Sunil G. Babu

Methylenetetrahydrofolate reductase C677T genetic polymorphisms and risk of leukaemia among the North Indian population.

BACKGROUND Leukaemia is a heterogeneous disease in which haematopoietic progenitor cells acquire genetic lesions that lead to a block in differentiation, increased self-renewal, and unregulated proliferation. The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR), involved in folate metabolism, plays a crucial role in cells because folate availability is important for DNA integrity. The aim of this case-control study was to evaluate the association of the C677T MTHFR gene polymorphism with acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML) and chronic lymphocytic leukaemia (CLL). MATERIALS AND METHODS A total of 275 leukaemia cases - including AML (n = 112), ALL (n = 81), CML (n = 43), CLL (n = 39) - and 251 age/sex-matched healthy control individuals participated in this study. MTHFR C677T polymorphisms in the cases and controls were evaluated by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). RESULTS The average MTHFR 677CC, 677CT, 677TT genotype frequencies of total leukaemia cases were 68.73%, 19.64%, and 11.64% in cases, and 71.71%, 24.30%, and 3.98% in healthy controls, respectively. The average frequency of the MTHFR 677T allele was 21.45% among the cases compared to 16.13% among the controls. CONCLUSIONS In the present case-control study we have observed a higher frequency of the MTHFR 677TT genotype in cases of leukaemia (AML, ALL, CML and CLL) as compared with controls; this might be due to ethnic and geographic variation. As per our findings, although the frequency of the MTHFR 677T allele is moderately high in AML, ALL and CLL, no statistically significant association was found; on the other hand statistically significant association was found in the context of CML cases.

Strategy for a generic resistance to geminiviruses infecting tomato and papaya through in silico siRNA search.

Use of siRNA is a powerful methodology to particularly knockdown the targeted genes in a sequence specific manner. The potential of siRNA can be harnessed for silencing specific geminiviral genes in papaya and tomato plant hosts, thus making them resistant to the respective viruses. The challenge is in designing exogenous siRNA which can trigger silencing of viral genes irrespective of the genetic variability in different viral isolates and at the same time the selected siRNA does not target any plant gene (off target silencing). In this study, we have designed siRNA from the most conserved regions of viral coat protein (AV1) and replicase (AC1) genes retrieved from different isolates of geminiviruses infecting papaya (PLCV), and tomato (TLCV & TLCV, Northern India), so as to give a broad spectrum resistance and efficient silencing as it is highly homology-dependent strategy. Software siRNA finder (Ambion) was used on the selected conserved sequences in order to select only those putative siRNA oligonucleotides which fulfill all the basic criteria required as per the algorithm. Finally, a cross search using BLAST was performed to confirm that the designed siRNAs do not have any homology to plant genome sequences. The putative siRNA sequences thus designed to target essential genes of geminiviruses and introduced into the plants may facilitate developing papaya and tomato crops with generic resistance to geminiviruses.

Role of HCMV miR-UL70-3p and miR-UL148D in overcoming the cellular apoptosis

The studies into the pathophysiology of viral miRNAs are still in infancy; the interspecies regulation at the miRNA level fuels the spark of the investigation into the repertoire of virus–host interactions. Reports pertaining to the viral miRNAs role in modulating/evading the host immune response are surging up; we initiated this in silico study to speculate the role of human cytomegalovirus (HCMV)-encoded miRNAs on human antiviral mechanisms such as apoptosis and autophagy. The results indicate that both the above mechanisms were targeted by the HCMV miRNAs, located in the unique long region of the HCMV genome. The proapoptotic genes MOAP1, PHAP, and ERN1 are identified to be the potential targets for the miR-UL70-3p and UL148D, respectively. The ERN1 gene plays a role in the initiation of Endoplasmic reticulum stress-induced apoptosis as well as autophagosome formation. This study shows that HCMV employs its miRNA repertoire for countering the cellular apoptosis and autophagy, particularly the mitochondrial-dependent intrinsic pathway of apoptosis. In addition, the homology studies reveal no HCMV miRNA bears sequence homology with human miRNAs.

The efficacy of diagnostic battery in Pott's disease: A prospective study

Background: The diagnosis of Potts disease is mostly based on clinicoradiological observations substantiated by the bacterial culture, staining and histopathology. Since, no single technique is enough to conclude Potts disease in diagnosis, the present study was undertaken to correlate the clinicoradiological, microbiological, histopathological and molecular method to evaluate the effectiveness in diagnosis of Potts disease. Materials and Methods: 62 clinicoradiologically suspected cases of Potts disease were included in this study. The specimens for diagnostic work up were collected either during surgery or by computed tomography guided fine needle aspiration. All these specimens were tested for tuberculosis (TB) through Ziehl-Neelsen (ZN) microscopy, BACTEC culture, histopathology and polymerase chain reaction (PCR). The final diagnosis was established by the results of performed tests and clinicoradiological improvement of cases at the end of 6 months on anti tubercular treatment. Results: Out of 62 cases, 7 were excluded from this study as these were turned out to be neoplastic lesions on histopathology. Amongst remaining 55 cases, the TB was diagnosed in 39 (71%) on histopathology, 37 (67.5%) on PCR, 27 (49%) on BACTEC culture and 20 (36.3%) on ZN microscopy. Ultimately 45 cases were tested as positive and 10 were detected as negative for TB in combination of ZN microscopy, BACTEC culture and histopathology. PCR was positive in 37 of 45 cases and 10/55 cases remained negative. On clinical analysis of these 10 cases, it was noted that these were cases of relapse/poor compliance. The combination of PCR and histopathology was also shown positive for TB in 45 cases. Hence, the PCR showed a fair positive agreement (Κc = 0.63) against the combined results of all performed traditional methods. Conclusions: The combination of PCR and histopathology is a rapid and efficient tool for diagnosis of Potts disease.

The Frequency of Mutations in Exon 11 of the c-kit Gene in Patients With Leukemia.

Objective: To determine the frequency of mutations in exon 11 of the c-kit gene in patients with leukemia. Material and Methods: The study included 50 leukemia patients (31 with acute myeloid leukemia, 5 with acutelymphoblastic leukemia, 9 with chronic myeloid leukemia, and 5 with chronic lymphocytic leukemia) that underwentPCR-SSCP, followed by direct DNA sequencing. Results: In all, 28 of the leukemia patients were male and 22 were female, with a mean age of 31.88 years (range: 2-65years). In total, 20 mutations in 19 patients were identified, including Lys550Asn, Tyr568Ser, Ile571Thr, Thr574Pro,Gln575His, Tyr578Pro, Asp579His, His580Gln, Arg586Thr, Asn587Asp, and Arg588Met, as well as novel point mutationsat codons Ile563Lys, Val569Leu, Tyr570Ser, and Pro577Ser. Ile571Leu substitution was observed in 2 patients andTrp582Ser substitution was observed in 3 patients. Conclusion: The results suggest that mutations in exon 11 of the c-kit gene might be useful as molecular geneticmarkers for leukemia

Identification of the c-kit gene mutations in biopsy tissues of mammary gland carcinoma tumor.

C-kit gene is a transmembrane tyrosine kinase that acts as type III receptor for mast cell growth factor and cellular migration, proliferation, survival of melanoblasts, haematopoietic progenitors and primordial germ cells. Apart from the scant information about the pathologies associated with loss-of-function mutations, few reports have proposed role of the c-kit gene in case of carcinogenesis. Apparently, in breast cancer the involvement of c-kit gene mutations has been considered as a rare phenomenon. Thus, we designed our study with aim to investigate the c-kit gene mutation in breast cancer, and their correlation with clinico-pathological findings. We performed mutational analysis of the c-kit gene in 58 cases of malignant breast cancer. With the aim to ascertain the variety of mutations at exon 8, 9, 11, 13, 15 and 17 of c-kit gene in breast cancer, we have done PCR-SSCP followed by DNA sequencing. In breast cancer the c-kit gene mutation rates were 3.44% (02/58) in exon 8, 5.17% (3/58) in exon 9, 5.17% (3/58) in exon 11, 3.44% (2/580 in exon 13, 3.44% (2/58) in exon 15 and 5.17% (3/58) in exon 17, respectively. The overall c-kit mutation frequency in exons 8, 9, 11, 13, 15 and 17 was determined to be 25.86% (15/58). Our study indicates to specify the role of c-kit proto-oncogene mutation in breast cancer. The result signifies that c-kit gene plays a poor role in prognosis of ductal and lobular carcinoma.

Abstract A44: Role of HLA‐G 14bp insertion deletion in cancer susceptibility and progression

Human leukocyte antigen ‐G (HLA‐G) is an immunosuppressive protein with multiple functions. The 14bp insertion / deletion in exon 8 of HLA‐G gene have been shown to play an important role in HLA‐G mRNA stability as well as expression level; and can be correlated with its immunosuppressive function. The role of HLA‐G in various cancers has already been demonstrated. Cancerous cells start expressing HLA‐G which in turn provides a shield for various immune responses. In the present study we have tested the possibility of the role of 14bp insertion / deletion polymorphism of HLA‐G in cancer progression and susceptibility. We have genotyped 59 cancer samples (26 benign and 33 malignant) and 79 matched controls for 14bp polymorphism of HLA‐G. Data showed increase of +14bp/−14bp and −14bp/−14bp genotype with both benign and malignant cancer. We observed significantly higher frequency of +14bp/+14bp genotype in control (p=0.01). This is consistent with both benign and malignant tumors as well as cancer types showing possibility of protective effect of +14bp/+14bp genotype in various cancers. However, further studies will be required with large sample size to test this hypothesis. The +14bp/−14bp and −14bp/−14bp genotypes have already been showed to be associated with higher expression of HLA‐G and its mRNA stability and might be providing favorable micro‐environment for cancer progression. There is no published work till date showing the role of HLA‐G 14bp polymorphism in various cancers. These preliminary data showed that HLA‐G 14‐bp genotypes might be playing an important role in pathogenesis of various cancers. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A44.