Genetic Variation and Disease II Gene testing and single base variation Flashcards
Revision
What is the central dogma?
describes the two-step process, transcription and translation, by which the information in genes flows into proteins: DNA → RNA → protein.
How much of the genome is exons and what does the rest do?
2-3% of the genome is exons.
The rest:
Regulation of genes
Spaces out genes - insulates genes fro promotors
Provides substrate to expand genome/add genes
What is the definition of a mutation?
A genetic variation that causes a disease.
What is the definition of polymorphism?
A genetic variation that is prevalent in the population and not, in itself, disease causing.
How do you get from cytosine to thymine?
There is a change in 1 base.
The change in molecular weight is negligible.
Focused testing: PCR
Allows us to select one small piece of the human genome from a patient (100 to 10000 bases) and amplify it - i.e. make lots of copies of one short stretch of the genome.
Where does a mutation act?
Promotor mutation - No, or reduced transcription - No, or reduced protein Splice consensus altered - most mRNA decays instead of carrying on and becoming a malfunctioning protein - Abnormal or absent protein Base change makes new stop - Base change alters amino acid sequence - Different or non-functioning protein - short or absent protein (mRNA decay)
What types of mutations occur in DNA sequences?
Wild Type (normal) - The cat sat on the mat
Stop - The cat
Missense - The car sat on the mat
Insertion - The cat spa to nth ema t
Deletion (out of frame) - The cas ato nt hem at
Deletion (in frame) - The cat on the mat
Triplet expansion - The cat cat sat on the mat
Predicting effects of point mutations
Normal (wild type): ATGAAACATTAG MetLysHisStop Mutant: ATGAAACAGTAG MetLysGlnStop c.9T>G c.9(number of bases from the left)T>G(a T base becomes a G base) p.His3Gln p.His3Gln (Histamine becomes glutamine)
What is the HGVS Mutation Nomenclature?
c. Effect on the mRNA (cDNA) sequence
p. Effect on the peptide sequence
c. 267G>A Substitution
c. 267delG Deletion
c. 267lnsA Insertion of an A
c. 267 + 2T>A Substitution of an intron
p. Ile122Ile No effect
p. Ile122Val Missense
p. Ile122Ter or p.Ile122* Premature stop/nonsense
p. Ile122Thrfs deletion causes Ile to Thr and frame shift.
(c. can be turned into a p.)
What are the effects of the different types of mutations?
Promotor and splice site sequence changes: Stop transcription or cause abnormal splicing
Base change causes an amino acid change:
Change in protein sequence
Not every base change causes disease
This may or may not reduce protein function
Some missense mutations make a protein work faster
Base change causing a premature stop codon
Insertion or deletion of bases:
remember that 3base pairs encode 1 amino acid
Mutations may be “in-frame” or “out of frame”
Trinucleotide repeat expansions:
replication of a trinucleotide.
What questions to ask to decide if something is a Polymorphism or disease causing mutation?
Does it match expected inheritance? Is it in the right gene? Has it been reported before - In the same phenotype - it may be causative - As a polymorphism - it's not causative What does it do to protein?
How do you filter through and find the mutation that matters?
Exclude if it is known to be a polymorphism. Keep if it affects gene function (stop, splice etc.). Is it in a gene that explains the phenotype? 1 mutation (on a good day)
What may a variant be?
A disease causing variant/mutation
A variant of uncertain significance
A polymorphsim
What is the definition of penetrance?
The likelihood of having a disease if you have a gene mutation
100% penetrance means you will always get the disease if you have the mutation. (e.g. If you have the gene for huntington’s disease you will eventually get the disease).
REALLY REALLY IMPORTANT
What are Mendelian Disorders?
Diseases that segregate in families in the manner by Mendel’s Laws
Essentially a disease that is predominantly caused by a change in a single gene (high penetrance).
What is an Autosomal dominant gene?
Disease seen in all generations.
50% risk of affected child if parent is affected.
Disease severity can be variable (individuals with a mutation may not show disease).
Males and females equally likely to be affected).
How can you describe a mutation?
Standard Nomenclature - Has to work everywhere.
What is genetic variation?
Genetic variation is described as a change from the normal
The most common sequence in Caucasian USA is that of Donald Trump
What does p. and c. mean?
p. Is the change in the peptide (protein) sequence
c. Is the change in the mature mRNA sequence
referenced to the first base or amino acid of the chain.
Normal (wild type): ATGAAACATTAG MetLysHisStop Mutant: ATGTAACATTAG MetStop What are the condensed summaries of what has happened?
c. 4A>T
p. Lys2Stop/(p.Lys2*)
What is the effect of Normal (wild type): ATGAAACATTAG MetLysHisStop Mutant: c.6delA
Mutant:
ATGAACATTAG
MetAsnIle…
p.Lys2Asnfs
What is the process of Next Generation Sequencing?
- Extracted gDNA
- gDNA is fragmented into a library of small segments that are each sequenced in parallel.
- Individual sequence reads are reassembled by aligning to a reference genome.
- The whole-genome sequence is derived from the consensus of aligned reads.
Alignment of multiple short reads shows that there is a different base in half of the reads. This is because half the reads come from a variant allele which contains either a polymorphism or a disease causing mutation.
You have approximately 3million polymorphisms (changes from the “normal” sequence)
If there is a mutation causing a genetic disease, there is only 1.
Is any genetic change you see a mutation?
No it can be:
- A disease causing mutation
- A polymorphism
- A variant of unknown significance.
What is mendelian inheritance?
Autosomal dominant Autosomal recessive X-linked (mitochondrial) It is a type of Multifactoral inheritance
What are the basic symbols for pedigree drawing. For: Male Female Unaffected Affected Miscarriage Dead
Male is a blank square Female is a blank circle Unaffected is blank Affected is filled in Miscarriage is a blank triangle Dead is a blank square with a line through it.
