Molecular Basis of Disease

Question 1

What is a pathogenic mutation?

Answer 1

-Cause of a disorder

Question 2

Describe the anatomy of a gene

Answer 2

• Exons and Introns
• Promoter, initiator and stop codons
• Polyadenylation signal

Question 3

What are the post transcriptional modifications to translation?

Answer 3

• Produces mature RNA
• Splicing (introns out of mRNA), maturation and polyadenylation
• Exported to cytoplasm to Golgi apparatus, translation from ATG initiator to TGA stop

Question 4

What is the Pathogenic Mutation Criteria?

Answer 4

-Does it affect the function of the protein?
-Is it in a conserved region of the protein?
=more likely to affect function if changes in a region conserved across species (orthologs) or between members of a gene family (paralogs)
=Indicative of critical function
-Does it co-segregate with the disorder in the family?
=Is the gene change only found in affected members
-Is the change seen in the normal population?
=Has a sample of the normal population been screened

Question 5

What are the types of mutation in DNA sequences?

Answer 5

-Deletions
=Ranges from 1bp to megabases
-Insertions
=Ranges vary can be as small as 1bp up to megabases
=Duplication and inversions
-Single base pair substitutions (point mutations)
-Frameshifts
=Caused by deletions, insertions or splice site errors
-Dynamic mutations
=Tandem repeats
=Triplet expansion

Question 6

How can the different types of mutations in genomic DNA cause disruption in messenger RNA?

Answer 6

-Insertion and deletion disrupts codon sequence
=frameshifts
-Nonsense= abrupt cessation of protein translation
-Missense= may still make sense, sometimes pathogenic, 1 bp changed so may change amino acid

Question 7

What are point mutations?

Answer 7

Can be classified according to their effect on the product of translation

• Synonymous
• Nonsynonymous

Question 8

What is a synonymous mutation?

Answer 8

• Changes a codon into another that specifies the same amino acid as the original codon
• Due to redundancies within genetic code

Question 9

What is a nonsynonymous mutation?

Answer 9

-Changes a codon into another that specifies a different amino acid to that of the original codon

Question 10

What are the types of nonsynonymous mutations?

Answer 10

-Missense mutations 
=Replace one amino acid with another
-Nonsense mutations 
=Replace an amino acid codon with a stop codon
-Splice site mutations 
=Create or destroy splicing signals

Question 11

Describe how missense mutations within the exon can be pathogenic

Answer 11

-Has it caused a change in amino acid?
=Some redundancy in the genetic code
=20 amino acids and 64 possible codons
-If there is a change in amino acid, has it caused a conserved or non-conservative change in amino acid?
=Change in polarity
=Change in hydrophobicity

Question 12

What is the Grantham Matrix?

Answer 12

• Method in calculating the significance of the amino acid substitution
• The bigger the score the more likely that the missense mutation has caused a change in the resultant protein structure

Question 13

What is segregation analysis?

Answer 13

-Determine whether there are 5 or more affected members within that family that carry the gene variant under investigation
=98% certainty of the event being pathogenic
=Highly unusual in modern day living
=Same degree of certainty if two families where two affected first degree relatives involved

Question 14

Describe splice site mutations

Answer 14

-4 nucleotides highly conserved across species (GUAG)
=Mutation within 4 nucleotides= abnormal splicing
-Nucleotides on either side of the group also conserved to a variant manner depending on gene and intron involved
=Disruptions of these cause alteration in splicing

Question 15

What happens if there is a mutation in the splice donor site?

Answer 15

• Inclusion of intron in mRNA
• Fails to recognise start of intron so failure to splice out
• Can be quite large so protein translation affected

Question 16

What happens if there is a mutation in the splice acceptor site?

Answer 16

• Exon skipping
• Fails to recognise end of intron so will splice all material
• Large exons= lots of sequencing codons= severely affected protein= pathogenic effect
• Small exons= small amount of sequencing codons= less affected

Question 17

How do mutations cause disease?

Answer 17

1. Loss of function (abolition) of gene product

2. Modification of gene product

Question 18

Describe how mutations lead to loss of function

Answer 18

-Due to non-functioning or truncated protein
=Usually due to intragenic mutations due to disruption of messenger RNA (Marfan syndrome, Duchennes muscular dystrophy)
-Haploinsufficiency
=Usually refer to submicroscopic chromosomal deletions, whole genes deleted (William syndrome)
-Dominant negative
=Deafness syndromes, Collagen disorders

Question 19

How do dominant and recessive disease manifest?

Answer 19

Threshold for dominant conditions higher than threshold for recessive conditions (above 50% vs less than 50%)

• Both genes contribute to gene product
• Dominant= one gene disrupted= only 50% of gene product so manifest when only one gene is disrupted
• Recessive= only when both copies lost there is no gene product= manifestation

Question 20

Describe Dominant negative mutations

Answer 20

-Mutation produces a non-functioning protein
-Nom-functioning protein interferes with the protein of the normal functioning homologous gene
=Resulting in no effective gene product
=Pathogenic effect

Question 21

Describe how mutations lead to modification of the gene product

Answer 21

-Creating a poorly functioning protein
=Becker’s muscular dystrophy (milder phenotype)
-Abnormal activation of protein (overexpression)
=Cancer genes
-Gain of function of protein (novel function)
=Huntington disease, cancer genes (philadelphilia chromosome-fusion protein)

Question 22

Describe the structure of DNA

Answer 22

• On average, a sequence of 16 bases is unique in the human genome.
• DNA methylation occurs most commonly at CpG dinucleotides.
• DNA is arranged in the chromosome in association with histones.

Question 23

What features does a gene have?

Answer 23

• A promotor region, this may contain a TATA box or CpG island.
• Exons, which contain the sequence transcribed to mRNA.
• Introns, which contain sequence that is removed by splicing after transcription.
• 5’ and 3’ untranslated regions.
• Start and stop codons.

Question 24

What factors control the amount of protein product from a gene?

Answer 24

• Rate of Transcription. Amount of mRNA produced.
• Splicing – controlled by splice consensus sequences at intron/exon boundaries.
• Stability of mRNA.
• Stability of protein product made.
• Correct localisation of protein product.
• Correct post-translational modification of protein product.

Question 25

What is a locus?

Answer 25

unique chromosome location, which defines a specific gene or DNA sequence.

Question 26

What is a polymorphism?

Answer 26

DNA variant present in the population at a significant frequency.
may be a single nucleotide polymorphism (SNP), insertion or deletion.

Question 27

What is an allele?

Answer 27

specific sequence variant at a particular locus.

Question 28

What are minisatellites and microsatellites?

Answer 28

repetitive elements that vary in repeat number

Question 29

What does the analysis of the gene sequence in an individual require?

Answer 29

Polymerase chain reaction
Gel Electrophoresis
DNA sequencing

Question 30

What is linkage?

Answer 30

tendency of two sequence variants to be inherited together.
because of their physical proximity on the same chromosome
-When two loci are close together on the same chromosome, so that the chance of crossing over between the two loci at meiosis is less than 50%, they are said to be linked.

Question 31

How can a mutation affect a protein?

Answer 31

Abolish protein product.
Abolish protein function. – this may lead to a dominant negative effect.
Reduce protein function.
Affect transport or post-translational modification of a protein.
Activate a protein.

Question 32

What is the difference between allelic and locus heterogeneity?

Answer 32

• The same disease phenotype is often caused by different mutations in the same gene= Allelic Heterogeneity.
• The same disease phenotype can be caused by mutations in different genes= Locus heterogeneity.

Question 33

What different screening techniques detect mutations?

Answer 33

• Unknown mutations can be detected by different screening techniques, including SSCP, Heteroduplex detection and Sequencing.
• Known mutations can be detected using different techniques, including RGPCR and ARMS.

Question 34

What can be used as markers for linkage?

Answer 34

Polymorphisms whose location in the genome are known

Question 35

Why is there linkage analysis?

Answer 35

• Linkage analysis allows location of unknown disease genes.
• It also allows tracking of a disease of known location through a family.
• It is not necessary to know the mutation in the gene for this, just the location of the gene involved.
• Analysis of several linked markers in one chromosomal region allows construction of haplotypes and localisation of crossovers which have occurred at meiosis.

Question 36

What are the problems with linkage analysis?

Answer 36

• Linkage is only useful for tracking disease in a family where markers are informative and phase can be determined.
• Analysis of DNA markers may demonstrate non-paternity, either intentionally, as in paternity testing, or incidentally during DNA analysis for disease.
• Locus heterogeneity limits the application of this technique.