a. Mapping and cloning disease-causing genes

No documento Peter St George-Hyslop (páginas 26-29)

Beginning in 1985, the author pioneered the use of molecular genetic strategies to dissect the causes and mechanisms of AD and other related de-mentias70,71. Multiple strategies have been developed to map and identify/

clone genes causing susceptibility to dementia.

One commonly-used strategy has been to investigate datasets with fami-lies multiply affected with dementia using genetic linkage and/or family-based association methods. Genetic linkage studies, using maximum likeli-hood (LOD score) methods (Figure 4, left panel) to assess co-segregation of dementia with anonymous polymorphic chromosomal markers (e.g. RFLPs, microsatellites, idels, etc.), have been highly successful when used in rare families with clear-cut monogenic Mendelian autosomal dominant inheri-tance of dementia. This genetic linkage approach has permitted the

chromo-TRANSLATING DISCOVERIES IN BASIC MOLECULAR BIOLOGY, CELL BIOLOGY, AND MOLECULAR GENETICS INTO TRANSFORMATIVE APPROACHES TO THE DIAGNOSIS AND TREATMENT OF CURRENTLY INCURABLE NEURODEGENERATIVE DEMENTIAS

somal mapping and then the subsequent positional cloning of multiple genes causing AD and FTLD - including the APP and PS1 genes (see below).

Family-based association studies, such as FBAT, which are better suit-ed to the analysis of families multiply affectsuit-ed by dementia, but where the inheritance pattern is less clear, have also been used. These studies led to the suggestion that, in AD for instance, there may be additional suscep-tibility loci in the pericentromeric region of chromosome 12 (Alzheimer Type 5)72, the long arm and pericentromeric region of chromosome 10q (Alzheimer Type 6)73, chromosome 20p74, 18q75, 15q2276, and 9p77. However, replications were less robust and the identities of the putative AD genes at those locations (if they really even exist) have not been de-fined for any of these loci.

A third strategy to identify dementia-causing genes has been to use cohorts of sporadic cases and age/sex matched controls in a case:control

LINKAGE BASED CASE : CONTROL ASSOCIATION

• Difficult to collect families

• Expensive

• Relatively few assumptions

• Robust directly observable results

• Easy to collect sporadic cases

• Cheap, quick

• Easy to mess up

• Requires assumption that cases and controls are from same founder population.

Figure 4: Genetic linkage studies, and to some extent family-based association studies rely on cosegregation of the trait and chromosomal markers to assign the location of a disease gene. In contrast, association-based methods (e.g. GWAS studies) look for differences in the frequency of a given allele in affected versus unaffected cohorts. This works best when the population is drawn from a homogeneous founder population so that all cases have the same genetic defect in the population. For admixed populations, where there are multiple different genetic variants causing the same disease, the power to detect association becomes less.

BASIC SCIENCE APPROACHES TO NEURODEGENERATIVE DEMENTIAS

association design (Figure 4, right panel). The two cohorts (cases versus controls) can be genotyped with genetic markers. These markers have recently typically been single nucleotide polymorphisms (SNPs) and/or insertion-deletion polymorphisms (indels). These SNPs or indels can be either within individual genes of interest (i.e. a candidate gene approach) or can be incorporated into arrays of 0.5 – 1.5 million SNPs randomly distributed across the genome. This latter approach - a Genome Wide Association Study (GWAS) is currently highly favoured as a tool for dissecting complex disorders like AD. It allows a robust, unbiased, hy-pothesis free survey for susceptibility genes across the entire genome. In doing so, it allows analysis of genes that would never be considered as candidates. Thus it allows consideration of genes whose function was un-known or whose function was in pathways that would not have suspected to be important in the disease biology. As described below, this approach recently indicted several genes involved in inflammation, and in doing so reversed previous dogma about the relative importance of inflamma-tion in these diseases. Previous ideas had suggested that inflammainflamma-tion was simply a late consequence of the disease. The discovery that inflam-mation pathway genes were in fact associated with susceptibility to the disease unequivocally revealed that inflammation was in fact a major and early component of the disease process. However, like all techniques GWAS approaches do have a number of important limitations includ-ing, most importantly, the fact that GWAS studies cannot detect genes in which there are multiple rare variants that occur on different SNP marker haplotypes.

The fourth approach, and one that is being increasingly pursued by the author’s lab and many other labs in the field, is the use of “next generation”

high-throughput DNA sequencing. This approach is well-suited to the dis-covery of rare variants that can frequently occur on different SNP haplotypes, and are thus not detected in standard GWAS studies. This high-throughput, deep sequencing technology can be focussed just on the coding sequenc-es (Whole Exome Sequencing), or on the entire genome (Whole Genome Sequencing). The author’s group together with colleagues78 has recently used this approach to identify disease-causing mutations in the TREM2 gene in affected members of several late onset AD families.

TRANSLATING DISCOVERIES IN BASIC MOLECULAR BIOLOGY, CELL BIOLOGY, AND MOLECULAR GENETICS INTO TRANSFORMATIVE APPROACHES TO THE DIAGNOSIS AND TREATMENT OF CURRENTLY INCURABLE NEURODEGENERATIVE DEMENTIAS

II.2.b. Clarifying aetiological heterogeneity and gene interactions

No documento Peter St George-Hyslop (páginas 26-29)