Human Genetic Variation in Health and Disease.

Question 1

What are the three levels of variation?

Answer 1

Inter-individual
Intra-individual
Inter-population

Question 2

What are the mechanism at play for global genetic variation?

Answer 2

Meiotic recombination, DNA replication and repair, Random genetic drift, natural selection, migration.

Question 3

What are the three main classifications of variation types?

Answer 3

Structural, sequence level, repetitive elements of various sizes.

Question 4

What are the structural forms of variation?

Answer 4

Copy number (deletions and duplications)
Positional (insertions, translocations)
Orientational (inversions)

Question 5

What are the sequence level forms of variation?

Answer 5

Single base substitutions
small deletions/ duplications
repetitive sequence (in tandem, or dispersed throughout the genome)

Question 6

What are the repetitive elements forms of variation?

Answer 6

Tandems (varying in length) or interspersed
Small or cytogenetically visible
May not be disease causing but may predispose to rearrangements, expansions or contractions particularly during cell dividing stages/ meiosis.

Question 7

What are the causes of variation?

Answer 7

DNA repair mechanisms to content with damage due to environmental agents (ionising radiation, UV, chemicals)
DNA replication errors (proof reading errors, fork-stalling)
Homologous DNA recombination during meiosis (allelic and non-allelic)
Retrotransposition.

Question 8

What are the consequential link between variation and phenotype?

Answer 8

No change in phenotype 
Alternative phenotype of no medical consequence
Disease susceptibility (penetrance)
Pathogenic.

Question 9

What is the significance of BRCA2 c.865A>C (p.Aasn289His):ExAC?

Answer 9

It is a common variant found in 5.2% of the population; once a variant reaches 5% threshold of the population, it is considered normal and unlikely to be disease causing if it is in that many people.

Question 10

What are complex diseases?

Answer 10

They are diseases caused by the interaction between variants in multiple genes and the environment and do not typically follow a recognisable inheritance pattern. An example type 2 diabetes which has an estimated heritability of 40%. At least 18 loci have been shown to confer risk to T2D; most common disease of human populations.

Question 11

What is the significance of DMD c.10412T>A(p.Leu3471*)?

Answer 11

It is a rare variant of the DMD gene; a nonsense mutation. It is not found in normal healthy individuals.

Question 12

What are the modes of inheritance?

Answer 12

Dominant, recessive, autosomal, X-linked, Y-linked.

Question 13

What are the main genomic locations where mutations can occur?

Answer 13

coding region, promoter/regulatory, splice site.

Question 14

What are the molecular changes of mutations and how do they vary?

Answer 14

Structural or sequence level:
Substitution (synonymous, non-synonymous, missense, nonsense) deletion/ insertions, expansion/contractions.
Homozygous, heterozygous, compound heterozygous, hemizygous.
Functional effects: loss/gain of function, haplo-insufficiency and dominant negative.

Question 15

Describe loss of function mutations.

Answer 15

They are the most common inherited disease. Recessive inheritance. Exhibit allelic heterogeneity. Exceptions include: haploinsufficiency and dominant negative gene.s

Question 16

What is allelic heterogeneity?

Answer 16

Lots of different variants within a given gene that can cause a loss of function.

Question 17

What is haploinsufficiency?

Answer 17

Loss of one allele results in an intolerable loss of function.

Question 18

What is dominant negative?

Answer 18

Non-functioning protein interferes with wildtype function.

Question 19

What are gain of function mutations?

Answer 19

Product acquires new abnormal function. Exhibit dominant inheritance and allelic homogeneity. Examples include over-expression of a protein, constitutive activation of a receptor, open conformation of a channel, aggregation of protein, novel protein, novel multimer.

Question 20

What is homogeneity?

Answer 20

Genes associated with this type of disease and gain of function, usually only small number of mutations that can cause the effect due to location requirements for that effect on the sequence.

Question 21

What are the characteristics of sequence level mutations?

Answer 21

Missense mutations: altered function due to changes in protein structure (functional domains and binding sites_
Non-sense and frame-shifting mutations: nonsense mediated decay or truncated protein.
Splicing mutations: exon skipping or inclusion of novel exons.

Question 22

How else can gene expression be affected by mutations?

Answer 22

Affecting interactions of the promoter with transcription enhancers/suppressors
Resulting in unstable mRNA that is rapidly degraded
Affecting splicing efficiency or translations.
Dynamic mutations (triplet repeats) & huntingtons

Question 23

Describe Cystic fibrosis in general terms

Answer 23

An example of a loss of function, autosomal recessive disease

Question 24

What are the symptoms of CF?

Answer 24

There is a broad phenotypic spectrum; largely associated as a respiratory disease. Also presents with GI (via compromised pancreas) problems, sinus, reproductive (absent vas deferens in men) reduced fertility in women. Blocked enzymes, poor weight gain, loose,fatty stools.

Question 25

How does CF arise?

Answer 25

A loss of function mutation in the CFTR gene (Cystic fibrosis conductance regulator) a chloride channel.

Question 26

How many common mutations are there for CF?

Answer 26

Three which account for 80% of CF in caucasians but over 2000 reported: allelic heterogeneity.

Question 27

What are the three common mutations for CFTR that cause CF?

Answer 27

1. pGly551Asp: missense mutation
2. p.F508del: deletion of amino acid in-frame
3. p.Gly524*: nonsense mutation.

Question 28

How do the three mutations cause CF pathogenesis?

Answer 28

All have different effects however they ultimately disrupt the protein from reaching the cell membrane at appropriate location or disruption of channel function.

Question 29

What is Hutchinson-Gilford progeria syndrome?

Answer 29

Violation of loss/gain of function rules. Accelerated ageing, death by 13 usually from atherosclerosis of coronary or cerebrovascular disease. Caused by mutations in LMNA gene: C>T in codon 608 (synonymous variant) observed in over 90% of patients. Autosomal dominant: displaying allelic homogeneity.
A synonymous change creates a cryptic splice site, creating a truncated protein and results in a dominant negative effect in disease.

Question 30

What are dynamic mutations and give an example.

Answer 30

Repetitive sequences that can expand or contract, thought to occur due to replication error introducing or removing repeats.
Huntingtons and Fragile X syndrome are examples.

Question 31

How does huntington’s disease arise?

Answer 31

The silencing of gene transcription due to methylation of promoter.
A mutant HTT protein is cleaved to generate toxic fragments, thought to have multiple interactions with cellular components to impair gene transcription, protein function and degradation. Expansion of the 3’-UTR of the DMPK gene results in production of toxic RNA (normal protein is produced)

Question 32

What is the genotype and phenotype correlation?

Answer 32

The link between a specific genetic mutation (genotype) and disease characteristic (phenotype). Observed genotype-phenotype correlations for loss of function mutations can sometimes be explained by considering residual protein function. Other important mechanisms that might explain the presence or absence of genotype-phenotype correlation include: mosaicism, modifier genes, change effects and environment.

Question 33

How are G-P correlations analysed?

Answer 33

Two people can have the same change and have the same phenotype - there is no phenotype. Another change that two different people share and the consequence can be widely different: there isnt a correlation between the two.

Question 34

How do G-P correlations affect CF?

Answer 34

The CF phenotype depends on the amount of normal CFTR protein produced.
R117H-7T is the CFTR mutation most commonly associated with CBAVD: 10% residual function.
P574H: an atypical CFTR mutation: maintains function of the pancreas however lung function is compromised.
DeltaF508: classic severe disease: no residual function of the protein: more sever disease.
Increasing pathogenesis with decreased CFTR production.

Question 35

What is mosaicism?

Answer 35

Post-zygotic mutations resulting in two or more genetically distinct cell lines: tumours are an example.
A cell mutating early in development will have descendants in many tissues. A mutation that arises later will be more localised. Results in a mix of normal and abnormal proteins: the phenotype will depend on the proportions of cells producing abnormal protein and the distribution of those cells. Mosacism typically results in milder disease.

Question 36

Name a specific example of a mosaic disease.

Answer 36

Neurofibromatoses
Autosomal dominant disorders:
NF1: freckling, cafe au lait spots, peripheral neurofibromas. Follow dermatomes carrying mutations.
NF2: schwannomas, meningiomas and other CNS tumours.

Question 37

What are copy number mutations?

Answer 37

they can be single gene, continuous gene and multiple gene syndromes and arise through: 
Gene dosage
Gene disruption
Gene fusion
Position effects
Unmasking of recessive alleles.

Question 38

Give an example of a copy number mutation syndrome.

Answer 38

Single gene duplication syndrome 17p12(PMP22 gene):
A single gene associated with a specific condition: gene dosage, deletion of more than one gene results in occurrence of more than one condition in the patient. Continuous gene deletion syndrome at Xp22.

Question 39

What is the symptomatology of Multiple gene deletion syndrome?

Answer 39

At 7q11.2 (Williams-Beuren syndrome) 
Features include: 
Cardiovascular disease (elastin arteriopathy) 
Distinctive facial features
Connective tissue abnormalities
Intellectual disability
Specific cognitive profile (strong in language and short term memory but weak in visuospatial construction) 
Unique personality
Growth abnormalities
Endocrine abnormalities.