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29 Cards in this Set
- Front
- Back
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Genetic testing / Genetic diagnosis |
Use of DNA technology to identify or exclude pathogenic mutations in individuals at risk for genetic disease. |
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Why test ? |
Establish diagnosis in symptomatic individual Risk of current pregnancy being affected Carrier risk to individual or couple Predictive testing for adult onset conditions, e.g. familial cancers, etc. |
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Direct [DNA] diagnosis |
Used to detect mutations in patients when the disease gene is known |
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Indirect [DNA] diagnosis |
Method of choice when disease gene has been localized to a specific chromosomal region BUT has not been cloned Or large gene & difficult to find mutations |
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Direct DNA testing |
Mutation scanning / screening Mutation testing / detection |
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Mutation scanning / screening |
Detect & localize unknown mutations |
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Mutation scanning / screening; Which method(s)? |
Determined by the type / locality of the mutation Frequency and nature of mutations vary within different genes and populations |
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Mutation testing / detection |
Detection of known or common mutations |
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Mutation detection: Detect specific mutation... found in same gene in all individuals? |
Some diseases - specific mutation in all affected Some diseases show limited range of mutations: Defined “set” of mutations that are common-Could be founder mutations Screen for common mutations (e.g. repeat expansions, a specific missense, deletion of an amino acid in CF the ΔF508 mutation, etc. |
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Methods of testing for specific mutation(s) |
PCR / Enzyme digestion ARMS (Amplification refractory mutation system) Allele specific oligonucleotides (ASOs) Multiplex PCR Long range PCR |
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Nature of mutations |
Point mutations, small insertion /deletions Large insertion/deletions Gross re-arrangements Expansion of triplet repeats |
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Frequencyof mutations; Point mutations, small insertion /deletions |
Together account for most mutations >50% point mutations C>T Chain terminating mutations common in APC, BRCA1 and BRCA2 (>80%), etc |
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Frequency of mutations; Large insertion/deletions |
Dystrophin (deletions) and PMP (duplications) |
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Frequency of mutations; Gross re-arrangements |
Haemophilia A |
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Frequency of mutations; Expansion of triplet repeats |
FraX A , HC, DM, Friedrich’s Ataxia,etc |
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Screening methods for detecting undefined mutations |
SSCP/HA: single strand conformation polymorphism /heteroduplex analysis DGGE: denaturing gradient gel electrophoresis PTT: Protein Truncation Test MLPA: Multiplex Ligation-dependent Probe Amplification |
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Criteria for designation of mutation as being of pathogenic significance |
Mutation type Amino acid affected Segregation Prevalence of mutation Functional analysis |
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Mutation type |
In coding regions causing truncation [frame-shift or nonsense] ~ likely to be phenotype modifying |
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Amino acid affected |
If Missense - more likely to be pathogenic when: •Located in functionally important part of protein •Evolutionary conserved A. acid – greater functional importance •Non-conservative A. acid change ~ greater effect |
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Segregation |
If mutation segregates with disease may be disease-causing |
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Prevalence of mutation |
Mutation = rare < 1% prevalence, likely to be pathogenic Polymorphic alleles are prevalent (> 1%) in population |
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Functional analysis |
Analysis of “mutant” gene by gene expression or measurement of biological activity = ultimate test / proof |
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Detection of Coagulation factor V mutation by: |
PCR & Restriction enzyme digestion analysis |
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Allele specific oligonucleotides (ASO) T > C mutation |
Hybridize with Normal ASO Hybridize with Mutant ASO |
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Multiplex PCR |
Using several sets ofprimers in a single reaction can amplify several targets simultaneously Exons of the dystrophin gene are analysed (for deletions) in one step |
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Long range PCR - Triplet repeat expansion - |
Modification of PCR to amplify >10 Kbp DNA.Wild-type genomic PCR products of 500 bases andlarge (>5kbp) expansions of a gene that gives rise to Friedreich’s ataxia |
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Variation in types of mutation and location / extent of genetic heterogeneity |
Determines which methods / approach |
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Indirect DNA diagnosis/testing [Gene tracking / tracing] |
Relies on genetic markers (polymorphisms) that are physically linked to a disease locus Used to track the inheritance of the defective gene in a family Co-inheritance of a specific marker allele with the disease phenotype provides evidence that permits the risk of a patient to be estimated |
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Gene Tracking – Three Stages [The logic of gene tracking] |
Distinguish the two chromosomes in the relevant parent(s) -i.e. find closely linked marker for which they are heterozygous Determine phase -i.e. work out which chromosome(s) carry disease allele(s) Work out which chromosome(s) the person who is being tested has inherited |
