L25 - Molecular and Genetic Aspects of Skeletal Disorders

Question 1

What are the 3 regions of the cartilage end of growing bone?

Answer 1

Epiphysis = secondary ossification centre: proliferative, endochondral ossification

Metaphysis = trabecular bone + Hypertrophic cells + Calcified cartilage

Diaphysis = cortical bone

Question 2

What bone ECM components for bone calcification are made by Chondrocytes?

Answer 2

Collagen II, collagen VI, collagen IX, collagen X, collagen XI

Aggrecan

(Others: CMP, COMP, syndecan 3 link protein)

Question 3

What factors are made by Chondroclasts/ Osteoclasts to modulate bone growth?

Answer 3

cathepsins, MMPs (Matrix metallopeptidase -)

Question 4

What molecules/factors for modulating bone calcification are made by Osteoblasts?

Answer 4

Collagen I

Osteocalcin,

osteopontin (Inhibitor of Mineralization, trigger by Calcitriol)

(Others: BSP, AP)

Question 5

Define Osteochondrodysplasia?

Answer 5

Inherited disorders that affect all parts of bones: Epiphysis Metaphysis, Diaphysis.

Causes Large numbers of murine and skeletal abnormalities

Question 6

What type of mutation causing bone deformity is most common?

Answer 6

Point mutation

Question 7

List some outcomes of point mutation?

Answer 7

1) Missense mutations: alter amino acid sequence = change protein function
2) Nonsense mutations: introduce premature termination (stop) codon
3) Alteration of promoter / enhancer (regulatory) sequences
4) mRNA splicing mutations: exon skips (loss of exon sequences)

Question 8

Name the disorder and affected tissue due to abnormal Collagen type I.

Answer 8

Osteogenesis imperfecta (OI), EDS (VII)

Bone, skin, tendon, cartilage

Question 9

Name the disorder and affected tissue due to abnormal Collagen type II.

Answer 9

Spondyloepiphyseal dysplasia (SED)

Cartilage

Question 10

List the symptoms and signs of Osteogenesis Imperfecta? think about the tissue affected

Answer 10

Fragile or brittle bone 
Hypermobile joints 
Tendon rupture 
Frequent hernias
Thin skin 
Thin, blue-color sclera 
Early onset deafness

Question 11

Rank the Sillence classification of OI in terms of severity?

Answer 11

Increasing severity:

I, IV (mild)&raquo_space; III (progressive)&raquo_space; II (lethal)

Question 12

List the clinical features of type I OI?

Answer 12

Normal stature with Little/no deformity

Blue sclerae

Hearing loss

Question 13

List the Sillence classification of OI in terms of inheritance pattern?

Answer 13

Type I = AD
Type II = AD, AR (rare)
Type III = AD, AR (rare)
Type IV = AD

All types predom. AD

Question 14

List the clinical features of type II OI?

Answer 14

Perinatal lethal:

• Marked long bone deformity: short limbs, small chests (causing underdeveloped lungs)
• Blue sclera
• Minimal calvarial
  mineralization (soft skulls)
• Numerous intrauterine fractures

Question 15

List the clinical features of type III OI?

Answer 15

 Very short stature
 Hearing loss (common)
 Dentinogenesis imperfecta (bow leg and scoliosis)

 Progressive deforming bones
 Scleral hue varies

Question 16

List the clinical features of type IV OI?

Answer 16

 Normal sclera ***
 Dentinogenesis imperfecta (common)
 Hearing loss

 variable short stature
 Mild to moderate bone deformity

Question 17

What genes are affected in Type I to IV of OI?

Answer 17

COL1A1 and COL1A2

Question 18

Compare the specific types of genetic mutation in type I to IV of OI?

Answer 18

All affect COL1A1 and COL1A2 in different ways:

I = Loss-of-function due to Haploinsufficiency (nonsense mutations)

II = Dominant-negative (amino acid substitutions)

III and IV = Dominant-negative (amino acid substitutions, deletions and splicing mutations)

Question 19

Describe the collagen formed in type I OI?

Answer 19

Haploinsufficiency = reduced amount of normal type I collagen in bone

Nonsense mutation = premature termination and truncated non-functional chains + selective degradation of mutant mRNA

Question 20

Describe the intracellular formation of collagen type I (individual chains)?

Answer 20

Gene code for mRNA

> Signal peptide guides chain into lumen of RER

> Post-translational hydroxylation by prolyl and lysyl hydroxylases (need oxygen, a-ketoglutarate, Vit.C as cofactor) : Proline hydroxylation for helix formation and stability

> Golgi for export

Question 21

Describe the extracellular formation of collagen type I?

Answer 21

Proteolytic processing: N- and C- proteinases cleave propeptide domains on procollagen

Fibril assembly and form covalent crosslinking by peptidyl lysine oxidase: zip 3 chains from C to N terminus

(need oxygen and copper ion)

Question 22

Why is type II OI more lethal than other types?

Answer 22

Collagen = Gly-X-Y repeats

• Glycine mutation
• Close to C- terminal (more upstream)

Question 23

Define the dominant- negative effect in OI?

Answer 23

In collagen, 2 a1 chains and 1 a2 chain form triple helix

If 50% of a1 and 50% of a2 chains are normal, when put into triple helix combination, only 25% of product collagen is normal, 75% abnormal

Question 24

Why is the dominant- negative effect in collagen type II more severe than in Type I?

Answer 24

Type II collagen has 3 identical chains: Homotrimer encoded by single gene COL2A1

Having 50% abnormal chains = almost none of the chains will be normal = more severe effects

Question 25

What is the result of abnormal collagen molecules in the ECM?

Answer 25

Overmodified collagen = alter collagen packing and cross linking = destabilize matrix

Question 26

Possible treatments for OI?

Answer 26

Reduce osteoclast activity by Bisphosphate inhibitors of TGFβ signaling

Question 27

What causes Marfan syndrome?

Answer 27

mutations in Fibrillin 1 >> impaired ECM function >> excessive TGFβ signaling

Question 28

Symptoms of Marfan syndrome?

Answer 28

```  Eye problems  Abnormal chest, heart, lung problems  short torso; tall, thin body frame  Bone overgrowth with long arms, legs, fingers  Joint laxity  Aorta, skin problems ```

Question 29

Briefly describe the formation of elastic fiber?

Answer 29

Fibrillin monomer>> fibrillin polymer >> elastic fiber or elastin-free microfibril Elastic fiber/ elastin-free microfibril = ECM proteins bound to assembled large latent complexes (LLCs) secreted by many cells

Question 30

What are the predisposing genes to OA and Intervertebral disc degeneration?

Answer 30

``` OA = Asporin IVD = cartilage layer immediate protein (CLIP) ``` Both regulate TGFβ signaling

Question 31

What is the difference between Null allele and Dominant negative effect?

Answer 31

Null allele = Reduced expression of normal collagen = quantitative defect Dominant- negative effect = expression of both normal and mostly mutated collagen = qualitative + quantitative defect

Question 32

How is TGFβ normally regulated?

Answer 32

Normally bind to ECM TGFβ bind non-covalently to Latency Associated Peptide (LAP) to form Small Latent Complex (SLC) SLC disulphide bonds to Latent TGFβ binding protein (LTBP) to form Large Latent Complexes (LLC) LLC binds to ECM via Fibrillin and Fibronectin and traps TGFβ

Question 33

Describe how TGFβ signalling is disrupted in Marfan syndrome?

Answer 33

- TGFβ + LAP = SLC - SLC + LTBP = LLC - LLC +ECM (fibronectin and fibrillin) = stabilize TGFβ If Fibronectin and Fibrillin is abnormal, ECM degradation occurs and MMPs are activated >> MMPs dissociate LAP and releases TGFβ >> More free TGFβ able to trigger cell functions i.e. cell death