Single Nucleotide Polymorphism

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Single Nucleotide Polymorphism

• ((SNP, snip

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Human Genome

• The human genome has 2.91 billion base pairs
• and approximately 35.000 genes.

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Single Nucleotide Polymorphism

• Its the difference of one base at specific base
  pair position.
• .e.g. from Cytosine ?Thymine
• Adenine ? Guanine
• One difference/1250 base pair/two unrelated
  individual
• Of total 2.3 million differences.
• 10-30million SNPs in the Human genome

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Effect of SNP

• Silent
• Alter the function of the protein
• Directly alter an amino acid sequence
• indirectly alter the function of the
  regulatory sequence

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Environmental Genome Project (EGP)
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Environmental Genome Project (EGP)

• It is a resequencing of the DNA
• for detecting the polymorphic sites that are
  representing at 1 or higher in the sampled
  population.
• the frequency of polymorphisms can vary in
  different population subgroups.

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SNPs of Clinical significance in

• Adverse Drug reaction
• Disease predisposition

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SNPs and Adverse Drug reaction

• SNPs Altering the function of drug metabolizing
  enzymes.
• Pharmacogenetic promise to develop individual
  medicine according to patient genotype

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Role of SNPs in Disease predisposition

• The Common disease are multifactorial
• The Genetic differences between human populations
  make one population more susceptible to
  particular disease.

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SNPs and Disease predisposition

• Epidemiological and biomedical researches are
  looking for comparing SNPs in patients and
  healthy controls in different population
  subgroup.
• SNPs have proven capable of locating genes
  involved in cancers, sickle cell anemia,
  psoriasis, migraine, Alzheimer and Diabetes.

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SNPs and Cancer
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SNPs and Cancer

• SNPs in genes involved in DNA repair and drug
  metabolizing enzymes which responsible for
  metabolism detoxification of Carcinogens can
  act as cancer susceptibility genes
• Through
• increase activation of chemical
  carcinogens
• decrease ability of cells to detoxify
  repair mutagenic damage

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• So
• identification of these genotypic variations
  and their association with cancer
• May help to
• - Elucidate cancer etiology.
• -predict disease risk and response to
  chemotherapy.

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SNP and Cancer

• There are many studies conducted over the past
  few years have identified variants alleles for
  number of drug metabolizing and DNA repair genes.
• e.g. MTHFR (methyl tetrahydofolate
  reductasesome)
• GST (Glutathione S-Transferases).
• NAD(p)H quinone Oxidoreductases.
• CytochromeP450 monooxigenases.
• N-Acetyle Transferases.
• change the enzyme activity with a relation to
  Cancer.

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MTHFR gene

• chromosome 1 (1p36.3).
• it direct folate metabolites towards
  remethylation of homocystien and away from
  DNARNA biosynthesis.

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• MTHFR gene

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MTHFR gene

• SNPs C677T, A1298C
• Protective Effect against DNA mutagenic damage.
• Reduced risk of childhood ALL.

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GST genes(GluthathhioneS-transferases)

• Superfamily of 4 major subfamilies.
• It catalyses the detoxification of exogenous and
  endogenous reactive substances.
• The deletion polymorphism (GST M1 null GST T1
  null) result in absence of enzyme activity.
• Which have increased risk of acute leukemia,
  colorectal adenocarcinoma, thyroid cancer,
  stomach, lung and pancreatic cancer in heavy
  smokers

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NAD(p)H quinone oxidoreductase

• locate in chromosome 16.
• protect the cells from oxidative damage.
• The SNP C609T results in decrease enzyme
  activity.
• ? risk of lung cancer and acute leukemia.

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Cytochrome P450 genes

• It is a superfamily of genes.
• Cytochrome monooxygenases associated in the
  metabolism of drugs, environmental pollutants and
  endogenous substances.
• Cyp1A1 polymorphism ? risk of acute leukemia and
  postmenopausal breast cancer in early smoking
  woman.

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N-Acetyltransferase genes

• NAT1 NAT2 gene on chromosome 8.
• N-acetylation (deactivation) and O-acetylation
  (activation) of aromatic and heterocyclic amine
  carcinogens.

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N-Acetyltransferase genes

• The slow acetylator polymorphism in NAT2
  (T341C) (C481T) SNPs associated with reduced
  enzyme activity.
• with increased risk of
• ALL
• Urinary bladder cancer in smokers

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Methods of identification of SNPs
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Methods of identification SNPs

• A) Detection of known SNPs
• B) Identification of new SNPs

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A) Detection of known SNPs

• a) Gel-Based genotyping methods
• 1-PCR with restriction enzyme coupled analysis.
• 2-Amplification refractory mutation system
  (ARMS).
• 3-Oligonucleotide ligation assay.
• 4-Minisequencing.

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1-PCR with restriction enzyme coupled analysis.

• Most common .
• Especially for known SNP that create or abolish a
  restriction site.
• different restriction digestion pattern can be
  recognized.

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1-PCR with restriction enzyme coupled analysis
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2-Amplification refractory mutation system (ARMS).

• primers matches the normal DNA sequence so
  amplification will occurs in normal individuals.

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3-Oligonucleotide ligation assay.

• hybridization with specific oligonucleotide
  probes

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A) Detection of known SNPs

• b) Non-Gel-based High throughput Genotyping
  Technologies
• 1-hybridization using fluorescence resonance
  energy transfer detection (TaqMan genotyping.
  Molecular beacons).
• 2-High-density chip array.

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B)Identification of new SNPs

• 2 steps
• 1- Conformation-based mutation scanning.
• 2-Direct DNA sequencing.

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Conformation-based mutation scanning

• Single-strand conformation polymorphism (SSCP).
• most widely used methods.
• Principle single strand DNA tend to fold into
  complex structure which determines the mobility
  of the DNA strand in non denaturating gel.

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Single-strand conformation polymorphism (SSCP).
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