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Molecular Diagnostics :

Hype or Hope ?

Patrick Willems

GENDIA, Antwerp, Belgium


We now know how God
wrote the book of life
Bill Clinton
But do we know
how to read the book ?
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
Diagnosis of genetic diseases

- Somatic rearrangements in cancer

- Genetic risk factors

- Pharmacogenetics

- Mutations in monogenic diseases


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:
Genetic Risk Factors
Genetic Risk Factors

Deep venous thrombosis

Cardiovascular disease

Alzheimer disease

Osteoporosis
Genetic Risk Factors

Most single risk factors

have NO clinical significance

in individual patients
Genetic Risk Factors

Deep venous thrombosis

Factor V
Factor II
MTHFR
Diagnosis of genetic diseases

- Somatic rearrangements in cancer

- Genetic risk factors

- Pharmacogenetics

- Mutations in monogenic diseases


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
Diagnosis of genetic diseases
- Somatic rearrangements in cancer

- Genetic risk factors

- Pharmacogenetics

- Mutations in monogenic diseases


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)
Diagnostic bottle necks

Number of diseases

Nature of disease mutation

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


Diagnostic bottle necks

Number of diseases

Nature of disease mutation

Technology

Cost

Number of samples

Organisation
Mutation Detection

1. Point mutations, frame shifts :


A. Sequencing
B. WAVE

2. Deletions : MLPA
Diagnostic bottle necks

Number of diseases

Nature of disease mutation

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
Diagnostic bottle necks

Number of diseases

Nature of disease mutation

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
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


Diagnostic bottle necks

Number of diseases

Nature of disease mutation

Technology

Cost

Number of samples

Organisation
Current Organisation
Small local labs : small portfolios ( < 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
Diagnostic bottle necks

Number of diseases

Nature of disease mutation

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 Sequencing

1980-1990 1990-2005 > 2005


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