Finding Disease Genes (Dr. Spritz L1)

Question 0

Why is our ability to make accurate predictions on the role of genes in most diseases actually pretty low?

Answer 0

With a few exceptions, it is usually the case that genes do not confer the same risk of disease to all individuals.

Many genes interact (along with environment) to result in disease

We calculate an odds ration to assist in predicting risk

Question 1

What is the rationale for finding disease genes?

Answer 1

1. provide clues to pathogenic mechanisms
2. new approaches to treatment
3. inference of environmental risk factors
4. disease prevention

Question 2

In order for personalized medicine to work, what do geneticists have to discover first?

Answer 2

1. risk genes for common diseases
2. specific risk variants
3. high-risk gene combinations

Question 3

Once genetic the genetic risk of an individual is known, what is the next step in the application of personalized medicine?

Answer 3

once a person’s genes are known, an accurate DNA-based predictive diagnostic process based on their individual genetic risk can be conducted.

Question 4

Once a diagnosis based on an individual’s genetic risk is made, what is the next step in personalized medicine?

Answer 4

Use the genetic diagnosis of disease susceptibilities and pharmacogenetic analysis of optimized drugs (optimal efficacy and specificity) to design an individual treatment and/or preventative.

Question 5

What do Odds Ratios contribute to the landscape of personalized medicine?

Answer 5

Since the odds ratios of “most” genes are low, accurate prediction of genetic risk, once an individual’s genome is known, is actually pretty low.

Question 6

What is Odds Ratio (OR)?

Answer 6

OR = (risk of disease with a gene variant) / (risk of disease without a gene variant)

Question 7

Describe positional cloning a bit more.

Answer 7

First, PC is the way genes are really mapped today.

Positional cloning is a way to map a gene by focusing your attention on a specific region of the genome. You carry out a systematic analysis of all the genes in the suspected (disease causing) area and look for mutations and/or variants that contribute directly to a disease. (p. 208, blue box)

Question 8

Explain the difference between Functional Cloning (FC) and Positional Cloning (PC) and what are they used for?

Answer 8

FC and PC are different pathways for finding a disease gene within the genome.

FC: Disease –> Function –> Gene –> Map
basically, you investigate genes that you already know (Dr. Spritz investigated hemoglobin and thalassemia)

PC: Disease –> Map –> Gene –> Function
basically, you now have to look for the gene and deduce its function because you don’t necessarily know the function up front.

Question 9

What is a polymorphic DNA marker?

Answer 9

A marker can be a SNP or a CNV (of which microsatellites are a subset) at a known genomic position which we can “score”.

In Positional Cloning, polymorphic markers are used as surrogates for disease mutations (which are harder to pinpoint). Due to linkage disequlibrium, knowing which markers a person carries implies which disease genes they are carrying. Sort of a 2-for-1 deal

Question 10

What is a microsatellite?

Answer 10

stretches of DNA containing units of 2, 3 or 4 nucleotides repeating

• are multi-allelic
• the number of repeats of the unit will vary from person to person allowing for DNA fingerprinting.

occur 1 / 30,000 bp’s roughly

Question 11

What is a VNTR?

Answer 11

variable number tandem repeats = MINI-satellite
is a stretch of 100 to 1000 bps that then repeats in tandem along the dna
also varies person-to-person in the number of repeats of the 100-1000bp unit.

Question 12

What is a SNP?

Answer 12

bi-allelic

1/50-300 bp
used for association
The occurence / allele frequencies differ in different ethnic groups / populations

Due to linkage, it is likely that a particular haplotype will be passed on to the next generation unchanged. So is useful for identifying if people are related (through many generations) vs. not related

Question 13

What is a haplotype?

Answer 13

A combination of alleles (DNA sequence) at adjacent locations on a chromosome that are inherited together.

Can be one locus, several loci or an entire chromosome depending on the number of recombination events that have occurred between a given set of loci.

Question 14

What did / does the 1000 Genome project do?

Answer 14

Sequenced 1000 genomes from different ethnic groups

catalog human genetic variations based on SNP’s
useful for analyzing sequence-based RARE VARIANTS that may be causal for common diseases

Question 15

What is the international HapMap project?

Answer 15

The mapping of haplotypes of the human gene.

Question 16

Why do close polymorphism genotypes carried on the same chromosome cluster into haplotype blocks and how large are these haplotype blocks?

Answer 16

Haplotypes blocks of 10-50kb are inherited as a cluster because recombination turns out to be not completely random

Question 17

Within a haplotype block, SNP alleles are in ___________.

Answer 17

linkage disequlibrium

Question 18

What does it mean for two (or more) SNP’s to be in linkage disequlibrium with one another?

Answer 18

Due to their “close proximity” within a haplotype block, SNP’s in LD with one another are generally co-inherited. Recombination within a haplotype block is uncommon.

Question 19

How do LD blocks vary among ethnic groups? Specifically African vs. Caucasian or Asian popuations?

Answer 19

Since African populations are simply more ancient than Caucasians or Asian populations, the African LD blocks are smaller (about half the size).

The LD blocks that have been around longer have undergone more recombination events so eventually SNP’s will be separated and no longer co-inherited.

Question 20

What is a CNV and how do they vary among individuals / ethnic groups?

Answer 20

copy number variant

bi-allelic, multi-allelic or unique

a common genomic deletion, 100’s to 10k’s bp long
occurance differs in different ethnic groups
individually rare (≤1%), collectively common
can be genes or not, can include genes
Not certain how often CNV’s are causal for human disease

Question 21

What does it mean to “score” a genetic variation?

Answer 21

to Assay. to determine the presence or absence of a genetic variation.

Question 22

Explain the difference between a Medelian and a Complex disease or trait.

Answer 22

Mendelian = single-gene; one gene is sufficient to cause disease phenotype

Complex = multi-gene; no one gene is sufficient to cause the disease.

Question 23

What would you rather be doing right now?

Answer 23

Driving to Lake City to take a hike up in the Uncompahgre plateau.
I bet it’s cold up there right now!

Question 24

What are the two broad categories of how one goes about finding a disease gene?

Answer 24

1. Hypothesis driven approach

2. Hypothesis-free approach

Question 25

What are the main categories of hypothesis driven approaches to finding a disease gene?

Answer 25

1. Candidate gene sequencing
   uses DNA sequencing to directly study a gene
2. Candidate gene association studies
   tests the gene - causal relationship indirectly

Question 26

What are the hypotheses that a candidate gene sequencing study might depend on?

Answer 26

1. Biological hypothesis
2. positonal hypothesis
3. a “hit” from a GWAS or other mapping method

basically: you think its a particular gene and you test the theory

Question 27

What sort of diseases is a candidate gene sequencing study useful for? Not useful for and why?

Answer 27

successful for mendelian gene diseases

not successful for complex disorders (unless already positive in a GWAS study) because often normal individuals will be positive for the suspected pathogenic variant

Question 28

What is a candidate gene association study? What does it depend on a priori?

Answer 28

Tests a gene or causal variant indirectly

• relies on a biological or positional hypothesis (candidate)
• most useful for common risk allelse with small to moderate effects (OR)

Unfortunately, most a priori biological hypotheses are wrong and there usually is some fatal flaw in the study that leads to a false positive

Question 29

In a genetic association study, what sort of statistical analysis might be performed?

Answer 29

Chi-square
Fischer exact test

look for p < 0.05, 0.01 etc.

multiple testing corrections applied in multiple variant testing

Question 30

In a genetic association study, what does real association imply?

Answer 30

NOT causation necessarily.

Does imply LD with a causal mutation

Question 31

What are two fatal flaws in gene-by-gene case-control design?

What may lead to a false positive in such a study?

Answer 31

1. Must include all tests that have been done in order to apply true multiple-testing corrections (and all tests may or may not be known to you / published) (e.g. the failures may not have been published!)
2. Background genetic variation may vary among populations. So the variation you are observing may be ethnic variation instead of case vs. control variation!
   * “Stratification” (occult pop. differences) may lead to false positives

Question 32

What percent of published “confirmed” gene-by-gene case-control studies have turned out to be false? Why?

Answer 32

96%
“comfirmed” means same results published 3 times independently
*Generally only “positive” studies are published and occult stratification of populations skews results

Question 33

What is a hypothesis-free genetic disease study?

Answer 33

By doing a linkage study one can study genes indirectly. The hypothesis-free does not start out with a particular gene of interest. rather the gene is uncovered as a result of the linkage study.

Question 34

What is the basis of study in a linkage study?

Answer 34

multiplex families, followed through many generations, in which there are many instances of a disease

best for mendelian traits (uncommon alleles with strong effects)

Question 35

What type of study was used to find the neurofibromatosis gene?

Answer 35

a genetic linkage analysis within families that shared the neurofibromatosis disease.

Question 36

In a linkage analysis test what is the statistical measure of choice?

Explain what the values of this measure mean?

Answer 36

LOD score (log of odds)

Significance level:
LOD ≥ 3 for Mendelian trait
LOD ≥ 3.3 for Polygenic trait

Question 37

What is the fundamental question in a GWAS?

Answer 37

Look at the entire genome and ask what is different in people with the disease vs. not the disease…genome-wide.

Study the gene indirectly b/c don’t have a single candidate gene that you are focusing on a priori

Question 39

How does a GWAS differ from a candidate gene case-control study?

Answer 39

Candidate Gene Case-Control = study gene directly, hypothesis dependent, usually wrong

GWAS = study gene indirectly (like association study), hypothesis independent, studies millions of SNP’s simultaneously