Lect 2

Ranchi: July 9, 2006

Impact of Genomics on human health Seyed E. Hasnain UNIVERSITY OF HYDERABAD

What distinguishes modern Human from its predecessors (300,000yrs)

Their ability to make and use complex tools ! The dazzling leap in technology influenced human evolution in every field

“(DNA technology) The most awesome and powerful tool acquired by man since the splitting of atoms” - The Time Magazine (USA)

CDFD

The Milestones in DNA Technology in the last 50 years: •1953: Double helix structure of DNA discovered. •1973: Cohen and Boyer “cut & paste” segments of DNA and reproduce (clone) the new DNA in bacteria. •1976: The first working synthetic gene was developed. •1977: The first human protein (somatostatin) was produced. •1982: Recombinant human insulin was marketed. •1983: Polymerase chain reaction (PCR) technique was conceived. •1985: Human growth hormone produced in bacterium was marketed. •1990: Human Genome Project (HGP), was launched. •1995: The first full gene sequence of a living organism was completed for the bacterium, Haemophilus influenzae. •2000: First draft of human genome sequence was completed. •2003: The final sequence of the human genome was produced.

The discovery that started it all!!

1953

Molecular Biology Overview Cell

Nucleus Chromosome

Protein

Gene (mRNA), single strand

Gene (DNA)

The Central Dogma of life Translation

DNA makes RNA makes Protein Transcription •DNA as gene •The concept of ‘junk’ DNA

From Chromosomes to Proteins

Recombinant DNA •Recombinant DNATechnology/ Transgenics/ GMOs and GM Food Technology and Sequencing •Molecular Diagnostics and Genetic Medicine •Stem cells •Cloning •DNA Fingerprinting or Genomic profiling

Fruits of the gene technology revolution

Speaking the language of DNA Recombinant Technology Cohen and Boyer gave the first break through in Recombinant technology

EcoRI

Ligase

PAUL BERG: A pioneer in the field of RECOMBINANT DNA TECHNOLOGY

Developed a Biochemical Method for Inserting New Genetic Information into SV40 DNA Circular SV40 DNA Molecules Containing Lambda Phage Genes and the Galactose Operon of E. coli.

Recombinant DNA Technology

Genetic Engineering of Microorganisms

GMO

Ananda Chakraborty and US patent

Applications of Genetic engineering Microbial systems • • • • • • • •

GMOs for Food and Dairy GMOs for environmenta Recombinant therapeutics Recombinant enzymes Therapeutic Biomolecules Recombinant vaccines Proteins, Neutraceuticals Cosmetic products

Transgenic animals • Biopharmaceuticals • Human proteins and humanised organs • Heterologous proteins of medical and surgical importance • Animal models of cancer, allergy and ageing

What does the DNA contain???? il fee offlif

T

no o toin a i t m a r o m f r n i o f nhe i he T

Revealing the information... Technological innovations to sequence the Human and other genomes ….. One of the largest endeavors in the history of mankind CDFD

1977- Maxam and Gilbert (US) and Sanger (UK) Independently developed methods for sequencing DNA

Slow rate of sequencing (250bp/day) and the

complex genomes

Improvement in technology was the key Advanced Molecular Biological techniques and computational Biology bridged the gap (6000 times enhancement in sequencing rate)

Technology in full swing sequencing by ‘synthesis nanotechnology’ approach Sequencing genomes in weeks and months

Sequencing genomes in hours!!

Genome Projects Model organisms

C. elegans (nematode) worm)

E. coli (bacteria) Saccharomyces cerevisae (brewer’s yeast)

50 years after the double helix story…...

Post-genomic issues Vast number of genes have no known function Human 40 - 60% Arabidopsis 48% Leishmania 52% H. pylori >50% M. tuberculosis 40% The question of numbers No clue as to which set of genes are expressed in a given time, given tissue, given environmental conditions Shift from qualitative biology to quantitative biology No clue as to how the genes cross-talk

Post Genomic Efforts: From data to knowledge High throughput technologies Rapid accumulation of biological data

Key Methods to analyse the vast amount of genomic data Structural genomics

Functional genomics

Comparative genomics Proteomics

Pharmacogenomics

Computational Biology

Harvesting the genomic data

Arrival of BIOINFORMATICS

Genomes

GeneProducts “ And that’s why we need a computer”

Structure and Function

• Scientific challenges • Algorithmic challenges

Pathways & Physiology

• Computational Challenges

Evolution of Informatics Tools Submission Target Identification

Hit Identification

Lead Identification

Lead Optimisation

CD Prenomination

Concept Testing

Development for launch

Launch Phase

Launch

BioSuit (India)

MouseBase

Biological Systems are several log More complicated than Physical Systems!

Make Connections: Build and Visualize Relationships Animal Models

Tissue Histopath

Disease

Projects

Time

Clinical Data

Biomarker Compounds

Literature

Genes

Target

Biomedial Image

-Omic tools -omic data

The Rise of Systems Biology

Anticipated Benefits of the human genome • Improved diagnosis of disease and intervention - PCG example • Earlier detection of genetic predispositions to disease • Rational drug design • Gene therapy • Pharmacogenomics or "custom drugs”: SNP Profiling

CDFD

•Recombinant DNATechnology/ Transgenics/ GMOs and GM Food

Diagnostics and Genetic Medicines •Molecular Diagnostics and Genetic Medicine •Stem cells •Cloning •DNA Fingerprinting or Genomic profiling

Fruits of the gene technology revolution

Molecular Diagnostics Another Breakthrough in Recombinant DNA Technology: The Invention of Polymerase Chain reaction (PCR) Invented by Kary Mullis in 1983. First published account appeared in1985. Awarded NobelPrize for Chemistry in 1993. Single molecule to 10 n molecules (n = number of PCR cycles)

Microarray /gene chips in diagnostics Measures global changes in gene expression Automated ultra-high throughput screening Technology for the future generation Can measure mRNA expression levels of 10,000 genes Can be used for genome wide mapping Can be used to monitor time kinetic expression of genes Useful for detecting SNPs, particularly important for allelically heterologous genes as BRCA1 Identification of disease genes - mismatch scanning (GMS) Identification of new drug targets

Microarrays: Biology's discovery platform

CDFD

Genomics for diagnostic markers Cancer tissue

Normal tissue

cells

cells

Biopsy

mRNA Subtractive methods

Only sequences that differ in Cancer tissue

Proteomics in generating Diagnostic markers Analytical methods Normal tissue Analytical methods Colon cancer tissue

2-D gel

Cumbersome Methods are under development

Marker for cancer

Origin of Laser Desorption (LD) During 1960’s it was demonstrated that the irradiation of low-mass organic molecules with a high-intensity laser pulse leads to the formation of ions that could be successfully mass analyzed – the origin of laser desorption (LD)

Origin of MALDI-TOF In 1987, Michael Karas and Franz Hillenkamp successfully demonstrated the use of a matrix in LD to circumvent the mass Limitation – origin of MALDI

Applications of MALDI-TOF • Mass detection of peptides • Peptide Sequencing • SNPs detection • Disease diagnosis

SELDI TOF – Surface Enhanced Laser Desorption Ionisation – Time of flight Varied chemical and biochemical surface allow differential capture of proteins based on the intrinsic properties of the proteins Patterns of masses are identified rather than actual identification of the proteins SELDI-TOF has been successfully used to detect peaks of proteins differentially expressed in sera of ovarian cancer patients compared to healthy individuals

Medicine in the Post Genomics era -

Better understanding of the disease process

-

Paradigm shift from preventive to predictive medicine

-

Generalized medicine vs personalized medicine - Genetic Medicine

Why are we so different? SNP…..ss??

• Single nucleotide variations that occur when a single nucleotide in the genome sequence is altered • Occur once in app. every 1000 bases • Two of every three SNPs involve in replacement of C with T • Occur both in coding and non-coding regions of the genome • Most SNPs have no effect on cell function but others predispose people to disease or influence their response to drug • About 300,000 SNPs in the human genome

Pharmacogenomics Study of the genetic basis for the differences between the individuals in responses to drug. • Optimum dose: Daily required dose varies (e.g. 40-fold for propanolol or 20-fold for warfarin) • Drug efficacy: (e.g. 30% of scizophrenics do not respond to antipsychotics, Interferon B/G is effective only in one out of three patients with multiple sclerosis or HCC) •Adverse drug reaction: In US alone, in 1994, 2.2.million of serious cases were reported and was the 5th leading cause of death

What can we gain? Markers for early detection Markers for efficacy Markers for safety

SNPs 0.1% frequency in the human genome Bases at the polymorphic site

Drug metabolism

Efficiency of medication

TACGTGG

Fast

High

TACATGG

Slow

Low

e. g. Cyp 2D6 locus vis-a-vis codein metabolism

Genetic Variation and drug responses

Gene CYP2C9 CYP2C19 UGT 1A1 5-LO Thiopurine methyl transferase Dopamine D3R CETP ADBR2

Drug

Consequences

Warfarin Omeprazole Irinotecan ABT-761

Increased bleeding Rapid metabolism and decreased efficacy in peptic ulcer Metabolism and toxicity of irinotecan Efficacy of ABT-761 in asthma

6-mercaptopurine Typ. neuroleptics Pravastatin Salbutamol

Toxicity and efficacy of 6-mercaptopurine in leukemia Development of tardive dyskinesia in schizophrenics Efficacy of pravastatin in coronaty atherosclerosis Efficacy of salbutamol in asthma

•Recombinant DNATechnology/ Transgenics/ GMOs and GM Food •Molecular Diagnostics and Genetic Medicine •Stem cellsCells Stem •Cloning •DNA Fingerprinting or Genomic profiling

Fruits of the gene technology revolution

Embryonic stem (ES) cells

Applications of Embryonic Stem Cells in Biotechnology and Medicine ¾ Single cell retains the ability to give

rise to precursor cells of different human cell types (Heart, Brain, Kidney, Lung, Ovary, Skin etc.)

•Recombinant DNATechnology/ Transgenics/ GMOs and GM Food •Molecular Diagnostics and Genetic Medicine •Stem cells •Cloning Cloning •DNA Fingerprinting or Genomic profiling

Fruits of the gene technology revolution

Cloning?

After Dolley...

Next..? Human Cloning ?

Medical benefits

How successful is it to CLONE ?

Green - success Yellow - very close Red - not yet

•Recombinant DNATechnology/ Transgenics/ GMOs and GM Food •Molecular Diagnostics and Genetic Medicine •Stem cells •Cloning •DNA Fingerprinting or Genomic profiling DNA Fingerprinting

Fruits of the gene technology revolution

DNA Fingerprinting or Genome Profiling DNA is the molecule of choice for forensic identification for the following four reasons 1. Discrimination No two people except identical twins (monozygotic) will share the same genetic blueprint. Identical twins are essentially clones. 2. Genetic Continuity Different biological sources of DNA found at the crime scene will share the identical pattern if they originate from the same person regardless of the biological origin 3. Sensitivity A few drops of blood, a buccal swab or a small semen stain will generally contain ample amounts of DNA for forensic analysis.

4. Stability extremely stable molecule capable of withstanding environmental insults both natural and manmade.

1

Case of the lost child Paternity disputes

A case of inclusion 1. Mother 2 Biological child 3. Lost in the “Mela” 4. Father

2

3

4

Mother is a mother is a fact, father is a father is a belief Maternity is also questioned!! Two mothers claiming parenthood for a single child!!

1. Biological Mother 2. Disputed Child 3. Suspected Mother

1

2 3

Locus

Blood stain recovered from revolver

Blood stain recovered from seat cover

D8S1179

15

17

15

17

D21S11

28

29

28

29

D18S51

17

17

17

17

D3S1358

15

16

15

16

vWA

14

19

14

19

FGA

20

23

20

23

D5S818

11

13

11

13

D13S317

10

11

10

11

D7S820

8

10

8

10

X

Y

X

Y

Amelogenin (Gender Marker)

One army officer was suspected to be involved In a murder case . Blood stain of the victim was recovered from the barrel of his revolver due to splashing of blood at the time when the deceased was shot

DNA on weapon

DNA Fingerprinting Criminal Identification and Forensics: cases of sexual abuse, murders, robbery etc (Madhumita murder case) Personal Identification: mass disaster and mass fatality incidents (Attack on World Trade Centre / The Kashmir issue: distinguishing civilians from terrorists)

In Civil Cases : The Basmati rice dispute

Non human DNA testing: The black buck case

DNA Technology: Not only justice but justice in time DNA analysis of 14,249 body parts of 2,795 people

Use of DNA in criminal investigations can drastically reduce the time consumed in judgement delivery

DNA technology Are we taking the human element out of it? Questions regarding GENES and PRIVACY ? What if your blood sample for medical check-up is used for research or other purposes without being informed? ? Genetic testing: what if it cause unnecessary stress and unwelcome changes in personal relationships ? Should genetic testing be carried out where there is no treatment available…as in albinism and dwarfism? ? What do you think about the following cases? - genetic testing for insurance - genetic testing for new employees - genetic databanks for forensic use - genetic databanks for institutionalized children ? Would it cause social biasness: what if people with ‘aggression genes’, ‘cancer genes’ or ‘mood-swing genes’ are considered the Bad Seeds!

We have recently advanced our knowledge of genetics to the point where we can manipulate life in a way never intended by nature. We must proceed with the utmost caution in the application of this new found knowledge. LUTHER BURBANK

Time for legal system and science to work together

DNA and the Law

n o y g o l o i m b e t w s e y n s f y o r t e c v i a l p e d m I e c i t s ju

The gene revolution is forcing judges to deal with science in a way they never had to before

Questions about the legal relationship between an egg donor, her husband, person being cloned, a surrogate mother and the resulting Child, legal rights , privileges and immunities a cloned child could claim in a jurisdiction that bans human reproductive cloning

While science in itself is often regarded as being neutral, the application and products of research may not be…

Make virus from written information- Bio terrorism!!

July, 2002: Polio virus “synthesized in the laboratory”, based on the sequence available in print The work raises alarming questions regarding Bio-terrorism Challenges the meaning of the terms “extinct/eradication” Other deadly viruses: Small pox, Ebola, Influenza - deadly weapons Vaccination should continue even though disease may no longer exist

Are we trying to play God or a Devil?

•Today it is Polio virus... •Tomorrow it could be still more deadly form of HIV or Plague •Day after tomorrow may be a human monster (Frankenstein?)

“It is humbling for me and awe-inspiring to realize that we have caught the first glimpse of our own instruction book, previously known only to God” - Francis Collins, NHGRI