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Molecular Diagnostics :Molecular Diagnostics :

Hype or Hope ?Hype or Hope ?

Patrick WillemsPatrick Willems

GENDIA, Antwerp, BelgiumGENDIA, 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

HPVChlamydia

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 LeukemiaInv(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 / ABLKITPDGFR A/BEGFR

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 diseasesNumber of diseases

• Nature of disease mutationNature of disease mutation

• Technology

• Cost

• Number of samples

• Organisation

Monogenic Diseases

> 4.000 monogenic diseases

> 2.000> 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 diseasesNumber of diseases

• Nature of disease mutationNature of disease mutation

• Technology

• Cost

• Number of samples

• Organisation

Disease Mutations

Easy tests : Single - common mutations

Difficult tests : Private mutations

Disease MutationsSingle mutations Fragile X

Sickle Cell Anemia

Common mutations Deafness Hemochromatosis

Panel of mutations Cystic Fibrosis

Private mutations Breast Cancer

Colorectal cancer

Easy testsDisease 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 diseasesNumber 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 diseasesNumber 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 diseasesNumber 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 BRCA2

Mutations in 23 exons Mutations in 28 exons

Complicated / expensive

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

Colorectal Cancer 1 / 25 1 / 1.000 MLH1 MSH2 MSH6 APC MUTYH

Mutations in 19 exonsMutations in 16 exons Mutations in 10 exons Mutations in 15 exons Mutations in 16 exons

Complicated / expensive

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

• F5 Leiden

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 diseasesNumber of diseases

• Nature of disease mutation

• 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

Diagnostic bottle necks

• Number of diseasesNumber of diseases

• Nature of disease mutationNature 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

GENDIAGENDIA

GENGENeticetic DIADIAgnosticgnostic NetworkNetwork

www.GENDIA.netwww.GENDIA.net

The GENDIA network

GENDIA Network

1000 Referral labs

1 Central lab

100 Test labs

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

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

Next generation sequencing

DNA SequencingDNA SequencingDNA SequencingDNA Sequencing

1980-1990 1990-2005 > 2005

Radio - gel Fluorescent - capillary Next generation

Thousand bp / day Million bp / day Billion bp / day