Sandhya Annamaneni

Association of vitamin D receptor gene start codon (Fok1) polymorphism with high myopia.

Background: High myopia caused primarily due to abnormal emmetropization and excessive axial ocular elongation is associated with sight-threatening ocular pathology. Muscular dysfunction of ocular ciliary muscles due to altered intracellular calcium levels can result in defective mechanotransduction of the eye and retinal defocus. The vitamin D3 receptor (VDR; a intracellular hormone receptor) is known to mediate calcium homeostasis, influencing the development of myopia. Materials and Methods: In the present study, a total of 206 high myopia, 98 low myopia and 250 control samples were analyzed for VDR gene Fok1 (exon 2 start codon) polymorphism using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Results: High myopia patients revealed decrease in the frequency of ff homozygotes (8.3%) as compared to control group (14.0%), with a corresponding increase in frequency of FF homozygotes (68.9% in high myopia vs. 62.8% in controls). The frequency of f allele carriers (Ff and ff) was increased in females of high myopia (35.6%) and low myopia cases (45.4%). Elevated frequency of f allele was found only in early age at onset cases of high myopia (0.227) and later age at onset (10–20 years) cases of low myopia (0.273) as well as in low myopia cases with parental consanguinity (0.458) (P 0.035; χ2 = 6.692*). Conclusion: The results suggest that VDR gene might not be playing a direct role in the development of myopia, but might contribute indirectly to the risk conferred by mechanical stress factors or growth/development related factors through its role in calcium homeostasis and regulation of ciliary muscle function.

Methylation status of CEBPA gene promoter in chronic myeloid leukemia

Abstract CCAAT/enhancer binding protein alpha is one of the crucial transcription factors for myeloid cell development that has been found to be involved in hematopoietic differentiation and leukemiogenesis. Recently, epigenetic regulation of CEBPA expression through DNA methylation has been demonstrated in leukemia. The aim of this study was to investigate the methylation status of CEBPA gene in chronic myeloid leukemia (CML) patients. The methylation status of CEBPA promoter was studied in 100 patients with CML and 98 normal healthy individuals from Hyderabad, India, using methylation-specific polymerase chain reaction. The aberrant methylation of CEBPA gene promoter was found in 32 of the 100 CML cases. A highly significant association was found between the frequency of CEBPA gene promoter hypermethylation and the CML stages (P = 0.017), but association with respect to age and gender of the patient was not found. The results suggest that aberrant methylation in the CpG island of the promoter region of this gene might be a common event in CML, and systemic expression studies will be needed to unfold the role of CEBPA promoter methylation in the development, progression, and prognosis of CML.

HIF-1α (1772C>T) polymorphism as marker for breast cancer development

Hypoxia-inducible factor 1α (HIF-1α) is an important transcription factor that regulates different cellular responses to hypoxia. HIF-1α is rapidly degraded by von Hippel–Lindau (VHL) protein under normoxic conditions and stabilized under hypoxia. A common variant of HIF-1α (1772C>T) (rs 11549465) polymorphism, corresponding to an amino acid change from proline to serine at 582 position within the oxygen-dependent degradation domain, results in increased stability of the protein and altered transactivation of its target genes. The present study was aimed to find the association between HIF-1α (1772C>T) (rs 11549465) polymorphism and breast cancer development. For this purpose, 348 primary breast cancer patients and 320 healthy and age-matched controls were genotyped through PCR-RFLP method. The genotype frequencies were compared between patients and controls, and their influence on clinical characteristics of breast cancer patients was analyzed. Our study revealed a significant increase of TT genotype in breast cancer patients compared to controls (p = 0.038). Further, TT genotype and T allele were found to be associated with progesterone receptor (PR)-negative status (p < 0.09). None of the clinical variables revealed significant association with HIF-1α (1772C>T) (rs 11549465) polymorphism.

Influence of BCL2-938 C>A promoter polymorphism and BCL2 gene expression on the progression of breast cancer

BCL2 (B-cell leukemia/lymphoma 2) gene functions as antiapoptotic regulatory element and known to be associated with tumorigenesis. The SNP-938 (C>A) (rs2279115), located in the inhibitory P2 promoter of the BCL2 gene, influences differential binding affinities of transcriptional factors thereby affecting BCL2 expression. The present study is an attempt to evaluate the association between BCL2(-938C>A) polymorphism and clinical characteristics of breast cancer patients as well as to analyze BCL2 expression and Ki67 proliferation index with respect to the genotypes. One hundred ten primary breast cancer tumor tissues were genotyped for -938 C>A polymorphism through PCR-RFLP method as well as evaluated for BCL2 expression and ki67 proliferation index by immunohistochemistry. Evaluation of apoptosis level was performed by flowcytometry. The results revealed that AA genotype was associated with an increased risk (AA Vs AC + CC) by 2.86-fold (p = 0.07) for breast cancer development which reflected in elevated A allele frequency also. AA genotype was found to be predominant among BCL2 positive tumors as compared to BCL2 negative tumors. Further, AA genotype was found to be associated with advanced stage tumors, node positive status, and high Ki67 proliferation index compared to CA and CC genotypes indicating that elevated expression of BCL2 gene in the presence of A allele might be associated with decreased apoptosis and enhanced proliferation rate. AA genotype of BCL2-938C>A polymorphism might influence BCL2 gene expression there by associated with elevated risk for breast cancer progression. Probably, failure of apoptosis due to enhanced expression and antiapoptotic protein BCL2 might promote malignant growth.

Significance of ATM Gene Polymorphisms in Chronic Myeloid Leukemia - a Case Control Study from India

BACKGROUND Development of chronic myeloid leukemia (CML) involves formation of double strand breaks (DSBs) which are initially sensed by the ataxia telangiectasia mutated (ATM) signal kinase to induce a DNA damage response (DDR). Mutations or single nucleotide polymorphisms in ATM gene are known to influence the signaling capacity resulting in susceptibility to certain genetic diseases such as cancers. MATERIALS AND METHODS In the present study, we have analyzed -5144A>T (rs228589) and C4138T (rs3092856) polymorphisms of theATM gene through polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) in 925 subjects (476 CML cases and 449 controls). RESULTS The A allele of -5144A>T polymorphism and T allele of C4138T polymorphism which were known to be influencing ATM signaling capacity are significantly associated with enhanced risk for CML independently and also in combination (evident from the haplotype and diplotype analyses). Significant elevation in the frequencies of both the risk alleles among high risk groups under European Treatment and Outcome Study (EUTOS) score suggests the possible role of these polymorphisms in predicting the prognosis of CML patients. CONCLUSIONS This study provides the first evidence of association of functional ATM gene polymorphisms with the increased risk of CML development as well as progression.

LCN2 Promoter Methylation Status as Novel Predictive Marker for Microvessel Density and Aggressive Tumor Phenotype in Breast Cancer Patients.

LCN2 (Lipocalin 2) is a 25 KD secreted acute phase protein, reported to be a novel regulator of angiogenesis in breast cancer. Up regulation of LCN2 had been observed in multiple cancers including breast cancer, pancreatic cancer and ovarian cancer. However, the role of LCN2 promoter methylation in the formation of microvessels is poorly understood. The aim of this study was to analyze the association of LCN 2 promoter methylation with microvessel formation and tumor cell proliferation in breast cancer patients. The LCN2 promoter methylation status was studied in 64 breast cancer tumors by methylation specific PCR (MSP). Evaluation of microvessel density (MVD) and Ki67 cell proliferation index was achieved by immunohistochemical staining using CD34 and MIB-1 antibodies, respectively. LCN2 promoter unmethylation status was observed in 43 (67.2%) of breast cancer patients whereas LCN2 methylation status was seen in 21 (32.8%). Further, LCN2 promoter unmethylation status was associated with aggressive tumor phenotype and elevated mean MVD in breast cancer patients.

Role of the MDM2 Promoter Polymorphism (-309T>G) in Acute Myeloid Leukemia Development

BACKGROUND The human homologue of the mouse double minute 2 (MDM2) gene is a negative regulator of Tp53. MDM2-309T>G a functional promoter polymorphism was found to be associated with overexpression thereby attenuation of Tp53 stress response and increased cancer susceptibility. We have planned to evaluate the possible role of MDM2-309T>G polymorphism with risk and response to chemotherapy in AML. MATERIALS AND METHODS A total of 223 de novo AML cases and 304 age and sex matched healthy controls were genotyped for the MDM2-309T>G polymorphism through the tetra-primer amplification refractory mutation system (ARMS)-PCR method. In order to assess the functional relationship of -309T>G SNP with MDM2 expression level, we quantified MDM2 mRNA in 30 primary AML blood samples through quantitative RT-PCR. Both the (-309T>G) genotypes and the MDM2 expression were correlated with disease free survival (DFS) rates among patients who have achieved complete remission (CR) after first induction chemotherapy. RESULTS MDM2-309T>G polymorphism was significantly associated with AML development (p<0.0001). The presence of either GG genotype or G allele at MDM2-309 confered 1.79 (95% CI: 1.12-2.86; p<0.001) and 1.46 fold (95%CI: 1.14-1.86; p=0.003) increased AML risk. Survival analysis revealed that CR+ve cases with GG genotype had significantly increased DFS rates (16months, p=0.05) compared to CR+ve TT (11 months) and TG (9 months) genotype groups. Further, MDM2 expression was also found to be significantly elevated in GG genotype patients (p=0.0039) and among CR+ve cases (p=0.0036). CONCLUSIONS The MDM2-309T>G polymorphism might be involved in AML development and also serve as a good prognostic indicator.

Abstract 3858: Genetic polymorphisms of DNA repair genes in the origin and progression of chronic myeloid leukemia

Background: The origin of Chronic Myeloid Leukaemia (CML) involves the formation of Double Strand Breaks (DSBs) which are initially sensed by the Ataxia Telangiectasia Mutated (ATM) signal kinase. Additionally, Bcr-Abl transformed CML cells produce high levels of Reactive Oxygen Species (ROS) that induce more DSBs. In such kind of cells, proper functioning of ATM and NHEJ repair mechanisms are required for ensuring the accurate repair of DSBs. Aim: The present study is planned to understand the role of DNA repair, specifically NHEJ repair in the occurrence of CML and its progression. Methodology: We have analysed seven genetic polymorphisms in ATM, XRCC5, XRCC6 and XRCC7 genes in 476 Ph+ve CML cases and 449 age and sex matched controls without family history of cancers. Direct Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) analyses were carried out using the DNA isolated from blood samples through non-enzymatic/salting out method. Statistical analyses were performed through SPSS (IBM SPSS Statistics 20) software. Results: The T allele of ATM C4138T polymorphism and A allele of ATM -5144A>T polymorphism were found to be significantly associated with enhanced risk of CML in our study. The CC genotype of XRCC6 -61C>G polymorphism was found to have independent effect with CML risk as confirmed by the LD and MDR analyses. Lower repeat (0R) of XRCC5 VNTR and TT genotype of XRCC7 6721G>T polymorphisms had shown borderline association with CML risk. Even though the XRCC5 2408G>A and XRCC6 -1310C>G polymorphisms did not show significant association with CML independently, the AA and GG genotypes of these polymorphisms have been found to confer enhanced risk for CML in combination with other SNPs of the respective genes in haplotype analysis. Interestingly, combined analysis demonstrated cumulative effect of risk genotypes in CML development as well as progression where the fold change increased with the number of adverse genotypes. Conclusion: The risk associated genotypes were known to influence the expression and functioning of genes in NHEJ pathway. Therefore, results of the present study suggested that defective DNA damage sensing and deregulated repair due to variations in ATM and NHEJ genes might influence CML development as well as progression. Key words: CML, ATM, NHEJ, DNA repair, polymorphisms Citation Format: Manjula Gorre, Prajitha Edathara Mohandas, Sailaja Kagita, Anuradha Cingeetham, Sugunakar Vuree, Sandhya Annamaneni, Raghunadharao Digumarti, Vishnupriya Satti. Genetic polymorphisms of DNA repair genes in the origin and progression of chronic myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3858. doi:10.1158/1538-7445.AM2015-3858