SNP Haplotypes as Diagnostic Markers

Shrish TiwariCCMB, Hyderabad

Introduction 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 Unambiguous assay techniques Slow mutation rate Amenable for high-throughput

genotyping

...C C ...C C AA T T G A C... T T G A C...

...C C ...C C GG T T G A C... T T G A C...

……G G G G TT A A C T G... A A C T G...

……G G G G CC A A C T G... A A C T G...

Single base-pair differences occurring in a population with a frequency of >1%

Single Nucleotide Polymorphism

Types of SNPs

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 dbSNP: (~3 million SNPs)

http://www.ncbi.nih.gov/SNP/

JSNP: (~195,000 SNPs) http://snp.ims.u-tokyo.ac.jp/

The SNP Consortium (TSC): http://snp.cshl.org/

Human Genome Variation Database http://hgvbase.cgb.ki.se/

SNP analysis

SNP discovery: by sequencing, by EST sequence comparison

SNP validation: SNAPShot, DHPLC, Sequenom

SNP allele frequency

SNP association

SNP discovery: Experimental approach 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 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 Use genomic sequence as reference

cluster and align all available EST sequences

remove repeats/paralogs

Use Bayesian statistics to 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

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

Sets of SNPs in linkage disequilibrium

SNP genotype data (4 loci): 00 01 01 11

Possible SNP haplotypes: 0001/0111 0011/0101

SNP-Haplotype Association

DNA Sequence

GATATTCGTACGGA-TGATGTTCGTACTGAATGATATTCGTACGGA-TGATATTCGTACGGAATGATGTTCGTACTGAATGATGTTCGTACTGAAT

SN

P

SN

P

1 2

3

4

5 6

AG- 2/6(BLACK EYE)GTA 3/6(BROWN EYE)

AGA 1/6 (BLUE EYE)

Haplotypes

PhenotypeBLACK EYE

BROWN EYE

BLACK EYE

BLUE EYE

BROWN EYE

BROWN EYE

Haplotype database

HapMap: http://www.hapmap.org/

The international HapMap Consortium, The International HapMap Project, Nature 426, 789-796 (2003)

Why SNP Haplotypes?

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 Association of allele and phenotype

Association can occur if 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 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

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