Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr....
-
Upload
therightdoctorsadmin -
Category
Health & Medicine
-
view
329 -
download
0
description
Transcript of Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr....
![Page 1: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/1.jpg)
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
![Page 2: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/2.jpg)
• Dept of Orthopaedics • Neuroscience Research Lab P.G.I.M.E.R., Chandiagrh, India.
![Page 3: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/3.jpg)
Risk factors for ACL tears
• Intrinsic – Age, Sex, BMI, Q angle……
• Extrinsic – Contact sport, game
specific ….
![Page 4: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/4.jpg)
Genetic risk factors in ACL tears ??
![Page 5: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/5.jpg)
• 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
![Page 6: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/6.jpg)
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.
![Page 7: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/7.jpg)
SNP ??
![Page 8: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/8.jpg)
What’s a SNP ??
• Our 23 chromosome pairs = GENOME
• 3 BILLION BASE PAIRS
![Page 9: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/9.jpg)
• Variation in a single base pair = SNP
![Page 10: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/10.jpg)
• 10 MILLION SNPs – make me different from the rest of the world !
![Page 11: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/11.jpg)
SNPs determine….
• How I look..• Response to diseases• Drug effects
![Page 12: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/12.jpg)
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
![Page 13: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/13.jpg)
• Most of the work - South African Caucasians, Swedes, Poles
• What about the rest of the world??
• No studies from the subcontinent..
![Page 14: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/14.jpg)
RESEARCH QUESTION:
Do single nucleotide polymorphisms in COL1A1 and COL12A1 genes pose a genetic risk for developing an ACL tear or not?
![Page 15: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/15.jpg)
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
![Page 16: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/16.jpg)
Inclusion criteria (Controls)
• Age matched• Trauma Patients with
closed fractures (single) of upper limb
• No history or clinical features suggestive of ACL tear
![Page 17: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/17.jpg)
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.
![Page 18: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/18.jpg)
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
![Page 19: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/19.jpg)
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.
![Page 20: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/20.jpg)
• 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.
![Page 21: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/21.jpg)
Results…
• 50 patients and 52 controls
• Females: 6% of the ACL tear population (n=3) and 13.5% of the control population (n=7).
![Page 22: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/22.jpg)
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
![Page 23: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/23.jpg)
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
![Page 24: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/24.jpg)
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).
![Page 25: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/25.jpg)
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
![Page 26: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/26.jpg)
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.
![Page 27: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/27.jpg)
• 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
![Page 28: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/28.jpg)
How is our study unique?
![Page 29: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/29.jpg)
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
![Page 30: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/30.jpg)
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
![Page 31: Genetic Polymorphisms in COL genes and Their Association with ACL Tears in the Indian Population-Dr. Sharad Prabhakar](https://reader035.fdocuments.net/reader035/viewer/2022062313/5582fd89d8b42a2c168b527b/html5/thumbnails/31.jpg)