Musculoskeletal diseases

Question 1

What genetic phenomenon do chondroplasias show?

Answer 1

Locus heterogeneity - defects in different genes can give the same clinical phenotype.

Question 2

In general what mutations usually cause chondroplasias?

Answer 2

Those in the collagen present in cartilage, affecting endochondral ossification.

Question 3

How many different types of chrondroplasia are there?

Answer 3

150 - variable severity

Question 4

Describe the process of endochrondral ossification

Answer 4

1. cartilage formation: mesenchymal cells divide and differentiate into chondroblasts, these secrete cartilage and become embedded in lacunae within the matrix.
2. Vascular invasion and longitudinal growth: ring of woven bone formed in the mid-shaft, osteoclasts allow vascular invasion if woven bone and cartilage, growth plate forms.

Question 5

What mutations are involved in achondroplasia?

Answer 5

Gain of function mutations in fibroblast growth factor receptor 3 (FGFR3).

Question 6

What are the effects of the causative mutation in achondroplasia?

Answer 6

Inhibits the transition between prehypertrophic and hypertrophic state of the chondrocytes, resulting in abnormal endochrondral ossification. Inhibits proliferation and terminal differentiation of growth plate chondrocytes and ECM synthesis.

Question 7

What is Vosoritide?

Answer 7

A drug used to treat achondroplasia. A C-natriuretic peptide (CNP - involved in endochonral ossification and longitudinal bone growth) analogue that inhibits the MAPK activity of the overactive FGFR3, increasing growth velocity.

Question 8

What are the 4 main causes of chondroplasias?

Answer 8

Extracelluar matrix protein defects
Metabolic pathway defects (enzymes, ion channels transporters)
Defects in folding, processing, transport and degradation of macromolecules

Question 9

Which types of collagen can be mutated in chondroplasias?

Answer 9

Types 2, 9, 10, 11

Question 10

Name and describe the syndrome caused by mutation in type II or XI collagen

Answer 10

Stickler syndrome: affects the eyes, ears, and skeleton. Cleft palate, hearing impairment, ligament laxity, irregular ossification of epiphyses, early onset osteoarthritis.

Question 11

Mutations in which genes cause Multiple Epiphyseal Dysplasia?

Answer 11

Type IX collagen (COL9A1, COL9A2, COL9A3), COMP (cartilage oligomeric matrix protein), Matrillin-3, DYDST (diastrophic dysplasia sulphate transporter)

Question 12

Describe Multiple Epiphyseal Dysplasia

Answer 12

Short limbs/bones, irregular epiphyses, abnormal knees, joint pain, premature osteoarthritis.
COMP mutations affect interaction of type IX collagen with types I and II.
Matrillin-3 mutations disrupt the organisation and structure of the growth plate.
DYDST mutations impair sulphation of of proteoglycans in the cartilage matrix, and so affect cartilage strength.

Question 13

Name and describe the condition caused by mutation in type X collagen

Answer 13

Schmid Metaphyseal Chondrodysplasia: affects the metaphyses (contains the growth plate) resulting in short stature, joint pain, bowing of lower limbs.

Question 14

Which collagen type maintains constant swelling pressure and how?

Answer 14

Type II: fibril network contains highly sulphated aggrecan, making the cartilage highly compressible - releases water which is drawn back in by the proteoglycans. This gives quick recovery from compressive, giving cartilage in joints some give and elasticity.

Question 15

What genetic phenomenon does osteogenesis imperfecta show?

Answer 15

Dominant negative effect - mutant gene product not only loses its own function but also prevents other gene products functioning correctly. Common in multimers. OI is Autosomal Dominant.

Question 16

Mutations in which genes cause osteogenesis imperfecta?

Answer 16

COL1A1, COL1A2 - those encoding type I collagen. Point mutations.

Question 17

Which type of osteogenesis imperfecta is the most severe?

Answer 17

Type II

Question 18

Which type of osteogenesis imperfecta is caused by mutations in just COL1A2?

Answer 18

Type IV

Question 19

What is meant by “included type” osteogenesis imperfecta?

Answer 19

The abnormal gene product is incorporated into the matrix. Results in reduced secretion of collagen but what is laid down in the matrix includes abnormal collagen molecules as well as normal. This type is more severe due to the dominant negative effect.

Question 20

What is meant by “excluded type” osteogenesis imperfecta?

Answer 20

Results from a null allele. The abnormal gene product is not incorporated into the matrix. There is only half the collagen laid down in the matrix but all of what is deposited is normal. This is less severe as we do better with less but normal collagen than more but mutated collagen.

Question 21

Which types of osteogenesis imperfecta are included type?

Answer 21

Types II, III, IV

Question 22

Which types of osteogenesis imperfecta are excluded type?

Answer 22

Type I

Question 23

Describe the 4 types of osteogenesis imperfecta?

Answer 23

Type I: mild, excluded. Little bone deformity, blue sclerae, hearing loss.
Type II: lethal, included. Ribs cannot support respiration due to patchy mineralisation, some limbs have no bones.
Type III: severe and progressive, included. Both II and III show short stature, deficient and defective matrix, dentinogenesis imperfecta, bone deformities.
Type IV: mild, included. Mutations in COL1A2 so although included symptoms are milder (only 1/3 helices is α2). Normal sclerae, slightly short stature, some hearing loss.

Question 24

What % of abnormal collagen will you have resulting from mutations in COL1A1 or COL2A2?

Answer 24

COL1A1: 75%
COL1A2: 50%

Question 25

Describe 4 factors affecting the severity of osteogenesis imperfecta?

Answer 25

1. Which gene is mutated: COL1A1 mutations are more severe than COL1A2 2. Nature of the mutation: which amino acid replaces glycine. Ala/Ser < Cys < Arg < Val < Glu/Asp 3. Position of the mutation: mutations at the 3' end of the gene result in a higher degree of post-translational modification, the triple helix of collagen assembled from the C-terminal end. Therefore mutations closer to the C-terminus are more severe than those at the N-terminus. 4. Is the mutated protein included or excluded?

Question 26

What is the result of over modification of the collagen?

Answer 26

Reduces the thermal stability of the collagen, resulting in increased intracellular degradation and slower secretion. Over-modification results in abnormal molecules which give abnormal fibrils - ineffcient cleavage of the propeptides affects fibrillogenesis. These abnormal fibrils are poor templates for mineralisation - an essential process in bone formation.

Question 27

Describe some options for treatment of OI

Answer 27

- Antiresporptive therapy: bisphosphonates reduce osteoclast activity which increases bone volume but cannot repairs all the mirco-cracks in the matrix, reduces fracture number in children. - Anabolic therapy: drugs that switch on bone formation. Teriparatide is effective in middle affected adults and only used for short term treatment due to associated risks. Anti-sclerostin antibodies activate wnt signalling but this has not yet been trialled in humans for OI. Recombinant GH can be used to alleviate short structure in children. - Orthropaedic surgery is currently the predominant treatment, pinning the bones in place. This is challenging due the weakness of the bones.

Question 28

What genetic phenomenon is demonstrated by Duchenne Muscular Dystrophy?

Answer 28

Mutational heterogeneity: different mutations in the same gene can result in the same clinical phenotype.

Question 29

What is the inheritance pattern of DMD?

Answer 29

X-linked recessive

Question 30

What mutations cause DMD?

Answer 30

Loss of function mutations in the dystrophin gene. 60-65% of patients have deletions - often multiple exons 5-15% of patients have duplications 20-30% have small mutations such as intron deletions or exon insertions. Mutations are generally frameshift mutations. Result in nonsense mediated mRNA decay, and so no detectable dystrophin expression.

Question 31

What is the result of frame neutral deletions in the dystrophin gene?

Answer 31

Beckers' Muscular Dystrophy - less severe symptoms due to a lower Mw form of dystrophin

Question 32

What has occurred in the only 20 females worldwide with DMD?

Answer 32

X-autosome translocations between Xp21 (dystrophin locus) and a critical region chromosome 21. Results in one normal X chromosome and the other X now has the critical region from chromosome 21. X-inactivation then occurs. If the X:21 chromosome is inactivated then one copy of the critical region is lost and the cells are not viable. If the normal X is inactivated, the cells are viable but will have DMD due to the disruption of the Xp21 dystrophin locus. Therefore in these 20 females X-inactivation favours DMD.

Question 33

What is the role of dystrophin?

Answer 33

A protein in muscle, links the dystrophin-associated protein complex at the sarcolemma to the cytoskeleton. Maintains the strength, flexibility and stability of the muscle fibres. Has some crucial domains - as long as these are maintained then the protein is functional.

Question 34

What happens in the muscle when the dystrophin-associated protein complex is lost?

Answer 34

Continual influx of inflammation, cycles of necrosis and regeneration, satellite cell depletion (skeletal muscle precursors), necrosis and fibrosis, muscle wasting, contraction induced injury in myofibres.

Question 35

How does DMD cause death?

Answer 35

Cardiac and respiratory failure

Question 36

What is the incidence of DMD?

Answer 36

1 in 5000 males

Question 37

Describe some problems with using gene therapy to combat DMD

Answer 37

The dystrophin gene is very large so vector and delivery are challenging Muscle cells are post mitotic so cannot use retro virus Adenovirus can be used but it is non-integrating so requires repeated treatments Requires delivery to all muscle cells

Question 38

Describe some solutions for using gene therapy to combat DMD

Answer 38

Use mini genes with the crucial domains Micro- and mini- dystrophin have been delivered using adeno associated viruses Adenovirus vectors - taken up by non-dividing cells Substitute dystrophin with utrophin Correct the reading frame with exon skipping

Question 39

How can exon skipping be used to combat DMD?

Answer 39

Antisense mediated exon skipping using antisense oligonucleotides: can correct the reading frame Targeted to the splice site of mutation-containing exon Exon 45 is commonly deleted in DMD; inducing skipping of exon 46 as well corrects the reading frame. This results in BMD as although the frame is corrected it still results in a truncated dystrophin protein but it is still partially functional so can alleviate the severe symptoms of DMD.

Question 40

What is utrophin?

Answer 40

A protein with similar function to dystrophin but expressed in development and switched off in adults. Switching it back on could alleviate the symptoms of DMD.

Question 41

Describe the genetic testing used for DMD

Answer 41

It is difficult - caused by many different mutations (various sites and natures) in a very large gene. There are mutation "hotspots" in exons 2-19 and 45-55. Multiplex PCR assays can be used to reveal 98% of all deletions.

Question 42

What has been achieved by cloning the dystrophin gene?

Answer 42

- Characterise the mutations associated with DMD - Predict the function of the protein encoded bu the gene - Reveal the molecular pathology underlying DMD - Generate diagnostic tests - Design new treatments to correct the effects of the mutations

Question 43

What method was used to identify the gene causing DMD?

Answer 43

Cytogenetics | Revealed visible deletions in the X chromosome at Xp21 and X:21 translocations in female with DMD