Genome sequencing + DNA sequence variants

Question 1

How are types of DNA variants described with respect to?

Answer 1

mRNA sequence
Proteins amino acid sequence
Reading frame
Exon and intron boundaries.

Question 2

Whats exon

Answer 2

Regions that code for protein
Will be part of the final mature RNA produced

Question 3

Whats Intron

Answer 3

Non-coding
Removed by RNA splicing during maturation of the final RNA product

Question 4

Start codon

Answer 4

Triplet of nucleotides which serves as the initiation point of transcription of the gene.
This is always an ATG sequence,

Question 5

WHats splicing?

Answer 5

Removal of introns from mRNA molecule

Question 6

Whats a codon

Answer 6

3 nucleotides together (codon) code for an amino acid.

Question 7

How does a reading frame start and end?

Answer 7

The Start ATG codon begins the reading frame = the sequence of codons required for correct amino acid sequence for the protein

Stop codon ends the reading frame

Question 8

State the types of DNA variants

Answer 8

STOP and START variants
Missense
Nonsense
Synonymous – does not alter amino acid
Splice site
duplications/deletions (frameshift)
May be single nucleotide change
May be multiple nucleotide changes

Question 9

Stop and start variants
What happens if variant in start codon?
WHat happens if variant in stop codon?
What does this produce?

Answer 9

DNA changes occuring in the start and stop codons.

If there is a variant in the start codon, in this examples and G to C change, transcription will not be initiated and there will be no protein product.

If there is a variant in the Stop codon, transcription will continue into non-coding DNA 5’ of the gene.

This will produce a protein with amino acids at the 5’ end that may interfere with structure and function and is likely disease causing.

Question 10

Missense variant
Give example

Answer 10

Cause a change in the amino acid that the DNA codon codes for.

Example, a G in a GGC codon is change to a A, producing a GAC codon
This causes a change of the glycine amino acid for an Aspartic acid.
This may or may not be pathogenic,

Question 11

Nonsense

Answer 11

DNA variants that alter the amino acid that the codon codes for.
Instead of a different amino acid, it can encode a stop codon - introducing premature stop codon.
Translation stops prematuraely
Shortened/incomplete protein.
Protein may lose function

Nonsense mediated decay

Question 12

Deletion variants

Answer 12

Loss of nucleotides - range from one to whole gene

Example - 2 nucleotides from a GTA codon are deleted result in altered reading frame. - frameshift

Frameshift often result in truncated protein
Any varient that causes a fraemshift is almost always disease causing

Question 13

Duplication variant

Answer 13

The addition of nucleotides alters the reading frame, causing a frameshift and an altered amino acid sequence. (new amino acid sequence)
And like deletions a premature stop codon often causes premature truncation of the protein.

Example - an additional T nucleotide is added.
You can see the triplet GTA is changed to a GTTA, therefore the reading frame is disrupted.
The GTA triplet is replaced by a GTT triplet follow by an ACC, then CGC.
Amino acid sequence altered compared to WT

Question 14

RNA splicing

Answer 14

Splicing, the removal of introns from the mRNA, is controlled by the splices sites that flank the intron.

The Acceptor splice site, is at the 5’ end of the intron and is always an A G dinucleotide.

The donor splice site, is at the 3’ end of the intron and is always a G T dinucleotide.

The splicing machinery identifies the donor splice site and begins cutting out the DNA from that point on.

This will only stop once the splice machinery identifies the acceptor splice site which is then follow by the exonic sequence.

The resulting mRNA strand contains sequences only derived from exons.

Note that in RNA, uracil replaces thymine.

Question 15

Donor splice site variants

Answer 15

Mutations that occur in introns

Example, the G of the GT donor splice is changed to an A.
This will mean that the splice machinery will not recognise the donor splice site.
Therefore it will not begin the removal of the intronic DNA.
The resulting mRNA will include the entire intron.
This will alter the proteins structure and possibly function.
It can also cause an alteration in the reading frame, causing a frameshift and as discussed with deletions and duplications leading to an inevitable prematurely truncated protein.
Such splice changes are often disease causing.

Question 16

Alternative splicing of genes

Answer 16

This process uses different splice sites within one gene to generate related but different protein products.
i.e one gene can produce a variety of different protein products dependent on which splice sites are used.

Question 17

Acceptor splice site varients

Answer 17

A mutation in the AG of an acceptor splice site has a similarly damaging effect.

However, rather than including the intronic DNA, the result it is that the exon is excluded.

The splice machinery recognises the donor splice and beings removed nucleotides,

But as the acceptor splice site is no longer recognised, it does not stop once it reaches the exon and continues to remove all DNA until the next active acceptor site is reached.

Removing an entire exon would be most likely disease causing.

The exon may encode vital parts of the protein, such as active sites, DNA or protein binding sites.

Question 18

Why is DNA variant Nomenclature important?

Answer 18

To describe variant so that others can understand
Share genetic knowledge
Chaos otherwise!
Similar to ISCN for chromosome karyotype description

Question 19

What p.Gly4Asp

Answer 19

Aminoacid position 4
Amino acid change Glycine to Aspartic

Question 20

c.11G>A

Answer 20

I misssense change
a G to A change at position 11.
11 nucleotides from the A of the ATG start codon

> indicates a chnage of nucleotide, therefore G>A

Question 21

How many alleles for each gene

Answer 21

2

Question 22

Varients can be…

Answer 22

Heterozygous - one allele has the varient
Homozygous - both alleles have the variant
Hemizygous (if X-likned and male carrier)

Question 23

How are nomanclature of two alleles separated

Answer 23

by a ;

Question 24

How are WT alleles represented?

Answer 24

by a =

Question 25

How are alleles indicated?

Answer 25

[]

Question 26

When both alleles are WT

Answer 26

c.[=];[=]

p.[=];[=]

Question 27

Describe c.11G>C and p.Gly4Asp assuming that the second alleles is WT

Answer 27

c.[11G>C];[=]
p.[Gly4Asp];[=]

Question 28

G to C change at position 20

Answer 28

c.20G>C

Question 29

c.59T>A

Answer 29

T to A change at position 59

Question 30

Two DNA changes in one gene

c.20G>C
c.59T>A
Write as if changes on different alleles, in trans

Also, write as if changes have unknown allelic relationship

Answer 30

c.[20G>C];[59T>A]

we may have this information following parental testing showing that each parent as one variant.

Unknown: c.[20G>C(;)59T>A]

Question 31

Two DNA changes in one gene
c.20G>C
c.59T>A
Write as if changes on same allele, in cis

Answer 31

c.[20G>C;59T>A];[=]

box the situation is described where we know that the variants are on the same allele, again we would only be able to know this following parental testing showing that one parent had both variants.

Question 32

Two amino acid changes in one gene
p.(Asp7Gly)
p.(Ala98Phe)

Write as if changes on different alleles, in trans
Also, write as if changes are unknown allelic relationship

Answer 32

p.[Asp7Gly];[Ala98Phe]

Unknown - c.[Asp7Gly(;)Ala98Phe]

Question 33

Two amino acid changes in one gene
p.(Asp7Gly)
p.(Ala98Phe)
Write as if changes on same allele, in cis

Answer 33

p.[Asp7Gly;Ala98Phe];[=]

Question 34

Deletion of nucleotides at postion 13 and 14

Answer 34

DNA deletion of two nucleotides
c.[13_14del];[=]

Question 35

Deletion of signle nucleotide position 14

Answer 35

c.[14del];[=]

Question 36

p.[Val5fs];[=]

Answer 36

Deletion = amino acid change
Chamnged reading frame resulting in frameshift

Frameshift occuered at valine at position 5.

Question 37

What is stop codon indicated by?

Answer 37

*

Question 38

Nonsence variant (changes create new premature stop codon)

T to A change at c.21, amino acid 7 is Gly

Answer 38

DNA = c.[21T>A];[=]
Protein = p.[Gly7*];[=]

Question 39

What are two things to think about when interpreting DNA variants?

Answer 39

Is the variant pathogenic?
Is the variant benign? many in human genome called polymorphisms

Question 40

How are the likely effects of DNA variants analysed?

Answer 40

follow the guidelines laid out by the Association of Clinical Genetic Science.

Question 41

Variant classification

Answer 41

1-5
1 Benign
2 Likely Benign
3 Unknown significance
4 Likely Pathogenic
5 Pathogenic

Question 42

What are the types of evidence?

Answer 42

Variant frequency vs disease incidence
Type of variant
Splicing variants e.g Adverse effect on splicing predicted
Functional information
Functional studies
Clinical information – does genes’ known disease associations fit patients phenotype?
Family study
How is gene inherited?

Question 43

Variant frequency vs disease incidence

Answer 43

Variant found in control normal populations? (se Gnomad database https://gnomad.broadinstitute.org)
If yes, less likely to be pathogenic,
If not, supports pathogenicity

Question 44

Evidance - Type of variance

Answer 44

e.g. Nonsense vs synonymous
Truncating changes more likely to be pathogenic
Missense - Is wild type amino replaced by one with different properties?
Is this amino acid change predicted to alter shape and or function of the protein?

Question 45

Splicing variants e.g. Adverse effect on splicing predicted?

Answer 45

If yes, likely to be pathogenic
If not, less likely t be pathogenic

Question 46

Functional information

Answer 46

Located in functional domains?
Protein active site?
Repeat motif?

Functional information is important, tis relates to where the variant is located in the gene and protein.
Is it in a functional protein domain with an important function, or is it in area that has only a minor role in the protein function and disruption of which is likely to have little adverse effect?
Variants may disrupt important repeat motifs, repeats of amino acid sequences, important for protein structure, disruption of such a repeat could cause loss of that function.

Question 47

Functional studies

Answer 47

Experimental assay shows adverse effect on protein function?
Or no effect?

Question 48

Clinical information – does genes’ known disease associations fit patients phenotype?

Answer 48

E.g. Referred with learning disability but gene only associated with short limbs
E.g. Referred with learning disability and gene commonly reported in patients with learning disability

Question 49

Family study

Answer 49

Is variant present in affected family members only – supports pathogenic
Is variant present in unaffected family members – supports benign
Is the variant de novo in patient or does parent carry it?
De novo supports pathogenic (unless carrier parent also affected)

Question 50

How is gene inherited?

Answer 50

If recessive, has a second variant been detected?
Does familial pattern of disease fit a recessive, dominant or X-linked inheritance?

Question 51

Literature searches & Disease database

Answer 51

Has variant previously reported as pathogenic or benign?