5.4 - Brittle Bones

Question 1

What is osteogenesis imperfecta?

Answer 1

• a disease where you are prone to repeated fractures of long bones, and malformed bones
• also affects eyes, teeth, skin, ears
• caused by a range of genetic disorders - most likely a mutation in glycine residues producing defective structural assembly of collagen

Question 2

Why does the mutation in the COL1A1 encoding the procollagen precursor of alpha1(I) collagen cause the altered electrophoresis pattern?

Answer 2

• sequence changes from glycine to cysteine
• cysteine forms disulphide bridges linking two alpha (I) chains together = migrate more slowly than individual chains in SDS-PAGE
• 2-mercaptoethanol (2-ME) breaks these disulphide bridges to get back the normal strands
• SDS-PAGE (sodium dodecyl sulphate poly acrylamide gel electrophoresis)

Question 3

Why are only some of the alpha1(I) collagen chains affected?

Answer 3

• since only one of the two copies of ColA1 are mutated, only some collagen molecules carry the mutation
• to form complex, need two copies of mutated protein to combine
• but it’s not 50% normal, 50% abnormal - because of differences in rates of transcription, translation and stability of mRNA/protein

Question 4

Predict possible biochemical consequences of the change on the assembly of type I collagen

Answer 4

• the repeating structure of collagen is -Gly, X, Y-
• glycine is usually at the centre of the triple helix as it is smallest
• other amino acids won’t fit at the centre of the triple helix = triple helix loses structure
• larger cysteine in mutant molecule will cause steric hindrance = generates kink in normally straight triple helix = causes defect in assembly of fibres

Question 5

The disorder has a dominant pattern of mutation in the patient’s family - explain this by reference to the structure of collagen

Answer 5

• gain of function mutation
• the mutation disrupts the activity of the normal version of ColA1
• only half of col-alpha1 protein mutated
• all fibrils affected due to packing
• dominant in most cases as triple helix contains two a1(I) chains and will be disrupted if only one is the mutant form

Question 6

Why might the predicted change cause skeletal abnormalities and brittle bones?

Answer 6

• initially skeleton laid down as collagen
• mineralisation - abnormal collagen structure leads to defects in mineralisation process –> skeletal abnormalities, weak bones
• if collagen is defective, then the bone is defective
• skeleton is an active tissue, whole skeleton turns over every 5-10 years

Question 7

Suitable prenatal diagnostic test to identify foetus who will suffer from OI

Answer 7

• need a sample derived from foetus e.g. amniocentesis chorionic villus sampling
• RFLP (restriction fragment length polymorphism) - mutation either removes or creates restriction site, digest DNA, gel electrophoresis, use a probe to region of DNA near mutation
• PCR - amplify region with mutation, gel electrophoresis, use a probe specific for mutation, sequence PCR product