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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




- 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 / ABLKITPDGFR A/BEGFR

Drug efficacity / toxicity

Cytochromes

CYP2D6

CYP2C9

CYP2C19




- Pharmacogenetics


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)




• Technology

• Cost


• 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



• Nature of disease mutation

• Technology

• Cost


• Organisation

Mutation Detection

1. Point mutations, frame shifts :

A. Sequencing

B. WAVE

2. Deletions : MLPA




• Technology

• Cost


• 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




• Technology

• Cost


• 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




• Technology

• Cost


• 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




• Technology

• Cost


• 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

http://www.usnews.com/usnews/images/news/photos/venter051022.jpg

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

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Molecular Diagnostics - Gendia | Home Page