‘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 two genomes
are roughly 99.9% identical, that still leaves
millions of differences among the 3.2 billion
base pairs.
It is precisely these differences that
account for heritable variation among
individuals, including susceptibility to
disease
 HUMAN GENETIC VARIATIONS
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)

Insertion or deletion of one or more nucleotide(s)

-Tandem Repeat Polymorphisms

-Insertion/Deletion Polymorphisms
 SINGLE 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 POLYMORPHISMS
Tandem repeats or variable number of tandem repeats (VNTR) are a
very 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)

Minisatellites
repeat 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 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 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 ?

Terminology for variation at a single

nucleotide position is defined by allele
frequency.
Polymorphism
A sequence variation that occurs
at least 1 percent of the time
(> 1%)
90% of variations are SNPs

Mutation
If the
variation is
present
less than 1
percent of
the time
(<= 1%)
 SINGLE NUCLEOTIDE POLYMORPHISMS
(SNPs)

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 fragment
...C C A T T G A C...
length polymorphisms (RFLPs)]
…G G T A A C T G...
clearly indicated the
existence of widespread ...C C G T T G A C...
subtle genome variation …G G C A A C T G...
LIFE CYCLE OF SNPs AND MUTATIONS
TRANSITIONS AND TRANSVERSIONS
SNPs include single base substitutions such as:

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

A G G A C T T C

Transversions
change of a purine (A,G) for a pyrimidine (C,T),
or vice versa

A C A T G C G T C A C G T A T G
TRANSITIONS AND TRANSVERSIONS

The higher level of C>T e G>A SNPs is probably partly

related to 5-methylcytosine deamination reactions that
are known to occur frequently, particularly at CpG
dinucleotides
TRANSITIONS 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 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.
CLASSIFICATION OF SNPs

Non-coding SNPs:
5’ and 3’ UTRs
Introns
Intergenic Spaces

Coding SNPs (subdivided into two groups):

Synonymous: when single base substitutions do not
cause a change in the resultant amino acid

Non-synonymous: when single base substitutions

cause a change in the resultant amino acid.
 NON - CODING SNPs
Example: Regulatory SNPs (rSNPs)

Two allelic variants of the same gene are transcribed in

different amounts as a consequence of an adjacent
polymorphism. In this example, allele G, located upstream of
the gene, has a higher transcript level than does allele T
 NON - CODING SNPs
Example: outside of gene SNPs can be used
as gene markers
 CODING SNPs
Example: Synonymous, mutation does not
change amino acid.
 CODING SNPs
Example: Non-synonymous, mutation change
amino acid seq.

rare mutations
that cause
medelian
diseases with
allele frequency
below 1%.
 SNPs DISTRIBUTION
1 SNP per 1 kb sequence, or nucleotide
diversity of 10 x 10-4 in the human genome

Highest (%)
of SNPs The number of SNPs seems to be correlated
with the length of the chromosomes

The distribution of SNPs among gene

Lowest (%)
structure categories depends on the GC content of the
of SNPs chromosomes
 SNPs DISTRIBUTION
On average, there were 22.59 SNPs per gene and 50.38 SNPs per intergenic regions
 SNP 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/
 dbSNP
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: STATISTICS
http://www.ncbi.nlm.nih.gov/SNP/snp_summary.cgi
09 Jan 2005
 SNP APPLICATIONS

• DISEASE MAPPING
Direct / Indirect Association Studies

• PHARMACOGENOMICS

• POPULATION GENETICS
 DISEASE MAPPING

• Linkage Analysis
– Within-family associations between marker
and putative trait loci

• Linkage Disequilibrium (LD)

– Across-family associations
 Linkage and
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 Disequilibrium (2)
• Linkage: 0   < 0.5
( = recombination fraction)
No linkage:  = 0.5
Perfect linkage:  = 0

• Linkage disequilibrium: 0   1
( = probability of allelic association)
Linkage equilibrium:  = 0
Complete linkage disequilibrium:  = 1
Linkage versus Linkage-Disequilibrium
Both linkage and linkage-disequilibrium (LD) measures a
correlation, or co-segregation, or association, between a
genetic marker and the disease affection status

1. Linkage focuses on a locus, Linkage-Disequilibrium focused on an allele

2. Linkage is resulted from recombination events in the last 2-3 generations,

Linkage-Disequilibrium is resulted from much earlier, ancestral recombination
events

3.Linkage measures co-segregation in a pedigree, Linkage-Disequilibrium

measures co-segregation in a population (essentially a huge pedigree)

4. Linkage is usually detected for markers reasonable close to the disease

gene (one centiMorgan/one Mb) , Linkage-Disequilibrium is detected for
markers even closer (0.01-0.02 centiMorgan/ 10-20 kB).
The complexity of common diseases
has made them largely refractory to
genetic analysis
In the face of this complexity,
geneticists agree that the
family-based
approaches that proved so
successful for the monogenic
diseases are
not up to the job
Instead, most favor association
studies, in which genetic and
phenotypic
variation is compared in large
population samples in order to
identify
correlations 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 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 ANALYSIS

Indirect association is the testing a dense map of SNPs for disease

association under the assumption that if a risk polymorphism exists it will
either be genotyped directly or be in strong LD with one of the genotyped
tagSNPs
The advantage of indirect association analysis is
that it does not require prior determination of
which SNP might be functionally important, but
the disadvantage is that a much larger number of
SNPs needs to be genotyped
 FROM SNP TO HAPLOTYPE

SNP

SNP
Phenotype
Black eye

1 2 3 4 5 6
GATATTCGTACGGA-T
Brown eye Haplotypes
GATGTTCGTACTGAAT
Black eye GATATTCGTACGGA-T AG- 2/6
Blue eye GATATTCGTACGGAAT GTA 3/6
Brown eye GATGTTCGTACTGAAT AGA 1/6
Brown eye GATGTTCGTACTGAAT

DNA Sequence

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 A
Set of SNP polymorphisms: a SNP haplotype
 HAPLOTYPE STUDY
Whole-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 PROJECT
The HapMap Home Page URL: http://www.hapmap.org/index.html.en

The goal of the International HapMap Project is to develop a haplotype

map 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
 PHARMACOGENOMICS
Pharmacogenomics 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
 PHARMACOGENOMICS
SNPs in genes encoding drug targets or drug metabolism pathways can
determine the therapeutic utility of pharmacologic agents

Most drugs show significant

interindividual variation in therapeutic
efficacy
 PHARMACOGENOMICS
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 reactions
Decrease failed drug trials,
Decrease the time for drug approved by government,
Decrease the time and the number of medication on patients
 POPULATION GENETICS
Population 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.
 Genetic polymorphisms can be used to predict the
population of origin of an individual

Global distribution of genetic diversity. Most genetic variations(shown here as

color) is found within individuals of the same population, with a small fraction
attributable to differences among populations
Population distribution of 37 582 SNPs discovered in
These differences are expected to have profound consequences
2036 genes. Degree of population sharing is

for the design of medical association studies and will be of vital

indicated.

importance when trying to identify the genetic

contribution to complex phenotypes such as aging

Population distribution of distinct haplotypes

discovered in 2036 genes. Degree of population sharing
Though they are small, these differences may be
used to partly understand differences in disease
risk among populations.
The next natural level -differences among
individuals- will pave
the way for personalized medicine