2 - Diagnosticul Molecular - Hype or Hope

Molecular Diagnostics :
Hype or Hope ?
Genetic Diagnostics
• Cytogenetic tests
• FISH
• Molecular tests
Molecular Diagnostics
-
Diagnosis of infectious diseases
- Genetic identification
- Diagnosis of genetic diseases

Diagnosis of infectious diseases
HPV
Chlamydia
Hepatitis
HIV
Toxoplasmosis
Genetic Identification
- Paternity Testing
- Forensics
Paternity Testing
Forensic testing
Diagnosis of genetic diseases
- Somatic rearrangements in cancer
- Genetic risk factors
- Pharmacogenetics
- Mutations in monogenic diseases

Rearrangements in Cancer Cells
Chromosomal breaks produce fusion genes
These cause leukemias and lymphomas
Diagnosis determines treatment and prognosis

Rearrangements in Cancer Cells
Lymphocytic Leukemia
t(9;22) : BCR - ABL
t(12;21) : TEL - AML1
t(1;19) : E2A - PBX1
t(4;11) : MLL - AF4
Myeloid Leukemia
Inv(16) : CBF - MYH11
t(8;22) : AML - ETO
t(9;22) : BCR - ABL
- Pharmacogenetics

Genetic Risk Factors
Monogenic diseases are caused by a

deleterious mutation in a single gene:
Disease-causing mutations
Multifactorial diseases are caused by a

combination of variations in multiple genes:
Deep venous thrombosis
Cardiovascular disease
Alzheimer disease
Osteoporosis
Most single risk factors
have NO clinical significance
in individual patients
Deep venous thrombosis
Factor V
Factor II
MTHFR
- Pharmacogenetics

Pharmacogenetic tests
• Drug specificity
• Drug efficacity - toxicity

Drug specificity
Herceptin : HER2
Tyrosine kinase inhibitors

BCR / ABL
KIT
PDGFR A/B
EGFR
Drug efficacity / toxicity
Cytochromes
CYP2D6
CYP2C9
CYP2C19
- Pharmacogenetics

Diagnostic bottle necks
•
Number of diseases
•
Nature of disease mutation
•
Technology
•
Cost
•
Number of samples
•
Organisation
Monogenic Diseases
> 4.000 monogenic diseases
> 2.000 disease genes isolated

Gene testing
• Most countries : limited number
(< 50 genes)
• Few countries : large number

(300-500 genes)
• Nowhere : network complete availability

(> 1000 genes)
•
Number of diseases
•
•
Technology
•
Cost
•
Number of samples
•
Organisation
Disease Mutations
Easy tests : Single - common mutations
Difficult tests : Private mutations

Disease Mutations
Single mutations Fragile X
Sickle Cell Anemia
Common mutations Deafness

Hemochromatosis
Panel of mutations Cystic Fibrosis
Private mutations Breast Cancer
Colorectal cancer
Easy tests
Disease Gene Mutation
Fragile X FMR1 Repeat
FRAXE FMR2 Repeat
Friedreich ataxia FRDA Repeat
Haw River DRPLA Repeat
Huntington type 1 HD Repeat
Kennedy AR Repeat
Myotonic dystrophy type 1 DMPK Repeat
Spinocerebellar ataxia SCA1,2, 3, 6, 7, 8,10, 12,17 Repeat
Alpha 1 antitrypsin PI 2 common mutations
Charcot-Marie-Tooth Type 1A PMP22 1 common mutation
Cystic fibrosis CFTR Common mutations
Deafness GJB2 1 common mutation
Hemochromatosis type1 HFE 2 common mutations
Hereditary neuropathy (HNPP) PMP22 1 common mutation
Sickle cell anemia HBB 1 common mutation
Spinal muscular atrophy SMN1 1 common mutation
Beta thalassemia HBB 1 exon
Difficult tests
Disease Gene Mutation
Breast cancer BRCA1 Private
BRCA2 Private
Colon cancer MLH1 Private
MSH2 Private
MSH6 Private
BRCA testing
BRCA1 : 23 exonen, 1863 AA, 6.200 bp
BRCA2 : 28 exonen, 3418 AA, 10.300 bp
Totaal : > 17.000 bp sequence

•
Number of diseases
•
•
Technology
•
Cost
•
Number of samples
•
Organisation
Mutation Detection
1. Point mutations, frame shifts :

A. Sequencing
B. WAVE
2. Deletions : MLPA
•
Number of diseases
•
•
Technology
•
Cost
•
Number of samples
•
Organisation
Cost
Single mutations : cheap (200 E)
Prevalent mutations : cheap (300 E)
Panel of mutations : moderate (300 E)
Private mutations : expensive (1000 E)

Cost
• Socioeconomic situation
• Social security
• Reimbursement by insurance
•
Number of diseases
•
•
Technology
•
Cost
•
Number of samples
•
Organisation
Common
Genetic Diseases
?
Common genetic diseases
Disease Frequency Mutation Genes Mutations Conclusion
Hemochromatosis 1 / 600 1 / 400 HFE 2 common mutations Easy / cheap
Breast Cancer 1 / 20 1 / 500 BRCA1 Mutations in 23 exons Complicated /

BRCA2 Mutations in 28 exons expensive
Hypercholesterolemia 1 / 500 1 / 750 LDLR Mutations in 16 exons Complicated /

expensive
Colorectal Cancer 1 / 25 1 / 1.000 MLH1 Mutations in 19 exons Complicated /

MSH2 Mutations in 16 exons expensive
MSH6 Mutations in 10 exons
APC Mutations in 15 exons
MUTYH Mutations in 16 exons
Cystic fibrosis 1 / 2.500 1 / 2.500 CFTR Common mutations Easy / cheap
Prelingual deafness 1 / 1.500 1 / 4.000 GJB2 1 common mutation Easy / cheap
Fragile X syndrome 1 / 5.000 1 / 5.000 FMR1 Only 1 mutation Easy / cheap
SMA 1 / 10.000 1 / 10.000 SMN1 Only 1 mutation Easy / cheap
Beta Thalassemia variable variable HBB Only 1 exon Easy / cheap

Most frequent DNA tests
• Thalassemia
• Cystic fibrosis
• Breast cancer
• Colorectal cancer
• FRAXE
• SCA
Usual portfolio of DNA tests
• Easy tests
• Common tests
• Research tests
Genetic testing in Europe
• inhabitants per country : 10 million
• births per year : 100.000
• disease frequency : 1 on 10.000
• new patients per year : 10
• genetic labs : 10
New patients per lab per year: 1

•
Number of diseases
•
•
Technology
•
Cost
•
Number of samples
•
Organisation
Current Organisation
• Small local labs : small portfolio’s ( < 50 tests )
• Same spectrum of tests : common + easy tests
• Majority academic labs : research -diagnostic setting
• Many academic labs give up diagnostic testing
• No (inter)national network
•
Number of diseases
•
•
Technology
•
Cost
•
Number of samples
•
Organisation
Gene testing
• Unreliable
• Expensive
• Slow
Unreliable
10 % mistakes in easy tests such as CF
Nature Genetics 2000; 25: 259 - 260

Expensive
RESEARCH DIAGNOSTICS
1 genome 1 gene
< 1000 USD 200 – 5.000 USD
Ratio : 25.000
Slow
RESEARCH DIAGNOSTICS
100 genomes 1 gene

in 10 days in 100 days
Ratio 25 million
Message in a bottle
• Many different tests

• Many uncommon tests
• Many esoteric tests
• Many expensive tests
• International network needed
Mission
A global network of diagnostic labs
• Large portfolio
• Reliable
• Fast
• Affordable
GENDIA
www.GENDIA.net
GENetic DIAgnostic Network

The GENDIA network
GENDIA Network
1000 Referral labs
1 Central lab
Advantages GENDIA
• 1 lab to send samples to
• 1 lab to get results from
• > 2.000 genetic tests

• Large portfolio
• Best first selection of test
• Best Reflex testing
Looking into the future
Next generation sequencing
Sequencing power : billion bp / day
Will rapidly multiply
Cost : 100.000 Euros

Will rapidly decrease to 1000 Euro
Whole genome sequencing of Watson and Venter
Sequencing all patients

DNA
DNA Sequencing
Sequencing
1980-1990 1990-2005 > 2005

Radio - gel Fluorescent - capillary Next generation
Thousand bp / day Million bp / day Billion bp / day

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Molecular Diagnostics :

- Diagnosis of genetic diseases

- Somatic rearrangements in cancer

- Genetic risk factors

- Mutations in monogenic diseases

Chromosomal breaks produce fusion genes

These cause leukemias and lymphomas

Diagnosis determines treatment and prognosis

- Somatic rearrangements in cancer

- Genetic risk factors

- Mutations in monogenic diseases

Monogenic diseases are caused by a

Multifactorial diseases are caused by a

Deep venous thrombosis

Most single risk factors

have NO clinical significance

Deep venous thrombosis

- Somatic rearrangements in cancer

- Genetic risk factors

- Mutations in monogenic diseases

• Drug efficacity - toxicity

Tyrosine kinase inhibitors

- Somatic rearrangements in cancer

- Genetic risk factors

- Mutations in monogenic diseases

> 4.000 monogenic diseases

> 2.000 disease genes isolated

• Few countries : large number

• Nowhere : network complete availability

Easy tests : Single - common mutations

Difficult tests : Private mutations

Common mutations Deafness

Panel of mutations Cystic Fibrosis

Private mutations Breast Cancer

Breast cancer BRCA1 Private

Colon cancer MLH1 Private

BRCA1 : 23 exonen, 1863 AA, 6.200 bp

BRCA2 : 28 exonen, 3418 AA, 10.300 bp

Totaal : > 17.000 bp sequence

1. Point mutations, frame shifts :

Prevalent mutations : cheap (300 E)

Panel of mutations : moderate (300 E)

Private mutations : expensive (1000 E)

Hemochromatosis 1 / 600 1 / 400 HFE 2 common mutations Easy / cheap

Breast Cancer 1 / 20 1 / 500 BRCA1 Mutations in 23 exons Complicated /

Hypercholesterolemia 1 / 500 1 / 750 LDLR Mutations in 16 exons Complicated /

Colorectal Cancer 1 / 25 1 / 1.000 MLH1 Mutations in 19 exons Complicated /

Cystic fibrosis 1 / 2.500 1 / 2.500 CFTR Common mutations Easy / cheap

Prelingual deafness 1 / 1.500 1 / 4.000 GJB2 1 common mutation Easy / cheap

Fragile X syndrome 1 / 5.000 1 / 5.000 FMR1 Only 1 mutation Easy / cheap

SMA 1 / 10.000 1 / 10.000 SMN1 Only 1 mutation Easy / cheap

Beta Thalassemia variable variable HBB Only 1 exon Easy / cheap

• births per year : 100.000

• disease frequency : 1 on 10.000

• new patients per year : 10

New patients per lab per year: 1

• Same spectrum of tests : common + easy tests

• Majority academic labs : research -diagnostic setting