Abstract
Purpose
Role of heritable blood clotting disorders, both thrombophilias and hypofibrinolysis in causing avascular necrosis (AVN) of femoral head have been studied in regions like Europe and U.S.A. This study was done to investigate the role of heritable thrombophilias in ethnic Indian population.
Materials and Methods
A case control study of 150 patients (100 cases and 50 age and sex matched controls) of Indian Ethnicity with clinico-radiographically documented idiopathic AVN of femoral head was done after ethics committee approval. DNA was extracted from the blood and PCR analysis was used to study heritable thrombophilic gene mutation (G1691A Factor V Leiden). Enzyme-linked immunosorbent assay (ELISA)-based assays, were utilized to measure antigen levels of protein C, antithrombin III levels and protein S.
Results
Nine cases out of 100 showed deficiency of Protein C (9%) while no control showed deficiency of Protein C (p value: 0.028—significant, Odds ratio: 9.791) Ten cases showed deficiency of Protein S (10%) in study population as compared to one case (2%) in control population (p value: 0.038—significant, Odds ratio: 5.44). ATIII deficiency was more prevalent in control group i.e. 22% compared to 11% in study group. Factor V mutation was present in 3% cases as compared to one (2%) in control group. (p value is 0.393—not significant).
Conclusion
Difference in thrombophilic mutations in various populations indicates possible effect of ethnicity on genetic profile in the development of AVN. This risk stratification will enable in near future early diagnosis and possible role of antithrombotics in disease prevention.
Keywords: Avascular necrosis (AVN), Osteonecrosis, Factor V mutation, Protein C, Protein S, Antithrombin III (AT III)
Introduction
Vaso-occlusion leading to infarction of articular surfaces and subchondral collapse of femoral head, results in arthritis of hip joint. Osteonecrosis primarily occurs in the third to fifth decade of life and unfortunately most of these patients need hip arthroplasty as mainstay of treatment which has finite survival rate in long term. This has significant social and economic implications on family and in turn on the society [1].
Femoral head osteonecrosis (FHO) is an ischemic bone disorder that can lead to joint degeneration and is associated with a variety of risk factors such as corticosteroid therapy, alcoholism, hemoglobinopathies, hip trauma, vasculitis, dysbarism, autoimmune diseases, neoplasia, irradiation, hyperuricemia and even pregnancy (secondary osteonecrosis of the femoral head) [2, 3]. In spite of various proposed mechanisms, etiopathogenesis of AVN is not yet completely understood and up to 40% of the cases are considered idiopathic in nature [4].
Osteonecrosis of the femoral head (ONFH) is characterized by intravascular coagulation and microcirculatory thrombosis. Various studies [5, 6], especially in Caucasian population, have analyzed the role of blood clotting disorders, both thrombophilia and hypo fibrinolysis in AVN. They have found significantly high presence of these inheritable thrombophilias in the patients with AVN, especially in those where no particular cause could be established and were labeled as idiopathic. The most frequent thrombophilic disorders include G1691A mutation in the factor V gene, protein C deficiency, protein S deficiency, antithrombin deficiency, G20210A mutation in the prothrombin gene, antiphospholipid antibody, increased serum homocysteine levels, polymorphism in the plasminogen activator inhibitor-1 gene (4G/5G polymorphism) [5–8]. Both thrombophilia (increased tendency to form thrombi) or/and hypo fibrinolysis (reduced ability to lyse thrombi) have been considered to play important role in occurrence of FHO in adults [5, 6]. The same thrombophilia/hypo fibrinolysis pathogenesis is found to be responsible for FHO in children (Legg–Calvé–Perthes disease) [9, 10].
This study tries to compare the occurrence of these thrombophilic risk factors in patients with osteonecrosis of femoral head, with regards to occurrence in general population.
Materials and Methods
We included group of 100 unrelated adult individuals in study group. All the patients were of Indian origin, coming from various parts of India to our tertiary referral institute located in the western part of the country in the city of Mumbai. Informed, written, valid consent was taken from all the subjects in the study after obtaining institutional ethics committee clearance. Patients were followed up in department of orthopedics for treatment avascular necrosis of femur. All cases had clinically and radiographically documented avascular necrosis of the femoral head (AVN) of idiopathic nature. Each patient had to qualify as having osteonecrosis of the head of the femur on the basis of a thorough history and physical examination, anteroposterior and frog-leg lateral radiographs of both hips, and magnetic resonance imaging (If required). Ficat and Arlet classification [11] was used for radiographic evaluation. MRI was performed to confirm the diagnosis of ONFH in patients without X-ray changes. MRI criteria were: (1) a focal bone signal anomaly with T1 and T2 hypo signal, (2) a peripheral medullar edema with T1 hypo signal (rising up after a gadolinium infusion) and T2 hypersignal separated by delimitation border. This definition allowed detection in the early stages or in the elimination of differential diagnosis in doubtful cases.
For study group, suitably matched control group was formed of 50 which were age and sex matched. The most important criteria were to select controls not affected by any renal or hepatic insufficiency, and without inflammatory syndromes, which could have influenced thrombophilia factors. The major part of controls was selected from the patients hospitalized for backache or lumbar radicular pain (degenerative spine disorders). Subjects were excluded from control groups if they were taking estrogens, raloxifene, tamoxifen, corticosteroids, or anticoagulants.
Laboratory Methods
After following due aseptic precautions, 10 ml blood was collected in 3.2% buffered sodium citrate (one-part citrate: nine parts blood). The samples were immediately transported and centrifuged to obtain platelet-poor plasma. The plasma was frozen and stored. The frozen samples were collectively run in batches for specialized tests.
Blood for polymerase chain reaction (PCR) analysis was drawn in tubes containing the appropriate anticoagulant (Ethylenediaminetetraacetic acid—EDTA), and the DNA was extracted for subsequent analysis. PCR analysis was used to study heritable thrombophilic gene mutation i.e. G1691A Factor V Leiden.
Commercially available enzyme-linked immunosorbent assay (ELISA)-based assays were utilized to measure antigen levels of protein C, antithrombin III levels and protein S. Protein C, total and free protein S, and antithrombin III levels below the fifth percentile for normal control subjects were considered abnormal [9].
Statistical Analysis
Data was entered into Microsoft Excel (Windows 7; Version 2007) and analyses were done using IBM SPSS Statistics software (International Business Machines Corp. Statistical Product and Service Solutions, Armonk, NY). Association between Variables was analyzed by using odds ratio and Mid-P exact test. Bar charts were used for visual representation of the analyzed data. Level of significance was set at 0.05.
Results
Sample size was calculated for given study using statistical package “Epiinfo” (open/free source software). Study group of 100 cases and control group of 50 was selected using convenient sampling without personal bias. Our sample size was adequate to ascertain patient-control differences in our key measures of thrombophilia with alpha = 0.05 and beta = 0.2.
We had 79 males and 21 Females in the cases group and 38 males and 12 females in the control group. There was no significant difference between both groups with respect to age and gender. Mean age in Study group was 39.9 years and control group was 41.3 years. Unilateral AVN was observed in 78 cases and 22 cases had bilateral involvement. We had 8 hips in Ficat–Arlet Class 1 group, 34 in Stage 2, 32 in stage 3, and 48 hips in Stage 4 (Total 122 hips).
Protein C Deficiency
9 cases out of 100 showed deficiency of Protein C (9%) whereas no control showed deficiency of Protein C (Table 1). The difference in proportion in Protein C deficiency in the study and control group was statistically significant since p value was 0.028 (Mid-P exact test) which is < 0.05. Odds ratio for developing AVN because of Protein C deficiency in study group is 9.791 times more than control group.
Table 1.
Indicates distribution of protein C deficiency among the cases and controls
| Protein C deficiency | Total | ||
|---|---|---|---|
| Present | Absent | ||
| Study group with AVN | 09 (9%) | 91(91%) | 100 |
| Control group | 0 (0%) | 50 (100%) | 50 |
| Total | 09 | 141 | 150 |
*p value: 0.028 (Mid-P exact test); Odds ratio: 9.791
Protein S Deficiency
10 cases showed deficiency of Protein S (10%) in study population as compared to 1 case (2%) in control population (Table 2).
Table 2.
Indicates distribution of protein S deficiency among the cases and controls
| Protein S deficiency | Total | ||
|---|---|---|---|
| Present | Absent | ||
| Study group with AVN | 10 (10%) | 90 (90%) | 100 |
| Control group | 1 (2%) | 49 (98%) | 50 |
| Total | 11 | 139 | 150 |
*p value: 0.038 (Mid-P exact test); Odds ratio: 5.44
The difference in proportion in Protein S deficiency in study and control group is statistically significant since p value is 0.038 (Mid-P exact test) which is < 0.05.
Odds ratio for developing AVN because of Protein S deficiency in study group is 5.44 times more than control group.
Antithrombin III Deficiency
ATIII deficiency was more prevalent in control group i.e. 22% compared to 11% in study group (Table 3).
Table 3.
Indicates distribution of AT III deficiency among the cases and controls
| AT III deficiency | Total | ||
|---|---|---|---|
| Present | Absent | ||
| Study group with AVN | 11 (11%) | 89 (89%) | 100 |
| Control group | 11 (22%) | 39 (78%) | 50 |
| Total | 22 | 128 | 150 |
As deficiency of ATIII was found to be more common in control group as compared to study group, hence, AT III deficiency cannot be considered to be causative factor.
Factor V Leiden Mutation
Factor V mutation was present in 3% cases as compared to 1 (2%) in control group (Table 4).
Table 4.
Depicts FACTOR V LEIDEN Mutation among cases and controls
| FACTOR V LEIDEN Mutation | Total | ||
|---|---|---|---|
| Present | Absent | ||
| Study group with AVN | 03 (03%) | 97 (97%) | 100 |
| Control group | 01 (02%) | 49 (98%) | 50 |
| Total | 04 | 146 | 150 |
*p value: 0.393 (Mid-P exact test)
The difference in proportion in in study and control group is not statistically significant since p value is 0.393 (Mid-P exact test) which is > 0.05.
Figure 1 depicts how the different parameters were present in the cases and control groups.
Fig. 1.
Depicts how different factors were distributed amongst case and control population
Discussion
Avascular necrosis of hip is one of the common reasons for middle age patients undergoing Total Hip Replacement (THR) in Indian subcontinent in contrast to western world, where AVN constitutes as indication in only 10% of the cases undergoing THR [12]. Various studies in literature mention corticosteroid induced AVN in approx. 30–40% of cases, alcohol use was found in 21–25% of cases, and the remainder of the cases are considered idiopathic [13]. Our study specifically looked for thrombophilic profile of this idiopathic group.
There are various naturally occurring plasma inhibitors which regulate the activity of coagulation factors. The most important inhibitors of the blood coagulation system are antithrombin III, protein C, and protein S. Activated protein C and protein S act by inhibiting the action of factor Va and factor VIIIa. Antithrombin III inhibits the serine proteases (factors II, IX, X, XI, and XII); heparin greatly increases its anticoagulant action. Protein C is activated by thrombin and thereby it halts the coagulation. Protein C requires protein S as co-activation factor. Hence it is a delicate balance between procoagulants and anticoagulants which is responsible for normal homeostasis of coagulation pathway and the deficiency of these proteins is associated with thromboembolic disease [14]. Factor V Leiden is allelic form of gene with a single amino acid substitution in Factor V through which APC (Activated protein C) mediates its action. This mutated allele is resistant to cleavage by APC at position 506. This leads to increased availability of factor V, leading to increased thrombin generation which ultimately leads to hypercoagulability [15]. This hypercoagulable state eventually leads to micro circulatory thromboses resulting in femoral head osteonecrosis.
In 1993, Glueck et al. reported twin brothers having osteonecrosis of femur and they were homozygous for the hypofibrinolytic 4G/4G polymorphism of the plasminogen activator inhibitor-1 gene (PAI-1 gene). This led to hypo fibrinolysis state in blood [16]. After this in their landmark study they compared both thrombophilic mutation and hypo fibrinolysis in osteonecrosis [5]. They suggested that these thrombophilic mutations result into an increase in interosseous venous pressure leading to impaired arterial flow, hypoxia culminating into osteonecrosis. They found that heritable thrombophilic disorders like high levels of factor VIII, Factor V Leiden mutation, and resistance to activated protein C, were risk factors for idiopathic as well as secondary osteonecrosis in femur.
Most of these studies on heritable thrombophilic mutations are region specific like specific part of Europe e.g.: Poland, Morocco and American context. There was no such study on thrombophilic mutations with respect to AVN femur in Indian context. We wanted to know whether thrombophilias play important role in Indian ethnicity or not. To the best of our knowledge this is the first study to seek association of thrombophilia with AVN femur in the national literature. Our study consisting of 100 cases of idiopathic AVN with no known risk factors along with 50 healthy controls highlighted important role of thrombophilic mutation in AVN of femur in Indian population. However, similar to the study by Glueck, we did not measure interosseous pressure. In contrast to most of the studies including abovementioned study, we included healthy controls. We did not include secondary risk factors of AVN as we believed that it would have resulted in confounding bias.
The most significant inhibitors of coagulation cascade are antithrombin, protein C, and protein S [17]. An inherited deficiency of either of them is seen in around 15% of the patients. Heritable thrombophilic mutation commonly found in the general population consists of G1691A mutation in the factor V gene (factor V Leiden mutation), protein S deficiency, protein C deficiency, antithrombin deficiency and G20210A mutation in the prothrombin gene [18].
Protein C deficiency is infrequent compared to the factor V Leiden or the prothrombin gene mutation with estimated prevalence of 0.2–0.5% in Caucasians [19]. Protein C deficiency has autosomal dominant inheritance. Activated protein C (APC) inactivates coagulation factors Va and VIIIa, thus, interfering with thrombin generation and factor X activation. The vitamin K dependant protein S markedly enhances inhibitory effect of APC on coagulation cascade. We found Protein C mutation in 9% of our study group with AVN femur. In contrast we did not find Protein C deficiency in control group. Protein C was found to be significant determinant in idiopathic AVN patients in our study. Thus, there was 9.79 times higher risk of finding Protein C deficiency in Idiopathic AVN in comparison to healthy controls.
Protein S is required by activated protein C. Protein S deficiency shows autosomal dominant inheritance. Thrombosis is seen in heterozygotes with functional protein S who are falling in the range of 15–50% of normal. In our study deficiency of Protein S was found in 10% of cases, in contrast to 2% found in control population. Thus, there was 5.44 times higher risk of finding Protein S deficiency in Idiopathic AVN in comparison to healthy controls.
Antithrombin (AT), a serine protease inhibitor along with its cofactor Heparin is a major determinant in process of coagulation. Antithrombin deactivates thrombin as its name suggests, and also inactivates factors IX, X and XI [20]. Contrary to expectation, in our study Antithrombin III was not found to have a significant mutation in AVN group as compared to control population.
Garcia et al. also found Protein C and S deficiency in patients with Idiopathic AVN group as compared to Secondary AVN group [21]. In contrast Lee et. al. in Korean population did not find thrombophilic factors like Protein C and S, AT III to be of significance in patients with Idiopathic AVN [22].
Factor V Leiden normally activates prothrombin activation and thrombin generation. Activated protein C (APC) inactivates factor V by proteolysis. The G1691A point mutation in the factor V gene leads to alteration of cleavage site which is responsible for degradation of factor V by APC. This leads to APC resistance and thrombophilia [22]. Prevalence of the factor V mutation in healthy Caucasians is found to be 4–5% [23]. In our study we found mutation in study group to be of 3% as compared to 2% in control group. Statistically, presence of Factor V Leiden mutation was not found to be significant in study group as compared to control group. In contrast to our study, Bjorkman et al. [7] reported a higher prevalence of the factor V Leiden mutation in patients with idiopathic osteonecrosis than in the general Swedish population. However, study done in Asian i.e. Korean population, similar to our study, showed neither the presence of Factor V Leiden Mutation nor the Prothrombin gene mutation in patients with Idiopathic AVN [24].
The presence of antiphospholipid antibodies (aPL), a potential risk factor for vascular thrombosis, has been proposed to predispose towards AVN. Tektonidou et al. could find AVN by MRI in 20% of patients with primary Antiphospholipid antibody syndrome [25] Mok et al. [26] investigated the role of antiphospholipid antibody status (IgM and IgG anticardiolipin antibodies and lupus anticoagulant) with adjustment for corticosteroid use as risk factors for the development of AVN. However, they were unable to show a link between the presence of aPL and the development of AVN in patients with SLE. Thus, the role of aPL remains controversial [26] and will need further studies to validate/refute the claim of its association with idiopathic AVN.
Homocysteine (HC) has also been implicated as a risk factor for development of AVN of Hip. Hyperhomocysteinemia is known to increase risk development of thrombosis and thus, can cause AVN hip. However, levels of homocysteine and its associated pathway metabolites have not been characterized [27]. Methylenetetrahydrofolate reductase (MTHFR) mutations can impact one-carbon metabolism and alter levels of homocysteine (HC). Narayanan et al. demonstrated elevated levels of homocysteine in plasma of AVN Hip patients [27]. Kutler et al. found association of MTHFR mutation to be significantly more common in patients with sickle cell anemia who developed AVN and those who did not develop AVN [28]. Meng-Ling et al. demonstrated association of hyperhomocysteinemia with AVN hip in Chinese population [29]. Although we could not test for the MTHFR mutation in our study due to constraint of resources and time, we strongly believe this is a potential area of future studies.
Our study has limitation of limited size of control group and chance of having Berkesonian bias as the control group was drawn from the hospitalized patients and not the healthy volunteers. Thus to conclude, the difference in thrombophilic mutations in various population indicates possible effect of ethnicity on the genetic profile in the risk for the development of AVN [7, 21–24]. Since, it is a complex interplay of both genetic and epigenetic/environmental factors in a disease like AVN, individual person’s susceptibility towards development of AVN will not only depend on environmental factors but also genetic constitutions of individual. This risk stratification will enable in near future early diagnosis and possible role of antithrombotics in disease prevention.
Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
Compliance with Ethical Standards
Conflict of interest
The Author(s) declare(s) that there is no conflict of interest.
Ethical standard statement
The study involved human subjects and approval of the Institutional Ethics Committee was taken for this study.
Informed consent
Informed, written and valid consent was obtained from each subject of the study and control group.
Footnotes
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