Genetic Polymorphisms in COL genes and their association with ACL tears

in the Indian population

Dr Sharad PrabhakarProf M.S. DhillonDr Akshay AnandDr Rakesh John

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

Risk factors for ACL tears

• Intrinsic – Age, Sex, BMI, Q angle……

• Extrinsic – Contact sport, game

specific ….

Genetic risk factors in ACL tears ??

• 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

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.

SNP ??

What’s a SNP ??

• Our 23 chromosome pairs = GENOME

• 3 BILLION BASE PAIRS

• Variation in a single base pair = SNP

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

SNPs determine….

• How I look..• Response to diseases• Drug effects

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

• Most of the work - South African Caucasians, Swedes, Poles

• What about the rest of the world??

• No studies from the subcontinent..

RESEARCH QUESTION:

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

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

Inclusion criteria (Controls)

• Age matched• Trauma Patients with

closed fractures (single) of upper limb

• No history or clinical features suggestive of ACL tear

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.

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

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.

• 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.

Results…

• 50 patients and 52 controls

• Females: 6% of the ACL tear population (n=3) and 13.5% of the control population (n=7).

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

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

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).

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

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.

• 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

How is our study unique?

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

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