Genetica umana - farmacogenetica

Le sfide del futuro: la farmacogenetica

Corso di genetica umana

Facoltà di Medicina e Chirurgia dell'Università di Torino

Alberto Piazza

L'identità genetica nella nostra specie Homo sapiens sapiens

• I vari genomi sono identici al 99.9%
• 3,200,000 nucleotidi sono differenti
• Tra ogni coppia di individui i genomi differiscono per circa 3 milioni di basi nucleotidiche
• Tra ogni coppia di individui i proteomi differiscono per circa 100,000 aa

Single Nucleotide Polymorphism (SNP)

GATTTAGATCGCGATAGAG

GATTTAGATCTCGATAGAG

L'SNP consiste in una posizione nel genoma in cui sono presenti in una popolazione due o più basi differenti, ciascuna con una frequenza >1%.

• Si tratta del polimorfismo più presente in natura
• Circa 10 milioni di SNPs negli uomini

Tipi di SNPs

• Genici, SNPs codificanti
  • Non-sinonimi: mantengono/alterano la struttura/funzione della proteina
  • Sinonimi: mantengono/alterano lo splicing
• Genici, SNPs non-codificanti
  • Regolatori: mantengono/alterano l'espressione genica
  • Intronici: mantengono/alterano l'espressione genica o lo splicing
• SNPs concatenati: comunemente intergenici

Gli aplotipi

Variazione nei genomi: insorgenza delle individuali mutazioni nel tempo entro una popolazione.

Disease Mutation

Common Ancestor

Whole Genome Associations

Disease Population Matched Control Population

N=500 N=500

~3,000,000 common SNPs across genome

221

Regions of association value

Chromosomal Location

Informatics to identify gene(s) mapped to associated SNP

Association studies

Control Disease Non-responder Responder

Allele 1 Allele 2

Marker A:

Allele 1 = Allele 2 =

Marker A is associated with Phenotype

Esempi di SNPs che causano malattie

• Primary hypomagnesemia
  • Patients unable to maintain sufficient levels of Mg²⁺ in their serum
  • Affected gene: Na/K ATPase g subunit (123G->A = Gly->Arg in transmembrane region) – mutation prevents targeting of the protein to cell membrane
• Mitochondrial SNPs
  • >50 known disease-causing SNPs in mtDNA
  • Most often affect tissues with high energy consumption
• BRCA1 (breast cancer-associated antigen 1)
  • Silent mutation in coding exon affects splicing (A. Krainer, CSHL)
  • Exons contain exonic splicing enhancers and exonic splicing silencers – mutations lead to exon skipping and aberrant inclusion, respectively

Esempi di SNPs che causano fenotipi alterati non patologici

• Glucose-6-phosphate dehydrogenase and favism
  • First enzyme in the oxidative branch of pentose phosphate pathway, which reduces NADP to NADPH+
  • Different mutations result in partially or completely inactive enzyme
  • People (10%) with inactive enzyme experience lysis of red blood cells when consuming fava beans (contains H₂O₂ – NADPH is needed to detoxify it)
• CytP450 mutations and drug responsiveness
  • Cytochrome P450 – activates many pre-drugs into active therapeutic compounds
  • Different people can be divided into typical, poor, and ultra-rapid metabolizers
  • Two genes in human:
  • 2D6 – required by more than 40 pre-drugs for activation; 12 known SNPs altering the gene’s activity
  • 2C19 – activates mephentyoin (epilepsy); 2-3% Caucasoids and 23% Asians are poor metabolizers

Studying the relationship of genetic variation and drug efficacy genome-wide: Pharmacogenomics

Drug dose (rel.)

Polymorphic drug metabolizing enzymes

Amplichip CYP450: The first commercial clinical test platform

Basel, 25 June 2003

Roche Diagnostics Launches the AmpliChip CYP450 in the US, the World’s First Pharmacogenomic Microarray for Clinical Applications

It is the first chip using Affymetrix technology that meets federal standards for clinical use

The route to a new drug…

is a long one

Exploratory Development Full Development Discovery Phase IV

Phase I Phase II Phase III

0 15 10 5 Years

11-15 Years Marketed Idea Drug

Patent life 20 years

…and an expensive one!

It costs >$800 million to get a drug to market

$ Millions spent in

3,332 9 months in 2001

2,660 2,487 2,281

1,916 1,955 1,740 1,645 1,499 1,402 1,116 934

SGP ABT AHP BMY LLY MRK PHA AZN AVE JNJ GSK

Pharmacogenomics defined

The study of genome-derived data, including human genetic variation, RNA and protein expression differences, to predict drug response in individual patients or groups of patients.

Pharmacogenomics includes Pharmacogenetics

Pharmacogenomics

Human Genetics

• SNPs
• Haplotypes
• Sequencing

Expression Profiling

• Specific transcript levels
• Total RNA profiling

Phenotype Prediction

• Drug response

Proteomics

• Disease
• Specific biochemical markers
• Protein profiling

Applying Pharmacogenomics

Discovery Development

• DISEASE TARGET SELECTING PHARMACO-GENETICS RESPONDERS GENETICS VARIABILITY
• Improving
• Choosing Better Predicting Early the Best Understanding Efficacy and Decision Targets of Our Targets Safety Making
• Goal: use genetics to broaden drug’s therapeutic index

Drug Efficacy in an Individual Patient

Efficacy: % patients cured at a given dose

Toxicity: % patients exhibiting side effects at a given dose

Therapeutic index: Dose range at which drug shows highest efficacy and low toxicity

Drug Efficacy in Patient Population

• Dose (mg/kg) 0 1 2 3 4 5 6 7 8 9
• Efficacy

Drug Efficacy in Patient Population

• Dose (mg/kg) 0 1 2 3 4 5 6 7 8 9
• Patient 1
• Patient 2
• Patient 3
• Patient 4
• Patient 5
• Patient 6
• Patient 7
• Patient 8
• Patient 9
• Patient 10

Drug Toxicity for Individual Patient

Dose (mg/kg) 0 1 2 3 4 5 6 7 8 9

Toxicity

Drug Toxicity in Patient Population

• Dose (mg/kg) 0 1 2 3 4 5 6 7 8 9