1
Q
Once a gene is mapped, it becomes possible to
A
- Obtain a clone copy of gene, obtain its DNA sequence and study function of the normal protein product.
- Understand molecular pathogenesis/ cause of genetic disease
- Develop diagnostic test
- Drug treatment eg. Manufacture normal gene product.
- Gene therapy: modify gene in affected individuals.
2
Q
Resources that advance mapping
A
- Human genome project
- Maps of millions of genetic markers across the genome, position of every marker is known.
- Having markers allows us to investigate the co-inheritance of locus with marker, as a proxy for where disease locus is located on a chr.
3
Q
Characterise linkage analysis
A
- Family-based mapping approach
- Uses pedigrees to follow inheritance of disease and makers .
- Typically for monogenetic disorder
4
Q
Characterise association analysis
A
- Population-based mapping approach
- Compare frequencies of alleles (haplotype) in affected individuals from population with that of unaffected controls.
- Typically for complex disease
5
Q
What are the several possible ways to Identify genes in monogenic disorders
A
- From a known protein product
- Candidates gene approach
- Positional-dependent strategies
- Mutation screening
6
Q
How do you identify genes in monogenic disorders from a known protein product.
A
- Enough protein can be purified to obtain partial aa sequence, screen cDNA libraries to obtain cloned copy of gene.
- Example: 1. Haemophilia A- blood clotting factor VIII
7
Q
How do you identify genes in monogenic disorders from candidate gene approach.
A
- Knowledge of biology of condition can suggest plausible gene candidate.
- E.g animal model with similar phenotype to human patients.
8
Q
How do you identify genes in monogenic disorders using positional-dependent strategies .
A
- Standard route
- Find the chromosomal position of disease-causing gene.
- Make use of genetic maps and linkage analysis
- Can sometime use chromosomal abnormalities.
9
Q
How do you identify genes in monogenic disorders using mutation screening.
A
Confirm disease-associated mutations in patients
10
Q
Define haplotypes
A
Series of alleles at linked loci that are co-inherited on a single chromosome.
11
Q
How does distance affect loci recombination ?
A
- Alleles at distantly spaced loci on the same chr are more likely to be separated by recombination .
- Alleles will not always be co-inherited
- Distance will influence how often the alleles are recombined.
12
Q
Relationship between unit of map distance is centimorgan (cM) and recombination.
A
1 cM ( 1 million bp(1Mb)) represents 1% recombination
13
Q
Detecting recombination events between loci require that?
A
A parent is heterozygous for both the loci.
14
Q
What is the first step toward identify a particular gene.
A
Gene mapping
15
Q
Computer programs that calculate max likelihood estimate calculate what two alternative probabilities.
A
- Likelihood of maker data if it is linked to disease locus at specific recombination fractions.
- Likelihood of marker data, assuming that marker is unlinked to disease locus.
16
Q
After disease gene is located what happens.
A
Identify all genes in region, select positional and functional candidates and systematically test all, e.g compare DNA of normal/ affected individuals to find the correct causative gene mutation.
17
Q
How to map a disease gene by linkage analyses.
A
- Obtain families in which genetic disease is segregating.
- Construct pedigree+ identify all affected individuals.
- Type all individuals for polymorphic markers scattered throughout the genome -> genome-wide linkage scan.
- Do finer mapping with markers in this region.
18
Q
Define autozygous
A
Markers or genes at a particular locus that are identical as a result of descent from a common ancestor.
19
Q
In a family if a region of autozygosity shared by all affected and none of unaffected, what does this mean?
A
Extremely likely disease gene present in shared region.
20
Q
Explain whole exome sequencing.
A
- Exome sequencing involves first capturing exons from the DNA of affected individuals, and then sequencing the captured DNA.
- In practice, exome capture is designed to capture exons with a little flanking intron sequence (to cover splice junctions) plus DNA sequences specifying some miRNAs; hybridization with a control set of cloned exon sequences allows capture of the desired exons
21
Q
Steps of exome sequencing
A
- Genomic DNA from subject are fragmented
- Cloned exon probe attached to biotin are added to fragments and hybridise to fragments of interest.
- Magnetic beads coated with streptavidin are added. The magnetic beads bind to the biotin attached to the fragments of interest.
- Unattached fragments are washed away.
- Elute the fragments
- Sequence the extracted exons
22
Q
How to sequence and analyse trios.
A
- Compare child’s variants with database of variants found in general population.
- Exclude common variants unlikely to cause a Rare disease.
- Select variants with pattern of inheritance consistent with the specific case. Such as dominant, recessive and sporadic cases.
- Evaluate candidates genes for relevance to child’s symptoms.
- Evaluate variants for likelihood of effect on gene or protein function.
23
Q
Explain what a trio analyses is ?
A
- Trio is made of two-parent and-child
- trios are used to assess the frequency of de novo mutations
24
Q
Explain why we see different phenotypes in monogenetic diseases.
A
The difference in phenotype can be explained if affected family members (who would be expected to have the same disease allele or alleles) have different alleles at one or more modifier loci. The product of a modifier gene interacts with the disease allele in some way: it may regulate expression of the disease allele, or it may interact in the same pathway as the product of the disease allele so as to affect its function.
25
Distinguish the difference between single-gene disorder vs polygenic disorder.
1. What distinguishes a single-gene disorder is that, although there may be minor effects from variants at other genetic loci, rare genetic variants at a primary gene locus have a very great effect on the phenotype.
2. polygenic disorder are one in which the genetic susceptibility to disease risk is not dominated by a primary locus where individual variants can have extremely strong effects on the phenotype.
26
What does it mean when many normal traits and common disorder show definite family tendency that cannot be explained by Mendelian inheritance patterns.
The traits and disorders are not determined by a single primary gene.
27
What two things affect complex (multi factorial) traits/ disease.
1. A complex disease is caused by many different genetic factors therefore shows polygenic inheritance, each gene makes a small contribution to expression of the phenotype.
2. Characteristically , a complex disease is also influenced by non-genetic factors: include environmental and chance factors.
28
What is the polygenic theory of complex diseases
1. Each gene that contributes to a polygenic theory has an additive effects.
2. Because of this we see continuous susceptibility to the disease within the population.
3. Which means the disease only manifests when certain threshold is exceeded .
29
What are the two main categories of multi factorial traits.
1. Continues [Quantitative] traits: everyone has trait , but differing degrees
2. Discontinuous/ dichotomous [qualitative ] traits : disease is either present or absent in individual.
30
Compare threshold/ liability model curves(bell curve) between the general population and relatives of affected.
We will see the relatives, curve shifted to the right. Meaning there will be a larger area exceeding threshold therefore susceptibility for disease is larger among relatives of affected because relatives share more high susceptibility alleles and/or adverse environment factors.
31
Explain what empiric risk is?
Risk based on observed outcomes in survey of families or certain groups of people.
E.g incidence of disorder in a UK population is 1/1500 people.
32
Empiric risk is influenced by?
1. Relatedness- the more closely related the higher the risk
2. Higher risk when there are more affected people
3. If there is a higher incidence in one particular sex, then the sex is at higher risk
4. If there is a severely affected pro and - there is a higher risk in relatives.
33
Define heritability [h2]
Proportion of variance attributable to genetic factors .
Value of h2 = 0 , no genetic factor
Value of h2 = 1 genetic factors exclusively responsible for phenotypic variance
34
Main characteristics of quantitative diseases affect individual cluster in families, why?
1. Indicates a genetic component, since family members share more alleles than average population therefore family members have certain pre-disposition for a qualitative disease because of shared alleles.
2. Families also share environmental influences eg, diet, sociology-economic status.
35
How do you Determine relative risk ratio.
1. Compare frequency of disease in relatives of affected to frequency off disease in general population.
2. Larger risk ratio = greater familial aggregation (depends on disease recurrence in family and population prevalence
3. If risk =1, indicates that recurrence risk for relatives of affected person is the same as for any other individual in general population.
36
Compare concordance between genes and environment
1. If genes play a role to predisposition -> frequency of disease concordance will increase with the degree of relatedness.
2. If the environment plays a significant role -> the concordance will be similar between all relatives, and to general population.
37
Define concordant
1. When two related individuals from same family have the same qualitative disorder.
2. Liability of both exceeds the threshold.
38
Define discordant
1. When one member of a pair of related individuals is affected with disorder and other one is not.
2. Affected person is above threshold, other is below threshold.
39
How to interpret results with twin studies .
1. If disease concordance <100% in monozygotes, evidence that non-genetic factors plays a role in disease.
2. If concordance rate in monozygotes is the same as in dizygotic, environment plays a very important role, not heritable
3. If greater concordance in monozygotes vs dizygotes, provides strong evidence of significant inherited genetic component to disease.
40
Limitation of twin studies
1. Monozygotes twins do not have identical gene expression(or genes), despite starting out with identical genotypes.
2. Assumes that both types of twins share similar experiences.
3. Better way would be to study twins separated at birth -adoption studies.
41
Explain what non-parametric linkage analysis
1. Model-free method that does not assume any particular mode of inheritance, it is used to analyse segregation patterns in complex disease.
42
Explain what non-parametric LOD (NPL) is
Allows mapping of genes in which variants contribute to susceptibility for diseases
43
NPL scores are based on ? And what are the types of NPL score testing?
1. Testing for excessive allele (or haplotypes sharing) sharing
Types:
2. Affected pedigree member method: analyse all affected family members in a family
3. Affected sib-pairs: analyse from multiple families
44
What are thresholds for statistical significance in affected sib-pair analysis .
1. Suggestive linkage : NPL score = 2.2
2. Significant linkage : NPL score = 3.6
3. Highly significant : NPL score = 5.4
45
Identifying disease-susceptibility gene
1. Need to find the condidtate chromosome region which can be hard because siblings share enlarge chromosomal segments.
2. Additional linkage disequilibrium mapping
46
Principles of linkage
1. Is a genetic phenomenon that relates to the position of loci on chromosome.
2. Based on co-segregation of marker and disease loci in families.
3. Different marker allele may co-segregate with disease locus in unrelated families.
4. Course mapping (>1cM)
47
Principles of Association
1. Is a statistical property of correlation between two factors.
2. Based on non-random co-occurrence of a specific allele with disease.
3. Studied by analysing samples from individuals within populations.
4. Fine mapping (<1cM)
48
Explain the case-control design
1. Analyse DNA of two groups in the population: individuals with disease and similar group of people without disease.
2. Statistical tests to verify that certain genetic variants are more prevalent I affected vs non-affected individuals
49
During the case-control design, if allele a1 at locus A is significantly more positively associated with complex disease.
Disease-susceptibility factor
50
During the case-control design if allele A1 at locus a is significantly more negatively associated with complex disease.
Disease-protective factor
51
What are the concerns of association studies.
1. Concerned with allelic combinations in haplotypes.
2. Looks at total independence (linkage equilibrium ) and non random association (linkage disequilibrium)
52
What will the frequency of haplotypes in a population that are in linkage equilibrium be.
(0.5 x 0.5)=0.25
53
Explain the method of Genome-wide association study ?
Scan genomes from many different people, look for markers that can be used to predict the presence of a disease.
54
What is the goal of the HapMap Project.
Develop complete maps of haplotypes and linkage disequilibrium in genome, using dense collection (millions) of SNPs across genome.
55
Common SNPs were genotyped in four geographically distinct groups.
1. European population
2. West African population
3. Han Chinese population
4. Population from Japan
56
What are haplotype blocks
1. SNPs very close together group into clusters on genome.
2. SNPs in any one cluster show high levels of linkage disequilibrium (LD) with each other, but not with SNPs outside that cluster.
3. Each block has limited genetic diversity
4. 10-20 Kb haplotype blocks
57
What is a Tag SNP
Single SNP can be used as marker for entire block.
58
Selecting haplotypes tag SNPs
1. Identify SNPs in DNA samples from multiple individuals
2. Adjacent SNP that are inherited together are compiled into haplotypes
3. Tag SNP within haplotypes are identified that uniquely identify those haplotypes.
59
Steps of typical GWAS
1. Selection of cases with disease and control group
2. DNA isolation and genotype
3. Statistical tests for association (SNPs and disease)
4. Replication of identify associations
60
Limitations of GWAS
1. GWAS is indirect
2. Must identify causal variant
3. Few variants show strong predisposition to disease
4. Majority of studies identify variants with a small (weak) effect on disease phenotype
61
Explain how GWAS is indirect
1. Mostly implicated SNP is associated with increased susceptibility but does not cause the disease.
2. Estimation of increased risk for developing the disorder NOT a prediction of disease.
62
Available GWAS data explain only same proportion of genetic variance of complex disease , raise what issues.
Missing heritability
63
Explain how large numbers of common variants with very weak effects (‘low penetrance’) is an explanation for missing heritability.
1. Many genuine susceptibility factors may be “missed” if they have a weak effect (OR<1.2)
2. To detect these variants-use much larger numbers of cases and controls in GWAS
3. Also do meta-analyses which aggregates data from multiple individuals studies.
64
Explain how rare variants of large effect explain missing heritability.
1. GWAS restricted to association with common variants.
2. Much of disease susceptibility may be due to a set of rare variants with individually strong effects
65
Explain how gene-gene interactions explain for missing heritability.
1. Concept of heritability assumes additive effects by different loci.
2. Proportion of heritability explained by known GWA variants does not take into account genetic interactions between loci.
66
Explain how gene - environment interactions explain for missing heritability.
1. Heritability separated into genetic and environmental components.
2. Simplistic because genes interact with the environment.
67
List the alternative explanations for missing heritability.
1. Large numbers of common variants with very weak effect (“low penetrance”)
2. Rare variants of large effect.
3. Gene-gene interactions
4. Gene-environment interactions
68
What are the two competing hypotheses for the relative contributions of common and rare variants to complex disease susceptibility.
1. common disease-common variant
2. Common disease - rare variant
69
Explain the common variant hypothesis.
1. Uses association studies (use SNPs) . Common variants of ancient origin shared by many individuals. Minor allele frequencies of selected SNPs > 0.05
2. Common disease susceptibility alleles have ancient origin, the alleles are mildly deleterious s(weal effects) therefore combinations of common variants increase disease risk.
3. Avoid elimination by natural selection because little or no effect on reproductive rates and/or have some selective advantage.
4. Therefore states that disease risk in specific individuals is due to aggregation of common variants therefore we’re a steep falling away of disease risk in relatives of a person affected with common disease. Common disease often appear as sporadic cases.
70
What is an example of common disease-common variant hypothesis.
Risk of late -onset Alzheimer’s disease
71
What is the gene and its isoforms are associated with late-onset Alzheimer disease susceptibility.
Apolipoprotein E gene- 19q: polymorphic with three major protein isoform.
1. E2,E3 and E4
2. E4- allele associated with increased risk of Alzheimer disease
3. E2- allele associate with decreased risk of Alzheimer’s disease
72
What are the difference between the proteins isoforms of the Apolipoprotein E gene.
APOE*E2= homozygous cysteine = low risk
APOE*E3=heterozygous arginine = intermediate
APOE*E4 = homozygous arginine= high
73
Explain the common disease-rare variant hypothesis
1. Rare DNA sequence variations are major contributors to genetic susceptibility of common disease likely only a few generations old, results of recent population growth.
2. Moderately rare variants may have moderate effects more deleterious than those of common susceptibility factors.
3. Very Rae variants are expected to be more deleterious, thus highly penetrant. Variants with strong effect would not persist for many generations in population, be eliminates by natural selection.
Are confined to groups of related families, will not appear on common haplotypes blocks of ancient origin, not currently detected by GWAS.
74
The basic concept of common disease-rare variant hypothesis.
The rare the variant the higher the effect (deleterious)
75
Example of protective factors.
1. FUT 2 non secretory allele.
2. Creates nonsense mutation in alpha(1,2)-fucosyltransferase gene.
3. Homozygote for non secretory allele do not present ABO antigens on cell surfaces of intestinal mucosa
4. Are strongly resistant to some strains of norovirus ( common cause of non-bacterial gastroenteritis)
5. But same person have increased risk for Crohn’s disease and type diabetes.
GTS 368
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