‘The Human Genome

Project’ has always been

something of a misnomer,

implying the existence of a

single human genome

Of course, every person on the planet with

the exception of identical twins has a unique

genome, and even though any twotwo genomesgenomes

are are roughlyroughly 99.9% 99.9% identicalidentical, that still leaves

millions of differences among the 3.2 billion

base pairs.

It is precisely these differences thataccount for heritable variation amongindividuals, including susceptibility todisease

HUMAN GENETIC VARIATIONSHUMAN GENETIC VARIATIONS

Insertion or deletion of one or more nucleotide(s)

-Tandem Repeat Polymorphisms

-Insertion/Deletion Polymorphisms

Primarily two types of genetic mutation events create all forms of variations:

Single base mutation which substitutes one nucleotide for another-Single Nucleotide Polymorphisms (SNP)

SINGLE NUCLEOTIDE POLYMORPHISMSSINGLE NUCLEOTIDE POLYMORPHISMS

Single nucleotide polymorphisms (SNP) are DNA sequence variations that occur when a single nucleotide (A,T,C,or G) in the genome sequence is altered. For example a SNP might change the DNA sequence AAGGCTAA to ATGGCTAA.

SNPS are the most common class of polymorphisms.

TANDEM REPEAT POLYMORPHISMSTANDEM REPEAT POLYMORPHISMS

Tandem repeats or variable number of tandem repeats (VNTR) are avery common class of polymorphism, consisting of variable length of sequence motifs that are repeated in tandem in a variable copy number.VNTRs are subdivided into two subgroups based on the size of the tandem repeat units.

Microsatellites or Short Tandem Repeat (STR)repeat unit: 1-6 (dinucleotide repeat: CACACACACACA)

Minisatellitesrepeat unit: 14-100

example:Spinocerebellar ataxia Type10 (SCA10) (OMIM:+603516) is caused by largest tandem repeat seen in human genome. Normal population has 10-22 mer pentanucleotide ATTCT repeat in intron 9 of SCA10 gene; where as SCA10 patients have 800-4500 repeat units, which causes the disease allele up to 22.5 kb larger than the normal one.

INSERTION/DELETION POLYMORPHISMSINSERTION/DELETION POLYMORPHISMS

Insertion/Deletion (INDEL) polymorphisms are quite common and widely distributed throughout the human genome.Sequence repetitiveness in the form of direct or inverted tandem repeat have been shown to predispose DNA to localized rearrangements between homologous repeats. Such rearrangements are thought to be one of the reason which create INDEL polymorphism.

example: Association between coronary heart disease and a 287 bp Indel Polymorphism located in intron 16 of the angiotensin converting enzyme (ACE) have been reported (OMIM 106180). This Indel, known as ACE/ID is responsible for 50% of the inter individual variability of plasma ACE concentration.

ESTIMATED NUMBERSESTIMATED NUMBERS

· SNPs appear at 0.3-1-kb average intervals, considering the size of entire human genome, which is 3X109 bp, the total number scales up to 5-10 million. (Altshuler et al., 2000)

· In silico estimation of potentially polymorphic VNTR are over 100,000 across the human genome.

· The short insertion/deletions are very difficult to quantify and the number is likely to fall in between SNPs and VNTR

VARIATION OR MUTATION ? VARIATION OR MUTATION ?

Terminology for variation at a single nucleotide position is defined by allele

frequency.

PolymorphismA sequence variation that occurs at least 1 percent of the time (> 1%)90% of variations are SNPs

MutationIf the variation is present less than 1 percent of the time (<= 1%)

SINGLE NUCLEOTIDE POLYMORPHISMSSINGLE NUCLEOTIDE POLYMORPHISMS((SNPsSNPs))

......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...

SNPs are single base pair positions in genomic DNA at

which different sequence alternatives (alleles) exist in

normal individuals in some population(s), wherein the least

frequent allele has an abundance of 1% or greater.

Almost two decades ago the original incarnation of SNPs[as restriction fragmentlength polymorphisms (RFLPs)] clearly indicated the existence of widespreadsubtle genome variation

LIFE CYCLE OF LIFE CYCLE OF SNPsSNPs AND MUTATIONSAND MUTATIONS

TRANSITIONS AND TRANSVERSIONSTRANSITIONS AND TRANSVERSIONS

SNPs include single base substitutions such as:

Transitionschange of one purine (A,G) for a purine, or a pyrimidine (C,T) for a pyrimidine

Transversionschange of a purine (A,G) for a pyrimidine (C,T),

or vice versa

A G G A C T T C

A C A T G C G T C A C G T A T G

TRANSITIONS AND TRANSVERSIONSTRANSITIONS AND TRANSVERSIONS

The higher level of C>T e G>A SNPs is probably partlyrelated to 5-methylcytosine deamination reactions thatare known to occur frequently, particularly at CpGdinucleotides

TRANSITIONS AND TRANSVERSIONSTRANSITIONS AND TRANSVERSIONS

In principle, SNPs could be bi-, tri-, or tetra-

allelic polymorphisms. However, in humans, tri-

allelic and tetra-allelic SNPs are rare almost to

the point of non-existence, and so SNPs are

sometimes simply referred to as bi-allelic

markers

CLASSIFICATION OF SNPSCLASSIFICATION OF SNPS

SNPs may occur at any position in the above gene structure and based on its location it can be classified as: intronic, exonic or promoter region etc.

NonNon--coding coding SNPsSNPs::5’ and 3’ UTRsIntronsIntergenic Spaces

Coding Coding SNPsSNPs (subdivided into two groups):Synonymous: when single base substitutions do not cause a change in the resultant amino acid

Non-synonymous: when single base substitutionscause a change in the resultant amino acid.

CLASSIFICATION OF CLASSIFICATION OF SNPsSNPs

NON NON -- CODING CODING SNPsSNPs

Example: Regulatory SNPs (rSNPs)

Two allelic variants of the same gene are transcribed in different amounts as a consequence of an adjacentpolymorphism. In this example, allele G, located upstream of the gene, has a higher transcript level than does allele T

NON NON -- CODING CODING SNPsSNPs

Example: outside of gene SNPs can be usedas gene markers

CODING CODING SNPsSNPsExample: Synonymous, mutation does not change amino acid.

CODING CODING SNPsSNPsExample: Non-synonymous, mutation change amino acid seq.

rare mutations that cause medeliandiseases with allele frequency below 1%.

SNPsSNPs DISTRIBUTIONDISTRIBUTION

1 SNP per 1 kb sequence, or nucleotidediversity of 10 x 10-4 in the human genome

Highest (%)of SNPs

Lowest (%)of SNPs

The number of SNPs seems to be correlatedwith the length of the chromosomes

The distribution of SNPs among genestructure categories depends on the GC content of the

chromosomes

SNPsSNPs DISTRIBUTIONDISTRIBUTIONOn average, there were 22.59 SNPs per gene and 50.38 SNPs per intergenic regions

SNP DATABASESSNP DATABASES

dbSNP

http://www.ncbi.nlm.nih.gov/SNP/index.html

Human Genome Variation Database (HGVbase)

http://hgvbase.cgb.ki.se/

TSC: The SNP Consortium

http://snp.cshl.org/

dbSNPdbSNP

URL: http://www.ncbi.nlm.nih.gov/SNP/index.html

The Single Nucleotide Polymorphism database (dbSNP) is a public- domain archive for a broad collection of simple genetic polymorphisms. This collection of polymorphisms includes:

Single-base nucleotide substitutions (also known as single nucleotide polymorphisms or SNPs)

Small-scale multi-base deletions or insertions (also called deletion insertion polymorphisms or DIPs)

Microsatellite repeat variations (also called short tandem repeats or STRs).

dbSNP: STATISTICSdbSNP: STATISTICS

http://www.ncbi.nlm.nih.gov/SNP/snp_summary.cgi

09 Jan 2005

SNP APPLICATIONSSNP APPLICATIONS

•• DISEASE MAPPINGDISEASE MAPPING

Direct / Indirect Association Studies Direct / Indirect Association Studies

•• PHARMACOGENOMICSPHARMACOGENOMICS

•• POPULATION GENETICSPOPULATION GENETICS

DISEASE MAPPINGDISEASE MAPPING

• Linkage Analysis

– Within-family associations between marker and putative trait loci

• Linkage Disequilibrium (LD)

– Across-family associations

Linkage and Linkage and Linkage Disequilibrium (1)Linkage Disequilibrium (1)

• Linkage: the tendency of genes or other DNA sequences at specific loci to be inherited together as a consequence of their physical proximity on a single chromosome.

• Linkage disequilibrium (allelic association): particular alleles at two or more neighboring loci show allelic association if they occur together with frequencies significantly different from those predicted from the individual allele frequencies.

Linkage is a relation between loci, but association is a relation between alleles.

Linkage and Linkage and Linkage Disequilibrium (2)Linkage Disequilibrium (2)

• Linkage: 0 ≤ θ < 0.5

(θ = recombination fraction)

No linkage: θ = 0.5

Perfect linkage: θ = 0

• Linkage disequilibrium: 0 ≤ ρ ≤1(ρ = probability of allelic association)

Linkage equilibrium: ρ = 0

Complete linkage disequilibrium: ρ = 1

Linkage versus LinkageLinkage versus Linkage--DisequilibriumDisequilibrium

Both linkage and linkage-disequilibrium (LD) measures a correlation, or co-segregation, or association, between a genetic marker and the disease affection status

1. LinkageLinkage focuses on a locus, LinkageLinkage--DisequilibriumDisequilibrium focused on an allele

2. LinkageLinkage is resulted from recombination events in the last 2-3 generations, LinkageLinkage--DisequilibriumDisequilibrium is resulted from much earlier, ancestral recombination events

3.LinkageLinkage measures co-segregation in a pedigree, LinkageLinkage--DisequilibriumDisequilibriummeasures co-segregation in a population (essentially a huge pedigree)

4. LinkageLinkage is usually detected for markers reasonable close to the diseasegene (one centiMorgan/one Mb) , LinkageLinkage--DisequilibriumDisequilibrium is detected for markers even closer (0.01-0.02 centiMorgan/ 10-20 kB).

The The complexitycomplexity of common of common diseasesdiseaseshashas mademade themthem largelylargely refractoryrefractory toto

geneticgenetic analysisanalysis

In the face of this complexity,geneticists agree that the

family-basedapproaches that proved so

successful for the monogenicdiseases are

not up to the jobInstead, most favor association

studies, in which genetic and phenotypic

variation is compared in largepopulation samples in order to

identifycorrelations implicating genetic

risk factors

Association studies compare the allele frequency of a polymorphic marker,

or a set of markers, in unrelated patients (cases) and healthy controls to

identify markers that differ significantly between the two groups

DIRECT ASSOCIATION ANALYSISDIRECT ASSOCIATION ANALYSIS

Direct association analysis is a direct test of association between a putatively functional variant and disease risk. Example: candidate SNP analysis of coding SNPs (cSNPs) that change amino acids

INDIRECT ASSOCIATION ANALYSISINDIRECT ASSOCIATION ANALYSIS

Indirect association is the testing a dense map of SNPs for diseaseassociation under the assumption that if a risk polymorphism exists it willeither be genotyped directly or be in strong LD with one of the genotypedtagSNPs

The advantage of indirect association analysis isthat it does not require prior determination of which SNP might be functionally important, butthe disadvantage is that a much larger number of SNPs needs to be genotyped

FROM SNP TO HAPLOTYPE

GATATTCGTACGGA-TGATGTTCGTACTGAATGATATTCGTACGGA-TGATATTCGTACGGAATGATGTTCGTACTGAATGATGTTCGTACTGAAT

SN

P

SN

P

1 2

3

4

5 6

DNA Sequence

AG- 2/6

GTA 3/6AGA 1/6

Haplotypes

PhenotypeBlack eyeBrown eyeBlack eyeBlue eyeBrown eyeBrown eye

Haplotype: A set of closely linked genetic markers present on one chromosome which tend to be inherited together (not easily separable by recombination). Each person has two haplotypes in a given region, and each haplotype will be passed on as a complete unit

G G A C ASet of SNP polymorphisms: a SNP haplotype

HAPLOTYPE STUDYHAPLOTYPE STUDYWhole-genome genotyping of 10 million SNPs•Technologically daunting•Prohibitively expensive

Researchers are trying to downsize the problem of genome-wide genotyping by studying haplotypes.

The term genotype can refer to the SNP alleles that a person has at a particular SNP, or for many SNPs across the genome

INTERNATIONAL HAP MAP PROJECTINTERNATIONAL HAP MAP PROJECTThe HapMap Home Page URL: http://www.hapmap.org/index.html.en

The goal of the International HapMap Project is to develop a haplotypemap of the human genome, the HapMap, which will describe the common patterns of human DNA sequence variation.

The HapMap will be a tool that will allow researchers to find genes and genetic variations that affect health and disease

PHARMACOGENOMICSPHARMACOGENOMICSPharmacogenomics is a science that examines the inherited variations in

genes that dictate drug response and explores the ways these variations

can be used to predict whether a patient will have a good response to a

drug, a bad response to a drug, or no response at all

PHARMACOGENOMICSPHARMACOGENOMICSSNPs in genes encoding drug targets or drug metabolism pathways can determine the therapeutic utility of pharmacologic agents

Most drugs show significantinterindividual variation in therapeutic

efficacy

PHARMACOGENOMICSPHARMACOGENOMICS

Better, Safer Drugs the First Time:Analyze a patient's genetic profile and prescribe the best available drug therapy from the beginning

More Accurate Methods of Determining Appropriate Drug Dosages:Drug dosage can be based on a person's genetics --how well the body processes the medicine and the time it takes to metabolize it.

Decrease in the Overall Cost of Health Care:Decrease adverse drug reactionsDecrease failed drug trials,Decrease the time for drug approved by government,Decrease the time and the number of medication on patients

POPULATION GENETICSPOPULATION GENETICSPopulation genetics is the study of the distribution of and change in allele

frequencies under the influence of the four evolutionary forces: natural

selection, genetic drift, mutation and migration. It also takes account of

population subdivision and population structure in space.

Global distribution of genetic diversity. Most genetic variations(shown here ascolor) is found within individuals of the same population, with a small fractionattributable to differences among populations

Genetic polymorphisms can be used to predict the population of origin of an individual

Population distribution of distinct haplotypesdiscovered in 2036 genes. Degree of populationsharing is indicated.

Population distribution of 37 582 SNPsdiscovered in 2036 genes. Degree of population sharing is indicated.

These differences are expected to have profound consequencesfor the design of medical association studies and will be of vital

importance when trying to identify the geneticcontribution to complex phenotypes such as aging

Though they are small, these differences may be Though they are small, these differences may be used to partly understand used to partly understand differences in disease differences in disease

risk among populationsrisk among populations. . The next natural level The next natural level --differences among differences among

individualsindividuals-- will pave will pave the way for the way for personalized medicinepersonalized medicine