Download Assessing Methods of Detecting Osteogenesis Imperfecta.

Assessing Methods of Detecting Osteogenesis
Imperfecta.
Lorren Kaeding, MSc.
University of Newcastle, Biomedical Sciences.
1st May, 2006
Abstract
Osteogenesis Imperfecta (OI) is a dramatic and painful genetic disorder in which a sufferer produces
either insufficient amounts of collagen, or collagen of poor quality. The result of which is bones that are
often malformed and are very easily broken.
Clinical diagnosis of OI is most common, however mild OI shares many of its symptoms with other
bone diseases and misdiagnosis is easy to make. Therefore we need scientific processes to accurately
identify the OI disorder so appropriate treatment can be commenced.
In this article two different methods of detecting OI are trialled for accuracy of differentiating OIaffected samples and a healthy control sample. These processes are Protein Analysis by gel filtration, and
genetic sequencing of the collagen gene for comparison to a healthy sequence.
Both methods returned a positive result for OI, the genetic sequencing returning a more definitive,
qualitative result, and the protein analysis a marginal, but still observable, positive result.
There are sources for error in both methods but none that can’t be reduced through further refining
of the methods and choice of separation mediums.
Despite genetic analysis having a much higher degree of accuracy and repeatability, protein analysis
was considered the more useful and practical in a real-life laboratory environment.
Introduction
Osteogenesis Imperfecta (OI), also knows an Brittle Bone disease or Ekman-Lobstein disease, is a
genetic defect that affects the production of the protein Collagen. Collagen is an integral component of
bone structure and the impairment causes the bones to be formed brittle and weak.
OI is characterized by bones that break easily, often from little or no apparent cause. A person with
OI may break a rib while coughing, or a leg by rolling over in their sleep. 1 Other symptoms include slight
to severe bone deformities, small stature, curvature of the spine and barrel-shaped rib cage, loose joints,
poor muscle tone in arms and legs, discolouration of the sclera (whites of the eyes), early hearing loss and
respiratory problems due to underdeveloped lungs. 2
OI falls into two broad categories; Type I, in which the quantity of collagen is inadequate for proper
bone and other tissue formation; and Type III and IV, in which the quality of the collagen produced is
inadequate, and was the focus of this article.
(Type II is an inadequacy of collagen quantity and quality and is the most severe form of OI with
most cases dying before adulthood.)
The poor ‘quality’ of the protein is due to a mutation in the gene for Type I collagen production, the
collagen utilised for bone construction. Normal collagen is formed by a triple helix of collagen amino acid
chains. Two α1 chains and one α2 chain. The mutation in the DNA sequence produces a deformed α1
amino chain, in which a cysteine is substituted for a glycine amino acid.
-2-
Fig1 Glycine (GLY)
Fig2 Cysteine (CYS)
The broad definition of OI implies Glycine substituted with Cysteine, but substitution by any other
amino acid will have a similar effect on collagen formation and is still classified as OI.
Collagen is a triple helix of collagen proteins. This structure has great tensile strength, similar to
wire cable, and this combined with the crystalline structure of bone minerals gives the structure a degree of
flexibility. This combination of rigidity and flexibility is what gives bones their strength.
Collagen has an unusual amino acid composition and sequence. Glycine (GLY) is found at almost
every third residue, and also contains large amounts of proline (PRO), as well as two uncommon post
translational amino acids not directly inserted during translation of mRNA: hydroxyproline (HYP) and
hydroxylysine.3
Glycine, and the regular occurrence of the amino acid in the protein sequence, plays an important
function in the assembly of the triple helix. Its position every third residue places it in the interior of the
triple helix when it is assembled, where there is no space for a sidegroup larger than Glycine’s hydrogen
atom.
When a Glycine is replaced by a Cysteine residue in one of the collagen strands it results in
abnormal helix formation as it is too large to fit in the inner axis of the triple helix. This forms a ‘kink’ in
the tertiary structure and in some cases terminating the protein chain resulting in a malformed or shortened
collagen strand. Inappropriate disulphide bonding between the Cysteine functional group and the other
strands in the triple helix, and unwanted post translational modifications due to the slow unfolding of the
helix and the strand’s resulting tertiary shape further add to the deformation of the triple helix.
It’s this malformation of the collagen strand that greatly reduces the tensile strength and flexibility of
the collagen fibre as a whole, in turn weakening the structure of the bone tissue.
There are many different methods for detecting and diagnosing OI. These include parent and patient
history, histology of bone samples, prenatal ultrasound, chorionic villus sampling (CVS, examining
placental cells for protein and genetic mutations) and amniocentesis (examining fetal cells shed into the
amniotic fluid for protein and genetic mutations).4
The two processes used here were protein analysis by SDS-PAGE (polyacrylamide gel
electrophoresis) and genetic sequencing using RT-PCR (Reverse Transcription – Polymerase Chain
Reaction).
The collagen and genetic samples were obtained from skin fibroblasts from a young boy suspected
of having OI, as this is far less invasive than obtaining collagen from placental or amniotic cells.
Protein analysis of collagen using PAGE is basically a matter of differentiating a control sample of
normal collagen and the suspected OI collagen based on their differences in size and shape. The OI
collagen, because of its irregular shape, makes it a physically larger molecule than the triple helix of the
normal collagen sample. When run through a polyacrylamide matrix, which effectively acts as a ‘sieve’, the
smaller normal collagen will move further through the gel than the OI-effected collagen in the same
amount of time. This presents as bands in the gel at different distances from the starting point, the OIeffected collagen having moved a shorter distance than the control sample.
Genetic analysis and sequencing is achieved by analysing collagen mRNA (messenger RNA). The
collagen-forming gene (the Type 1, α1 collagen-forming gene, COL1A1) is first isolated from the
fibroblast cells, the RNA is amplified by the RT-PCR process, processing with ddNTPs (di-deoxiribose
nucleotide tri phosphate) which dyes each specific nucleotide with a different fluorescent colour which is
detected by an optical laser. The sequence of colours represents the gene sequence which is then compared
to a sequence from a normal COL1A1 gene for any differences.
A single different nucleotide is all it takes for an amino acid substitution to take place, producing a
defective collagen strand.
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Method
Protein Analysis
Fibroblasts from two skin samples were obtained, one from a subject with OI and the other from a
healthy subject to be used as the control sample.
Prior to starting the processes, the fibroblasts were treated for 4 days with ascorbic acid to stimulate
the production and processing of collagen.
Step 1 - Extraction of Collagen from Skin Fibroblasts.
Preparation and required solutions.
Saturated Ammonium Sulphate solution (pH 7.5):
22.802g of solid ammonium sulphate [(NH4)2SO4] was gradually added to 30mL of deionised water
stirred with a small magnetic stirrer. Not all of the ammonium sulphate was dissolved even with constant
stirring (as was expected) indicating the solution was saturated.
This produced a slightly acidic solution with a pH of around 6. To give the solution the required 7.5
pH, 1M ammonium hydroxide solution was added in small ( < 10µL ) increments to get the required pH.
Extraction of Collagen
200µL of 0.5M EDTA (pH 7.5) and 200µL of 100x Protease inhibitor was added to 20mL of the
fibroblast cell medium in two falcon tubes (one containing the affected sample, the other containing the
control), kept at 4˚C by immersion in ice. They were mixed well and 5mL of the saturated ammonium
sulphate was added to each, which causes the protein to precipitate out.
The falcon tubes were then gently mixed overnight at 4˚C, during which a precipitate was formed
containing the collagen and possibly other proteins. The precipitate was centrifuged and the supernatant
discarded in preparation for Step 2.
Step 2 – Solubilisation and Digestion of Procollagen
Preparation and required solutions.
100% Ethanol
0.1mg/mL Pepsin in 0.5M acetic acid
“Buffer A” solution (pH buffer)
A 50mL solution made up of
 50mM TRIS (2-amino-2-hydroxymethyl-1,3-propanediol) / HCl (pH buffer)
 0.15M NaCl
 5mM EDTA (ethylenediaminetetraacetic acid) pH 7.5 (Buffer and detergent)
 100x protease inhibitor (inhibits protein-damaging enzymes)
The following quantities were added to 40mL of deionised water
-4-



0.303g of solid TRIS/HCL (mol weight: 121.138g)
0.0075L of 0.15M NaCl solution
0.05L 5mM EDTA solution (pH 7.5 - some 1M HCl needed to be added to the
sample to give the required pH)
 0.5mL protease inhibitor
And then topped up with more deionised water to form a 50mL solution
All procedures carried out at 4ºC (by keeping samples on ice as much as possible) unless otherwise
stated
Resuspension and Digestion
250µL of Buffer A (a pH buffer) was added to each falcon tube containing the pelletised proteins
from Step 1 and dissolved by gently pipetting the solution and down so as to avoid frothing until the pellet
was fully dissolved.
200µL of each sample were transferred to separate microfuge tubes and 600µL of 100% Ethanol
added to each to make a 75% Ethanol solution, which dehydrates the protein and removes any remaining
cell material.
The samples were immersed in ice for 45 minutes and a white precipitate was formed. They were
then microfuged for 5 minutes and the supernatant discarded.
200µL of pepsin solution was added to each pellet and resuspended. The samples were then left
overnight at 4ºC to denature (separate from the triple helix into individual strands), after which they were
concentrated using an evaporative centrifuge.
Step 3 – Electrophoresis of Collagen
Preparation and required solutions.
1 x 5% pre-cast acrylamide gel pack
S-20 sample buffer. (Protein standard)
Reducing Sample Buffer made from
 1 part 60mM DTT (Dithiothreitol, a reducing agent)
 5 parts S-20 sample buffer
Electrode Tank Buffer
The following were added to deionised water to form a 500mL solution
 1.5g TRIS
 7.2g Glycine
 0.5g SDS
Gel Stain.
Electrophoresis
A pre-made polyacrylamide gel was used. The gel pack was opened top and bottom, and placed in
the tank. Electrode tank buffer was poured into the tank enough to fully submerge the gel pack.
20µL of non-reducing S-20 sample buffer was added to the concentrated samples and boiled in a
heating block for 5 minutes at 90°C.
After checking that the pellets had fully dissolved (some gentle pipetting was required to fully
dissolve the control sample), a 5% dilution of the protein solutions was made from 1µL of each protein
-5-
sample and 19µL of reducing sample buffer. The solutions were then placed in heating blocks for 5 minutes
at 90°C before 15µL of each sample was loaded into the polyacrylamide gel wells.
Unused wells were filled with non-reduced S-20 sample buffer to prevent “bowing” of the tracks.
The electrophoresis tank was closed run at 60mA for 45 minutes, until the dye front had reached the
bottom of the gel. The power was disconnected, the gel pack removed and the plastic plates gently prised
off the gel, which was placed into a staining box filled with deionised water. The gel was washed for 15
minutes on the shaking platform (replacing the water every 5 minutes) then immersed in Coomasie Blue
stain for 60 minutes with gentle agitation on the shaking platform.
The gel was then rinsed in water for 30 minutes (again replacing the water every 5 minutes) and then
photographed for analysis.
Method
Genetic Analysis
GLOVES MUST BE WORK AT ALL TIMES WHEN ISOLATING AND
PROCESSING RNA - THE SKIN SURFACE IS A MAJOR SOURCE OF RNAse
ACTIVITY.
Rather than working two separate samples, only the OI-affected cells were analysed in this process.
The ‘control’ in this process came from a gene sequence database and was compared to the genetic
sequence found in the sample cells.
Step 1. Isolation of RNA from Fibroblast cells.
Preparation and required solutions.
RNA lysis
 250g guanidinium thiocyanate
 293ml water
 17.6ml 0.75M Na Citrate, pH7
 26.4ml 10% sarcosyl
Isolation of RNA
The fibroblast cells were lysed in RNA lysis solution and 700µL of the lysed cell solution was
placed in a falcon tube. 200µL of pH4, 2M sodium acetate was added and gently mixed by inversion of the
tube. 700µL of H2O-staurated phenol was added and gently mixed, followed by 400µL of chloroform
(CH3Cl) and gently mixed.
The solution was centrifuged at 13000rpm for 7 minutes at room temperature. This separates the
solution into layers of cellular proteins at the bottom suspended in the phenol, genomic DNA in the middle
suspended in the chloroform, and RNA in the top layer suspended in the sodium acetate. The centrifuging
separates the cellular components based on molecular weight. Proteins, being larger and heavier then DNA
or RNA are forced to the bottom of the tube and RNA, being smaller and lighter than DNA settles at the
top (see Fig3).
-6-
Fig 3. Lysed cell components post-centrifusion.5
The RNA / Na-acetate layer was carefully pipetted out down to within 0.5cm of the DNA layer and
placed in a fresh falcon tube. The tube containing the DNA and protein was discarded.
1mL of isopropanol was added to the RNA solution, gently inverted and placed in -20ºC for 15
minutes before being centrifuged at 13000rpm for 15 minutes at 4ºC. The supernatant was carefully and
quickly removed and placed in a waste tube, thus removing any residual DNA.
The pellet resuspended in 500µL lysis solution and 1mL of 100% ethanol, mixed by gentle inversion
and replaced in -20°C for another 15 minutes. It was then centrifuged at 13000rpm for 15 minutes at 4°C.
The supernatant was again quickly removed and discarded, removing any tRNA or rRNA and leaving just
the mRNA.
500µL cold 70% ethanol was added and the solution and pellet immediately centrifuged at
13000rpm for 3 minutes and 4°C and the supernatant discarded.
The open falcon tube was placed in a 50°C heating block until the pellet was dry, resuspended in
20µL sterile 1 x TE and the closed tube placed on the 50°C heating block for 10 minutes until the pellet
was fully dissolved. It was then centrifuged briefly to force all the liquid to the bottom and then stored at 20°C until the next stage was set up.
Before it was stored, 2µL was taken and diluted in 498µL (1:250) of deionised water, then placed in
a cuvette and the optical density was measured at a wavelength of 260nm. An optical density of 360µg/mL
was observed.
Step 2. Assessing the integrity of the RNA by Agarose Gel Electrophoresis
Preparation and required solutions.
10 x MOPS Buffer
 0.2M 3-(N-morpholino)propanesulfonic acid
 0.05M Na Acetate, pH 7
 0.01M Na2 EDTA
RNA Loading Buffer
 50% glycerol
 0.1mg/mL bromophenol blue
Agarose powder
Formaldehyde
Formamide
Ethidium Bromide
Assessing the RNA
Note: Do not use all of the RNA sample for the electrophoresis! 9-10µL was set aside
and kept on ice for the RT-PCR in the next part of the process.
-7-
0.5g of Agarose powder was weighed out into a 250mL conical flask and 36.5mL of sterile water
and 5mL of 10 x MOPS buffer was added and swirled gently to mix. This was placed into a microwave
oven set to ‘high’ and the time set to about 5 minutes. Under careful observation the Agarose mixture was
heated and the oven stopped as the mixture started to boil. The flask was taken out (caution, HOT!) and
swirled to help the Agarose powder dissolve. This was repeated as necessary until the Agarose was fully
dissolved, care taken not to let the solution boil over out of the flask.
Once the Agarose was dissolved, the flask was taken out and allowed to cool until it was able to be
handled comfortably by hand.
While the Agarose was cooling, the tray for the gel was prepared by taping the ends of the tray and
inserting the sample comb 1-2mm above the bottom of the tray.
In a fume hood, 8.5mL of formaldehyde and 1µL of ethidium bromide was added and swirled to
mix. The agarose was immediately poured into the tray, as once the formaldehyde is added it will start to
solidify.
When the gel had set, the comb and the tape were removed and the tray placed in the electrophoresis
tank. Enough 1 x MOPS buffer (1:10 10xMOPS and deionised water) was added to completely cover the
gel.
To prepare the RNA sample, it was diluted to 100µL with sterile water then 10µL 3M Na Acetate,
pH5.2 and 300µL 100% ethanol was added. The solution was mixed gently and placed in -20°C for 15
mins before being centrifuged at 13000rpm and 4°C for 15 mins.
The supernatant was removed and 300µL of ice-cold 70% ethanol was added and spun at 13000rpm
and 4°C again for 3 mins. The supernatant was removed, dehydrating the RNA and removing any
remaining foreign substances, and the tube was place on a 65°C heat block for 5 mins until the pellet was
dry, then 6µL of sterile water was added.
A separate tube of 6µL standard RNA sample was also obtained for comparison.
To each tube, 12.5µL of formamide, 2.5µL of 10 x MOPS buffer and 4µL of formaldehyde was
mixed and placed on a 65°C heating block for 5 mins, then immediately placed on ice. This minimises as
much as possible any breakdown and fragmentation of the RNA strands.
2.5µL of RNA loading buffer was added to each and centrifuged briefly to ensure all the contents
were at the bottom of the tubes.
The two samples were loaded into the gel and the electrophoresis run at 80V until the dye was
approx. two thirds of the way down the gel. The gel was then photographed using UV illumination.
The reason for assessing the RNA is to make sure that enough whole RNA strands are intact for the
following steps to be useful. Without the distinct bands expected to be visible in the gel, the preceding steps
may need to be repeated.
Step 3. Reverse Transcription – Polymerase Chain Reaction (RT-PCR)
Preparation and required solutions.
RT Master Mix
 2µL 10 x buffer
 4µL 25mM MgCl2
 0.5µL 40U/µL RNAse inhibitor
 1µL 9U/µL AMV reverse transcriptase
 2µL 10mM dNTP mix
 1µL 0.5µg/µL oligo d(T)
 1µL 0.5µg/µL random primer mix
PCR Master Mix
 10µL 10 x PCR buffer
 6µL 25mM MgCl2
 1µL 10mM dNTP mix
 1µL 100µM forward primer
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

1µL 100µM reverse primer
1µL 9U/µL Taq polymerase
RT-PCR
8.5µL of RNA solution was pipetted into a 1.5mL tube and placed onto a 70°C heating block for 5
minutes, then immediately placed in ice. After 5 minutes, while keeping the tube in ice as much as possible,
11.5µL of RT Master Mix was added.
The tube was centrifuged briefly to mix the solution and make sure it was all at the bottom of the
tube. The tube was then placed on a 42°C heat block for 15 minutes then placed back in ice.
A sterile 0.2mL tube was placed in the ice, where 75µL of sterilised water, 5µL of the RNA / RT
solution and 20µL of PCR Master Mix were added into it, gently tapping the bottom of the tube briefly to
mix it.
The tube was kept on ice until the thermocycler was programmed and pre-heated. It’s important to
keep the sample on ice right up until it is placed into the thermocycler because if the solution warms up the
RT process will partially begin and render the RNA unusable.
The thermocycler was set for 35 cycles of the following sequence:
 1 minute at 94°C (mRNA / cDNA denatures)
 1 minute at 65°C (primers attach to cDNA)
 2 minutes at 72°C (Reverse transcription polymerisation occurs)
Step 4. Analysis of PCR Products by Agarose Gel Electrophoresis
Preparation and required solutions.
1 x TAE Buffer
 4.84g TRIS base
 0.744g Na2 EDTA
 1.142mL glacial acetic acid
Made up to a 1L solution using deionised water.
Agarose powder
Ethidium bromide
Analysis of PCR products
A 2% agarose gel was prepared by dissolving 3g agarose in 150mL 1 x TAE buffer by heating in a
microwave (as described above), and 1µL of ethidium bromide added when cooled
The gel was poured into a tray (as described above) and placed into the tank when set. Enough 1 x
TAE buffer was added to cover the gel.
10µL of the PCR product was placed in a 1.5mL tube, along with 5µL sterile water and 2.5µL
loading buffer. The PCR sample was loaded into the gel and the electrophoresis run at 90V until the dye
was two thirds down the gel.
The gel was then photographed under UV light.
The reason behind Assessing the PCR product is to ascertain the quality of the sample before it goes
through the process of DNA sequencing. If the RNA, and consequently the cDNA was fragmented at some
stage, then the PCR product will not contain the full 355bp needed for sequencing and the process will need
to be redone before you can proceed.
Step 5. Sequencing of the cDNA
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The actual sequencing was done by a 3rd party and the results returned displayed below. The
following is a brief summary of the sequencing process.
The materials and methods used for genetic sequencing are the same for the RT-PCR, except along
with the dNTP mix a small, calculated amount of ddNTP is also added. ddNTPs have the affect of
terminating the transcription when one is attached, leaving cDNA strands of all different lengths, each
terminating in one of the ddNTPs.
Each different kind of ddNTP is tagged with a different fluorescent colour, so when the product is
run through a denaturing agarose gel and the strands separate into bands of different lengths, if forms bands
of distinct colours (each corresponding to a different nucleotide) which is read by the optical laser and the
sequence printed out (See Fig 4).
Fig 4. Optical (Fluorescent) Laser Sequencing. 6
Results
Protein Analysis.
The results of the protein analysis can be seen in the polyacrylamide gel electrophoresis.
1
2
3
4
Track
1
2
3
4
Fig 5. PAGE of Proteins
Contents
Protein standard
Control sample
Protein standard
Affected sample
Table 1. Contents of PAGE wells
- 10 -
Tracks 2 and 4 are the two protein samples. The results we are looking for are a difference in how
far the bands (indicated, Fig 4) have moved down the gel. According to the hypothesis, the control collagen
should move further down the gel than the OI-affected collagen due to its smaller molecular size.
The difference is slight however the sample in Track 2, the healthy collagen protein, has moved
further than the OI collagen protein has in Track 4 (see Fig 5).
1
2
3
4
Fig 6. Close up of Collagen Protein Bands.
On an enlarged photograph the difference is more obvious. The control collagen extends further
down than the OI collagen, although it may also be argued that the lower band of the control collagen was
‘pushed’ further down the gel because of the thickness of the bands (possibly due to a higher concentration
of control collagen loaded into the gel than the OI collagen).
Despite this, the protein analysis for detecting OI would, in this case, return a positive result.
Genetic Analysis
Assessing the Integrity of the RNA
The integrity of RNA is it a matter of minimising the fragmentation of the RNA strands.
Fragmentation shows up on the acrylamide gel as a ‘haze’ near the bottom of the gel, representing all the
randomly-sized small pieces of RNA that move down the gel at a relatively fast but un-uniform rate. RNA
in that state cannot be used for genetic sequencing.
An RNA sample with good integrity should show up on a gel as 3 distinct bands at the 28s, 18s and
5s locations (with reference to a RNA standard run in the same gel)
1
2
3
Track 1
Track 2
Track 3
Fig 7. rRNA in an Agarose gel.
RNA Standard
N/A
RNA Sample
Table 2. Contents of Agarose wells.
- 11 -
The gel obtained from the method above yielded this gel (Fig 6, Table 2). Note the two faint bands
indicated on the left, and how they are level with the two bands of the RNA standard on the left. The two
bands of the standard represent the 28s and 18s (5s not shown) and the two bands of the sample RNA
indicate the presence of numerous RNA strands of those two molecular sizes.
This means there are significant amounts of whole, intact RNA strands in the sample above the
amounts of other random lengths of RNA, represented by the ‘smear’ appearance of the gel track, so the
RT-PCR has a good starting point to work with.
Assessing the PCR Product.
The DNA Standard in track 1 is marked every 100bp (by molecular size), the brighter band in the
middle indicating 500bp and each band below it decreasing by 100bp.
1
2
3
1
2
3
Fig 8. Agarose gel of DNA Standard and PCR Product
100bp DNA Ladder
N/A
PCR Product
Table 3. Contents of Agarose Gel
The PCR product in this sample was expected to be 355bp, given the specific forward and reverse
primers used, and as can be seen in Fig 7 the indicated band on the PCR product (track 3) is midway
between the 300bp and 400bp.
The presence of the band at about 350bp mark tells us that the RT-PCR worked as expected, and the
DNA is good for sequencing.
Genetic Sequencing
The sequence of the sample DNA template (the PCR product) shows a definite nucleotide
substitution of the 142nd base pair (see arrow) of the PCR sequence or the 3580th nucleotide of the COL1A1
gene sequence. The absence of a stroke indicates a mismatch between that nucleotide of the sample DNA
sequence and what the nucleotide should be for a normal sequence.
A Guanine nucleotide has been substituted by an Adanine. The triplet codon that should read GGA
(bottom sequence) instead reads AGA. This means that where there’s supposed to be a Glycine in the
protein chain, Arginine is there in its place. (see Fig 9)
- 12 -
Fig 9. Comparison of Normal Collagen Gene Sequence (bottom) and the Sequence of the Sample DNA (top)
A glycine is substituted for another amino residue, therefore the diagnosis of Osteogenesis
Imperfecta is positive. The broad definition of OI says it should be substituted by Cysteine, but substitution
by any other amino will have a similar effect on the collagen and the subject.
Any amine residue other than glycine on the inner axis of the collagen triple helix will cause a
deformation of the structure due to its larger size not being able to fit in the same area. The inability to
unfold into its proper structure causes additional sporadic post-translational modification regardless of
which amine residue is substituted.
- 13 -
Table 4. List of Amino Acids and their Triplet Codons.
Fig 10. Arginine (ARG)
Discussion
Both Protein Analysis and Genetic Sequencing returned positive results to Osteogenesis Imperfecta,
but they both have advantages and disadvantages in their method.
The SDS-PAGE of the collagen proteins (Figs 4 & 5), although providing a positive result,
demonstrated very little differentiation between the normal and OI-affected proteins. The slight difference
may be accounted for by the apparently higher concentration of control sample added to the PAGE well
which may have ‘pushed’ the lower of the two bands slightly further down the track than the affected
sample giving a false positive. The upper of the two bands were identical. An optical density test and
dilution prior to loading the gel would remove this possible source of error.
Another problem is the denaturing pepsin. This needs to be monitored carefully as if left too long it
starts to break down the collagen into smaller pieces, instead of simply separating the collagen strands from
its triple helix structure. This results in loos of collagen to analyse and a ‘messy’ gel track rather than well
defined bands.
A more definitive differentiation between the affected and normal collagen would be possible if the
electrophoresis was allowed to run longer, even to the point of allowing some of the smaller proteins to
flow out of the gel completely. Contamination of the tank buffer shouldn’t be significant as both the dye
and the proteins would be attracted to the electrode at the bottom of the tank.
Using a coarser acrylamide gel than the 5% used (perhaps a 3% gel) would also magnify the
different distances travelled through the gel.
The Genetic sequencing is by far the more accurate way of detecting OI, but it is a very timeconsuming and involved process compared to protein analysis, and in a commercial laboratory environment
may not always be the most suitable method except as a “second opinion” test following an ambiguous or
questionable result from the initial protein analysis.
The Taq Polymerase may also be seen as a source of error, not having the proof-reading ability to fix
any mismatched nucleotides during PCR7. Pfu Polymerase could possibly be used instead as it has this
proof-reading characteristic and is far more accurate, however it is also slower to process the DNA in PCR
and is much more expensive8. The nature of the optical sequencing also doesn’t require that high an
accuracy rate, as any single mismatched nucleotide will be easily masked by the numerous correct ones for
that particular base pair.
Osteogenesis Imperfecta can be detected using either of these two laboratory methods. Genetic
sequencing is the more accurate and quantitative method of the two, but is the more expensive in terms of
expense and man-hours.
Protein Analysis is the less accurate and more qualitative method, but a confidently positive result
was achieved at the first attempt, so an experienced laboratory staff should have no troubles repeating and
interpreting a result to a reasonably high level of accuracy, even moreso once the method and gels can be
refined and optimised. So dispite its lower precision and a method more prone to error and influence,
- 14 -
protein analysis would be the preferred method of detecting Osteogenesis Imperfecta, with genetic analysis
still available as a highly accurate and definitive backup.
References
Note: Dates in brackets after URLs indicate date of submission or last update, if known. All websites were
viewed between 17/4/6 and 30/4/6.
Steiner, Robert D, et al. GENEReviews – Osteogenesis Imperfecta
http://www.genetests.org/profiles/oi/index.html (28/1/05)
1
2
Wikipedia. Osteogenesis Imperfecta. http://en.wikipedia.org/wiki/Brittle_bone_disease (5/4/06)
3
Wikipedia. Collagen. http://en.wikipedia.org/wiki/Collagen#Synthesis (23/4/06)
4
National Institute of Health - Osteoporosis and Related Bone Diseases. OI Issues: Genetics
http://www.osteo.org/newfile.asp?doc=i114i&doctitle=Genetics&doctype=HTML+Fact+Sheet (4/02)
5
Scott, Dr J. Lectures on Nucleic Acids. B. Biomedical Science, Newcastle University. 2005
6
Scott, Dr J. Lectures on Nucleic Acids. B. Biomedical Science, Newcastle University. 2005
7
Wikipedia. Taq Polymerase. http://en.wikipedia.org/wiki/Taq_polymerase (18/4/6)
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Wikipedia. Pfu DNA Polymerase. http://en.wikipedia.org/wiki/Pfu_DNA_polymerase (28/4/6)
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