Albert PinhasovDepartment of Molecular Biology

Ariel University Center

Actual Issues of laboratory medicine of the XXI centuryKazakhstan

May 22, 2012

Genome-based Diagnostics:Hype, Hope or Reality?

We now know how God wrote the book of life

Bill Clinton

But…..

Do we know how to read the book !?

Genetic code

Human Genome contains about 3-billion nucleotides

Approximately 99.9% of our DNA sequence is identical the 0.1% difference between any two individuals

But…………

At the DNA level, this 0.1% difference translates into 3 million sites of genomic variation

DNA Sequencing

“Sequencing” DNA is simply the elucidation of the order of the bases in an organism’s DNA strand

Early techniques were developed in the 1970’s

A variety of approaches now exist

The biggest limitation to sequencing is that the genome is big

DNA Sequencing

First generation sequencing

Next generation sequencing

Third generation sequencing

Sanger sequencing’ has been the only DNA sequencing method for 30

years

Next Generation Sequencing

Miniaturization enables massively parallel analysis

Carrying out millions of sequencing reactions simultaneously in each of 10 million tiny wells

Computer analysis of huge amounts of information allows “assembly" of a given sequence

Cost of Human Genome

Next Generation Sequencing

Efforts are made

REVEAL

Individualized therapy

Individualized therapy

Integrated healthcareIntegrated healthcare

Pharmacoge-nomics

Pharmacoge-nomics

Translational medicine

Translational medicine

Predictive medicine

Predictive medicine

Genotype-based therapy

Genotype-based therapy

Customized drug therapyCustomized

drug therapy

Information-based

medicine

Information-based

medicineSystems medicineSystems

medicinePharmacoprot

eomicsPharmacoprot

eomics

Personalized Medicine

Personalized Medicine

What is it?

What does it mean to us?

How it impacts our life?

How it changes our future?

Personalized Medicine Vision vs. Reality

• Advances in science and technology

• Convergence of molecular biology,

genetics, biotechnology, bioinformatics

• Transformational changes in medicine

– Shift towards prevention

– Reclassification of disease

– Integration and coordination

• Health as a national asset

• Symptom based care

• Fragmented, lack of coordination

• Inefficient use of information

• Huge gaps provider knowledge re

genomics

• Powerful investors in current

system resist change

• Costs growing and unsustainable

Molecular Diagnostics are Transforming Medicine

13

Pre-natal testing

Disease predisposition

Disease detection

Drug selection

Recurrence monitoring

Key questionsIs the baby healthy? “

What diseases is this patient at

risk for?

Has this patient a disease?

What drugs should I

prescribe?

How has the disease returned?

Molecular diagnostics is >a many

billion market

growing at >20%

annually

Current challenges in genomic diagnostics

Personal training

Equipment

Data analysis

Standardization

Role of Central laboratories in development of genomic resources

Developing algorithms

Writing standard operating procedure (SOP)

Quality Control Scheme organization

Training courses at peripheral level

Peripheral laboratories need

Well trained people

SOP and quality assurance culture

Appropriate laboratory equipment

Sample collection and transportation experience

Data collection and registration experience

Clinical and Molecular Diagnostics of neurodegenerative and

psychiatric conditions

Neurodegenerative disorders

Alzheimer’s Disease (AD)

Parkinson Disease (PD)

Epilepsy

Migraine

Multiple sclerosis (MS)

Alzheimer Disease

Signs & Symptoms: Memory loss for recent events Progresses into dementia and almost total

memory loss Inability to converse, loss of language ability Affective/personality disturbance Death from opportunistic infections, etc.

Confirmation of Diagnosis

Neuronal (b-amyloid) plaques

From http://www.rnw.nl/health/html/brain.html

Neurofibrillary tangles

From http://www.rnw.nl/health/html/brain.html

Brain Atrophy

AD: Lab diagnostic’s view

There is currently no biomarker of AD for early detection.

To date there is no definitive blood test available

that can discriminate dementia patients from healthy individuals.

A combination of characteristic plaque markers tau and amyloid b (Ab) may constitute a specific and sensitive cerebrospinal fluid marker for AD.

AD: genetic markers

Type 1 Type 2 Type 3 Type 4

Gene Mutations in APP gene on chromosome 21

Apo E polymorphism

Mutations in the presenilin1 gene

Mutations in the presenilin2 gene

Effect Abnormal b- amyloid fragment

Allele dependent susceptibility

Overproduction of the b-amyloid fragment

Overproduction of the b-amyloid fragment

Genotype: Controls AD

E2/E2 1.3% 0%

E2/E3 12.5% 3.4%

E2/E4 4.9% 4.3%

E3/E3 59.9% 38.2%

E3/E4 20.7% 41.2%

E4/E4 0.7% 12.9%

Jarvik G, Larson EB, Goddard K, Schellenberg GD, Wijsman EM (1996) Influence of apolipoprotein E genotype on the transmission of Alzheimer disease in a community-based sample. Am J Hum Genet 58:191-200

Prevalence of APOE genotypes in Alzheimer’s disease (AD) and controls

Farrer et al., JAMA, 1997

Odds of Alzheimer’s Disease by APOE and Age

Cupples et al., Genetics in Medicine, 2004Christensen et al., Genetics in Medicine, 2008

Clinical laboratory Diagnostics of

Depression

Neuropeptides Neurotrophins

Depression

PACAP BDNF

Research Strategies

GDPGTP

TM1

TM5

TM4TM3

TM2Asp -

PACAP

N

C

PACAP Receptor

TM7 TM6

Gs protein GDP

αs

AC

i3 loop

GTP

α s

TM1

TM5

TM4TM3

TM2

N

TM7 TM6

Asp -

βγ

ATP

cAMP

GDP

αs

ACβγ

cAMP

cAMPcAMP

Cytoplasm

Extracellular space

RNA pol II

C

CcA

MP

cAMP

cAMP

cAM

P

PKA

C

C

R

R

C

C

C

TATATBP

DNACREB

CREB

PO4

PO4

C

CBP

CBP

CREBCREB

PO4

PO4

CBPCBP

Transcription of BDNF gene

Nucleus

Cytoplasm

1 2 3 4 1 2 3 40

5

10

15

20

25

MDD Control

**

Weeks in Experiment

BDNF in MDD patients

Pre-symptomatic Genetic Testing: Shifting the Emphasis from

Reaction to Prevention

Pre-symptomatic testing

Genetic and biochemical analysis of healthy individuals who are at increased risk for a specific disorder.

Aims of Pre-symptomatic testing

Early detection of disease manifestation

To improve clinical outcome in positive cases

Huntington Disease: A Paradigm for Presymptomatic Genetic Testing

Heritable neuropsychiatric disorder First described by George Huntington in 1872 Prevalence vary among different populations

Highest -5–10 per 100,000 Onset –typically fourth decade of life. death usually occurring in the sixth decade.

No known medical cure No preventive measures available.

Huntington Disease: cause

Single-gene disorder Autosomal dominant

One mutated copy of the gene is necessary for an individual to be affected– each child of an affected parent is at 50% risk to inherit the

disease since only

Responsible Gene – HTT

Huntington Disease: symptoms

• Movement abnormalities – (e.g., chorea, dystonia,

bradykinesia

• Psychiatric disturbances – (e.g., depression and

anxiety)

• Cognitive impairment– (e.g., inattention and

executive dysfunction)

Huntington disease - a triplet repeat disease

The pathogenic mutation in the HTT gene is unstable, expanded CAG trinucleotide repeat in the first exon which encodes an abnormal polyglutamine tract

A run of > 34 glutamine residues causes the protein to aggregate in the brain cells and cause progressive cell death

CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG …... CAG

HD alleles categorization

Normal: 26 or fewer CAG repeats. Intermediate: 27–35 CAG repeats.

These individuals will not develop HD, but may be at risk of having a child with an HD-causing allele.

Reduced penetrance: 36–39 CAG repeats. These individuals are at risk for, but may not develop,

HD more likely to have later onset disease.

Full penetrance: 40 or more CAG repeats. These individuals will develop HD.

Advantages of predictive testing for Huntington disease

Uncertainty of gene status removed. If negative:

concerns about self and offspring reduced

If positive: make plans for the future arrange surveillance/treatment if any inform children/decide whether to have children

Disadvantages of predictive testing for Huntington disease

If positive: removes hope introduces uncertainty (if and when) known risk to offspring impact on self/partner/family/friends potential problems with insurance/mortgage.

If negative: expectations of a ‘good’ result ‘survivor’ guilt.

Potential outcomes of pre-symptomatic HD genetic testing.

Severe Psychological Distress

International testing protocols were established to protect at-risk individuals and to provide guidance for clinical and laboratory personnel

Our Expectations

Greater effectiveness of health care delivery

Improved health

Improved quality of life