Association Analysis

Question 1

Define the term “Genetic Association”

Answer 1

The presence of an allele at a higher frequency in unrelated subjects with a particular trait, compared to those that do not have the trait

Question 2

How do we determine whether variants in the genome are associated with a disease?

Answer 2

If we substitute the word “disease” for “trait”.
- With disease = cases
- Without disease = Controls

Question 3

Describe how a genetic association study is conducted

Answer 3

• Cases are subjected with the disease of interest E.g obesity
• Definition of the disease must be applied consistently
• Controls must be as well (similarly) matched as possible for the non disease traits
• such as age, sex, ethnicity, location

Question 4

What other factors need to be catered for?

Answer 4

Match for all other risk factors
- affected/unaffected cases
- Measure genetic loci of interest
- Statistical analysis which genetic loci are associated with disease
- Identify genes/genomics region

Question 5

How would you make the study fair?

Answer 5

• Use a large number of well defined cases
• use an Equal number of matched controls
• Reliable genotyping technology (SNP microarray)
• Standard statistical amalysis (PLINK)
• Positive associations should be replicated

Question 6

How does using a genetic marker fit well in a genetic association study?

Answer 6

Individuals in a population are genetically far more diverse than individuals in a single family

Question 7

How is this genetic diversity captured?

Answer 7

• This genetic diversity is captured through reliable genetic markers
• Genetic markers are alleles that we can genotype and assess whether they are associated with the disease
• Association means <100kb from a casual variant

Question 8

What is the ideal Genetic Marker?

Answer 8

If it is
- Polymorphic
- Randomly distributed across the genome
- Fixed location in genome
- Frequent in genome
- Frequent in population
- Stable with time
- Easy to assay

Question 9

How is a Single Nucleotide Polymorphism (SNP) used in a genetic association study?

Answer 9

• Common in the genome ~1/300 nucleotides
• 12 million common SNPs identified in human genome
• Generated by mismatch repair during mitosis

Question 10

Where could SNP’s be found?

Answer 10

In the gene (Coding region)
- No amino acid change (synonymous)
- Amino acid change (non-synonymous)
- New stop codon (nonsense)

Question 11

Where else could SNP’s be found?

Answer 11

In the gene (Non coding region)
- Promoter: mRNA and protein level changed
- Terminator: mRNA and protein level changed
- Splice site: Altered mRNA, altered protein

Could also be found in the intergenic region (98% of genome)

Question 12

Describe what a dbSNP is

Answer 12

• An online database at NCBI, database of SNP’s
• The rs number is a unique identifier given to each SNP
• Has two unique flanking sequences between a single polymorphism

Question 13

Describe what a minor Allele Frequency is (MAF)

Answer 13

SNP’s have two forms. The major and minor form.
- The less common allele is called the minor allele
- Major allele frequency + Minor allele frequency = 1

Question 14

Why are SNP’s chosen for genetic association studies?

Answer 14

• SNP’s are chosen on the basis of their MAF
• Common diseases are likely to be caused by common variants
• SNPs with MAF >0.05 (5%) are usually used in association studies - GWAS
• Exceptions are known monogenic disease SNPs

Question 15

How is a Genome Wide Association Study carried out?

Answer 15

• Recruit large numbers of cases and controls
• Genotype markers across the whole genome
• Look for association between disease and alleles of each marker (Chi squared test)
• Positive association is at p<5x10-8 (multiple testing correction)

Question 16

How are the GWAS results plotted? VD

Answer 16

• Presented as a single graph called a Manhattan plot
• All results are plotted typically for >1M SNPs
• X-axis Is the position of the SNP on the chromosome
• Y-axis is -log10 (p-value) of the association

Question 17

View the results from the Wellcome Trust Case Control Consortium study. How are these results interpreted?

Answer 17

• The peak of association often does not identify the gene causing the disease
• The peak identifies the genomic region associated with disease and this is usually smaller than 100kb.

Question 18

When is a meta analysis used?

Answer 18

• When is it difficult to do large studies (>1K cases/controls)
• Easier to combine smaller studies
• Meta analysis allows the statistical combination of regulars from multiple studies

Question 19

What are the problems with GWAS?

Answer 19

• GWAS has identified associations that are statistically strong and reproducible
• However their contribution to the genetic component of disease is estimated to be low (<5%)

Possible answers
- Many common SNPs of very small effect
- Rare SNPs
- Copy Number Variation
- Epigenetic variation

Question 20

What are some of the medical complications of obesity?

Answer 20

• Pulmonary disease
• Coronary Heart Disease
• Severe pancreatitis
• Stroke
• Cancer

Question 21

How strongly is Obesity linked to genetics?

Answer 21

• Twin studies: 70-80% of body shape is genetically determined
• Adoption Studies: 30-40%
• Family Studies: 40-60%

Question 22

What was the results of the large scale meta analysis done on obesity?

Answer 22

• BMI meta analysis in 322k subjects
• 97 BMI associated loci
• 125 separate studies
• > 60l authors and >2000 collaborators

Question 23

What are some advantages of GWAS?

Answer 23

• They have already highlighted hundreds of loci that are associated with obesity or BMI related traits
• Results are reproducible
• however there’s still a long way to understand how all these genes contribute to obesity

Question 24

What is the relationship between linkage analysis and recombination?

Answer 24

• During meiosis, recombination occurs at random points along the chromosome
• Recombination reduces the linkage between the two loci over time in proportion to the distance between them

Question 25

What is Linkage Disequilibrium?

Answer 25

• LD is when two alleles are inherited together more often than expected by chance
• This is usually because they are close together in the genome
• Alleles that are physically close together are rarely separated by recombination

Question 26

How is Linkage disequilibrium used in disease gene mapping?

Answer 26

• If a genetic marker allele and an undiscovered allele for disease susceptibility are in LD then the genetic marker will be associated with disease
• Genotype many marker alleles and we can find the regions of associations with disease - GWAS
• Remember that the marker alleles are close together in a GWAS so we would expect to see many alleles associated with disease in a region due to LD