molecular investigation tet2, saxl1 runx1,runx1, ras genen

1. Molecular investigation in TET2, SAXL1, RUNX1, RAS Gene,
TP53 and Expression studies of associated TLRs of
Myelodysplastic Syndrome (MDS).
1
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.

2. CONTENTS
 INTRODUCTION &
LITERATURE REVIEW
 HYPOTHESIS
 OBJECTIVES
 MATERIALS AND METHODS
 METHODOLOGY
 STUDY PLAN
 EXPECTED SIGNIFICANCE
 REFERENCES
2

3. 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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4. 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.
4

5.  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.

6.  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.

7.  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.

8.  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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9.  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.
9
OBJECTIV
E

10. MATERIALS & METHODS
MATERIALS
MDS Sample
Reagents
Antibodies
Chemicals
Primers
TLRs Primers &
Probes
Rnase kit
Instruments
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METHODS
RNA 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)

11. METHODOLOGY
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Blood
collection
mRNA
Extraction
Protein
Extraction
qRT-PCR
ELISA,
Western Blot &
FACS
Statistical
Analysis
DNA
Extraction
PCR &
GEL DOC

12. STUDY PLAN
12
Blood
collection PCR ELISA WB RT-PCR &
FACS
SA
2- Months 1- Month 1- Month 1- Month 1- Month 1- Month

13. 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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14. 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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