Genomi 7 Aplotype Con il termine aplotipo si definisce la combinazione di varianti alleliche lungo un cromosoma o segmento cromosomico contenente loci in linkage disequilibrium, cioè strettamente associati tra di loro, e che in genere, vengono ereditati insieme. Haplotype: A series of polymorphisms that are close together in the genome. The distribution of alleles at each polymorphic site is nonrandom: the base at one position predicts with some accuracy the base at the adjacent position. Persons sharing a haplotype are related, often very distantly. Haplotypes in Europeans are generally of the order of tens of kilobases long; older populations, such as those of West Africa, tend to have shorter haplotypes, since a longer period of evolutionary time means more meiotic events and a greater chance of population admixture, both of which result in shorter haplotypes. Aplotype Over the course of many generations, segments of the ancestral chromosomes in an interbreeding population are shuffled through repeated recombination events. Some of the segments of the ancestral chromosomes occur as regions of DNA sequences that are shared by multiple individuals. These segments are regions of chromosomes that have not been broken up by recombination, and they are separated by places where recombination has occurred. These segments are the haplotypes that enable geneticists to search for genes involved in diseases and other medically important traits. The fossil record and genetic evidence indicate that all humans today are descended from anatomically modern ancestors who lived in Africa about 150,000 years ago. Because we are a relatively young species, most of the variation in any current human population comes from the variation present in the ancestral human population. Also, as humans migrated out of Africa, they carried with them part but not all of the genetic variation that existed in the ancestral population. As a result, the haplotypes seen outside Africa tend to be subsets of the haplotypes inside Africa. In addition, haplotypes in non-African populations tend to be longer than in African populations, because populations in Africa have been larger through much of our history and recombination has had more time there to break up haplotypes. As modern humans spread throughout the world, the frequency of haplotypes came to vary from region to region through random chance, natural selection, and other genetic mechanisms. As a result, a given haplotype can occur at different frequencies in different populations, especially when those populations are widely separated and unlikely to exchange much DNA through mating. Also, new changes in DNA sequences, known as mutations, have created new haplotypes, and most of the recently arising haplotypes have not had enough time to spread widely beyond the population and geographic region in which they originated. Linkage e linkage disequilibrium • Linkage: l’associazione fisica degli alleli sui cromosomi • Linkage disequilibrium: l’associazione non casuale degli alleli di diversi loci nei gameti LINKAGE EQUILIBRIUM: indica una combinazione casuale di alleli a loci associati. Consideriamo per esempio il caso di due loci associati 1 e 2 con 2 possibili alleli ciascuno (A e a per il locus 1 e B e b per il locus 2). Gli aplotipi possibili in una determinata popolazione (AB, Ab, aB, ab) si verificheranno con una frequenza che è il prodotto delle frequenze dei singoli alleli per ciascun aplotipo. LINKAGE DISEQUILIBRIUM: indica una combinazione non casuale di alleli a loci associati. Il linkage disequilibrium è spesso la conseguenza di un effetto "founder" (fondatore), cioè di una mutazione in un singolo individuo. Perchè l'effetto fondatore sia evidenziabile in una popolazione è necessario che i due loci siano vicini, in maniera tale che gli eventi di ricombinazione siano rari tra i due loci, e che non sia trascorso abbastanza tempo dalla comparsa del fondatore poichè la ricombinazione puo' ristabilire nel tempo l'equilibrio. Linkage e linkage disequilibrium Il linkage disequilibrium (LD) indica la presenza di associazione statistica tra specifici alleli relativi a due o più loci, che costituiscono di solito un particolare aplotipo ancestrale, diffuso nella popolazione in cui è rilevato perché trasmesso lungo la discendenza da un comune progenitore. Per questo motivo il linkage disequilibrium è maggiore in popolazioni omogenee, cioè originate da un nucleo di individui fondatori come le popolazioni sarda o finlandese. Infatti le migrazioni recenti generano substruttura ed eterogeneità genetica e aplotipi differenti associati allo stesso tratto tendono ad abbassare i valori di significatività. Il linkage disequilibrium è un importante strumento per individuare regioni cromosomiche di limitata ampiezza in cui si collocano i geni per una data malattia (mappaggio ad alta risoluzione) e si avvale dell'analisi molecolare di varianti alleliche (per lo più SNPs) che costituiscono aplotipi in pazienti tra loro apparentemente non imparentati. Infatti è prevedibile che pazienti che hanno ereditato lo stesso segmento cromosomico, definito dal medesimo aplotipo, abbiano ereditato anche la stessa mutazione in esso contenuto. Origini del linkage disequilibrium (LD) Alla sua comparsa, una nuova mutazione è in LD (grigio) con tutti I loci dello stesso cromosoma. Attraverso le generazioni la ricombinazione riduce progressivamente l’area di LD. Contano soprattutto: 1. Tasso di ricombinazione 2. Numero di generazioni Attraverso le generazioni si riduce anche l’area di LD Nature Reviews Genetics 4; 701-709 (2003) Linkage and Linkage Disequilibrium Within a family, linkage occurs when two genetic markers (points on a chromosome) remain linked on a chromosome rather than being broken apart by recombination events during meiosis, shown as red lines. In a population, contiguous stretches of founder chromosomes from the initial generation are sequentially reduced in size by recombination events. Over time, a pair of markers or points on a chromosome in the population move from linkage disequilibrium to linkage equilibrium, as recombination events eventually occur between every possible point on the chromosome. Association Studies and Linkage Disequilibrium • If all polymorphisms were independent at the population level, association studies would have to examine every one of them… • Linkage disequilibrium makes tightly linked variants strongly correlated producing cost savings for association studies This diagram shows two ancestral chromosomes being scrambled through recombination over many generations to yield different descendant chromosomes. If a genetic variant marked by the A on the ancestral chromosome increases the risk of a particular disease, the two individuals in the current generation who inherit that part of the ancestral chromosome will be at increased risk. Adjacent to the variant marked by the A are many SNPs that can be used to identify the location of the variant. Il genoma umano è diviso in blocchi di aplotipi, al cui interno la ricombinazione è rara Nove diversi blocchi di aplotipi in questa regione Genome-wide association study (GWAS) Genome-wide association study (GWAS) Genome-wide association study (GWAS) is an approach used in genetics research to associate specific genetic variations with particular diseases. The method involves scanning the genomes from many different people and looking for genetic markers that can be used to predict the presence of a disease. Once such genetic markers are identified, they can be used to understand how genes contribute to the disease and develop better prevention and treatment strategies. Genome-wide association study (GWAS) A variety of resources have been developed to catalog common single nucleotide polymorphisms (SNPs). More recently, data from the 1000 Genomes Project have begun to be used to catalog variants in the 1% frequency range. In order to test whether these common SNPs are associated with risk of disease, commercial ‘SNP chips’ or arrays have been developed that capture most, although not all, common variation in the genome. These genotyping arrays can genotype hundreds of thousands of SNPs in a single experiment, at a cost of several hundred US dollars per sample. Contemporary GWASs use these arrays to measure the frequency of alleles in cases compared with controls. If the difference in allele frequency reaches a stringent level of statistical significance that corrects for the fact that there are about 1,000,000 independent common SNPs in the human genome (this significance level is about P < 5 × 10-8), then the allele is said to be ‘associated’ with disease. GENOME WIDE ASSOCIATION STUDY Christensen and Murray 356 (11): 1094, Figure 1 March 15, 2007 Joel N. Hirschhorn & Mark J. Daly Nature Reviews Genetics 6, 95-108 (February 2005) Example calculation illustrating the methodology of a case-control GWA study. The allele count of each measured SNP is evaluated, in this case with a chi-squared test, in order to identify variants associated with the trait in question. The numbers in this example are taken from a 2007 study of coronary artery disease (CAD) which showed that the individuals with the G-allele of SNP1 (rs1333049) were overrepresented amongst CAD-patients Published Genome-Wide Associations through 12/2012 Published GWA at p≤5X10-8 for 17 trait categories NHGRI GWA Catalog www.genome.gov/GWAStudies www.ebi.ac.uk/fgpt/gwas/ The Human Reference Genome The Genome Reference Consortium http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/