is a clinically heterogeneous chronic, inflammatory autoimmune disorder that affects multiple organs where exact etiology of the disease is not yet clearly understood. Various evidences suggest that genetic polymorphisms in inflammatory mediators like cytokines and chemokines may influence development of the disease. Here, we investigated whether functional polymorphism at the Monocyte Chemoattractant Protein-1 (MCP-1) regulatory region associates with disease phenotype in Indian SLE patients. This case control study included 200 SLE patients and 201 ethnically matched healthy controls. Genotyping of MCP-1 (-2518 A/G) polymorphism was performed using PCR-RFLP method. Serum MCP-1 levels were detected by bead-based multiplex immunoassay. Serum MCP-1 levels were found to be higher in patients compared with healthy individuals (p<0.0001). A significant difference for MCP-1G allele frequency (OR=1.9, 95%CI=1.4-2.6, p<0.0001) was observed among SLE patients against healthy individuals. A significant difference in the distribution of MCP-1 -2518GG (OR=3.0, 95%CI=1.4-6.7, p=0.0041) and AG+GG genotypes (OR=2.0, 95%CI=1.4-3.0, p=0.0005) was also noted among SLE patients when compared with healthy individuals. A significant association was observed between A/G and G/G versus A/A genotypes with renal manifestations (p<0.0001, Pc<0.001). Serum MCP-1 levels in active LN patients were found to be significantly higher than inactive LN (p=0.0059), mild LN (p=0.0061) as well as non-LN patients (p=0.0001). These findings suggest that -2518G allele of MCP-1 -2518 A/G polymorphism is associated with renal disorders and may influence MCP-1 gene expression among Indian SLE patients.

Effect of the Hemochromatosis Mutations on Iron Overload among the Indian ß Thalassemia Carriers.

BACKGROUND: Hereditary hemochromatosis is a disorder of iron metabolism characterized by increased iron absorption.HFE gene mutations C282Y and H63D are responsible for the majority of hereditary hemochromatosis cases. METHODS: We tried to look at the effect of HFE mutations on the iron status. A total of 100 ß thalassemia traits (BTT) with 100 normal individuals were screened for the C282Y and H63D mutations using PCR-RFLP. The serum ferritin levels were determined using ELISA kit. RESULTS: We did not find the C282Y mutation in our study group. The allelic frequencies for H63D mutation did not differ significantly between ß-thalassemia traits (8.5%) and normal controls (9%). ΒΤΤ with H63D genotype of H/D (143.16 ± 80.3 ng/ml) and D/D (504 ng/ml) showed higher ferritin levels as against H/H genotype (88.64 ± 92.43 ng/ml). The statistically significant difference was observed in the mean serum ferritin levels among the individuals showing H/H and D/D genotypes (P < 0.002) and H/D and D/D genotype (P < 0.01) in both the groups. CONCLUSION: This suggests that iron load in BTT tends to aggravated with the co-inheritance of the H63D mutation. The mutant H63D gene showed the presence of haplotype 6 which is reported in the European population suggesting a common origin.

Diverse phenotypes and transfusion requirements due to interaction of ß-thalassemias with triplicated α-globin genes.

Co-inheritance of triplicated α-genes can alter the clinical and hematological phenotypes of ß-thalassemias. We evaluated the phenotypic diversity and transfusion requirements in ß-thalassemia heterozygotes, homozygotes, and normal individuals with associated α-gene triplication. Clinical and hematological evaluation was done and the ß-thalassemia mutations characterized by a covalent reverse dot blot hybridization/amplification refractory mutation system. Alpha-globin gene triplication was assessed by multiplex PCR. During the last 2.5 years, 181 ß-thalassemia patients and ß-thalassemia carriers with an unusual clinical presentation were referred to us for screening for the presence of associated α-globin gene triplication. Twenty-nine of them had associated α-gene triplication (3 ß-thalassemia homozygotes or compound heterozygotes and 26 ß-thalassemia heterozygotes). One ß-thalassemia compound heterozygote [IVS 1-5 (G → C) + CD 41/42 (-CTTT)] was anemic at birth and required blood transfusions unusually early by 6 weeks of age. The second patient (4.5 years) was also clinically severe and became transfusion dependent in spite of having one mild ß-thalassemia mutation [Capsite +1 (A → C)]. The third case (3.5 years) who was homozygous for a mild ß-gene mutation [-88 (C → T)] with α gene triplication was untransfused. The 26 ß-thalassemia heterozygotes with associated triplicated α-genes presented variably, with a ß-thalassemia intermedia-like presentation. While screening the family members of all these cases, we found another 10 ß-thalassemia heterozygotes and 9 normal individuals with α-globin gene triplication; however, all of them were asymptomatic. Beta-thalassemia carriers, homozygotes, and compound heterozygotes with an unusual presentation should be screened for the possible presence of associated α-globin gene triplication which could influence the clinical and hematological presentation.

Newborn screening for haemoglobinopathies by high performance liquid chromatography (HPLC): diagnostic utility of different approaches in resource-poor settings.

BACKGROUND: Sickle cell disease is a major health burden in India. The aim of the study was to compare the diagnostic utility of two different approaches on automated high performance liquid chromatography (HPLC) for newborn screening for sickle cell disorders and other haemoglobinopathies in India. METHODS: Newborn babies of sickle heterozygous mothers were tested by HPLC using two different kits, the ß-thal short kit, which is routinely used for screening for haemoglobinopathies in most laboratories, and the sickle cell short kit which is specific only for neonatal samples. Confirmation of the sickle and α genotypes was done by molecular analysis. RESULTS: Of the 601 babies tested, 276 were normal, 284 were sickle heterozygous and 41 were sickle homozygous using the ß-thal short kit. Using the sickle cell short kit, a discrepancy was seen in one newborn sample where a normal baby was identified as a sickle heterozygous baby. α-Genotyping was done in 42 babies and 16 of them had α gene deletions. The presence of α thalassaemia could be suspected in 15 of these 16 babies based on a spike at the start of the chromatogram using the ß-thal short kit. In comparison, using the sickle cell short kit the diagnosis of α thalassaemia was difficult based on the percentage of the FAST peak. Further, other rare α chain Hb variants were also missed. CONCLUSIONS: The ß-thal short kit was more versatile than the sickle cell short kit for screening for haemoglobinopathies in newborns in our population.

Effect of proinflammatory cytokines (IL-6, TNF-α, and IL-1ß) 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-1ß) 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-1ß 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-1ß (P = 0.0002). Correlation between IL-6, TNF-α, and IL-1ß 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.

Masking of a ß-thalassemia determinant by a novel δ-globin gene defect [Hb A2-Saurashtra or δ100(G2)Pro→Ser; HBD: c.301C>T] in Cis.

Abstract The molecular basis of ß-thalassemia (ß-thal) syndromes have been well documented, while the spectrum of mutations causing δ-thalassemia (δ-thal) has not been well characterized. δ-Thalassemia has no clinical symptoms but its coinheritance with heterozygous ß-thal may cause misdiagnosis, especially in countries with a high prevalence of ß-thal where prevention programs have been implemented. The coinheritance of ß- and δ-globin mutations in India is not common. This association may interfere with correct diagnosis and genetic counseling of ß-thal in screening programs. Here we report two families showing borderline Hb A2 levels belonging to the Koli Community, indigenous to the Saurashtra Province of Gujarat, India. They were referred to us for thalassemia molecular screening as they had children clinically presenting before 2 years of age and requiring regular blood transfusions. Interestingly, both families carried a novel δ-globin gene mutation at codon 100 (C > T) linked to a polyadenylation (polyA) site [AATAAA > A(-AATAA)] 5 bp deletional ß-thal mutation, never before reported in the Indian population. This report highlights the importance of considering δ-globin gene analysis during ß-thal screening to avoid false-negative results in the detection of at-risk couples. It also highlights how incomplete diagnosis of a borderline or normal Hb A2 level may lead to the probable birth of a ß-thal major (ß-TM) child. This has important implications in prenatal diagnosis.

Variable presentation of HB H disease due to homozygosity for the rare polyadenylation signal A T(Indian) (AATAAA>AATA- -) mutation in four Indian families.

The aim of this study was to identify the molecular defects leading to the variable clinical and hematological presentation of four patients with Hb H disease. Investigations included a complete blood count, high performance liquid chromatography (HPLC) analyses, cellulose acetate electrophoresis (pH 8.9), heat stability test, α genotyping by multiplex gap polymerase chain reaction (gap-PCR) to screen for the eight common α-globin gene deletions and DNA sequencing to detect the other deletional and nondeletional α-globin gene mutations. Two patients aged 15 and 5.5 years had a mild clinical presentation. The first patient aged 3 years had a severe presentation requiring regular transfusions. This patient also had an enlarged spleen and had to undergo splenectomy. The third patient, aged 5 years, also had severe anemia, had been transfused once and had a spleen of 4.5 cms. The hemoglobin (Hb) levels in the four patients ranged from 4.2 to 8.2 g/dL and they all had reticulocytosis (10.0 to 31.0%). Cellulose acetate electrophoresis at pH 8.9 showed a fast moving band that ranged from 18.0 to 25.9%. All the four patients were homozygous for the polyadenylation signal A (polyA) T(Indian) (AATAAA>AATA-) mutation. This mutation has been seen in Eastern India but not from Maharashtra and Uttar Pradesh where our patients originated.

Is the poly A (T>C) mutation a causative factor for misdiagnosis in second trimester prenatal diagnosis of ß-thalassemia by fetal blood analysis on high performance liquid chromatography?

We report the problems in diagnosis faced by two families referred for prenatal diagnosis of thalassemia where cordocentesis and fetal blood analysis by high performance liquid chromatography (HPLC) had to be done. The Hb A levels of the fetal blood measured by HPLC on the VARIANT™ Hemoglobin Testing System were 1.2 and 6.7%, respectively, suggestive of a heterozygous ß-thalassemia (ß-thal) fetus in the first case and a normal fetus in the second case. In one family, one of the parents had a borderline Hb A(2) level and in the other, one parent had normal RBC indices. However, DNA sequencing, done later, showed that in the first case the fetus was a compound heterozygote for the IVS-I-5 (G>C) and the polyadenylation signal site [poly A (T>C)] mutation, while in the second case, the fetus was homozygous for the poly A mutation. This emphasizes that characterization of ß-thal mutations must be done whenever one of the parents has a borderline Hb A(2) level or normal RBC indices, and one should not rely on fetal blood analysis by HPLC for prenatal diagnosis of ß-thal so as to avoid misdiagnosis.

Newborn screening shows a high incidence of sickle cell anemia in Central India.

There is limited data on the incidence of sickle cell anemia in Central India; we therefore conducted a study to estimate the incidence of this disease in Central India. Mothers who delivered a live baby at the Government Medical College, Nagpur, India were screened for the presence of the sickle cell hemoglobin {Hb S: [ß6 (A3) Glu→Val, GAG>GTG]} using the solubility test within 48 hours of delivery. Infants of mothers who showed the presence of Hb S then underwent Hb analysis by high performance liquid chromatography (HPLC). A total of 8243 mothers was screened, 1178 of whom were positive. One thousand, one hundred and sixty-two infants of mothers with a positive solubility test underwent Hb analysis by HPLC; 530 infants were normal, while 536 were heterozygous for Hb S (sickle cell trait), 88 babies were homozygous for Hb S (sickle cell anemia), while another eight babies had other Hb abnormalities. The incidence of sickle cell anemia was highest in the Scheduled caste group (1:50). We concluded that the incidence of sickle cell anemia is high in central India.

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

BACKGROUND: Sickle cell-ß thalassemia (HbS-ß thalassemia) is a sickling disorder of varying severity, which results from compound heterozygosity for sickle cell trait and ß thalassemia trait. The present study was undertaken to determine the genetic factors responsible for the clinical variability of HbS-ß thalassemia patients from western India. MATERIALS AND METHODS: Twenty-one HbS-ß thalassemia cases with variable clinical manifestations were investigated. The α and ß 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 ß 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 ß(S) chromosomes were linked to the typical Arab-Indian haplotype (#31). Framework (FW) linkage studies showed that four ß thalassemia mutations were associated with different ß 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-ß 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.

Recent advances in screening and diagnosis of hemoglobinopathy.

Introduction: Hemoglobinopathies are important causes of inherited disorders with substantial mortality and morbidity across the world. Therefore, proper utilization of available screening and diagnostic techniques are important for its diagnosis and management.Areas covered: In this review, the authors attempt to summarize clinical presentations, give a brief account of existing techniques, and discuss evolving and advanced techniques for detection and screening of the condition. As prevention of the disease condition is an important community measure to control the disease, techniques involving newborn screening, antenatal diagnosis, and point of care tests have been described in addition to more advanced molecular and protein diagnostics. The literature search in this area is covered between 1980 and 2018 with PubMed as the main source along with authors' own research in this area.Expert opinion: Screening and detection of hemoglobinopathy is best accomplished by a hierarchical approach with the optimum blend of old and newer techniques. Starting with point of care techniques through the commonly used HPLC and high voltage capillary electrophoresis, or modern and high throughput molecular biology and mass spectroscopic techniques can be used depending on specific situations. Every country needs to optimize its techniques depending on the frequency of the problem and available resources.

Role of MMP-2 and its inhibitor TIMP-2 as biomarkers for susceptibility to systemic lupus erythematosus.

Aim: To investigate the possible association between MMP-2 (-1575 G/A, -1306 C/T) and its inhibitor TIMP-2 (-418 G/C) functional polymorphisms with development of severity in systemic lupus erythematosus (SLE) patients. Materials & methods: 150 SLE patients and matched healthy controls were recruited. Polymorphisms were detected by PCR-RFLP and serum levels by ELISA. Results: Mean MMP-2 and TIMP-2 serum level and mRNA expression were significantly increased in SLE cases as compared with controls (p < 0.0001). The concomitant presence of both MMP-2 1575A and its inhibitor TIMP-2 418C alleles synergistically increased the risk of SLE by 3.25-fold (CI: 1.44-7.34, p = 0.003). Conclusion: MMP-2, TIMP-2 and MMP-2/TIMP-2 ratios may act as biomarkers for susceptibility to SLE.

The phenotypic and molecular diversity of hemoglobinopathies in India: A review of 15 years at a referral center.

INTRODUCTION: The hemoglobinopathies pose a significant health burden in India. Apart from the ß thalassemias and sickle cell disorders, α thalassemias and structural hemoglobin variants are also common. Here we have reviewed the phenotypic and molecular diversity of hemoglobinopathies encountered at a referral center in western India over a period of 15 years. MATERIALS AND METHODS: Screening for hemoglobinopathies was done using HPLC and cellulose acetate electrophoresis. Molecular characterization was done using Covalent Reverse Dot Blot Hybridization (CRDB), Amplification Refractory Mutation System (ARMS), GAP PCR and direct DNA sequencing. RESULTS: The study includes 31 075 individuals who were referred for diagnosis of hemoglobinopathies and prenatal diagnosis. Of these 14 423 individuals showed various hemoglobin abnormalities. Beta genotyping in 5615 individuals showed the presence of 49 ß thalassemia mutations. 143 ß thalassemia heterozygotes had normal or borderline HbA2 levels. We identified three δ gene mutations (HbA2 Pellendri, HbA2 St.George, HbA2 Saurashtra) in ß thalassemia heterozygotes leading to normal HbA2 levels. The commonest defects among the raised Hb F determinants were Gγ(Αγδß)0 Indian inversion and the HPFH-3 Indian deletion. A total of 312 individuals showed the presence of α thalassemia, of which 12.0% had a single α gene deletion (-α/αα). HbH disease was identified in 29 cases with 10 different genotypes. Alpha globin gene triplication was seen in 2.1% of ß thalassemia heterozygotes with a thalassemia intermedia phenotype. Seven unusual α chain variants and eight uncommon ß chain variants were identified. CONCLUSION: The repertoire of molecular defects seen in the different globin genes will be valuable for management and control of these disorders both in India as well as in other countries where there is a huge influx of migrant populations from India.

A novel G143D mutation in the NADH-cytochrome b5 reductase gene in an Indian patient with type I recessive hereditary methemoglobinemia.

We report a novel homozygous mutation responsible for NADH-b(5)R deficiency in a family from Ratnagiri district in western India with recessive congenital methemoglobinemia (RCM) type I. The propositus was a 20-year-old female with a history of increasing cyanosis exacerbated by fever and weakness. There was no history of cardiac illness or exposure to drugs and chemicals. The methemoglobin level was 38.0% in the propositus with 70% reduction in NADH-b(5)R activity. Spectroscopic analysis of the hemolysate showed normal peaks suggesting absence of Hb-M. There was no hemoglobin instability and G6PD activity was normal. This novel G-->A homozygous mutation at codon 143 in exon 5 was identified by SSCP followed by DNA sequencing and results in a glycine to aspartic acid substitution in the cytochrome b(5) reductase protein. This mutation, which is located outside the FAD and NADH binding domain, leads to mild cyanosis. Investigations of the family members revealed that both the parents and a brother of the propositus were heterozygous for the G143D mutation.

Evaluation of the use of monoclonal antibodies and nested PCR for noninvasive prenatal diagnosis of hemoglobinopathies in India.

Our purpose was to develop and evaluate isolation and enrichment of fetal erythroblasts and a nested polymerase chain reaction (PCR) approach using fetal erythroblasts for detecting the beta-globin gene mutations for a noninvasive prenatal diagnosis of hemoglobinopathies. Maternal blood at different periods of gestation was layered on a Percoll density gradient for enrichment of fetal nucleated RBCs (NRBCs). A combination of 3 monoclonal antibodies (CD45-peridinin chlorophyll protein, glycophorin A-phycoerythrin, and anti-hemoglobin F-fluorescein isothiocyanate) was used for flow cytometric sorting of fetal NRBCs from enriched cells. Different nested PCR-based approaches were used for identification of fetal mutations. Owing to heterogeneity of beta-thalassemia mutations in the population of India, we had to screen for 12 mutations and were able to give an accurate diagnosis in 84 (84.0%) of 100 cases when compared with chorionic villus sampling or cordocentesis and DNA analysis.This nested PCR approach enabled amplification of small quantities of DNA from fetal erythroblasts, providing a cost-effective method for noninvasive diagnosis.

Impact of TNF-α and LTα gene polymorphisms on genetic susceptibility in Indian SLE patients.

The promoter polymorphisms of tumour necrosis factor-α (TNF-α) and intronic Lymphotoxin-α (LTα) have been implicated as genetic risk factors for systemic lupus erythematosus (SLE) in various ethnic groups. The aim of this study was to investigate an impact of TNF-α (-308G/A; 238G/A) and LTα (+252A/G) gene polymorphisms in disease susceptibility among Indian 200 SLE patients along with 201 healthy controls. The gene polymorphisms were studied by using direct DNA sequencing and Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) methods. Serum levels were measured by multiplex assay. Allelic frequencies of TNF-α -308A (OR=2.3, p=0.0001, Pc=0.0003) and LTα +252G (OR=2.1, p<0.0001, Pc<0.001) were significantly higher in SLE patients. Frequency of haplotype-AGG was found to be higher in patients than controls (OR=12.2, p=0.0050). Serum levels of TNF-α and LTα also were found to be significantly higher in patients showing variant alleles. TNF-α -308G/A+A/A genotypes (p<0.01) and LTα +252 A/G+G/G genotypes (p<0.02) were significantly associated with renal disorders and haematological manifestations. SLE patients with -308G/A+A/A genotypes showed higher prevalence of anti-dsDNA antibodies (OR=3.9, p=0.0014, Pc=0.0098) and anti-Sm antibodies (OR=4.1, p=0.0002, Pc=0.0014). The present study suggests TNF-α -308A and LTα +252G as risk alleles for disease susceptibility associated with higher serum levels of TNF-α and LTα and concomitant discrete clinical features among Indian SLE patients.

Secretion and Expression of Matrix Metalloproteinase-2 and 9 from Bone Marrow Mononuclear Cells in Myelodysplastic Syndrome and Acute Myeloid Leukemia.

BACKGROUND: Matrix metalloproteinase -2 (gelatinase-A, Mr 72,000 type IV collagenase, MMP-2) and -9 (gelatinase-B, Mr 92,000 type IV collagenase, MMP-9) are key molecules that play roles in tumor growth, invasion, tissue remodeling, metastasis and stem-cell regulation by digesting extracellular matrix barriers. MMP-2 and -9 are well known to impact on solid cancer susceptibility, whereas, in hematological malignancies, a paucity of data is available to resolve the function of these regulatory molecules in bone marrow mononuclear cells (BM-MNCs) and stromal cells of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). OBJECTIVES: The present study aimed to investigate mRNA expression and gelatinase A and B secretion from BM-MNCs in vitro and genotypic associations of MMP-2 (-1306 C/T; rs243865), MMP-9 (-1562 C/T; rs3918242), tissue inhibitor of metalloproteinase -1 (TIMP-1) (372T/C; rs4898, Exon 5) and TIMP-2 (-418G/C; rs8179090) in MDS and AML. RESULTS: The study covered cases of confirmed MDS (n=50), AML (n=32) and healthy controls (n=110). MMP- 9 mRNA expression revealed 2 fold increased expression in MDS-RAEB II and 2.5 fold in AML M-4 (60-70% blasts). Secretion of gelatinase- B also revealed the MMP-9 mRNA expression and ELISA data also supported these data. We noted that those patients having more blast crises presented with more secretion of MMP-9 and its mRNA expression. In contrast MMP-9 (-1562 C/T) showed significant polymorphic associations in MDS (p<0.02) and AML (p<0.02). MMP-9 mRNA expression of C/T and T/T genotypes were 1.5 and 2.5 fold increased in MDS and AML respectively. In AML, MMP-2 C/T and T/T genotypes showed 2.0 fold mRNA expression. Only MMP-9 (-1306 C/T) showed significant 4 fold (p<0.001) increased risk with chemical and x-ray exposed MDS, while tobacco and cigarette smokers have 3 fold (p<0.04) risk in AML. CONCLUSIONS: In view of our results, MMP-9 revealed synergistic secretion and expression in blast crises of MDS and AML with 'gene' polymorphic effects and is significantly associated with increased risk with tobacco, cigarette and environmental exposure. Release and secretion of these enzymes may influence hematopoietic cell behavior and may be important in the clinical point of view. It may offer valuable tools for diagnosis and prognosis, as well as possible targets for the treatments.