1. Drs. C Glueck, N Khan, S Khanal, P Wang; University of Cincinnati (Jewish Hospital Dr. R McMahon; Oral Surgery Group, Chesterton Indiana Dr. J.E. Bouquot, University of Texas at Houston Dental Branch Contact: 713-500-4420 (Phone); 713-500-4416 (Fax); Jerry.Bouquot@uth.tmc.edu IDENTIFYING TREATABLE ETIOLOGIES FOR ISCHEMIC JAW DISEASE Charles J. Glueck MD, Naseer A Khan MD, Suraj Khanal MD, Ping Wang PhD The Cholesterol Center, Jewish Hospital of Cincinnati, University of Cincinnati Robert E McMahon DDS, The Oral Surgery Group, Chesterton, Indiana Jerry E. Bouquot DDS, MS, University of Texas Dental Branch at Houston, Texas Supported in part by the Lipoprotein Research Fund of the Jewish Hospital and the Jewish Hospital Medical Research Fund

2. Platelet/fibrin aggregates in dilated marrow vessel in jaw Platelet/ aggregate extruding from nonviable marrow vessel  Risk factors for osteonecrosis of the hip: -- Gene mutations leading to thrombophilia (increased tendency to develop thrombosis) -- Gene mutations leading to hypofibrinolysis (reduced ability to lyse thrombi) -- Endothelial nitric oxide synthase (eNOS) T-786C gene polymorphisms that reduce nitric oxide (NO) production  Heritable thrombophilia and hypofibrinolysis have been reported to have a pathoetiologic role in the development of osteonecrosis of the alveolar bone of jaws

3. InheritedThrombophiliaFactor V Leiden gene (heterozygotic) InheritedHypofibrinolysisIncreased plasminogen activator inhibitor (PAI) InheritedThrombophilia Decreased stimulated tissue plasminogen activator InheritedThrombophiliaDecreased Protein C or Protein S InheritedThrombophiliaResistance to activated Protein C InheritedHypofibrinolysisIncreased lipoprotein a InheritedThrombophiliaMethylene tetrahydrofolate reductase (MTHFR) InheritedThrombophiliaAnticardiolipin antibody InheritedThrombophiliaProthrombin gene mutation Inherited?ThrombophiliaLupus anticoagulant antibody Hypercoagulation states are found in 6-15% of Western populations Redbook, June, 2005:

4. We have previously reported:  Thrombophilic Factor V Leiden mutation in 89 patients with alveolar osteonecrosis of the jaws: -- 16/76 (21%) of women -- 5/13 (39%) of men  Factor V Leiden mutation in 209 healthy/normal controls: -- 3/101 (3%) of women (p =.001) -- 4/108 (3.7%) of men (p =.001) Subcortical void in posterior mandible Perineural osteonecrosis of posterior mandible

5. Increased plasminogen activator inhibitor (PAI)18 Decreased stimulated tissue plasminogen activator22 Decreased Protein C or Protein S8 Resistance to activated Protein C18 Increased lipoprotein A36 Factor V Leiden gene (heterozygotic)23 Methylene tetrahydrofolate reductase (MTHFR)65 Total (including multiple coagulopathies):78% * Data from coagulation labs of the University of Cincinnati and Indiana University, 2002 Hypercoagulation states are found in up to 78% of ischemic jaw disease

6. * Reference: Bouquot, et al. J Oral Pathol Med 2002; 31:290 Platelet aggregate completely plugging marrow vessel in jaw  G1691A mutation of the Factor V Leiden gene  G20210A mutation of the prothrombin gene (Factor II)  C677T & A1298C mutations of the gene responsible for the production of the enzyme methylene tetrahydrofolate reductase (MTHFR)

7. * Reference: Bouquot, et al. J Oral Pathol Med 2002; 31:290 Fibrin aggregate completely plugging marrow vessel in jaw  The gene responsible for production of plasminogen activator inhibitor-1 (PAI-4G4G)  The gene responsible for production of lipoprotein (a) Additionally: Gene polymorphisms associated with: -- Reduced nitric oxide (NO) production -- Endothelial nitric oxide synthase (eNOS) T-786C -- Stromelysin 5A6A) can lead to vasoconstriction, platelet aggregation, and thrombosis, and may also have a pathogenic role in osteonecrosis of the hip

8. Anticoagulation with Enoxaparin for the same duration as used in deep vein thrombosis in the leg has been reported to stop the progression of idiopathic Ficat stages I-II unifocal osteonecrosis of the head of the femur in patients with thrombophilia-hypofibrinolysis, decreasing the frequency of total hip replacement We hypothesize that a similar protocol will either diminish the pain of ischemic jaw disease or improve the prognosis with or without additional surgery

9. Thromus  17 patients with painful ischemic jaw disease (biopsy proven)  51 – 68 age/gender matched controls  No use of bisphosphonates, etc.  Detailed history & physical exam  Blood drawn (fasting, seated) for assessment of thrombophilia, hypofibrinolysis, the T-786C eNOS and stromelysin 5A6A mutation  Of the 17 Caucasian cases: -- 13 women and 4 men -- Mean age: 50 years  13SD -- 4 of the females were taking exogenous estrogens -- None took corticosteroids -- None were cigarette smokers -- 1 was a recovered alcoholic Platelet aggregate plugging marrow vessel in jaw Region of ischemic marrow disease in mandible

10. PCR assays for mutant genes:  -- G1691A Factor V  -- G20210A Prothrombin  -- C677T-A1298C MTHFR  -- 4G4G plasminogen activator inhibitor-1  -- eNOS T-786C  -- Stromelysin 5A6A Platelet aggregate plugging marrow vessel in jaw Atrophic neurovascular bundle in cavitational area in mandible Platelet thrombus plugging marrow vessel in jaw

11. Serologic tests:  Resistance to activated protein C (RAPC)  Protein C  Protein S (free S)  Antithrombin III  Homocysteine  Anticardiolipin antibody IgG  Anticardiolipin antibody IgM  Lupus anticoagulant  Factor VIII  Factor XI  Plasminogen activator inhibitor activity  Lipoprotein(a) Platelet aggregate in marrow vessel of jaw Fibrin aggregate in marrow vessel of jaw

12.  Distributions of abnormalities of thrombophilia and hypofibrinolysis and the eNOS and stromelysin polymorphisms were compared in patients and matched controls by Chi square analyses or Fisher’s exact tests  Risk ratios with 95% confidence intervals were reported  The confidence intervals for sensitivity and specificity were calculated by asymptotic normal distribution for binomial variables, when cell frequency was 0 using continuity adjustment correction +0.5  Mantel-Haenszel X 2 tests were used to compare the distribution of eNOS T786C and stromelysin 5A6A genotypes in patients versus gender-race matched normal controls

13. N Not Normal Normal Fisher’s p Risk ratio, 95% CI Factor V Leiden heterozygosityCase17 3 (18%) 3 (18%)26.1(1.4-482) Control66 0 ( 0%) 0 ( 0%).0074 Resistance to Activated Protein C Case 17 17 5 (29%) 5 (29%)5.98(1.6-22.5) Control 61 61 3 ( 5%) 3 ( 5%).011 ENOS T786C mutation * Case15 3 MM (20%) 5 NN, 7 MN Fisher’s p=.013 Mantel-Heanszel Χ 2 =8.26, p=.004 20.1(1.1-369) Control45 0 MM (0%) 29 NN, 16 MN Stromelysin 6A mutation * Case15 5 MM (33%) 1 NN, 9 MN Fisher’s p=.12 Mantel-Heanszel Χ 2 =4.1, p=.042 2.5(0.89-7.0) Control45 6 MM (13%) 11 NN, 28 MN * Abnormal, mutant allele (M), Normal, wild-type normal allele (N) Table 1. Case-control differences in the Factor V Leiden and eNOS T-786C mutations.

14. N Risk ratio, 95% CI Sensitiv ity, 95%,CI Specificity, 95%,CI Factor V Leiden heterozygosityCase1726.1(1.4-482)18%, (0-36%) (0-36%) Control66100%, (98-100%) (98-100%) Resistance to Activated Protein C Case 17 175.98(1.6-22.5)29%,(8-51%) Control 61 61 95%, 95%, (90-100%) (90-100%) ENOS T786C mutation Case1520.1(1.1-369)20%(0-40%) Control45100% (97-100%) (97-100%) Stromelysin 6A mutation Case152.5(0.89-7.0)33%(9-57%) Control4587%(77-97%) Table 2. Sensitivity and specificity

15. 1) Chronic facial pain patients with biopsy and imaging-defined alveolar osteonecrosis of the jaw differed from matched normal controls, being more likely to have: -- Heterozygosity for the thrombophilic Factor V Leiden mutation -- Thrombophilic resistance to activated protein C -- The eNOS T-786C polymorphism associated with reduced NO production. 2) The increased prevalence of the Factor V Leiden mutation in the current study is consistent with our previous reports that the Factor V Leiden mutation was pathoetiologic for osteonecrosis of the jaw and for osteonecrosis of the hip in children (Legg-Calve-Perthes disease) 3) Anticoagulation with Enoxaparin for the same duration as used in deep vein thrombosis in the leg should provide improvement in the facial pain and/or prognosis of jaw osteonecrosis patients.