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1Director and Professor, Institute of Orthopedics and Traumatology, Coimbatore Medical College and Hospital,
Coimbatore, Tamil Nadu, India
2Associate professor, Institute of orthopaedics and traumatology, Coimbatore Medical College and Hospital, Coimbatore, Tamil Nadu, India
3Junior resident, Institute of Orthopaedics and traumatology, Coimbatore Medical College and Hospital, Coimbatore,
Tamil Nadu, India
4Research Scientist, Multi-Disciplinary Research Unit, Coimbatore Medical College and Hospital, Coimbatore,
Tamil Nadu, India
Background: Avascular necrosis of the femoral head (ANFH) is a multifactorial disorder in which Genetic predisposition may have a role in the onset and progression. The pathophysiology of ANFH has been linked to polymorphisms that impact vascular control and endothelial function. Hence, this study designed to determine the association of the 27-bp repeat polymorphism in intron 4 and the G894T polymorphism in exon 7 with ANFH. Methods: In this case-control study, a total of 100 subjects (50 ANFH patients and 50 age and sex matched healthy controls) were included. PCR and PCR-RFLP was performed to genotype the 27-bp repeat polymorphism and the G894T polymorphism, respectively. Comparison of Genotype and allele frequencies was done using chi-square tests. Estimation of Odds ratios (ORs) with 95% confidence intervals (CIs) was done using binary logistic regression. Results: Both the screened genes exhibited significant difference in genotype and allele frequency between ANFH patients and controls (p < 0.05). The a allele of the 27-bp repeat polymorphism and the T allele of the G894T polymorphism was significantly more frequent in ANFH patients than in controls (25.0% vs 3.0%; 33.0% vs 12.0%) and was strongly associated with ANFH risk (adjusted OR = 10.41, 95% CI = 2.95–36.7, p < 0.001; adjusted OR = 3.51, 95% CI = 1.67–7.39, p = 0.001). Similarly, it was clearly studied that the risk alleles were also more prevalent in advanced disease stages, showing a potential association with the disease severity. Conclusion: The findings demonstrate that the 27-bp repeat polymorphism in intron 4 and the G894T polymorphism in exon 7 have an independent association with increased risk of ANFH. These polymorphisms may contribute to disease susceptibility and progression and could serve as potential genetic markers for early identification of individuals at increased risk.
Osteonecrosis of the femoral head (ONFH), commonly known as avascular necrosis (AVN), is a progressive disease which develop due to insufficient blood supply to the subchondral region of the femoral head. Disruption of the local blood circulation leads to ischemia, osteocyte death, and subsequent weakening of the trabecular bone structure which eventually leading to collapse of the articular surface and secondary degenerative arthritis of the hip joint. ²˒⁶˒⁸ Because ONFH predominantly affects individuals in early and middle adulthood, the associated pain, functional impairment, and loss of mobility affects substantial personal and socioeconomic burdens. ¹˒⁶ Although total hip arthroplasty remains an effective and proven treatment for advanced-stage disease, its longevity in younger patients is limited, often requiring revision procedures later in their life.⁶ Therefore, current management strategies emphasize on early diagnosis and joint preserving interventions such as core decompression, bone grafting, and osteotomies.⁶˒⁷˒¹⁰ These interventions are most successful when performed earlier before the structural collapse, highlighting the importance of early risk detection.²˒⁸ The vascular supply of the femoral head renders it particularly susceptible to ischemic injury. Its blood supply depends primarily on terminal branches of the medial circumflex femoral artery with limited collateral circulation. ⁶˒²³ ONFH is broadly classified into traumatic and non-traumatic forms. Traumatic cases are common in patients with femoral neck fractures or hip dislocations, whereas non-traumatic ONFH is due to multifactorial mechanisms involving metabolic, vascular, and genetic causes. ⁶ Prolonged corticosteroid therapy and chronic alcohol consumption are well-established non-traumatic risk factors.⁶˒¹⁰˒²⁸ Steroid-induced osteonecrosis causes alterations in lipid metabolism leading to increased intraosseous pressure and causing microvascular compromise.⁵˒¹¹˒²⁹ Similarly, alcohol-related cases also causes disturbances in the lipid metabolism and vascular regulation.²⁸ However, only a small proportion of individuals exposed to these risk factors have been found to develop ONFH, suggesting that environmental factors alone are not sufficient to fully explain susceptibility to the disease.³˒⁶ Increasing evidence supports a role for inherited thrombophilia and endothelial dysfunction in the pathogenesis of avascular necrosis.³˒¹³˒¹⁵ Abnormalities in coagulation pathways and impaired microvascular regulation may predispose individuals to vascular occlusion within the femoral head.³ This has led to the “multi-hit” hypothesis, in which genetic predisposition amplifies the harmful effects of external triggers such as corticosteroids or alcohol.⁶ Nitric oxide (NO) is a key regulator of vascular homeostasis. NO is synthesized by the endothelial nitric oxide synthase (eNOS), NO promotes vasodilation, inhibits platelet aggregation, and maintains endothelial integrity. ¹⁶˒²² Reduced NO bioavailability has been implicated in hypertension, coronary artery disease, and other vascular disorders. ¹⁴˒¹⁷˒¹⁸ Additionally, nitric oxide influences bone remodeling by modulating osteoblast and osteoclast activity, thereby supporting a link between vascular dysfunction and skeletal pathology. ¹⁶ the eNOS gene has several polymorphic regions capable of altering enzyme expression or activity. The 27-base pair variable number tandem repeat (VNTR) in intron 4 has been associated with altered nitric oxide production and cardiovascular complications.¹²˒¹⁵ Another widely studied variant is the G894T polymorphism in exon 7 (Glu298Asp), which may affect enzyme stability through increased proteolytic susceptibility.¹⁶˒²² These polymorphisms have been implicated in cardiovascular and thrombotic diseases,¹⁷˒¹⁸ and several studies have investigated their association with osteonecrosis of the femoral head, yielding inconsistent findings across populations.⁹˒¹⁹˒²⁰˒²⁵ Given these biologically plausible mechanisms and conflicting epidemiological data, further investigation is necessary to clarify the contribution of eNOS polymorphisms to ONFH susceptibility. Therefore, the present case–control study was undertaken to evaluate the association between the intron 4 (27-bp VNTR) and exon 7 (G894T) polymorphisms of the eNOS gene and its associated risk in avascular necrosis of the femoral head.
MATERIALS AND METHODS
2.1 Study Design and Ethical Considerations
This molecular case-control study was conducted at the Institute of Orthopedics and Traumatology.
Ethical committee approval
The study protocol adhered strictly to the principles of the Declaration of Helsinki and was approved by the Institutional Ethical Committee (IEC Approval No. 123/2025) of Coimbatore medical college on 04.04.2025. Written informed consent was obtained from all participants after a detailed explanation of the study's genetic nature and potential implications.
2.2 Study Population
A total of 100 participants were recruited for this investigation, divided into two distinct groups:
Exclusion Criteria: Patients with post-traumatic osteonecrosis (e.g., following femoral neck fracture), congenital hip dysplasia, Perthes disease, sickle cell anemia, or any history of major hip surgery were strictly excluded to ensure the homogeneity of the genetic analysis.
2.3 Sample Collection and Genomic DNA Extraction
From each participant, approximately 5 mL of venous blood was drawn under aseptic conditions into sterile vacutainers containing Ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Samples were stored at 4°C and processed within 24 hours. Genomic DNA was isolated from leukocytes using a commercial DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s spin-column protocol. The purity and concentration of the extracted DNA were verified using a NanoDrop spectrophotometer, ensuring an A260/A280 ratio between 1.8 and 2.0.
2.4 Genotyping of the 27-bp VNTR Polymorphism (Intron 4)
The variable number tandem repeat in intron 4 was analyzed using Polymerase Chain Reaction (PCR).
2.5 Genotyping of the G894T Polymorphism (Exon 7)
The G894T polymorphism was genotyped using PCR followed by Restriction Fragment Length Polymorphism (RFLP) analysis.
2.6 Statistical Analysis
Data management and analysis were performed using IBM SPSS Statistics for Windows, Version 24.0 (Armonk, NY: IBM Corp). Continuous variables (age) were expressed as mean ± standard deviation (SD) and compared using the Student's t-test. Categorical variables (sex, genotype, allele frequency) were presented as counts and percentages and compared using Pearson’s Chi-square test. The Hardy-Weinberg equilibrium (HWE) was tested for the control group to ensure representative sampling. Binary logistic regression analysis was utilized to determine the independent effect of genotypes on ANFH risk, calculating Odds Ratios (OR) with 95% Confidence Intervals (CI). A two-tailed p-value of <0.05 was considered statistically significant.
RESULTS
Baseline characteristics
The demographic and clinical characteristics of the study population were summarized (Table 1). A total of 50 patients diagnosed with ANFH and 50 healthy controls were included based on the inclusion criteria. The mean age of ANFH patients was comparable to that of controls (46 ± 6.9/48 ± 5.3 years), exhibits no statistically significant difference (p > 0.05). Similarly, sex distribution did not differ significantly between the two groups (ANFH: 22 m/28 f; controls: 23 m/27 f; p > 0.05). Among ANFH patients, disease staging revealed 4 patients in stage I, 17 in stage II, 18 in stage III, and 11 in stage IV.
Table 1: Baseline Characteristic of study subjects
|
|
Controls (n=50) |
ANFH (n=50) |
p-value |
|
Age (years) |
48 ± 5.3 |
46 ± 6.9 |
> 0.05 |
|
Sex (M/F) |
23 / 27 |
22 / 28 |
> 0.05 |
|
Stage |
NA |
I=4, II=17, III=18, IV=11 |
|
Genotype and allele distribution of the 27-bp repeat polymorphism
The genotype and allele distributions of the 27-bp repeat polymorphism in intron 4 are Tabulated (Table 2 & 3). A significant difference in genotype distribution was observed between control and ANFH patients (χ² = 19.4, p < 0.001). The incidence of b/b genotype was found to be low among ANFH patients than control (58.0% vs 94.0%). The genotypes b/a and a/a were found to be more prevalent in ANFH patients. In determining allele frequency, ‘a’ allele demonstrates significantly higher prevalence in ANFH patients that among controls (25.0% vs 3.0%). It is obvious that, the ‘a’ allele had a substantially increased risk of developing ANFH (OR = 10.78, 95% CI = 3.12–37.1, p < 0.001). Genotype distribution in controls conformed to Hardy-Weinberg equilibrium (p > 0.05).
Table 2. Genotype and allele distribution of 27-bp repeat polymorphism
|
Genotype distribution |
||||
|
Genotype |
Controls n (%) |
ANFH (%) |
χ²/OR (95% CI) |
p-value |
|
b/b |
47 (94.0) |
29 (58.0) |
19.4 |
0.001 |
|
b/a |
3 (6.0) |
17 (34.0) |
||
|
a/a |
0 (0) |
4 (8.0) |
||
|
Allele distribution |
||||
|
b |
97 (97.0%) |
75 (75.0%) |
10.78(3.12-37.1) |
0.001 |
|
a |
3 (3.0%) |
25 (25.0%) |
||
Genotype and allele distribution of the G894T polymorphism
Genotype and allele distributions of the G894T polymorphism in exon 7 was determined (Table 3). The results exhibit sight difference between two groups (χ² = 7.9, p = 0.005). The incidence of T/T genotype was found notably predominant among ANFH group (18 %) when compared to control group (2 %). While calculating allele frequency the T allele was significantly higher in ANFH group than in controls (33.0% vs 12.0%). The association of the T allele demonstrates significant increase in risk among ANFH patients (OR = 3.62, 95% CI = 1.75–7.47, p = 0.001).
Table 3. Genotype and allele distribution of G894T polymorphism
|
Genotype distribution |
||||
|
Genotype |
Controls n (%) |
ANFH n (%) |
χ²/OR (95% CI) |
p-value |
|
G/G |
39 (78.0) |
26 (52.0) |
7.9 |
0.005 |
|
G/T |
10 (20.0) |
15 (30.0) |
||
|
T/T |
1 (2.0) |
9 (18.0) |
||
|
Allele distribution |
||||
|
G |
88 (88.0%) |
67 (67.0%) |
3.62 (1.75-7.47) |
0.001 |
|
T |
12 (12.0%) |
33 (33.0%) |
||
Genotype distribution and progression of disease stage
The association of genotype and other demographic variable was determined using logistic regression analysis (Table 4). It is evident that the a allele of the 27-bp repeat polymorphism and the T allele of the G894T polymorphism were strongly associated with the susceptibility of individuals to ANFH (adjusted OR = 10.41, 95% CI = 2.95–36.7, p < 0.001; OR = 3.51, 95% CI = 1.67–7.39, p = 0.001). These alleles associated with risk was also found to be elevated in patients with advanced ANFH stages (III and IV) when compared with early stages (I and II). The significance association with the presence of risk allele and increased disease severity was demonstrated in Chi-square test (p < 0.05).
Table 4. Multivariable logistic regression analysis (adjusted ORs)
|
Polymorphism |
Genetic Model |
Adjusted OR |
95% CI |
p-value |
|
27-bp repeat |
a vs b |
10.41 |
2.95–36.7 |
<0.001 |
|
27-bp repeat |
a-carriers vs b/b |
10.92 |
3.01–39.6 |
<0.001 |
|
G894T |
T vs G |
3.51 |
1.67–7.39 |
0.001 |
|
G894T |
T-carriers vs G/G |
3.18 |
1.35–7.50 |
0.007 |
DISCUSSION
The present study studied whether functional polymorphisms within the endothelial nitric oxide synthase (eNOS) gene contribute to susceptibility of avascular necrosis of the femoral head. Our findings demonstrate a clear association between both the intron 4 variable number tandem repeat and the exon 7 G894T variant and the occurrence of disease. Individuals carrying the risk alleles showed significantly higher odds of developing ANFH compared with matched healthy controls. These results are consistent with previous reports suggesting a role for eNOS polymorphisms in osteonecrosis. ⁹˒¹⁹˒²⁰˒²⁵ Among the two variants studied, the intron 4 polymorphism exhibited the strongest relationship with the disease risk. Carriers of the shorter repeat allele showed approximately a tenfold increase in susceptibility after adjustment for demographic factors. Although located in a non-coding region, this variant is believed to influence gene regulation rather than affecting the protein structure. Reduced transcriptional efficiency and lower nitric oxide production have been reported in association with this allele.¹²˒²² From a pathophysiological perspective, diminished nitric oxide availability may impair baseline vasodilation within the already decreased microcirculation of the femoral head.⁶˒²³ Even small reductions in perfusion could promote venous stasis, intraosseous hypertension, and microthrombus formation, ultimately resulting to ischemic bone injury.³ Our results therefore support the concept that impaired endothelial function is a key contributor to the vascular compromise underlying osteonecrosis.³˒¹⁵ The exon 7 G894T polymorphism also demonstrated a significant, though comparatively smaller effect size. This variant results in a Glu298Asp amino acid substitution that may alter enzyme stability and increase susceptibility to proteolytic cleavage. ¹⁶ Consequently, nitric oxide synthesis may be reduced at the functional level. Evidence linking this polymorphism to vascular disorders such as coronary artery disease and hypertension further supports its biological relevance.¹⁷˒¹⁸ While the magnitude of risk associated with this polymorphism was lower than that of the intronic variant, its association with disease presence is in agreement with prior studies examining this locus in osteonecrosis populations.⁹˒¹⁹˒²⁰ Together, these two polymorphisms appear to influence nitric oxide biology through complementary mechanisms—one affecting gene expression and the other affecting protein integrity. Our findings align with the broader hypothesis that osteonecrosis is the result of multiple interacting factors rather than a single cause. ⁶ Environmental exposures such as corticosteroid therapy and chronic alcohol use are well-established triggers, ⁶˒¹⁰˒²⁸ yet they do not uniformly produce disease. Genetic predisposition affecting coagulation pathways or endothelial function may determine which individuals are unable to compensate for these vascular stresses. ³˒¹³˒¹⁵ The eNOS pathway represents a plausible biological link connecting vascular regulation with bone viability. ¹⁶˒²² By identifying polymorphisms that modify this pathway, the present study provides additional support for the “multi-hit” model of osteonecrosis pathogenesis. ⁶ from a clinical perspective, these results has significant practical implications. Recognition of genetic susceptibility markers will help stratify patients who are expected to receive high-dose corticosteroids or those who possess other known risk factors for osteonecrosis. ⁶˒¹¹˒²⁹ Earlier imaging surveillance or preventive strategies aimed at preserving endothelial function might be considered for individuals identified as high risk. Although such applications require further validation, integration of genetic data into risk assessment models may ultimately facilitate earlier diagnosis and improved joint preservation. ⁷ In summary, our findings indicate that polymorphisms affecting endothelial nitric oxide synthase are significantly associated with susceptibility to avascular necrosis of the femoral head. Variants that reduce nitric oxide bioavailability may predispose the femoral head to microvascular compromise and ischemic injury. ³˒¹⁶ These observations support the role of endothelial dysfunction in disease pathogenesis and highlight the potential utility of genetic markers in identifying individuals at increased risk. Further research is necessary to confirm these associations and to explore their implications for preventive and personalized treatment strategies.
Study Limitations and Future Directions
While our study offers robust statistical significance, several limitations must be acknowledged. First, the sample size of 100 subjects, while sufficient for a pilot study, lacks the power to perform detailed haplotype analyses (examining the interaction between the two polymorphisms). It is possible that individuals carrying both risk alleles suffer from a "super-additive" risk, but a larger sample study will confirm this. Secondly, we did not measure serum NO levels or eNOS enzyme activity directly. While the genetic association is strong, functional validation—correlating the genotype with actual physiological deficits in our specific patients—would strengthen the causal link. Also, we did not account for other genetic factors such as polymorphisms in the P-glycoprotein (MDR1) or coagulation factors (Factor V Leiden), which are known to interact with the eNOS pathway [15, 29].
CONCLUSION
In conclusion, this study confirms that the 27-bp repeat polymorphism in intron 4 and the G894T polymorphism in exon 7 of the eNOS gene are significant, independent risk factors for the development and progression of Avascular Necrosis of the Femoral Head. These genetic anomalies likely predispose the femoral head to vascular failure by compromising Nitric Oxide bioavailability and endothelial integrity. Future research should focus on integrating these genetic markers into predictive clinical models to facilitate early intervention and prevent the devastating sequelae of joint collapse.
ACKNOWLEDGEMENT
Authors would like to thank all the members who have participated in the research. We would also like to acknowledge Multidisciplinary Research Unit, Department of Health Research (DHR) for providing infrastructure for doing the study.
Conflicts of Interest
There are no conflicts of interest.
Funding
No funding was received for this study.
REFERENCES
Vetrivel Chezian Sengodan*, S. Marimuthu, D. R. Hariharan, A. S. Vijay Krishnan, B. Anbuvigneshwaran, Vishnu K., Divakar R., Anees B., Anurag Satish Vaishnav, Sureshkumar Muthusamy, Detection of Enos Gene Polymorphism Among Avascular Necrosis of Femoral Head- Our Experience, Int. J. Med. Pharm. Sci., 2026, 2 (4), 195-203. https://doi.org/10.5281/zenodo.19642968
10.5281/zenodo.19642968