Brittle Bones

Question 1

What effect does SDS have on the electrophoresis of collagen?

Answer 1

The speed of migration of the proteins in the collagen depend on their size not charge

Question 2

What is SDS?

Answer 2

sodium dodecyl sulphate

Question 3

How do you culture fibroblasts from a patient?

Answer 3

from a skin biopsy

Question 4

What is collagen I made of?

Answer 4

2 alpha-1 polypeptide chains, 1 alpha-2 polypeptide chain

Question 5

What is 2-mercaptoethanol?

Answer 5

A sulfhydryl reagent which reduces the disulphide bond between the cysteine residues in proteins

I.e. -S-S- becomes 2-S-H

Question 6

What is the relationship between weird alpha-1 chain and 2-mercaptoethanol in brittle bones?

Answer 6

Low electrophoretic mobility in the absence of 2-mercaptoethanol
But co-migrates with normal alpha-1 chains in the presence of the reagent

Question 7

What does the COL1A1 gene code for?

Answer 7

encodes the procollagen precursor of alpha-1 collagen

Question 8

What is the point mutation and its effect of the point mutation which occurs in the brittle bones case?

Answer 8

(guanine to thymine)

Glycine is replaced by cysteine - this causes a different amino acid to be coded for, meaning a different secondary structure will form - hence protein will fold up differently and different bonds will form

Question 9

Why does the change cause the altered electrophoresis pattern?

Answer 9

Sequence changes from glycine to cysteine
Cysteines can form disulphide bridges linking 2 chains tgt
2-Mercaptoethanol breaks these disulphide bridges

Question 10

What is the effect on the protein of a different, larger amino acid being coded for in a protein sequence?

Answer 10

The larger amino acid will cause steric hindrance - this generates a kink in the normally straight chain resulting in defective protein forming

Question 11

Which is heavier cystine or glycine?

Answer 11

Cysteine

Question 12

Why are only some of the alpha-1 collagen chains affected?

Answer 12

(if heterozygous) Since only one of the 2 copies of COLA1 gene are mutated only some collagen molecules carry the mutation
To form complex needs 2 copies of mutated protein to combine

Question 13

Why is the pattern of inheritance basically dominant for osteogenesis imperfecta?

Answer 13

Gain of function mutation
The mutation disrupts the activity of the normal version of ColA1
Only half of ColA1 protein mutated
All fibrils will be affected due to packing

Question 14

Why does abnormal collagen production result in the symptoms and signs seen in osteogenesis imperfecta?

Answer 14

Abnormal collagen structure leads to defects in the mineralisation process of bone as hydroxyapatite has nothing to be laid onto

Question 15

Why might the predicted change cause skeletal abnormalities and brittle bones (aka OI)?

Answer 15

Initially skeleton laid down as collagen
Later stage mineralisation
If collagen is defective then the bone is defective
Skeleton is an active tissue, whole skeleton turns over every 5-10 years

Question 16

Suggest a suitable prenatal diagnostic test to identify a foetus who may be at risk of osteogenesis imperfecta?

Answer 16

Amnio or CVS (chorionic villus sampling)

Question 17

What is restriction fragment length polymorphism?

Answer 17

When specific probes that are complementary to the part with the mutation are set under the right conditions of temperature and ionic strength - only becomes hybridised if the sequence is exactly complementary which allows mutant and normal sections to be identified

Question 18

After amnio/ CVS (apart from RFLP) what else can you do?

Answer 18

PCR- amplify region with mutation
Gel electrophoresis
Use a probe specific for mutation
Sequence PCR product

Question 19

Why does a substitution of glycine for cysteine result in defective assembly of collagen fibres?

Answer 19

The larger amino acid in the mutant molecule will cause steric hindrance which generates a kink in the normally straight triple helix, with a resulting defect in the assembly into fibres.

Glycine will be in the centre of the triple helix, so other AA won’t fit

Question 20

What does cysteine contain in its side chain?

Answer 20

a reactive sulphydryl group

Question 21

What is the effect of cysteine having a reactive sulphydryl group in its side chain?

Answer 21

inappropriate disulphide bonds between the two α1(I) chains in the helix, resulting in a cross linked polypeptide chain

Question 22

What effect does a cross-linked polypeptide chain have on the speed of migration on an agar plate?

Answer 22

polypeptide chains will migrate much more slowly than the individual chains when examined by gel electrophoresis in the presence of SDS

Question 23

Why is directly sampling the patients collagen protein not a suitable pre-natal testing method?

Answer 23

Sampling collagen from a foetus would be impractical and risky

Question 24

What is collagen?

Answer 24

An extracellular matrix protein synthesised by and secreted from a variety of cells
Such as fibroblasts, and organised into insoluble fibres
These fibres are a major part of the ECM giving mechanical strength and rigidity to tissues and organs

Question 25

What does collagen provide TENSILE strength to?

Answer 25

skeletal tissues including bone, cartilage, tendons and ligaments

Question 26

What do all 5 major types of collagen have in common?

Answer 26

triple helix structure
- associated with this is the unusual AA composition with its high conc of glycine
- glycine is the smallest AA of the AA and occurs at every 3rd position in collagen
- It faces the interior of the helix

Question 27

What is the main feature of OI and what is the misunderstanding usually caused by it?

Answer 27

repeated fracture of long bones- easily misdiagnosed with child abuse

• there are also malformed bones (whole range of disorders which can lead to the disease)

Question 28

Where can there also be problems with OI?

Answer 28

eyes, teeth, skin, ears