DNA mutations and genetic testing Flashcards
What is a polymorphism
the presence of 2 or more variant forms of specific DNA sequence that occur among different individuals or populations - most common type is single nucleotide polymorphism (SNP) but they can be larger involving longer stretches of DNA. Usually benign and helps to maintain variation in a population
what are the stop codons
UAA, UGA, UAG
what is a mis-sense variant
A single base substitution which changes the type of amino acid in the protein, can be both pathogenic and non-pathogenic. It could also be a polymorphism of no functional significance
what are the different types of variation
- Duplications (of whole or parts of a gene)
- ## Deletions (of whole genes or some exons)
what is an out of frame mutation
the removal or addition of one or more nucleotide which severely disrupts the production of the protein as the shift of bases causes different amino acids to be coded for e.g. duchenne muscular dystrophy
ATC GT(C) TTA CGC
to
ATC GTT TAC GCG
what is an in-frame mutation
A deletion where the reading frame of the gene is preserved - 3 nucleotides (1 codon) are removed - only 1 amino acid is no longer made and the other codons are unaffected
ATC (GTC) TTA CGC
to
ATC TTA CGC TGC
what is an exon
Segment of a DNA or RNA molecule containing information coding for a protein or peptide sequene
What is an intron
A segment of a DNA or RNA molecule which doesn’t code for proteins and interrupts the sequence of genes
what is a splice-site variant
It affects the accurate removal of an intron to make mature RNA containing only exons, so instead the intron is translated into the protein rather than being removed. This can also cause the loss of exons (or part of an exon) so they aren’t incorporated into the protein
where is a splice site
at the boundary of an exon and an intron
what is a non-sense variation
An out of frame deletion produces a stop codon at a deletion site or further along - causes the protein construction to be stopped prematurely - non-sense mediated decay
how do you tell between a pathogenic variant or a polymorphism
- changes an amino acid which had been conserved through evolution (pathogenic)
- Disrupts the active site or splice site (pathogenic)
- Not seen in a large number of normal individuals
seen previously in individuals with the same condition and shown to segregate with the disease (pathogenic) - functional studies showing an effect on protein function (Pathogenic)
Whats is a tri-nucleotide repeat (TNR) expansion
tri-nucleotide repeat (TNR) expansion is when the number of triplets (e.g. huntingtons = CAG) present in a mutated gene is greater than the number found in a normal gene, the number of repeats in the diseased gene increases as it is inherited so in the next generation the symptoms develop earlier and are more severe (anticipation)
what is anticipation
repeat gets bigger when transmitted to the next generation and so symptoms develop earlier and are more severe
what is allelic heterogeneity
lots of different variants in one gene e.g. cystic fibrosis
what is locus heterogeneity
variants in different genes give the same clinical condition e.g. hypertrophic cardiomyopathy
In which state does the dominant variant manifest the disease phenotype
heterozygous state - i.e. the condition occurs if there is one variant and one normal gene
In which state does the recessive variant manifest the disease phenotype
homozygous state - i.e. there has to be variants in both alleles for it to be expressed, the majority of pathogenic variants are recessive
what is a loss-of-function variant
Only 1 allele is functioning - most loss-of-function variants are recessive. If a pathway in the body is sensitive to the amount of gene product produced and only half is produced then it will not be able to function and this will cause a problem
what is haplo-insufficiency
The situation that occurs when one copy of a gene is inactivated or deleted and the remaining functional copy of the gene is not adequate to produce the needed gene product to preserve normal function.
what are gain of function variants
A type of mutation in which the altered gene product possesses a new molecular function or a new pattern of gene expression. Gain-of-function mutations are almost always Dominant - they can cause a new trait to appear in inappropriate tissues or at inappropriate times in development. A variant may occur at the recognition site for protein degradation leading to an accumulation of undegraded protein within the cell
what is a dominant-negative variant
where the protein from the variant allele interferes with the protein from the normal allele
what are the different clinical contexts for genetic testing
- Diagnostic
- Predictive (plan for the future if going to be affected)
- carrier (helps with decisions about bearing children)
- pre-natal (to check the baby is affected or not - abortion?)
- Pre-implantation genetic diagnosis (IVF)
- Screening
- susceptibility
why would a patient get a diagnostic test
if they are showing signs and symptoms suggesting a diagnosis and so they have a test to confirm a clinical diagnosis. the issues involved are informed consent as other people may be affected by the results
what is predictive testing used for
testing at risk family members for a previously identified familial variant - often dominant. The amount of intervention depends on the disease
what is carrier testing used for
Autosomal recessive and X-linked disorders - usually done for couples as testing individuals in isolation is not that helpful for reproductive decision making
what is pre-natal testing used for
it is used when there is an increased risk of a specific condition affecting the fetus e.g. a chronic villous test for downs, edwards or patau syndromes. Often they are chromosonal or DNA tests if a specific variant has been identified in the family. Issues with this involve counselling and decision making
what is Pre-implantation Genetic Diagnosis (PGD)
they take an 8 cell embryo and under gentle suction with a pipette, one cell is removed making it free for analysis
when is genetic screening used
the target population is for non-high risk families/an asymptomatic population to help identify those who are at high risk early to give them the best prevention and treatment odds or provide reproduction advice and options
what is susceptibility testing used for
offered to individuals who have a family history of a genetic disorder and to people in certain ethnic groups with an increased risk of specific genetic conditions
what are the roles of genetic testing
- to confirm a clinical diagnosis
- to give info bout prognosis
- to inform management
- to allow predictive testing relatives
- carrier testing
- give accurate recurrence risks
- prenatal diagnosis
what do you have to think about when undergoing genetic testing
- is the test available
- what type of test (carrier etc)
- is consent full and informed, who else will it affect
- what are the other implications ie employment, insurance etc
What is FISH an what does it do
Fluorescence In Situ Hybridisation - used to test for abnormalities of chromosome number and chromosome microdeletions/duplications
what does targeted mutation analysis test for
single nucleotide changes
what do multi-gene panels test for
single nucleotide changes
What does chromosome analysis test for
abnormalities of chromosome number and of chromosome structure
what does chromosomal microarray analysis test for
abnormalities of chromosome number and chromosome microdeletions/duplications
what is sanger sequencing
uses PCR to amplify regions of interest followed by sequencing of products - useful for single gene testing. Allows for 800 bps to be read - accurate and simple but slow and expensive
what is next generation sequencing
Allows rapid sequencing of targeted gene panels, has improved speed over sanger (due to parallel analysis - enhances sequencing speed - can sequence whole genome in a day) and reduced man power and cost
Sanger vs NGS
Sanger -
Single start point (primer)
Single DNA fragment sequenced
High cost per gene
Time consuming
Simple analysis (read the sequence)
Very accurate
The gold standard
NGS -
Library of DNA fragments
Massively parallel sequencing
Low cost per gene
Fast
Huge amounts of raw data to interpret
Moderately accurate
Issues with NGS data analysis
NGS generates millions of short DNA fragments - need to be filtered for quality and aligned to a reference sequence, then you need to identify and interpret the variants - this all takes time - sifting through the noise of many different variants
what does VUS mean
Variant of Unknown Significance
what are some examples of databases of disease mutations
Decipher, OMIM, ClinVar
what are incidental/secondary findings
findings that could have potential health and clinical significance that are beyond the aims of the original test
Advantages and disadvantages of targeted panels
- you select specific genes to sequence
- less noise
- fewer variants of uncertain significance
- the panels need to be updated often
how much of the genome gets sequenced by whole genome sequencing
95%< at a high cost and lower depth of coverage than whole exome sequencing and targeted sequencing
how much of the genome gets sequenced by whole exome sequencing
~1.5% (protein coding regions) at a lower cost than whole genome and in more depth
how much of the genome gets sequenced by targeted sequencing
0.005% - 0.1% (100s - 1000s of genes) at a lower cost and in more depth than whole genome and whole exome sequencing
