Dr Rana Bazzi

PhD in Genetics

University of Nottingham, UK

Applied genomics: toward

personalized medicine

Contents

• Introduction to the HG

• DNA polymorphisms

• SNP microarrays

• Genetic polymorphism and pharmacogenetics

The sequence of the human genome

• Draft annouced in 2000 by IHGSC and celera

• The euchromatin part was sequenced (92%)

• Difficulties to sequence heterochromatin parts

The Human Genome

• human genome = 3.3 billion bases

• For most organisms, less than 5% of the total genome has a

genetic function.

• Only 1.2% DNA encodes protein!

• Many genes produce more than one transcript

• Junk DNA-what a rumour?!

Genomic revolution

• Discovery of the double-helical structure of DNA 1953

• The Human Genome Project (HGP) 2003:the “project Apollo of biology".

• Advances in genome-based analysis methods increases our ability to explore genome function:

Genomic sequence database, cDNA sequencing, proteomics, databases and bioinformatics.

• Genomics has become a central discipline of biomedical research

• Applying the genomic information to improve human health is a big challenge!!!

Genomic disciplines

Toxicogenomics

Structural Genomics

Chemical Genomics

Gene therapy

Functional Genomics

Pharmacogenomics

Comparative

Genomics

Nutrigenomics

Bioinformatics

Proteomics

Genomic variations

Relationship between genomic variation, variation in RNA

and protein expression and the biological effects and clinical

effects. Some – but not all – mechanisms of regulation are

also indicated

Online Mendelian Inheritance in Man (OMIM)

OMIM reported:

• > 387 human genes of known sequence with a known

phenotype, and

• > 2,310 human phenotypes with a known molecular basis.

• >1,621 confirmed Mendelian phenotypes for which the

molecular basis is not known.

• > 2,084 phenotypes for which a Mendelian basis is suspected

but has not been fully established, or that may exhibit overlap

with other characterized phenotypes

• Ensembl is one of the world's primary resources for

genomic research

• Completely sequenced genomes as of January,

2009) http://www.genomesonline.org.

• http://www.ensembl.org/info/website/tutorials/cour

sebook.pdf

Structure of a human gene

Human DNA polymorphisms

• Usually involve population “polymorphisms”

• – occurrence at a locus of identifiable alternative

alleles each at an appreciable frequency in the

population

• Polymorphic markers

• – Two or more alternative alleles each at frequency >

1%

• – marker alleles can be used to follow the alternative

forms of a “trait / phenotype / disease” gene in family

and population studies

• Alleles <0.01 are “rare variants“

• Potential genetic markers

Human DNA polymorphisms

•Genetic polymorphisms have been studied at various levels:

•Phenotypic assessment (such as tests of colour vision)

•Cellular and serological tests (such as the determination of blood groups and HLA types ),

•Metabolic analysis (such as the acetylator status ),

•Direct examination of the nuclear and mitochondrial DNA itself.

•Drug response

•Disease susceptibility

DNA Microarray

• Microarray analysis involves the use of what are commonly

called "gene chips" to determine the expression of a large set of

genes at the same time in a single experiment.

• Gene chips can be purchased from several different companies,

eg Affymetrix, or they can be custom prepared in laboratories

with the proper equipment.

• Affymetrix gene chips are created through the covalent

attachment of synthetic oligonucleotides (oligos) to a small

surface. In general, there are 20 or more different oligos on the

chip corresponding to different regions of each gene to be

analyzed. In addition, a set of oligos that each contain a

nucleotide mismatch are included as negative controls for each

gene. The technology of creating gene chips is such that there can

be 10's of thousands of different genes represented on a single

chip approximately 2cm square.

Gene Chip

Genetic polymorphism

• By definition, a polymorphism is the coexistence of more than one genetic variant in a population and is typically described in terms of wild-type versus mutant alleles.

• Polymorphisms result from heritable mutations that become stable genotypes in a population and appear at an appreciable frequency (>1%).

• Pharmacogenetic polymorphism occurs when the 1% frequency of the mutant allele causes a different drug response or phenotype.

• Functional polymorphism: the genetic variants have functional importance in that they are linked with a variant phenotype.

Microsatellites

• Also known as simple tanem repeats (STRs)

• Consist of multiple copies of tandemly repeated sequences

of two (TGTG … TG), three (CAACAA … CAA), or four

nucleotides (AAATAAAT … AAAT)

• They tend to occur in non-coding regions of the DNA and to

a less extent in coding regions

• >10 000 microsatellite polymorphic loci are known

randomly distributed throughout the human genome

• Being small in size they can be readily genotyped (PCR)

The use of microsatellites in Genetics

• Being highly polymorphic and highly specific,

they are useful genetic markers that allows:

- Mapping genomes (genetic linkage)

- Biomedical diagnosis (markers for certain disease

conditions).

- DNA testing in forensics

- Parentage analysis/ relatedness of individuals,

etc..

STR

Microsatellites and

fingerprinting

Single Nucleotide Polymorphisms (SNPs)

• Single nucleotide polymorphisms

• More frequent than microsatellites

• Frequencies>1%

• Random distribution / codominant

• Less variable than microsatellites

• Generally only two alleles per site

• Can be identified by various methods (comparisons of

sequences/heteroduplex hybridizations/PCR/DNA chips

• Go to dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/)

and find the "dbSNP

And their number is still increasing!

• 2001…… 1.42 million SNPs

• 2004……. 4

• 2009…….10 million

• 2011…….13 million!

SNP characteristics

• It has the following three characteristics

1) very common in the human genome (a SNP

occurs every 100 to 300 bases along the 3-

billion-base human genome)

2) among the SNPs, two of every three SNPs are the

variations from cytosine (C) to thymine (T)

3) very stable from generation to generation.

Applications

• This technique is used:

- In forensics medicine,

- Paternity testing

- Fingerprinting

- Gene mapping (genetic linkage)

- Biomedical diagnostics (markers for certain genetic

disorders)

- Pharmacogenomics

SNPs

• Partial DNA sequence of exon 7 of African–American patient (top) and wild type

CYP2C9 (lower). Direct sequencing of the sense strand of exon 7 shows the patient

is heterozygous for a G1078A mutation (numbering based on the cDNA).

HapMap Project

• The international HapMap Project provides many

kinds of data for researchers, such as the HapMap

genotype data and the phased haplotype data.

• The phased haplotype data describes SNP alleles

on a chromosome inherited from one of father and

mother, while

• The genotype data describes SNP alleles on both

chromosomes inherited from parents