Genetics - Brittle Bones Flashcards
What is the name for brittle bone disease?
Osteogenesis imperfecta
What is the main feature of brittle bone?
- Repeated fracture of long bones
- Malformed bones
What is the distinct property/feature of collagen?
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)
What are collagens for?
- Give mechanical strength and rigidity to tissues and organ
- Give tensile strength to skeletal tissues (bone, cartilage, tendon, ligament)
What is the mutation of brittle bone disease?
Point mutation in collagen gene Type I
- Substitution of glycine with cysteine
What is the effect of substituting glycine with cysteine in collagen gene?
- 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
What chains do collagen type I contain?
2 [a1(I)] + 1 [a2(I)]
Why is there an unusual band in the absence of 2-ME but not in the presence of 2-ME?
2-ME breaks disulphide bridges so the bands return to ‘normal’ co-migrate
What is 2-ME?
2-Mercaptoethanol used in gel electrophoresis
How does gel electrophoresis separates the bands?
SDS-PAGE (sodium dodecyl sulfate)
- causes denature of protein
- forms layer around protein making it negative [per length of protein]
- migration due to size difference
*Cysteine forming larger protein w/ disulphide bridges
Why are only some [a1(I)] collagen chains affected?
- only one of two copies of CoA1 gene mutated
- Requires 2 copies of mutated protein to form complex (larger size protein)
How does the structure/biochemical of Type I collagen change?
Gly-x-y —> Cys-x-y
- Side chain will not fit into centre, so from kink
What genetic pattern does osteogenesis imperfecta follow?
Dominant pattern, one gene mutated will disrupt normal activity
- Gain-of-function mutation
Why does the triple helix inside ER contain propeptides?
Don’t want fibrils to assemble within cell –> cell death
How does malformed collagen cause skeletal abnormalities & Brittle bone?
- Skeleton (hydroxyapatite - a form of calcium phosphate) is laid on collagen matrix then mineralised
- If collagen defective, bone defective
What are the prenatal tests identify genes in foetus?
- RFLP - restriction fragment length polymorphism
2. PCR - polymerase chain reaction
How do we acquire samples of DNA from foetus?
Amniocentesis / Chorionic villus sampling
- only if parents are carriers
What does RFLP reply on?
Method replies on the mutation destroying known restriction site OR creating one
// which allow restriction endonuclease to recognise/remove the site
How do we identify mutated gene using PCR?
Amplify region of mutation
1a. Gel electrophoresis - use probe to bind to mutation
1b. Sequence PCR product to detect
How do we treat Brittle bone?
Disorder cannot be treated, only managed [depending on severity &type]
eg use bisphosphonates
How do we identify mutated gene using RFLP?
- Take DNA sample, digest it with appropriate restriction enzyme
- 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