Pattern of vitamin d receptor polymorphism

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  • extraction of DNA using salting out technique followed by amplification of extracted DNA at exon 2 for FOKI & intron 8 for BSMI and amplified DNA was then cut by restriction enzyme FOKI for detection of FOKI polymorphism and by enzyme BSMI for detection of BSMI polymorphism and then electrophoresis of the DNA was done on agarose gel.
  • Pattern of vitamin d receptor polymorphism

    1. 1. <ul><li>Pattern of Vitamin D Receptor Polymorphism In Postmenopausal Osteoporosis </li></ul>
    2. 2. Introduction <ul><li>Bone mineral density (BMD) decreases with advancing age among both women and men. Women in particular, suffer accelerated bone loss after menopause. </li></ul>
    3. 3. OSTEOPOROSIS <ul><li>Osteoporosis is a common disease that is characterized by low bone mass with micro architectural disruption and skeletal fragility, resulting in an increased risk of fracture </li></ul>
    4. 4. Age-related loss of trabecular bone
    5. 5. <ul><li>The World Health Organization has defined diagnostic thresholds for low bone mass and osteoporosis based upon bone mineral density ( BMD ) measurements compared with a young adult reference population ( T-score ). </li></ul><ul><li>most clinicians and investigators defined osteoporosis solely on a bone mass measurement more than 2.5 standard deviations below young normal reference ranges </li></ul>
    6. 6. Functions of the Vitamin D Endocrine System
    7. 7. Introduction <ul><li>Osteoporosis is a multifactorial disease caused by hormonal and genetic factors. </li></ul><ul><li>One of the suggested candidate gene involved in the pathogenesis is polymorphism of vitamin D receptor (VDR). </li></ul><ul><li>The gene encoding the VDR is located on chromosome 12q and has several allelic variants including BSMI, FOKI, TaqI and APaI. </li></ul>
    8. 8. Schematic diagram of the human vitamin D receptor gene
    9. 9. Aim of Work <ul><li>to determine the distribution of VDR gene polymorphism (FOKI and BSMI) in postmenopausal Egyptian females, </li></ul>
    10. 10. PATIENTS AND METHODS <ul><li>50 postmenopausal osteoporotic females compared to 20 age matched healthy postmenopausal non – osteoporotic females </li></ul><ul><li>All women were subjected to Bone mineral density measurement by DEXA for both femoral neck and lumbar spine (L2 – 4). </li></ul>
    11. 11. Identification of Vit. D receptor [FOKI, BSMI] polymorphism by PCR technique followed by RFLP analysis <ul><li>Extraction of genomic DNA from peripheral blood leucocytes </li></ul><ul><li>DNA Amplification (for FOKI&BSMI ) </li></ul><ul><li>Mutation detection by restriction analysis </li></ul>
    12. 12. DNA Amplification with the Polymerase Chain Reaction (PCR) Make copies ( extend primers ) Starting DNA Template 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ Add primers ( anneal ) 5’ 3’ 3’ 5’ Forward primer Reverse primer Separate strands ( denature ) 5’ 5’ 3’ 3’
    13. 13. In 35 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created PCR Copies DNA through Multiple Thermal Cycles Original DNA target region Thermal cycle Thermal cycle Thermal cycle
    14. 14. Mutation detection by restriction analysis 7 6 5 4 3 2 1 Ladder 69 bp-- 196 bp-- 265 bp--
    15. 15. 7 6 5 4 3 2 1 Ladder 69 bp-- 196 bp-- 265 bp--
    16. 16. 8 7 6 5 4 3 2 1 Ladder 200 bp 650 bp 850 bp
    17. 17. RESULTS
    18. 18. Body mass index of osteoporotic and controls
    19. 19. Distribution of different genotypes of FOKI polymorphism & alleles frequency in patients & controls: FF genotype Ff genotype ff genotype Allele (%) F f Patients (n=50) N (%) 34 (68) 9 (18) 7 (14) 72.9 27.1 Controls (n=20) N (%) 20 (100) 0 (0) 0 (0) 100 0
    20. 20. Distribution of different genotypes of BSMI polymorphism & alleles frequency in patients & controls: BB genotype Bb genotype bb genotype Allele (%) B b Patients (n=50) N (%) 27 (54) 15 (30) 8 (16) 64.6 35.4 Controls (n=20) N (%) 1 (5) 2 (10) 17 (85) 13.6 86.4
    21. 21. Comparison of the studied parameters in FOKI 0.07 0.68±.088 .74 ± .140 BMD lumbar (g/cm 2 ) 0.000 0.68±.040 0.77 ± .129 BMD femoral (g/cm 2 ) 0.04 -2.11 ±0 .91 -1.54 ± 1.14 T score lumbar 0.3 -1.21 ± 1.14 -.94 ± .983 T scorefemoral P-value Ff+ff (N=16) Mean ± SD FF (N=54) Mean ± SD Variables
    22. 22. BMD lumbar of osteoporotic patients
    23. 23. Comparison of the studied parameters in BSMI 0.000 0.67±0.10 0.84 ± 0.11 BMD lumbar (g/cm 2 ) 0.000 0.70 ±0 .07 0.85 ±0 .13 BMD femoral (g/cm 2 ) 0.001 -2.00 ± 1.02 -1.08 ± 1.05 T score lumbar 0.10 -1.14 ± 1.06 -0.75 ± 0 .92 T scorefemoral P-value Bb+bb (N=45) Mean ± SD bb (N=25) Mean ± SD Variables
    24. 24. BMD lumbar of osteoporotic patients
    25. 25. Combined genotypes of BSMI and FOKI of osteoporotic patients
    26. 26. Combined genotype of BSMI and FOKI of controls
    27. 27. O.R ratio of FOK1genotypes of patients and controls:   (32) (0) (%)   16 0 Count Ff+ff   (68) (100) (%)   1.588(1.29-1.95) 0.004 34 20 Count FF   cases control FOK1   OR (95% CI) P value GROUP
    28. 28. O.R ratio of BSM1 genotypes of patients and controls: (16) (85) (%) 8 17 Count bb (84) (15) (% ) 29.75(7.04-125.8) 0.000 42 3 Count BB+Bb cases control BSM1
    29. 29. Conclusion <ul><li>VDR gene ( FOKI and BSMI ) polymorphism plays an important role in osteoporosis in Egyptian post menopausal females. </li></ul>
    30. 30. <ul><li>The FF genotype was predominant among post menopausal females and ff genotype was associated with osteoporosis. The BB genotype was higher in patients than controls and the genotype is a protective genotype. </li></ul>
    31. 31. Thank you

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