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SNP Haplotypes as
Diagnostic Markers
Shrish Tiwari
CCMB, Hyderabad
Introduction
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Genomic variation mostly (90%) as
single nucleotide differences (2/3 of
which are transitions)
Non-uniformly distributed (on an
average 1 difference for every 1000
bp)
Useful as genetic markers
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Unambiguous assay techniques
Slow mutation rate
Amenable for high-throughput genotyping
Single Nucleotide
Polymorphism
Single base-pair differences occurring in a
population with a frequency of >1%
...C C A T T G A C...
…G G T A A C T G...
...C C G T T G A C...
…G G C A A C T G...
Types of SNPs
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SNPs can occur in protein coding or
non-coding DNA
The coding region SNPs can either
be in exons (cSNPs), introns or
regulatory regions
cSNPs may either be silent or
cause a change in amino acid
SNP databases
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dbSNP: (~3 million SNPs)
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JSNP: (~195,000 SNPs)
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http://snp.ims.u-tokyo.ac.jp/
The SNP Consortium (TSC):
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http://www.ncbi.nih.gov/SNP/
http://snp.cshl.org/
Human Genome Variation Database
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http://hgvbase.cgb.ki.se/
SNP analysis
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SNP discovery: by sequencing, by
EST sequence comparison
SNP validation: SNAPShot, DHPLC,
Sequenom
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SNP allele frequency
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SNP association
SNP discovery:
Experimental approach
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Obtain DNA sequence around SNP
PCR amplify the segment of
interest
Identify SNP by sequencing the
segment from different individuals
Map the marker onto chromosome
Compute the allele frequency in
population
SNP discovery: in silico
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Align EST’s to themselves or to
cDNAs
Find variations in ESTs that align
with 95% identity over >100bp
Distinguish true SNPs from
sequencing errors by checking the
base quality using Phred/Phrap set
of programs
The PolyBayes Program
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Use genomic sequence as reference
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Use Bayesian statistics to
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cluster and align all available EST
sequences
remove repeats/paralogs
distinguish polymorphic sites from
artifacts
estimate likelihood
Marth, GT, Korf, I, Yandell, MD, Yeh, RT, Gu, Z, Zakeri, H, Stitziel, NO, Hillier, L, Kwok, P-Y,
Gish, WR: A general approach to single-nucleotide polymorphism discovery. Nature
Genet. 1999; 23:452-456.
SOME OTHER SNP PREDICTION & SNP
FINDING SOFTWARE
 SEAN:
Search for localized SNPs
and predict SNPs
(http://zebrafish.doc.ic.ac.uk/Sean/)
 SNPpipeline: Mine SNPs stored in gene
databases
 SNP Finder: For analyzing user-submitted
trace data
(http://gai.nci.nih.gov/)
SNP Applications
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Disease diagnosis
Identification of factors for
increased susceptibility to disease
May be responsible for the different
responses of individuals to drugs
Will help in developing personalised
medicines
SNP Haplotypes
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Sets of SNPs in linkage
disequilibrium
SNP genotype data (4 loci):
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00 01 01 11
Possible SNP haplotypes:
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0001/0111
0011/0101
BLACK EYE
BROWN EYE
BLACK EYE
BLUE EYE
BROWN EYE
BROWN EYE
DNA Sequence
1 2 3 4 5 6
Phenotype
SNP
SNP
SNP-Haplotype Association
GATATTCGTACGGA-T
GATGTTCGTACTGAAT
GATATTCGTACGGA-T
GATATTCGTACGGAAT
GATGTTCGTACTGAAT
GATGTTCGTACTGAAT
Haplotypes
AG- 2/6(BLACK EYE)
GTA 3/6(BROWN EYE)
AGA 1/6 (BLUE EYE)
Haplotype database
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HapMap:
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http://www.hapmap.org/
The international HapMap Consortium, The
International HapMap Project, Nature 426,
789-796 (2003)
Why SNP Haplotypes?
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More reliable than single SNPs as
markers
Usually less in number than the
number of SNPs in the set
Haplotype tagging SNPs further
reduce genotyping costs
Association study
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Association of allele and phenotype
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Association can occur if
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Allele is in linkage disequilibrium with
the mutation responsible for the
phenotype
Allele is responsible for the phenotype
For association study a control and
a case population is necessary
SNP haplotypes as
diagnostic markers
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Identify SNPs for gene associated
with disease
Find the different sets of SNPs in a
control population
Find different sets of SNPs in
affected population
Look for unique sets of SNPs in
affected population
References
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A.J. Brookes, The essence of SNPs,
Gene 234, 177-186 (1999).
Pui-Yan Kwok and Zhijie Gu, Single
Nucleotide Polymorphism libraries:
why and how are we building them,
Mol. Med. Today 5, 538-543 (1999).
SNPs Science Primer:
http://www.ncbi.nlm.nih.gov/About/primer/snps.html