Sameer Guru

Epigenetic Silencing of DAPK1 Gene is Associated with Faster Disease Progression in India Populations with Chronic Myeloid Leukemia

Background: One of the major epigenetic changes in human cancer is DNA methylation of tumour suppressor genes which leads to silencing of gene leading to disease progression. Therefore, DNA methylation status of such genes may serve as the epigenetic biomarker for prognosis of human Chronic Myeloid Leukemia. Material and methods: We used MSP-PCR technique for the analysis of aberrant promoter DAPK1 methylation on 200 CML venous blood samples. Stastical analysis was done for evaluating differences between different parameters using SPSS 16.0 version. Results: We could detect 91/200 promoter methylation (45.5%) in CML patients. Percentage of methylation detected was seen higher in blast phase (63.07%) and in accelerated phase (48.1%) than in chronic phase (29.6%). A significant correlation was seen between CML stages and DAPK1 aberrant methylation. We also found a significant association of DAPK1 methylation in gender and in haematological resistance CML patients. However no correlation was found between DAPK1 promoter methylation and other clinical parameters like age, BCR-ABL type and Thrombocytopenia. Conclusion: In summary we concluded that methylation status of DAPK1 gene is associated with advanced phase of CML and may be related to disease progression in chronic myeloid leukemia. Further study on a more number of patients is needed to explore the role of DAPK1 methylation in the prognosis of CML.

Simple multiplex RT-PCR for identifying common fusion BCR-ABL transcript types and evaluation of molecular response of the a2b2 and a2b3 transcripts to Imatinib resistance in north Indian chronic myeloid leukemia patients.

INTRODUCTION Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome, an abnormally shortened chromosome 22. It is the result of a reciprocal translocation of chromosomes 9 and 22, creating BCR-ABL fusion transcripts, b3a2, b2a2, and e1a2. The aim of our study was to determine the type of BCR-ABL fusion transcripts for molecular diagnosis and investigate the frequency of BCR-ABL fusion transcripts in CML patients by multiplex RT-PCR in CML. MATERIALS AND METHODS A single reaction with multiple primers multiplex PCR was used to detect and investigate the type and frequency in 200 CML patients among which 116, 33, and 51 were in CP, AP, and BC phase, respectively. RESULTS The study included 200 CML patients, among whom breakpoints in b3a2, b2a2 transcripts were detected in 68% and 24%, respectively, while 8% of the patients showed both b3a2/b2a2. A statistically significant difference was seen between frequency of BCR-ABL fusion transcripts and gender (P = 0.03), molecular response (P = 0.04), and hematological response (P = 0.05). However, there was no correlation found between frequencies of BCR-/ABL fusion transcripts and other clinicopathological parameters like age, type of therapy, thrombocytopenia, and white blood cell count. CONCLUSION Multiplex reverse transcriptase-polymerase chain reaction is useful and saves time in the detection of BCR-ABL variants; the occurrence of these transcripts associated with CML can assist in prognosis and treatment of disease.

Inactivation of RIZ1 Gene by Promoter Hypermethylation is Associated with Disease Progression and Resistance to Imatinib in Indian Chronic Myelogenous Leukemia Patients, First Study from India

Background: The epigenetic impact of DNA methylation in chronic myelogenous leukemia (CML) is not completely understood. RIZ1 expression and activity are reduced in many cancers. In CML, blastic transformation is associated with loss of heterozygosity in the region where RIZ1 is located. RIZ1 is a PR domain methyltransferase that methylates histone H3 lysine 9, a modification important for transcriptional repression. In CML blast crisis cell lines RIZ1 represses insulin-like growth factor-1 expression and autocrine signaling. Together these observations suggest that RIZ1 may have a role in the chronic phase to blast crisis transition in CML. Methods: To examine whether promoter methylation is involved in the disease development and progression of CML, we investigated promoter methylation status of RIZ1 gene in 100 chronic myeloid leukemia’s (CML) patients and 50 controls by MSP method. Results: The RIZ1 methylation was studied in 100 CML patients, 9 were cases were methylation positive cases, six of nine were in blastic phase, 2 in chronic phase and one patient in accelerated phase. It was seen that RIZ1 methylation was increased significantly from early to advanced phase. The higher frequency of RIZ1 methylation was reported in haematologically resistant cases (42% vs 2%) and molecularly resistant cases (16.77% vs 1.92%) than the responders. The higher frequency of RIZ1 methylation was found in CML patients who were treated with interferon initially followed by imatinib treatment. Also RIZ1 hypermethylation was associated with faster disease progression p<0.003 than the non methylated cases. No correlation was found between RIZ1 gene methylation with age, thrombocytopenia, types of bcr/abl transcripts of CML patients. Conclusion: We conclude that epigenetic silencing of RIZ1 gene is associated with CML progression and imatinib resistance. Early detection of RIZ1 methylation could be a predictive marker for imatinib resistance and disease progression in CML.

A deletion polymorphism in the RIZ gene is associated with increased progression of imatinib treated chronic myeloid leukemia patients

Abstract RIZ1 encodes a retinoblastoma (Rb)-interacting zinc finger protein, is commonly lost or expressed at reduced levels in cancer cells. The RIZ1 gene locus commonly undergoes LOH in many cancers. Here, we analyzed Proline insertion–deletion polymorphism at amino acid position 704 in the RIZ1 gene and its association with CML. The RIZ1 pro-704 LOH genotypes were determined by AS-PCR in 100 CML patients among which 50 were in CP-CML, 25 in AP-CML, and 25 in BC-CML. Pro704 ins/del polymorphism (LOH) was detected in 27% CML patients. Proline ins–ins homozygosity, del–del homozygosity and ins–del heterozygosity was detected in 9%, 18%, and 73% CML patients compared with 3%, 3%, and 94% in healthy controls, respectively (p < .0003). A four-fold increased risk was found to be associated del-del genotype. We found a statistically significant association between RIZ1 LOH and stage (p > .01) and hematological resistance (p > .001). However, there were no correlations found with other clinical parameters like age, gender, thrombocytopia, type of BCR–ABL, and molecular response. Our findings suggest that proline 704 del–del homozygosity phenotype can play an important role in progression of CML.

Biological and Clinical Implications of Exon 8 P53 (R282W) GeneMutation in Relation to Development and Progression of Chronic MyeloidLeukaemia Patients in India Population

Background: TP53, located on chromosome 17p13, is one of the most mutated genes affecting many types of human cancers .To establish an association between the incidence of exon 8 p53 (R282W) gene and progression of the disease in CML and also to correlate the presence of mutation with the clinicopathological features of the disease. Methods: p53 status was investigated by studying mutations in the p53 gene at exon 8 region after confirming the diagnosis by BCR-ABL. 100 CML samples were analyzed using the Allele-Specific Oligonucleotide PCR assay. Mutations occurred in 58% of the cases in exon 8 codon 282 region of the p53 gene. C : T transitions occurred at a high frequency with a statistically significant result (p=0.03). Results: Of the 100 clinically confirmed specimens, 58% tested positive for the mutation. Also, the mutation was found to be higher in the progressed stages (88.2% in accelerated phase and 60.0% in blast crisis) of CML compared to the chronic stage (35.2%). A statistically significant association (p=0.001) was found between the occurrence of p53 R282W mutation and the clinical phase of CML with chronic, accelerated and blast crisis phases. The mutation was detected in a vast majority (88.2%) of patients in the accelerated and the blast crisis phase (60.0%) indicating that this mutation might play a critical role in predicting the progression of disease in CML. Clinicopathological correlation with TLC, platelet count and the haematological response elicited a significant association with patients harboring the mutation with (p=0.01), (p=0.001) and (p=0.01) respectively. Conclusion: Our study suggests that p53 mutations in the exon 8 region might have a strong influence on disease progression and poor response of imatiib (Tyrosine kinase inhibitor) in CML patients.

Role of SCN1A and SCN2A Gene Polymorphisms in Epilepsy Syndromes-A Study from India

Introduction: Epilepsy is the most common heterogeneous neurological disorder affecting approximately 42 million people worldwide. Juvenile myoclonic epilepsy (JME) is a common form of idiopathic generalized epilepsy representing 5-10% of all epilepsy cases. Lennox-Gastaut syndrome (LGS) is one of the most severe epileptic encephalopathies of childhood onset, the cause of which may be symptomatic, i.e., secondary to an underlying brain disorder or cryptogenic, i.e., with no known cause. Sodium channels are integral membrane proteins which play a central role in neuronal membrane excitability and action potential generation. Alpha subunit of voltage gated sodium channels encoded by SCN1A, SCN2A and other genes is pivotal for neuronal signalling. It was planned to analyse the roles of SCN1A Thr1067Ala and SCN2A Arg19Lys polymorphisms in the pathophysiology and risk JME and LGS in the Indian population. Methods: A total of 50 JME patients, 50 LGS Patients and 100 age and sex matched healthy volunteers were recruited in this study. The genotyping of SCN1A Thr1067Ala i.e., 3184 A>G (rs2298771) and SCN2A Arg19Lys i.e., 56 G>A (rs17183814) polymorphism was performed by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) analysis. Results: The SCN1A Thr1067Ala polymorphism genotypic distribution in LGS was significantly different from the normal population (P=0.008), with mutant homozygous (GG) plus heterozygous (AG) genotypes’ percentage in LGS patients (16%) being lower than in healthy controls (24%). Frequency of the mutant ‘G’ allele of this SNP in LGS patients was 0.1, while it was 0.2 in control subjects (P=0.04). These observations which suggest a protective role of SCN1A Thr1067Ala polymorphism in LGS, were in sync with computation of an odds ratio of 0.21 (95% CI 0.07 to 0.66, p=0.005) for the GG genotype in LGS patients. Though no correlation of SCN1A Thr1067Ala SNP with the severity of disease phenotype in LGS viz. frequency/duration of seizures etc. was noted, a conflicting finding was the significant association of its mutant genotypes with an early age of onset of the syndrome (p=0.007). Contrary to the findings in SCN1A Thr1067Ala, in case of SCN2A Arg19Lys polymorphism, though a significantly different genotypic distribution was present in LGS, in comparison to normal population (p=0.03), the mutant homozygous (AA) and heterozygous (GA) combined percentage in LGS patients (16%) was greater than in healthy controls (11%). This was complemented by observation of an odds ratio of 4.24 (95% CI 1.15 to 15.55, p=0.029, in case of LGS patients with heterozygous (GA) genotype, indicative of an increased disease susceptibility. Unlike LGS, in JME patients no significant differences in genotypic/allelic frequencies of SCN1A Thr1067Ala and SCN2A Arg19Lys polymorphisms were noted and the associated odds ratios for mutant genotypes were also non-significant. Conclusion: The SCN1A Thr1067Ala and SCN2A Arg19Lys polymorphisms may play contrary roles in the pathophysiology of LGS. Inheritance of SCN1A Thr1067Ala mutant allele decreases the susceptibility for LGS occurrence, and may hamper Na+ channels opening and neuronal excitability. On the other hand, the mutant allele of SCN2A Arg19Lys polymorphism confers an increased risk for development of LGS, consequent to a likely facilitatory effect on action potential generation and misfiring in neurons. Neither of these two SNPs appears to influence the pathogenesis/susceptibility to JME.

PO-470 hOCT1 gene polymorphism M420del is associated with decreased response to imatinib in CML patients and amp; its effect is counteracted by M408V polymorphism

Introduction Human organic cation transporter1 (hOCT1,SLC22A1),an influx transporter,is responsible for the uptake of Imatinib into chronic myelod leukaemia (CML)cells. Some patients fail to achieve optimal molecular response to Imatinib, defined as major molecular response (MMR) i.e. BCR-ABL 1≤0.1% within 12 months of therapy. Variation in clinical response to Imatinib has been observed with two nonsynonymous SNPs in hOCT1 gene, namely M420del and M408V in some populations. Material and methods 30 newly diagnosed BCR-ABL positive CML patients in chronic phase,and 30 healthy control subjects, all ethnic Indians, were recruited in the study. M420del and M408V SNPs were examined by allele specific PCR(AS-PCR) in DNA from PBMCs.After initiation of imatinib therapy, haematological response was monitored at regular intervals, and molecular response (BCR-ABL1/ABL1 ratio) assessed after 6 or 12 months. Results and discussions Minor allele frequencies for M420del were 0.18 and 0.1 in CML patients and controls; for M408V 0.4 and 0.27 respectively, closely paralleling those reported in western population. No significant association between different genotypes of M420del and M408V was observed with either time to achieve complete haematological response (CHR) (p=0.341 for both SNPs),or presence of optimal/sub-optimal molecular responses(p=0.125, 0.629 for M420del and M408V respectively). To analyse the combined effect of these two SNPs, CML cases were divided into 4 groups.Patients with mutant (homo/heterozygous) M420del and wild type homozygous M408V, failed to achieve an optimal molecular response to imatinib, unlike those with mutant genotypes (homo/heterozygous) for both SNPs (p=0.02). Conclusion Mutant M420del allele may be linked to poor outcome of imatinib treatment in CML,however simultaneous presence of mutant M408V allele appears to circumvent this effect.These SNPs in hOCT1 gene occur at reasonable frequencies in Indian population, to be of clinical interest as predictors of response to imatinib in CML.

PDGFRα promoter polymorphisms and expression patterns influence risk of development of imatinib-induced thrombocytopenia in chronic myeloid leukemia: A study from India

Platelet-derived growth factor receptor has been implicated in many malignant and non-malignant diseases. Platelet-derived growth factor receptor-α is a tyrosine kinase and a side target for imatinib, a revolutionary drug for the treatment of chronic myeloid leukemia that has dramatically improved the survival of chronic myeloid leukemia patients. Given the importance of platelet-derived growth factor receptor in platelet development and its inhibition by imatinib, it was intriguing to analyze the role of platelet-derived growth factor receptor-α in relation to imatinib treatment in the development of imatinib-induced thrombocytopenia in chronic myeloid leukemia patients. We hypothesized that two known functional polymorphisms, +68GA insertion/deletion and −909C/A, in the promoter region of the platelet-derived growth factor receptor-α gene may affect the susceptibility of chronic myeloid leukemia patients receiving imatinib treatment to the development of thrombocytopenia. A case-control study was conducted among a cohort of chronic myeloid leukemia patients admitted to the Lok Nayak Hospital, New Delhi, India. A set of 100 patients of chronic myeloid leukemia in chronic phase and 100 age- and sex-matched healthy controls were studied. After initiation of imatinib treatment, the hematological response of chronic myeloid leukemia patients was monitored regularly for 2 years, in which the development of thrombocytopenia was the primary end point. Platelet-derived growth factor receptor-α promoter polymorphisms +68GA ins/del and −909C/A were studied by allele-specific polymerase chain reaction. Platelet-derived growth factor receptor-α messenger RNA expression was evaluated by quantitative real-time polymerase chain reaction. The messenger RNA expression results were expressed as 2−Δct ± standard deviation. The distribution of +68GA ins/del promoter polymorphism genotypes differed significantly between the thrombocytopenic and non-thrombocytopenic chronic myeloid leukemia patient groups (p < 0.0001). Moreover, +68GA del/del and ins/del genotypes in imatinib-treated chronic myeloid leukemia patients were associated with an increased risk of developing thrombocytopenia, with odds ratios 6.5 (95% confidence interval = 2.02–0.89, p = 0.001) and 6.0 (95% confidence interval = 2.26–15.91, p = 0.0002), respectively. Similarly, −909C/A promoter polymorphism genotype distribution also differed significantly between thrombocytopenic and non-thrombocytopenic chronic myeloid leukemia patient groups (p = 0.02), and a significantly increased risk of imatinib-induced thrombocytopenia was associated with −909C/A polymorphism mutant homozygous (AA) genotypes the odds ratio being 7.7 (95% confidence interval 1.50 to 39.91, p = 0.009). However, no significant risk of imatinib-induced thrombocytopenia was found to be associated with heterozygous genotype (−909C/A) with odds ratio 1.9 (95% confidence interval = 0.86–4.56, p = 1.14). Platelet-derived growth factor receptor-α messenger RNA expression was significantly higher in chronic myeloid leukemia patients compared to controls (p = 0.008). Moreover, patients with imatinib-induced thrombocytopenia had a significantly lower platelet-derived growth factor receptor-α messenger RNA expression, compared to patients without thrombocytopenia (p = 0.01). A differential expression of platelet-derived growth factor receptor-α messenger RNA was observed with respect to different +68 GA ins/del and −909C/A polymorphism genotypes. The +68GA deletion allele and −909A allele were significantly associated with lower expression of platelet-derived growth factor receptor-α messenger RNA. The platelet-derived growth factor receptor-α +68GA del/del, +68GA ins/del, and −909AA genotypes are associated with an increased risk of developing thrombocytopenia in imatinib-treated chronic myeloid leukemia patients. A significantly lower platelet-derived growth factor receptor-α messenger RNA expression accompanies the +68GA deletion allele in an allele dose-dependent manner. Platelet-derived growth factor receptor-α −909AA genotype is also associated with lower expression of platelet-derived growth factor receptor-α. The downregulation of platelet-derived growth factor receptor-α expression may play a causative role in imatinib-induced thrombocytopenia, a common side effect, in the subset of chronic myeloid leukemia patients with platelet-derived growth factor receptor-α +68 GA ins/del, +68 GA del/del, and −909C/A genotypes.