Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar

1. Genetic Polymorphisms in COL
genes and their association with ACL
tears in the Indian population
Dr Sharad Prabhakar
Prof M.S. Dhillon
Dr Akshay Anand
Dr Rakesh John

2. • Dept of Orthopaedics
• Neuroscience Research Lab
P.G.I.M.E.R., Chandiagrh, India.

3. Risk factors for ACL tears
• Intrinsic – Age, Sex, BMI,
Q angle……
• Extrinsic – Contact sport,
game specific ….

4. Genetic risk factors in ACL tears ??

5. • Serendipitous observation
of Harner et al (1994) -
individuals with a family
history of ACL tear were
twice as likely to have an
ACL tear
• Flynn et al ( 2005) - a
patient with an ACL tear
was twice as likely to have
a relative with an ACL tear

6. Breakthrough !
• Khoschanau et al (2008)
found the first specific
genetic element - the
functional COL 1A1 Sp1
binding site
polymorphism, to be
positively associated with
ACL tears.

7. SNP ??

8. What’s a SNP ??
• Our 23 chromosome
pairs = GENOME
• 3 BILLION BASE PAIRS

9. • Variation in a single base
pair = SNP

10. • 10 MILLION SNPs –
make me different
from the rest of the
world !

11. SNPs determine….
• How I look..
• Response to diseases
• Drug effects

12. Posthumus et al (2008-09)
• Reported that the AA genotype of
COL12A1 AluI Restriction Fragment
Length Polymorphism (RFLP) was
significantly over-represented in
females with ACL injury
• Established that the TT genotype of
the COL 1A1 Sp1 binding site
polymorphism is under-represented
in patients with ACL tears

13. • Most of the work -
South African
Caucasians, Swedes,
Poles
• What about the rest of
the world??
• No studies from the
subcontinent..

14. RESEARCH QUESTION:
Do single nucleotide
polymorphisms in
COL1A1 and COL12A1
genes pose a genetic
risk for developing an
ACL tear or not?

15. Materials and Methods
INCLUSION CRITERIA
(Cases)
• Patients of either sex
between 18-45 years of
age taken up for
arthroscopic ACL
reconstruction.
• No co-morbidities
• No evidence of multi
ligament injuries
• Informed written consent

16. Inclusion criteria (Controls)
• Age matched
• Trauma Patients with
closed fractures (single)
of upper limb
• No history or clinical
features suggestive of
ACL tear

17. Methodology…
• Lymphocyte extraction was carried
out from blood of these patients.
• ACL remnant tissue was removed at
the time of arthroscopy and stored for
DNA extraction
• Venous blood samples were taken
from both cases and controls while
ACL tissue samples were taken from
cases only
• DNA was isolated using commercial
kits.

18. Points to note…
• Genomic DNA was isolated using
QIAGEN DNeasyt blood and
tissue kit (catalogue no-69504).
The eluted DNA was quantified
using UV spectrophotometer
(Backman Coulter) and run on
agarose gel (Biorad) to verify the
quality of DNA

19. Points to note…
• Unique standardised
freeze – thaw – freeze ACL
disruption method
• Real time PCR performed
in the 48 wells model Step
oneTM (Applied
Biosystems Inc, Foster
City, CA)using published
TaqMan SNP/ SybrGreen
Genoytyping assays.

20. • By using Real Time PCR
amplification COL12A1 genes
were analyzed for SNPs using
specific primers.
• The Real Time PCR
amplification products were
imported by Sequence
Detection System (SDS)
Software for the detection of
single nucleotide
polymorphism and the results
were correlated clinically.

21. Results…
• 50 patients and 52
controls
• Females: 6% of the ACL
tear population (n=3)
and 13.5% of the control
population (n=7).

22. COL 1A1
• There was no
statistically significant
difference in the
genotype or allele
frequencies between
ACL and control
groups for rs1800012
(the Sp1 binding site
polymorphism) or
rs1107946 region in
both blood and tissue
samples

23. So what about the TT genotype?
• Four different studies in 3
different ethnic populations
have previously reported a
positive association between
risk of ACL tear and gene
polymorphisms at the
functional Sp1 binding site
region of COL1A1 - Khoshnau
et al (Swedish), Ficek et al
(Polish), Posthumus et al (South
African Caucasians) , Sladowska
et al

24. Results….COL 12A1
• The AG and GG genotypes
were significantly under-represented
in patients
with ACL tears in both
blood and tissue samples in
rs970547 region of
COL12A1 gene (p=0.0361
and p=0.0374 respectively
in PBMC; p=0.0315 and
p=0.0374 in tissue).

25. We are Different??
• In our study, the AG and GG
genotypes were significantly
under-represented in
patients with ACL tears
• Posthumus et al stated that
the AA genotype over-represented
in females with
ACL injury

26. Substitution..
• The rs970547 SNP located
within exon 65 of
chromosome 6
(Chr.6:75797302,
missense, COL12A1) is a
non-synonymous coding
variant, which changes the
amino acid from a serine
to a glycine at position
3058.

27. • Under-representation
of AG and GG
genotypes may result
in an altered type 12
collagen protein which
may lead to an
alteration of the
biomechanical
properties of the
collagen fibrils

28. How is our study unique?

29. How is our study unique?
• This is the first study in
the world where ACL
tissue was genotyped in
addition to blood in
contrast to other
studies where DNA
isolation and
genotyping was done
either from blood or
oral epithelial cells only

30. Take home message…
• There was no statistically
significant difference in the
genotype or allele frequencies
for polymorphisms involving the
COL1A1 gene
• The AG and GG genotypes were
significantly under-represented
in patients with ACL tears in
both blood and tissue samples in
rs970547 region of COL12A1
gene