18.03.07 AD - Dominant negative effect

Question 1

What is the effect of a dominant negative mutation?

Answer 1

Only seen in heterozygotes where they have a more severe effect than a null effect of the same gene.

The product of the variant allele, (which can be non-functional), interferes with function of the normal allele.

Question 2

Which mechanism likely evolved to protect again dominant-negative effects of abnormally truncated products?

Answer 2

NMD

Better to have no product of the mutant gene than to have a product that interferes with function.

Question 3

Which types of protein are more susceptible to dominant-negative effects?

Answer 3

Proteins with multimeric structures are particularly vulnerable to dominant-negative effects as they are dependent on oligomerisation for activity.

e.g. one subunit with intact binding domains and altered catalytic domain will effect the function of the whole multimer

Question 4

Give three examples of conditions which exhibit a dominant negative effect and the genes with which they are associated.

Answer 4

1. GJB2 (13q12.1) - Non-syndromic hearing loss
2. COL1A1 (17q11.33)/ COL1A2 (17q21.3) - osteogenesis imperfecta
3. CLCN7 (16o13.3) - Osteopetrosis
4. CLCN1 (7q34) - Myotonia congenita
5. FBN1 (15q21.1) - Marfan syndrome

Question 5

Describe the DFNB1 locus.

Answer 5

Contains the Gap Junction Beta 2 and 6 genes which encode connexin 22 and connex 30 (Cx26/Cx30).

These are the major gap junciton proteins expressed in the human cochlea.

Question 6

Describe the structure of connexins and how they form connexons.

Answer 6

Membrane proteins containing 4 transmembrane domains.

6 connexins oligomerise to form a connexon.

Connexons align symmetrically with those of neighboruting cells to create continuous aqueoys pores (gap junctions) which functionall connect adjacent cells facilitating transport of small molecules and ions.

Question 7

What is the structure and function of connexons?

Answer 7

Can be homopolymeric or heteropolymeric which gives them physiological characteristis.

Cx26 and Cx30 and involved in K+ ion recycling in the ear.

The molecular composition of gap junctions determines their conductance and gating properties as well as influencing the permeability and intracellular trafficking of hemi-channels.

Question 8

What are K+ ions required for in the ear?

Answer 8

Release of neurotransmitters from the hair cell in the cochlea.

Question 9

What is the most common inheritance pattern for GJB2 mutations?

Answer 9

AR - which prevents full range of functional gap junction formation when homozygous.

Heterozygous = phenotypically normal

Question 10

How can mutations in GJB2 show a dominant negative effect?

Answer 10

Dominant missense Cx26 pathogenic variants produce full-length structurally abnormal Cx26 molecules.

These form gap junction plaques also containing wild type Cx26 and Cx30 forming connexons with impaired permeability to K+ions and other small moleculares resulting in hearing loss in heterozygous individuals through a dominant-negative effect.

Question 11

What are fibrillar collagens?

Answer 11

Major structural proteins of connective tissue consisting of triple helices of homo- or hetero-trimeric polypeptide chains that are assembled into close packed cross-linked arrays ro form rigid fibrils.

Question 12

What is the structure of preprocollagen?

Answer 12

N- and C-terminal globular pro-domains flanking a central repeat sequence (Gly-X-Y)n, where every third residue if a glycine due to spatial constraints of triple helix formation.

Question 13

How is mature collagen formed?

Answer 13

Three preprocollagen chains associate and wind into a triple helix under the control of the C-terminal globular domain.

Mature collage formed by cleavage of the C-terminal domain.

Disrupted in OI

Question 14

In which genes do 90% of osteogenesis imperfecta patients have mutations in?

Answer 14

COL1A1 and COL1A2 which encode type I procollagen chains.

Question 15

What are the proposed disease mechanisms underlying the different types os osteogenesis imperfecta?

Answer 15

Type 1 = Haploinsufficiency
Null variants in COL1A1 decrease the amount of type 1 procollagen produced. NMD results in reduced mRNA production. Milder disease.

Types 2, 3 and 4
Dominant negative effect. 80% of pathogenic variants replace glycine residues in the triple helix domains of COL1A1 or COL1A2 leading to the production of abnormal type I procollagen molecules. THis dirupts the triple helix formation resulting in severe disease.

Question 16

Which gene is associated with osteopetrosis? What is the phenotype of this disorder?

Answer 16

ADO is an inherited metabolic bone disorder that results from ineffective osteoclast-mediated bone reabsorption.

Mutations in CLCN7.

Question 17

How do mutations in CLCN7 give rise to osteopetrosis?

Answer 17

pH control is vital for osteoclast function as it balances the acidic environment that osteoclasts use to dissolve bone tissue.

CLCN7 regulates the pH of osteoclasts as it is a chloride/proton antiporter - Cl- ions transported out of the osteoclast for each H+ in.

Question 18

How do pathogenic variant in CLCN7 result in both AD and AR inheritance patterns?

Answer 18

ARO - null pathogenic variants (mostly nonsense) leading to loss of the chloride channel function. In most severe cases chloride channel protein 7 is absent (CIC-7)

ADO - less severe osteopetrosis. Incomplete inactivation of CIC-7 resulting in altered electrophysiological properties of the channel and reduced chloride conductance.
Dominant negative eggect

Question 19

Which gene is associated with AD myotonia congenita (Thomsen-type myotonia)? What is the phenotype of this disorder?

Answer 19

Commonest genetic skeletal muscle ion channelopathy.

Caused by pathogenic variant in CLCN1 which encodes the skeletal muscle voltage-gated chloride channel CIC_1

Question 20

What is the structure and function on CIC-1?

Answer 20

CIC-1 is a homodimer each forming a separate ion conduction pathway = protopore

Function of the channel = stabilise cell’s electrical charge preventing muscles from contracting abnormally.

Question 21

What are the two forms of myotonia congenita and what inheritance patterns are they associated with?

Answer 21

AD myotonia - dominant negative
Inactive channel dimers have both WT and variant CIC-1. Less common and less severe than AR form. Variant scattered across the channel but may be more common at the dimer interface (exon 8)

AR myotonia (Becky-type)
Nonsense or missense variant. 
LoF through NMD. Impaired transport to the membrane of inability to form dimers.

Question 22

In myotonia congenita the same pathogenic variants may occurs in families with both types of inheritance. How can this be explained?

Answer 22

Reduced penetrance of dominant-negative variants
Incomplete dominance
Presence of unidentified second pathogenic variant.

Question 23

Which gene is associated with Marfan syndrome? What is the phenotype of this disorder?

Answer 23

Connective tissue disorder caused by mutations in FBN!

Syndromic condition where morbidiy and mortality results from aortic aneurysm and dissection.

Characteristic fascies - long narrow face
Cardiovascular disease
Skeletal malformations

Question 24

What is the role of fibrillin-1?

Answer 24

Structural role in the wall of the large arteries

Question 25

The mutation type in Marfan syndrome may predict disease severity and direct treatment options. Give examples.

Answer 25

1. Dominant negative FBN1 variants.
   Missense and exon skipping variants that result in a stable but altered protein. Distrubed interaction between variant and wt filbrilin 1 and other proteins results in disorganised ECM.
2. HI FBN1 variants
   Null variants leading to NMD
   Decreased fibrilin-1 leads to thinner fibrillin-1 matrix with consequences in aortic wall strength.
   Increased risk of cardiovascular death compares d with DN variants.
   Potentially mroe responsive to losartan therapy for inhibition of aortic root dilatation.

Question 26

When is the p53 pathway triggered?

Answer 26

By a wide variety of DNA damage signals which lead to the stabilisation, post-translational modification and recruitment of p53 to binding sites in chromatin.

Question 27

Describe the role of p53.

Answer 27

Transcription fatcor that mediated changes in gene expression that promote apoptosis senescence or a reversible and protective cell cycle arrest; eliminating the damaged cells and suppress tumorigeneiss.

Question 28

What proportion of p53 pathogenic variant are missense variant in the DNA-binding domain? What is the effect of this mutation type?

Answer 28

74%

Affect amino acids directly involved in the sequence-sequence recognition of DNA or located at distant sites and repsumably inactivate p53 function by altering in protein confromation.

Question 29

How can p53 mutations lead to cancer?

Answer 29

By exerting a dominant negative effect on wt p53 (can also exert GoF activity.

Dominant negative effect arises because p53 binds DNA as a tetramer consisting of a dimer of dimers

Wr and pathogenic variant p53 proteins form heteroligomers with impaired DNA association and transcriptional activity.

p53 oligomerisation domain lies at the C-terminus, therefore truncated forms lacking this dominant cannot bind WT and don’t act in a dominant-negative fahsion.