BCH 4101 Lecture Notes - Lecture 6: Genome-Wide Association Study, Linkage Disequilibrium, Reverse Genetics
28 Apr 2018
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October 16, 2017
Genome Wide Association Studies (GWAS)
Finding a Disease-Causing Gene
*Forward genetics: identify a gene that confers a phenotype (even if very little is known about the phenotype)
-Have a phenotype and try to identify the gene that is responsible
•Establish Mendelian inheritance
•Obtain DNA samples from members of affected family
•Pedigree analysis to determine linkage between disease gene and mapped DNA markers
•Look in that particular area for a list of possible genes that are responsible for the disease
-Based on the principles of linkage disequilibrium
•Based upon the principle that the gene and the marker are less likely to be separated during recombination
*Reverse genetics: start with a gene sequence and try to determine the associated phenotype (figure out what a gene
does)
-Study expression patterns
•Determine what phenotype or areas of the body that the gene is expressed in
-Search for homologs - BLAST
•If a gene is important enough that a single mutation can cause a disease, it is most likely has a very important
function and is conserved in other organisms
•Allows you to determine possible functions of the gene if the gene function is known in other organisms
•Further narrows down the list
-Analyze DNA sequence of affected and unaffected individuals
•Sequence the DNA for the genes of interest and try to find mutations that appear more frequently in the affected
individuals but are absent in the healthy individuals
-***NEED TO KNOW DIFFERENCE BETWEEN FORWARD AND REVERSE GENETICS
Ex. The case of BRCA1 - mutation increases probability of getting breast cancer
-Found that it was linked to an allele of the marker D17S74 and were able to map it to 17q21
-Pedigree analyses with additional markers
•Mapped between markers D17S1321 and D17S1325
•Markers are approximately 600 kb apart
-Used positional cloning/mapping to identify the genes (done before the humane genome project)
•Identify particular markers within the 600 kb stretch
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October 16, 2017
•Make BAC clones covering the 600 kb region that will act as probes for mRNA sequences
•Hybridize BAC clones to cDNA library to see which sections are being expressed
•Sequence the cDNAs and see if there were any corresponding the mutations in the isolated genes in the
affected patients
-NB: Now, we can use a genome browser instead in order to find the genes located between the 2 markers
•Use reverse genetics to narrow down the list of candidate genes
-Most likely candidate gene: BRCA1
•100 kb gene (22 exons, 1863 aa protein)
Complex Traits/Diseases
Complex traits/diseases examples: coronary heart disease, osteoporosis, high blood pressure, certain types of
cancer, eye colour, height, hair type
Complex traits: more complex hereditary patterns than Mendelian inheritance
-Complex traits are not coded by a single locus, so they do not follow a Mendelian inheritance pattern
-Can’t use pedigree data to link the disease to the DNA markers with any degree of certainty because there are
several genes that come into play
Hereditary patterns include:
-Incomplete penetrance (predisposition): mutant genotype does not necessarily result in a mutant phenotype
•Makes you more susceptible to get the disease, but does not mean that you will get the disease
-Phenocopy: mutant phenotype not caused by an inherited mutation
•Phenotype, but not genotype
•Not a familial mutation, but it mimics the same phenotype (i.e. spontaneous mutation mimics the same
phenotype)
-Genetic heterogeneity: mutations at 1+ locus cause the same phenotype
•Different genotypes, same phenotypes
-Polygenic heredity: 2+ genes interact to influence expression of the phenotype
Dissection of Complex Traits
How do we find the loci responsible for the disease phenotype? —> use haplotype association analysis
-Haplotype: “haploid genotype”
•Set of SNPs (2+) present on a chromosome and genetically (statistically) linked (i.e. inherited from the same
parent)
•Blocks of SNPs that are very rarely separated by recombination
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October 16, 2017
-Want to find haplotypes that are associated with healthy and diseased states
-The genetic distance occupied by multiple loci of a haplotype has to be short enough to ensure that the alleles
remain associated throughout the generations (1-100 kb)
•Need to be short enough to ensure that it won’t be broken up by recombination)
•A distance of 100kb ~ 0.1 cM —> 1/1000 chance that the haplotype will be broken up by recombination
•Recombination in a haplotype of 1 kb will occur at a frequency of 1/100 000 generations
-Ex.
-Majority of SNPs have neighbours that are almost perfectly correlated (SNP genotypes predicts genotype of
neighbouring SNP)
-Therefore, a few SNPs can become “representatives" of a particular haplotype - cuts down on the need to
genotype
•Use tag SNPs for analysis in order to determine haplotype: representative SNPs
-A combination of SNPs that allow you to distinguish between the haplotypes
•Need to only genotype the positions of the tag SNPs in order to determine haplotype
•Allows us to rapidly determine haplotype without needing to sequence the whole sample
•Ex. Only need to look at 3/5 SNPs
-Only need to sequence 3 positions
Haplotypes and Evolutionary Time
Haplotypes are used to track the inheritance over many many generations
-Over time the mutations will accumulate, but people with the same haplotype more likely inherited it from the same
ancestor
•Unlikely that people will spontaneously gain the exact same combination of SNPs
-Ex. Used to test the Out of Africa theory
20#SNPs#that#span#
6kb#of#DNA
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Document Summary
*forward genetics: identify a gene that confers a phenotype (even if very little is known about the phenotype) Based on the principles of linkage disequilibrium: based upon the principle that the gene and the marker are less likely to be separated during recombination. *reverse genetics: start with a gene sequence and try to determine the associated phenotype ( gure out what a gene does) Study expression patterns: determine what phenotype or areas of the body that the gene is expressed in. Analyze dna sequence of affected and unaffected individuals: sequence the dna for the genes of interest and try to nd mutations that appear more frequently in the affected individuals but are absent in the healthy individuals. ***need to know difference between forward and reverse genetics. The case of brca1 - mutation increases probability of getting breast cancer. Found that it was linked to an allele of the marker d17s74 and were able to map it to 17q21.
