Brittle Bones Flashcards

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1
Q

What effect does SDS have on the electrophoresis of collagen?

A

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

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2
Q

What is SDS?

A

sodium dodecyl sulphate

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3
Q

How do you culture fibroblasts from a patient?

A

from a skin biopsy

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4
Q

What is collagen I made of?

A

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

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5
Q

What is 2-mercaptoethanol?

A

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

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

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6
Q

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

A

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

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7
Q

What does the COL1A1 gene code for?

A

encodes the procollagen precursor of alpha-1 collagen

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8
Q

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

A

(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

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9
Q

Why does the change cause the altered electrophoresis pattern?

A

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

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10
Q

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

A

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

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11
Q

Which is heavier cystine or glycine?

A

Cysteine

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12
Q

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

A

(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

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13
Q

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

A

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

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14
Q

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

A

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

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15
Q

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

A

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

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16
Q

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

A

Amnio or CVS (chorionic villus sampling)

17
Q

What is restriction fragment length polymorphism?

A

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

18
Q

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

A

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

19
Q

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

A

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

20
Q

What does cysteine contain in its side chain?

A

a reactive sulphydryl group

21
Q

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

A

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

22
Q

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

A

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

23
Q

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

A

Sampling collagen from a foetus would be impractical and risky

24
Q

What is collagen?

A

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

25
Q

What does collagen provide TENSILE strength to?

A

skeletal tissues including bone, cartilage, tendons and ligaments

26
Q

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

A

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

27
Q

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

A

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)
28
Q

Where can there also be problems with OI?

A

eyes, teeth, skin, ears