Genetics - Brittle Bones

Question 1

What is the name for brittle bone disease?

Answer 1

Osteogenesis imperfecta

Question 2

What is the main feature of brittle bone?

Answer 2

• Repeated fracture of long bones

- Malformed bones

Question 3

What is the distinct property/feature of collagen?

Answer 3

All types of collagen have triple helix structure - with glycine at every third position of collagen facing interior of helix

• modified amino acid (hydroxyproline & hydroxylysine)

Question 4

What are collagens for?

Answer 4

• Give mechanical strength and rigidity to tissues and organ
• Give tensile strength to skeletal tissues (bone, cartilage, tendon, ligament)

Question 5

What is the mutation of brittle bone disease?

Answer 5

Point mutation in collagen gene Type I

- Substitution of glycine with cysteine

Question 6

What is the effect of substituting glycine with cysteine in collagen gene?

Answer 6

• Larger amino acid generates kink in straight triple helix (defect in assembly)
• Cysteine contains reactive sulphydryl group forming disulphide bond between 2 [a1(I)] chain - polypeptide chain will migrate more slowly in electrophoresis

Question 7

What chains do collagen type I contain?

Answer 7

2 [a1(I)] + 1 [a2(I)]

Question 8

Why is there an unusual band in the absence of 2-ME but not in the presence of 2-ME?

Answer 8

2-ME breaks disulphide bridges so the bands return to ‘normal’ co-migrate

Question 9

What is 2-ME?

Answer 9

2-Mercaptoethanol used in gel electrophoresis

Question 10

How does gel electrophoresis separates the bands?

Answer 10

SDS-PAGE (sodium dodecyl sulfate)

1. causes denature of protein
2. forms layer around protein making it negative [per length of protein]
3. migration due to size difference

*Cysteine forming larger protein w/ disulphide bridges

Question 11

Why are only some [a1(I)] collagen chains affected?

Answer 11

• only one of two copies of CoA1 gene mutated

- Requires 2 copies of mutated protein to form complex (larger size protein)

Question 12

How does the structure/biochemical of Type I collagen change?

Answer 12

Gly-x-y —> Cys-x-y

- Side chain will not fit into centre, so from kink

Question 13

What genetic pattern does osteogenesis imperfecta follow?

Answer 13

Dominant pattern, one gene mutated will disrupt normal activity
- Gain-of-function mutation

Question 14

Why does the triple helix inside ER contain propeptides?

Answer 14

Don’t want fibrils to assemble within cell –> cell death

Question 15

How does malformed collagen cause skeletal abnormalities & Brittle bone?

Answer 15

1. Skeleton (hydroxyapatite - a form of calcium phosphate) is laid on collagen matrix then mineralised
2. If collagen defective, bone defective

Question 16

What are the prenatal tests identify genes in foetus?

Answer 16

1. RFLP - restriction fragment length polymorphism

2. PCR - polymerase chain reaction

Question 17

How do we acquire samples of DNA from foetus?

Answer 17

Amniocentesis / Chorionic villus sampling

- only if parents are carriers

Question 18

What does RFLP reply on?

Answer 18

Method replies on the mutation destroying known restriction site OR creating one

// which allow restriction endonuclease to recognise/remove the site

Question 19

How do we identify mutated gene using PCR?

Answer 19

Amplify region of mutation

1a. Gel electrophoresis - use probe to bind to mutation
1b. Sequence PCR product to detect

Question 20

How do we treat Brittle bone?

Answer 20

Disorder cannot be treated, only managed [depending on severity &type]

eg use bisphosphonates

Question 21

How do we identify mutated gene using RFLP?

Answer 21

1. Take DNA sample, digest it with appropriate restriction enzyme
2. Run on gel electrophoresis
   - separate DNA fragments
   - use probe to bind to region of mutation

\ only either normal/mutated DNA would be cleaved by enzyme