cap.21 genetica delle malattie complesse; le nanotecnologie

Trieste 15/10/2003LA GENETICA DELLEMALATTIECOMPLESSE E LE “NANOTECNOLOGIE”

Caratteri Mendeliani e Caratteri Multifattoriali

Ereditarietà “semplice”, correlazioneGenotipo-fenotipo

Caratteri Complessi: interazionePoligeni + ambiente

THE BEEP BEEP GENES…

A FRUSTRATEDGENESHUNTER…

EREDITA’ MULTIFATTORIALEE TRATTI QUANTITATIVI

Ogni singolo gene hascarsa penetranza,espressività variabileed un piccolo impatto sul fenotipoA differenza delle malattie ereditarie POLIGENICHE o OLIGOGENICHE, i fattori “AMBIENTALI” giocano un ruolo fondamentale nella comparsa di un fenotipo patologico.

Tecnologie utilizzate perl’dentificazione della componente geneticanelle malattie Multifattoriali:Analisi di Linkage senza modellistudi di Associazione supopolazioni giovani, di recentefondazione ed isolate (Islanda, DeCodeGenetics): pochi crossing over, aplotipiAncestrali conservati

Microsatelliti e SNPs

17G1T

17C 2G

16G

3G 3A16C

18G

4T19C

4A

20G20A

31T

6A22G 7T

21G29A21A

29G

23T

10G

24C

25G25A11A

32G26A

9C

27A

12C13A13G

28C

30T30A14G5A

$$$$$$$$OB!!!

La Genetica della Celiachia

I fattori genetici di rischio nella patogenesi

della malattia

La Celiachia Come Malattia Genetica Complessa

• La celiachia (MIM 212750) e’ una comune malattia autoimmune caratterizzata da malassorbimento in seguito a danni alla mucosa intestinale.

• La malattia colpisce piu’ frequentemente i famigliari rispetto la popolazione sana (λs varia da 20 a 60).

• Nei gemelli omozigoti il tratto mostra una concordanza del 75%.

• La celiachia e’ una malattia autoimmune con una forte componente genetica

Regioni Genomiche Associate Alla Celiachia Mediante Studi

Di Linkage

HLA

HLA: HUMAN LEUKOCYTE ANTIGENSHLA: HUMAN LEUKOCYTE ANTIGENS

•• LocusLocus genico dell’HLA UMANO Cromosoma 6 genico dell’HLA UMANO Cromosoma 6 braccio cortobraccio corto

Genotipizzazione HLA mediantetecnica SSP

DQA1*0102 ;*0501

DQB1*0201; *05

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

1 2 3 4 5 6 7 8 1 2 3 4 5 6

0

0,2

0,4

0,6

controls

aea+ttg+

*010

1*0

102

*010

3*0

104

*010

5

*020

1*0

301

*030

2*0

303

*040

1*0

501

*050

2*0

503

*050

4*0

505

*060

1

*

Frequenza alleli HLA-DQA1nella Celiachia

Nella maggior parte delle popolazioni studiateil 90% o più dei celiaci contro circa il 20-30%della popolazione generale presenta l’aplotipo

DQ2 (a1*0501, b1*0201),

il 5-8% l’aplotipo DQ8 e il 2-5% non presenta

né l’aplotipo DQ2 né l’aplotipo DQ8.

Geni non HLA-linked

CTLA4

CD14 Tmp1 IL12B Tim

MBL: Molecole Deputate Alla Difesa Immunitaria Aspecifica Nella Celiachia

• MBL (Mannose Binding Lectin) e’ una molecoladeputata alla difesa aspecifica dell’ospite

• Tre polimorfismi al primo esone (raggruppati nell’allele 0) sono responsabili di bassi livelli serici della proteina

Melting Temperature Assay per lo studio delle varianti alleliche del gene

MBL

Frequenza dei polimorfismi nel gene MBL

A/A A/0 0/0

“Real Time PCR”0,5

4 0,6

0,33

0,35

0,13

0,05

0%

20%

40%

60%

117 CD 130 controls

0/0 0/A A/A

** **

0,71 0,77

0,29

0,23

0%

20%

40%

60%

80%

117 CD 130 controls

mutant alleles

Wild type

Perche’ MBL e’ coinvolto nella patogenesi della malattia?

• Varianti alleliche del gene MBL sono gia’ state associate a malattie autoimmuni (SLE, UC, RA, Dermatomiosite)

• MBL potrebbe essere coinvolta nel combattere le infezioni a livello intestinale

• MBL e’ una proteina importante nella “clearance” delle cellule apoptotiche e necrotiche

RT in situ PCR dimostra espressione del gene MBLnelle mucoseintestinali dei celiaci

Un altro gene coinvolto nell’immunita’ naturale: CD14

• CD14 e’ una molecola deputata alla difesa aspecificadell’organismo stabilizzando il legame LPS- TLRs,

• Molecola importante nel riconoscimento e nella fagocitosi delle cellule apoptotiche

• 4 SNPs nel promotore del gene che regolano la quantita’ di CD14 serico (sCD14) e di CD14 attaccato alla membarana dei monocitie macrofagi (mCD14)

• Studio mediante tecnica PCR-RFLP di due polimorfismi –159 C/T e –1359 G/T

0,8 0,77 0,84

0%

50%

100%

115 CD

HLAt

37 CD

HLAa

180

controlli

T G

0,67 0,67 0,7

0,270,19

0,28

0%

20%

40%

60%

80%

115 CD

HLAt

37 CD

HLAa

180

controlli

T/T G/T G/G

** **

Proteine coinvolte nel processo di apoptosi potrebbero giocare un ruolo

chiave nella patogenesi della celiachia?

Gene Expression e celiachiaAssay on demand ABI

5’

5’

3’ 5’

3’5’

forwardprimer

R = Reporter

Q = Quencher

. Cleavage. . CleavageCleavage

RR

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Q = Quencher

. Cleavage. . CleavageCleavage

RR

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“Real Time” PCR quantitativa: 5’ fluorogenic assay

Valutazione similtanea dell’espressione genica in CD Gene Expression Card

Genetic factors involved in HIV-1 vertical transmission and AIDS progression in Brazialian children

Collaborazione con l’IMIPInstituto Materno Infantildo Pernambuco (Recife)

AIDS as a “complex poligenicdisease”

• AIDS as an “immunological” disease: do the host characteristics influence HIV infectionand AIDS progression?

• The immunogenetists focused their attention in the last years on the analysis of differentgenes involved in HIV infection and diseaseprogression

• Which are the genes responsible for the susceptibility to the infection and which are the environmental factors of risk?

AIDS as a “complex poligenicdisease”

• Could we use genetic polymorphisms asprognostic elements to investigate AIDS progression in children?

• Are genetic polymorphisms related to differentresponses to drug treatment?

GENETIC SCREENING IN ITALIAN AND BRAZILIAN CHILDREN

Italian registry of AIDS patients: 301 infected children and 100 healthycontrols (blood donors)Collaboration with the I.M.I.P (Recife, PE): 114 infected children and 100 healthy controls (blood donors)

Why children???• The moment of the

infection is known• Classification on the

basis of diseaseprogression in rapidprogressors (RP), slow progressors (SP) e Long-Term-Non progressors(LTNP)

Genetic polimophisms related to HIV-1 infection

• HLA• Allele D32 in CC-CKR5 gene

encoding for chemokyne receptor • Allele CCR2b-64I in the gene

encoding for CCR2 chemokyne receptor

• Allele 3’A in the promoter region of SDF-1 (Stromal-Derived Factor1) gene

• Polymorphisms MBL2 gene encodingfor Mannose Binding Protein

• Delta-32 allele of CCR5 gene was not involved in resistence to HIV infection

• Children carrier of Delta-32 allele of CCR5 gene showed a slower diseaseprogression

3’A SDF-1 gene promoterpolimorphism is related to rapid progression of the infection in Italian children

Polymorphism at codon 54 of mannose-binding protein geneinfluences AIDS progression but not HIV infection inexposed children

RP vs SP: p=.024RP vs HIV-: p=.01

RP vs Controls: p=.025

0,74 0,91 0,92

0,89

0,26

0,09

0,08 0,11

0%

20%

40%

60%

80%

100%

25 RP 27 SP 27

Exposed

HIV-

41

HealthycontrolsGly54Asp Wild type

Low or very low MBL serum levels

RP vs SP: p=.024RP vs HIV-: p=.01

RP vs Controls: p=.025

0,74 0,91 0,92

0,89

0,26

0,09

0,08 0,11

0%

20%

40%

60%

80%

100%

25 RP 27 SP 27

Exposed

HIV-

41

HealthycontrolsGly54Asp Wild type

0,74 0,91 0,92

0,89

0,26

0,09

0,08 0,11

0%

20%

40%

60%

80%

100%

25 RP 27 SP 27

Exposed

HIV-

41

HealthycontrolsGly54Asp Wild type

Low or very low MBL serum levels

Polymorphisms in the MBL promoter correlated with risk of HIV-1 vertical transmission and AIDS progression

We found that the polymorphism at position –550 influences the risk of HIV-infection and AIDS progression. Also a 6 bp deletion at position –328 was correlated with HIV-1 infection.This study indicates that the promoter of the MBL2 gene influences vertical transmission of HIV and the course of perinatal infection.

HIV-1 SNPs Card

Genotipizzazione polimorfismi medianteAssay by design ABI

TETTET TAMRAFAMFAM TAMRA

AlleleAllele 22

AlleleAllele 11TETTET TAMRAFAMFAM TAMRA

TETTET TAMRATETTET TAMRAFAMFAM TAMRAFAMFAM TAMRA

AlleleAllele 22

AlleleAllele 11TETTET TAMRATETTET TAMRAFAMFAM TAMRA

Sonde allele-specifiche5’ fluorogenic assay

Paziente genotipizzato per i polimorfismi associatiall’infezione da HIV-1 o alla progressione dell’AIDS 96 wells HIV-1 SNPs card

Versatile Defensins(Tomas Ganz. SCIENCE VOL 298 1 NOVEMBER 2002; 977-8)

Defensins (see the figure) are a family of antimicrobial peptides abundant in immune cells, white blood cells(specifically neutrophils), intestinal Paneth cells, and barrier epithelial cells thatengage in host defense. Thereis increasing evidence that in these settings defensins (and other antimicrobialsubstances) directlycontribute to the killingof microbes

DEFENSINS AND HIV• Zhang et al. now show that CD8+ T cells from long-

term nonprogressor HIV patients secrete α -defensins-1, -2, and -3 when stimulated.

• These α -defensins account for much of the antiviral activity of CAF. The authors used neutralizing antibodies to human α - defensins-1, -2, and -3, and to the β chemokines MIP-1 α, MIP-1β, and RANTES in order to calculate the specific contributions of α -defensins and β- chemokines to CAF activity.

• These experiments demonstrate that HIV strains using the CXCR4 chemokine receptor for entry into host target cells are predominantly blocked by the α-defensins in CAF.

β-DEFENSINS POLYMORPHISMS: COULD BE INVOLVED IN HIV-1

INFECTION AND AIDS PROGRESSION?• 18 SNPs known in β-

defensins• SNPs detection by direct

sequencing or by 5’-fluorogenic assay

• Possibility of new synthetic peptide design and synthesis

• Test avaliable forantimicrobicity

MICROARRAYS, STUDIO DEI PROFILIDI ESPRESSIONE DI GENI COINVOLTIIN MALATTIE COMPLESSE

Overview of the process for the RT labeling KitAAAAAAA 3’ mRNA

5’

TTTTTTT 5’ DIG labeled cDNA+ mRNA

+ mRNA

UDIG

UDIG

U

DIG

Reverse transcription

+ mRNA3’

TTTTTTT 5’ purified DIG labeled cDNAUDIG

UDIG

U

DIGRNA degradation+ DNA purification

3’

= DigoxigeninDIG

Overview of the process for the RT /IVT labeling KitAAAAAAA 3’ mRNA

TTTTTTT 5’ cDNA+ mRNA

+ mRNA

+ mRNA

TTTTTTT 5’ cDNA

AAAAAAA 3’ cDNA

+ mRNA+ mRNA

+ mRNA

TTTTTTT 5’ cDNA

AAAAAAA 3’ cDNA

5’

Reverse transcription

3’

2nd strand DNA synthesis

3’

5’

RNA degradation

3’DNA purification

5’

Overview of the process for the RT /IVT labeling Kit: cont.TTTTTTT 5’ cDNA

AAAAAAA 3’ cDNA

UUUUUUU 5’ purified DIG labeled cRNA

3’

DIG

UDIG

UDIG

U

UUUUUUU 5’ purified DIG labeled cRNA

3’

DIG

UDIG

UDIG

U

UUUUUUU 5’ purified DIG labeled cRNA

3’

DIG

UDIG

UDIG

U

UUUUUUU 5’ purified DIG labeled cRNA

3’

DIG

UDIG

UDIG

U

UUUUUUU 5’ purified DIG labeled cRNA

3’

DIG

UDIG

UDIG

U

UUUUUUU 5’ purified DIG labeled cRNA

3’

DIG

UDIG

UDIG

U

UUUUUUU 5’ purified DIG labeled cRNA

3’

DIG

UDIG

UDIG

U

UUUUUUU 5’ purified DIG labeled cRNA

3’

DIG

UDIG

UDIG

U

UUUUUUU 5’ purified DIG labeled cRNA

3’

DIG

UDIG

UDIG

U

UUUUUUU 5’ purified DIG labeled cRNA

3’

DIG

UDIG

UDIG

U

UUUUUUU 5’ purified DIG labeled cRNA

3’

DIG

UDIG

UDIG

U

UUUUUUU 5’ purified DIG labeled cRNA

3’

DIG

UDIG

UDIG

U

3’

5’ 1. Amplification cRNA synthesis2. cRNA purification

= DigoxigeninDIG

Applied Biosystems 1700 Microarray Assay

DIG Antibody –alkaline phosphatase conjugate

DIG Labeled cDNA, cRNA

Immobilized Oligo

= Alkaline Phosphatase

= Digoxigenin

= Anti-digoxigenin AntibodyAB

DIG

AP

DIG DIG ABA

P

Applied Biosystems 1700 Microarray Assay

DIG AB

AP

EnhancerEnhancer

DIG AB

AP

DIG AB

AP

ChemiluminescentSubstrate

EnhancerEnhancer

EnhancerEnhancer

Light

Chemiluminescent time coursedemonstrates that features don’t spread and the light isstable

25 second exposure after 15 mins

25 second exposure after 30 mins

25 second exposure after 45 mins

Same array imaged in time course at 35°C

Applied Biosystems Microarray has Removed Most Pseudogenes improving

probe specificity

1414

27828

148

31097

pseudogenes

Non-pseudogenesNon-pseudogenes

Celera Applied Biosystems Microarray

Confirmation/Diagnosticuse of published

Gene Set

Validation of hits by Real Time PCR

Interpretation & Understanding

Bio-informaticsStatistical analysis

Gene Expression WorkflowThe future model

Whole GenomeScreening

calpain x

LocusLink ID and Gene Symbols

Panther Classification: Function and Process

Public Location

Celera Gene ID

Public RefSeq and GenBank transcripts

Assays-on-DemandGene Expression

Assays-on-DemandSNP Genotyping

Public RefSeq Alternative Transcripts

Public GenBank Alternative Transcripts

Selected Assay

Transcripts targeted by selected Assay

Location shown graphically

Genome – Chr. 2

Assays-on-DemandSNP Genotyping

Three Assays-on-Demand Gene Expression

Selected Sequence

Selected gene

Transcription Factor Binding Sites

Celera Transcripts

SNP and GEx Assays

Selected ExonCelera & Public SNPs

Mouse blocks Mouse SNPs

Human and Mouse Assays

Mouse Homologs

CDS in Action

ACMEMicroarrays

Tools… !BEEP BEEP GENES LOCKED!!!

The happy end!!!