Molecular investigation in TET2, SAXL1, RUNX1, RAS Gene, TP53 and Expression studies of associated TLRs of

Myelodysplastic Syndrome (MDS).

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Presented by:

Venkatesh V. Naik M.S. Pharm. (Biotechnology), NIPER, Guwahati

Under Supervision of:Dr. Ranadeep Gogoi, Asst. Professor, Dept. of Biotechnology, NIPER, Guwahati.

Co-Supervision of: Dr. (Mrs.) Jina Bhattacharyya, Prof. & HOD, Dept. of Haematology, GMC.

CONTENTS INTRODUCTION &

LITERATURE REVIEW HYPOTHESIS OBJECTIVES MATERIALS AND METHODS METHODOLOGY STUDY PLAN EXPECTED SIGNIFICANCE REFERENCES

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INTRODUCTION &

LITERATURE REVIEW Myelodysplastic syndrome (MDS) represents a

heterogeneous hematopoietic disorder in which mature blood cells are derived from an abnormal multipotent progenitor cell.

The disease is characterized by morphologic features of dyspoiesis, a hyperproliferative bone marrow, and peripheral-blood cytopenias involving one or more lineages.

The generally accepted mechanism of primary MDS pathogenesis involves an initial deleterious genetic event within a hematopoietic stem cell.

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Cont.... The phenotypic heterogeneity in MDS is likely due to the

variety of genetic lesions that contribute to disease pathogenesis.

Unraveling the genetic complexity of MDS promises to elucidate the pathophysiology, refine the taxonomy and prognostic scoring systems, and provide novel therapeutic targets.

More sensitive technologies such as single nucleotide polymorphism microarrays can detect copy number changes or acquired uniparental disomy in as many as 75% of patients with MDS.

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Moreover, the majority of patients have mutations that alter the sequence and function of oncogenes or tumor suppressor genes.

In addition, patients with MDS commonly have abnormal epigenetic profiles, resulting in aberrant gene expression.

TET2 is the most frequently mutated gene identified in MDS to date. Mutations of TET2 are present in nearly 20% of patients with MDS and are also seen in myeloproliferative neoplasms (MPN) (10%), chronic myelomonocytic leukemia (CMML; 30% to 50%), and secondary AML (25%). In MDS, the impact of TET2 remains unclear.

Mutations in the additional sex-comb like-1/ (ASXL1) gene have been described in roughly 10% of MDS and MPN, 17% of AML, and >40% of patients with CMML. The prognostic significance of ASXL1 mutations in MDS has yet to be determined. RUNX1 is a member of the transcriptional core-binding factor gene family (also known as CBFA2 or AML1) and is the second most commonly mutated gene in MDS. In MDS, they are present in 7% to 15% of de novo patients and at a higher frequency in therapy-related disease.

Patients with MDS are more commonly characterized by activating mutations of the downstream RAS genes. NRAS mutations are present in 10% to 15% of patients with another 1% to 2% having KRAS mutations.

The TP53 gene, located on chromosome 17p, is a prototypical tumor suppressor gene.

In MDS, TP53 is mutated in 5% to 15% of de novo patients and more frequently in patients who have had prior exposure to alkylating agents or radiation.

Toll-like receptors (TLRs), which represent a key molecular link between tissue injury, infection, and inflammation play pivotal role in disease progression & proliferation.

After discussion the abovementioned observations, it is proposed to have study on the Expression profiling of Associated genes and TLRs in MDS population of Assam.

Most patients with high-risk MDS (i.e., French-American-British [FAB] subtypes with excess blasts) die within 1 year from progressive bone marrow failure attributable to hemorrhage or infection.

The presence of detectable cytogenetic abnormalities in approximately 40–70% of patients with primary MDS and over 80% of those with secondary MDS, as well as the validated prognostic value of cytogenetic data have been considered to support the theory of an inciting genetic event.

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To measure the expression and mutation level of TET2, SAXL1, RUNX1, RAS Gene, TP53 genes in MDS patients.

  To determine the expression of Toll like receptors (TLR1-

10) at mRNA level.

 To determine the expression of Toll like receptors (TLR1-10) at protein level.

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OBJECTIVE

MATERIALS & METHODS

MATERIALSMDS SampleReagents AntibodiesChemicalsPrimersTLRs Primers &

ProbesRnase kitInstruments

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METHODSRNA Extraction (NIPER)cDNA Synthesis (NIPER)Reverse Transcriptase-Polymerase Chain Reaction (NIPER)Quantitative Real Time PCR (GU/GMC)ELISA (NIPER)Western Blotting (NIPER)Flow Cytometry (GMC)

METHODOLOGY

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Blood collection

mRNAExtraction

Protein Extraction

qRT-PCRELISA,

Western Blot & FACS

Statistical Analysis

DNA Extraction

PCR & GEL DOC

STUDY PLAN

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Blood collection PCR ELISA WB RT-PCR &

FACS SA

2- Months 1- Month 1- Month 1- Month 1- Month 1- Month

EXPECTED SIGNIFICANCE

This study may explore the probable expression and mutation pattern of target genes in MDS Patients along with it’s progression in Assam population.

The expression of TLRs may help in controlling the growth and proliferation of MDS.

The observation may supports the probable therapy and modulation or approach for the MDS disease patients.

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REFERENCES

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Silverman L: The Myelodysplastic Syndrome, in Holland J, Frei EI, Bast RJ, et al (ed): Cancer Medicine. Hamilton, Canada, BC Decker, , pp 1931-1946, 2000.

Look AT: Molecular pathogenesis of MDS. Hematology Am Soc Hematol Educ Program 156-160, 2005.

Gondek LP, Haddad AS, O’Keefe CL, et al: Detection of cryptic chromosomal lesions including acquired segmental uniparental disomy in advanced and low-risk myelodysplastic syndromes. Exp Hematol 35:1728-1738, 2007.

Bejar R, Ebert BL: The genetic basis of myelodysplastic syndromes. Hematol Oncol Clin North Am 24:295-315, 2010.

Issa JP: Epigenetic changes in the myelodysplastic syndrome. Hematol Oncol Clin North Am 24:317-330, 2010.

Delhommeau F, Dupont S, Della Valle V, et al: Mutation in TET2 in myeloid cancers. N Engl J Med 360:2289-2301, 2009.

Kosmider O, Gelsi-Boyer V, Ciudad M, et al: TET2 gene mutation is a frequent and adverse event in chronic myelomonocytic leukemia. Haematologica 94:1676-1681, 2009.

Gelsi-Boyer V, Trouplin V, Ade´ laïde J, et al: Mutations of polycomb-associated gene ASXL1 in myelodysplastic syndromes and chronic myelomonocytic leukaemia. Br J Haematol 145:788-800, 2009.

Boultwood J, Perry J, Pellagatti A, et al: Frequent mutation of the polycomb-associated gene ASXL1 in the myelodysplastic syndromes and in acute myeloid leukemia. Leukemia 24:1062-1065, 2010.

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Carbuccia N, Murati A, Trouplin V, et al: Mutations of ASXL1 gene in myeloproliferative neoplasms. Leukemia 23:2183-2186, 2009.

Chen CY, Lin LI, Tang JL, et al: RUNX1 gene mutation in primary myelodysplastic syndrome: The mutation can be detected early at diagnosis or acquired during disease progression and is associated with poor outcome. Br J Haematol 139:405-414, 2007.

Steensma DP, Gibbons RJ, Mesa RA, et al: Somatic point mutations in RUNX1/CBFA2/AML1 are common in high-risk myelodysplastic syndrome, but not in myelofibrosis with myeloid metaplasia. Eur J Haematol 74:47-53, 2005.

Tang JL, Hou HA, Chen CY, et al: AML1/RUNX1 mutations in 470 adult patients with de novo acute myeloid leukemia: Prognostic implication and interaction with other gene alterations. Blood 114: 5352-5361, 2009. Bacher U, Haferlach T, Kern W, et al: A comparative study of molecular mutations in 381 patients with myelodysplastic syndrome and in 4130 patients with acute myeloid leukemia. Haematologica 92:744-752, 2007.

Christiansen DH, Andersen MK, Desta F, et al: Mutations of genes in the receptor tyrosine kinase (RTK)/RAS-BRAF signal transduction pathway in therapy-related myelodysplasia and acute myeloid leukemia. Leukemia 19:2232-2240, 2005.

Christiansen DH, Andersen MK, Pedersen- Bjergaard J: Mutations with loss of heterozygosity of p53 are common in therapy-related myelodysplasia and acute myeloid leukemia after exposure to alkylating agents and significantly associated with deletion or loss of 5q, a complex karyotype, and a poor prognosis. J Clin Oncol 19:1405-1413, 2001.

Jahangir Abdi, Tuna Mutis, Johan Garssen, Frank Redegeld, Characterization of the Toll-like Receptor Expression Profile in Human Multiple Myeloma Cells, April 2013 , Volume 8, Issue 4, e606-671.

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