Study Unit 4 Flashcards
Once a gene is mapped, it becomes possible to
- 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.
Resources that advance mapping
- 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.
Characterise linkage analysis
- Family-based mapping approach
- Uses pedigrees to follow inheritance of disease and makers .
- Typically for monogenetic disorder
Characterise association analysis
- Population-based mapping approach
- Compare frequencies of alleles (haplotype) in affected individuals from population with that of unaffected controls.
- Typically for complex disease
What are the several possible ways to Identify genes in monogenic disorders
- From a known protein product
- Candidates gene approach
- Positional-dependent strategies
- Mutation screening
How do you identify genes in monogenic disorders from a known protein product.
- 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
How do you identify genes in monogenic disorders from candidate gene approach.
- Knowledge of biology of condition can suggest plausible gene candidate.
- E.g animal model with similar phenotype to human patients.
How do you identify genes in monogenic disorders using positional-dependent strategies .
- Standard route
- Find the chromosomal position of disease-causing gene.
- Make use of genetic maps and linkage analysis
- Can sometime use chromosomal abnormalities.
How do you identify genes in monogenic disorders using mutation screening.
Confirm disease-associated mutations in patients
Define haplotypes
Series of alleles at linked loci that are co-inherited on a single chromosome.
How does distance affect loci recombination ?
- 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.
Relationship between unit of map distance is centimorgan (cM) and recombination.
1 cM ( 1 million bp(1Mb)) represents 1% recombination
Detecting recombination events between loci require that?
A parent is heterozygous for both the loci.
What is the first step toward identify a particular gene.
Gene mapping
Computer programs that calculate max likelihood estimate calculate what two alternative probabilities.
- 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.
After disease gene is located what happens.
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.
How to map a disease gene by linkage analyses.
- 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.
Define autozygous
Markers or genes at a particular locus that are identical as a result of descent from a common ancestor.
In a family if a region of autozygosity shared by all affected and none of unaffected, what does this mean?
Extremely likely disease gene present in shared region.
Explain whole exome sequencing.
- 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
Steps of exome sequencing
- 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
How to sequence and analyse trios.
- 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.
Explain what a trio analyses is ?
- Trio is made of two-parent and-child
- trios are used to assess the frequency of de novo mutations
Explain why we see different phenotypes in monogenetic diseases.
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.
Distinguish the difference between single-gene disorder vs polygenic disorder.
- 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.
- 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.
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.
What two things affect complex (multi factorial) traits/ disease.
- 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.
- Characteristically , a complex disease is also influenced by non-genetic factors: include environmental and chance factors.
What is the polygenic theory of complex diseases
- Each gene that contributes to a polygenic theory has an additive effects.
- Because of this we see continuous susceptibility to the disease within the population.
- Which means the disease only manifests when certain threshold is exceeded .
What are the two main categories of multi factorial traits.
- Continues [Quantitative] traits: everyone has trait , but differing degrees
- Discontinuous/ dichotomous [qualitative ] traits : disease is either present or absent in individual.
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.
