brittle Bones

Question 1

explain why a mutation in the COL1A1 gene shows a change in electrophoresis results for alpha 1 collagen chain

how does 2-Mercaptoethanol affects this

Answer 1

Sequence changes from glycine to cysteine

Cysteines can form disulphide bridges linking two chains together

in the absence of 2-Mercacaptoethanol, the cross linkage from disulphide bonds results in very low electrophoretic mobility

2-Mercaptoethanol breaks these disulphide bridges allowing mutated chain is separated by molecular weight, so the mutated collagen co transports with normal alpha 1 chains

Question 2

Why are only some of the alpha chains affected?

Answer 2

Since only one of the two copies of ColA1 gene are mutated, only some collagen molecules carry the mutation.

other allele is making the normal version

To form the complex we need two copies of the mutated protein to combine

Question 3

what are the two biochemical consequences of the change on the assembly of type I collagen and why

Answer 3

-Gly, X, Y structure- Glycine is in centre of triple helix. Other AA wont fit into the centre of the chain

cysteine is a larger amino acid molecule that generates a kink in the fibre, disrupting collagen assembly

inappropriate dissulphide bonds are formed as well due to cysteine

Question 4

Why does OI disorder have a dominant pattern of mutation in the patient’s family. Explain this by reference to the structure of collagen.

what kind of mutation is this

Answer 4

the triple helix is disrupted even when only one chain is disrupted

so even if only half of col α1 protein mutated , All fibrils will be affected due to packing

Gain of function mutation

Question 5

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

Answer 5

skeleton is laid down as hydroxyapatite on an ordered scaffold of collagen 1

abnormal collagen structure leads to defects in mineralisation process

so skeletal abnormalities occur and generally weak bones

issues in eyes, teeth, skin and ears as well

Skeleton is an active tissue - whole skeleton turns over every 5-10 years

Question 6

Where do you get fetal DNA from?

Answer 6

Need a sample derived from foetus e.g. amniocentesis, chorionic villus sampling.

Question 7

How is RFLP used to identify foetus who will suffer from OI?

Answer 7

RFLP – restriction fragment length polymorphism

mutation either removes or creates restriction sites
Digest DNA using restriction endonucleases
Gel electrophoresis
visualisation of bands
or use probe to region of dna near mutation

Question 8

what point mutation causes osteogenesis imperfecta

Answer 8

G replaced for T
so glycine is replaced by cysteine
in alpha 1 chain of type 1 collagen

Question 9

How are probes used to tell if foetus has OI?

Answer 9

probes are complementary to part of sequence where mutation is known to occur

under right conditions of temperature and ionic strength probe will bind to DNA if it has the same complementary sequence

so normal and mutant DNA can be distinguished

Question 10

how could you diagnose OI if a restriction enzyme recognition site has been altered/ added or removed

Answer 10

if site is altered only the normal DNA will be cleaved by enzyme to shorter fragments

if added or removed, there will be different length fragments to fragments of normal DNA

Question 11

what alpha chains does type one collagen contain

Answer 11

two alpha one peptides

one alpha two peptide

Question 12

how do you use PCR to see if foetus suffers from OI

Answer 12

amplify region with mutation

gel electrophoresis

use probe specific to mutation

or sequence PCR product