The Journal of Bone & Joint Surgery

The Journal of Bone & Joint Surgery, Volume 90, Issue 10

Scientific Articles | October 01, 2008

Thrombophilia, Hypofibrinolysis, the eNOS T-786C Polymorphism, and Multifocal Osteonecrosis

Charles J. Glueck, MD¹; Richard A. Freiberg, MD²; Swapna Boppana, MD¹; Ping Wang, PhD¹

¹ Cholesterol Center, Jewish Hospital of Cincinnati, ABC Building, 3200 Burnet Avenue, Cincinnati, OH 45229. E-mail address for C.J. Glueck: glueckch@healthall.com
² Orthopedic Surgery Service, Cincinnati Veterans Administration Hospital, 3200 Vine Street, Cincinnati, OH 45220

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Disclosure: The authors did not receive any outside funding or grants in support of their research for or preparation of this work. Neither they nor a member of their immediate families received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, division, center, clinical practice, or other charitable or nonprofit organization with which the authors, or a member of their immediate families, are affiliated or associated.

Investigation performed at the Jewish Hospital of Cincinnati, Cincinnati, Ohio

The Journal of Bone and Joint Surgery, Inc.

J Bone Joint Surg Am, 2008 Oct 01;90(10):2220-2229. doi: 10.2106/JBJS.G.00616

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Abstract

Background: We examined the hypothesis that thrombophilia, hypofibrinolysis, and the endothelial nitric oxide synthase (eNOS) T-786C polymorphism are common, potentially treatable, and similar pathophysiologic causes of multifocal (three sites or more) and unifocal (single-site) osteonecrosis.

Methods: We prospectively evaluated twenty-six consecutively referred adults with multifocal osteonecrosis, who included thirteen with idiopathic multifocal osteonecrosis and thirteen with secondary multifocal osteonecrosis (resulting from steroid therapy in ten and alcoholism in three). We compared these patients with race, sex, and age-matched normal control subjects and with patients with idiopathic unifocal and secondary unifocal osteonecrosis, respectively. Using polymerase chain reaction and serologic measures, we studied thrombophilic and hypofibrinolytic mutations and the eNOS T-786C polymorphism.

Results: The total number of polymerase chain reaction and serologic thrombophilic-hypofibrinolytic abnormalities and the eNOS T-786C polymorphism did not differ between patients with idiopathic (p > 0.5) or secondary (p > 0.5) multifocal and unifocal osteonecrosis. The frequency of low free protein-S levels (<66%) in patients with secondary multifocal osteonecrosis (four of eleven patients) was higher than that in the control subjects (one of fifty-nine) (risk ratio = 21.5; 95% confidence interval, 2.6 to 174; p = 0.0016, Benjamini-Hochberg adjusted p [Bp] = 0.004). Factor-V Leiden heterozygosity was present in two of thirteen patients with secondary multifocal osteonecrosis compared with none of sixty-four control subjects (p = 0.027, Bp = 0.008). For eleven patients with secondary multifocal osteonecrosis, the eNOS T-786C polymorphism was present in nine of twenty-two alleles compared with eight of forty-four alleles in twenty-two normal control subjects (risk ratio = 2.3; 95% confidence interval, 1.0 to 5.0; p = 0.047, Bp = 0.016). The frequency of homocystinemia (>13.5 µmol/L) was higher in patients with idiopathic multifocal osteonecrosis (two of thirteen patients) than in normal controls (none of fifty-one) (p = 0.039, Bp = 0.004). A high level of factor VIII (>150%) was seen in four of eight patients with idiopathic multifocal osteonecrosis and in seven of forty-eight normal controls (risk ratio = 3.4; 95% confidence interval, 1.3 to 9.1; p = 0.04, Bp = 0.008). The eNOS T-786C mutant allele was present in seven of twelve alleles in the six patients with idiopathic multifocal osteonecrosis who were tested, compared with twenty-five of 108 alleles in fifty-four control subjects (risk ratio = 2.5; 95% confidence interval, 1.4 to 4.5; p = 0.015, Bp = 0.008).

Conclusions: Limited by the small numbers of patients with multifocal osteonecrosis, this exploratory study suggested that thrombophilia was associated with both idiopathic multifocal osteonecrosis and secondary multifocal osteonecrosis, as was the eNOS T-786C polymorphism. Multifocal and unifocal osteonecrosis are similarly associated with thrombophilia, hypofibrinolysis, and the eNOS T-786C polymorphism, which are potentially treatable pathophysiologic conditions, requiring further study.

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Introduction

Introduction | Materials and Methods | Results | Discussion | References

Multifocal osteonecrosis, which is categorized as involving three or more anatomic sites^1,2, is usually thought to be secondary to corticosteroid therapy^2-4 or alcoholism⁵, but it can be associated with thrombophilia-hypofibrinolysis^2,6,7. Approximately 3% of all patients with osteonecrosis² have multifocal osteonecrosis. Beyond corticosteroids^2-4, it is not clear what pathoetiologies distinguish multifocal from unifocal (single anatomic site) osteonecrosis.

We^8-10 and other investigators^6,7,11-30 have hypothesized that osteonecrosis of one or both hips is caused by familial or acquired thrombophilia-hypofibrinolysis and that venous thrombosis in the femoral head leads to increased intraosseous venous pressure and, thence, to impaired arterial flow, osseous hypoxia, and bone death.

Koo et al.³¹ showed that the 4a polymorphism on intron 4 of the endothelial nitric oxide synthase (eNOS) gene is associated with idiopathic osteonecrosis, probably by means of a reduced production of nitric oxide. In another study³², we showed that the T-786C polymorphism of the eNOS gene is associated with idiopathic osteonecrosis, also probably mediated by reduced nitric oxide production.

In twenty-six consecutively referred patients with multifocal osteonecrosis (thirteen with idiopathic and thirteen with secondary disease), we examined the hypothesis that thrombophilia, hypofibrinolysis, and the eNOS T-786C polymorphism are potentially treatable^9,32 and similar pathophysiologic causes of both multifocal and unifocal osteonecrosis. Limited by the small numbers of patients with multifocal osteonecrosis, our hypothesis-generating study is exploratory.

Materials and Methods

Introduction | Materials and Methods | Results | Discussion | References

Our institutional review board approved the research protocol, and signed informed consent was obtained from each patient.

Study Design

By current convention, osteonecrosis of the hip is categorized either as secondary (resulting from long-term use of high-dose corticosteroids, alcoholism, lupus erythematosus, hip dislocation or fracture, chemotherapy, human immunodeficiency virus-acquired immunodeficiency syndrome [HIV-AIDS], dysbarism, hormone replacement therapy, or pregnancy) or as idiopathic when there is no known etiology^11,33-35. Of the 224 adult patients referred to our center for the diagnosis and treatment of osteonecrosis (between 1995 and 2007), we prospectively studied twenty-six consecutive patients with multifocal osteonecrosis. None of the cases of these twenty-six patients with multifocal osteonecrosis have been previously reported¹⁰. Thirteen patients with idiopathic multifocal osteonecrosis and thirteen patients with secondary multifocal osteonecrosis (resulting from the use of steroids—about 3000 to 4000 mg of prednisone or its equivalent^34,35—in ten patients and from alcoholism in three patients) were compared with race, sex, and age-matched normal control subjects and with race, sex, and age-matched patients with idiopathic unifocal (one-site) osteonecrosis and those with secondary unifocal osteonecrosis, respectively (Tables I and II). To reduce etiological variability, we restricted the study of secondary multifocal osteonecrosis to patients in whom it was associated with steroid therapy or alcoholism, and we did not study the cases of patients with other causes of secondary osteonecrosis^11,33-35 such as chemotherapy, HIV-AIDS, and/or a history of fracture or dislocation of the hip.

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TABLE I Data on Patients with Idiopathic and Secondary Multifocal Osteonecrosis; Age, Sex, and Race-Matched Patients with Idiopathic and Secondary Unifocal Osteonecrosis; and Age, Sex, and Race-Matched Normal Controls

	Multifocal Osteonecrosis	Unifocal Osteonecrosis*					Normal Controls†
	No. of Patients	Matched for Race and Sex	Age Difference of =5 Yr	Age Difference of >5 Yr	Matched for Race and Sex	Age Difference of =5 Yr	Age Difference of >5 Yr
Idiopathic
White male	4	12	12	0	16	16	0
White female	9	27	25	2	36	36	0
Total	13	39	37	2	52	52	0
Secondary
White male	5	20	17	3	25	23	2
Black male	1	4	1	3	5	0	5
White female	7	28	20	8	35	32	3
Total	13	52	38	14	65	55	10

Patients with idiopathic multifocal osteonecrosis were matched three to one with patients with idiopathic unifocal osteonecrosis, and patients with secondary multifocal osteonecrosis were matched four to one with patients who had unifocal osteonecrosis.Normal controls were matched four to one with patients with idiopathic multifocal osteonecrosis and five to one with patients who had secondary multifocal osteonecrosis.

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	Multifocal Osteonecrosis	Unifocal Osteonecrosis*					Normal Controls†
	No. of Patients	Matched for Race and Sex	Age Difference of =5 Yr	Age Difference of >5 Yr	Matched for Race and Sex	Age Difference of =5 Yr	Age Difference of >5 Yr
Idiopathic
White male	4	12	12	0	16	16	0
White female	9	27	25	2	36	36	0
Total	13	39	37	2	52	52	0
Secondary
White male	5	20	17	3	25	23	2
Black male	1	4	1	3	5	0	5
White female	7	28	20	8	35	32	3
Total	13	52	38	14	65	55	10

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TABLE II Comparisons Between Patients with Idiopathic Multifocal Osteonecrosis and Normal Controls, Between Patients with Secondary Multifocal Osteonecrosis and Normal Controls, and Between Patients with Secondary Multifocal Osteonecrosis and Secondary Unifocal Osteonecrosis

Tests	No.	No. (%) with Abnormality	P Value*	Risk Ratio (95% Confidence Interval)	Sensitivity (95% Confidence Interval) (%)	Specificity (95% Confidence Interval) (%)
Comparison of patients with idiopathic multifocal osteonecrosis and normal controls
High homocysteine (>13.5 µmol/L)
Patients	13	2 (15)			15 (0-35)
Controls	51	0 (0)	0.039/0.004			100
High factor VIII (>150%)
Patients	8	4 (50)		3.4 (1.3-9.1)	50 (15-85)
Controls	48	7 (15)	0.040/0.008			85 (75-95)
eNOS T-786C allele (no. of alleles)
Patients	12	7 (58)		2.5 (1.4-4.5)	58 (30-86)
Controls	108	25 (23)	0.015/0.008			77 (69-85)
Comparison of patients with secondary multifocal osteonecrosis and normal controls
Low free protein S (<66%)
Patients	11	4 (36)		21.5 (2.6-174)	36 (8-65)
Controls	59	1 (2)	0.0016/0.004			98 (95-100)
Factor-V Leiden
Patients	13	2 (15)			15 (0-35)
Controls	64	0 (0)	0.027/0.008			100
High plasminogen activator inhibitor activity (>21.1 U/mL)
Patients	13	3 (23)		4.7 (1.1-20.7)	23 (0-46)
Controls	61	3 (5)	0.063/0.008			95 (90-100)
eNOS T-786C allele (no. of alleles)
Patients	22	9 (41)		2.3 (1.0-5.0)	41 (20-61)
Controls	44	8 (18)	0.047†/0.016			82 (70-93)
Comparison of secondary multifocal osteonecrosis and secondary unifocal osteonecrosis
High lipoprotein (a) (=35 mg/dL)
Multifocal osteonecrosis	13	1 (8)		0.2 (0.03-1.4)	8 (0-22)
Unifocal osteonecrosis	45	17 (38)	0.046/0.008		38 (24-52)
Low free protein S (<66%)
Multifocal osteonecrosis	11	4 (36)		5.2 (1.4-20.0)	36 (8-65)
Unifocal osteonecrosis	43	3 (7)	0.026/0.004		7 (0-14)

The values are given as the Fisher p value/Benjamini-Hochberg adjusted p value⁴³.Chi square.

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Tests	No.	No. (%) with Abnormality	P Value*	Risk Ratio (95% Confidence Interval)	Sensitivity (95% Confidence Interval) (%)	Specificity (95% Confidence Interval) (%)
Comparison of patients with idiopathic multifocal osteonecrosis and normal controls
High homocysteine (>13.5 µmol/L)
Patients	13	2 (15)			15 (0-35)
Controls	51	0 (0)	0.039/0.004			100
High factor VIII (>150%)
Patients	8	4 (50)		3.4 (1.3-9.1)	50 (15-85)
Controls	48	7 (15)	0.040/0.008			85 (75-95)
eNOS T-786C allele (no. of alleles)
Patients	12	7 (58)		2.5 (1.4-4.5)	58 (30-86)
Controls	108	25 (23)	0.015/0.008			77 (69-85)
Comparison of patients with secondary multifocal osteonecrosis and normal controls
Low free protein S (<66%)
Patients	11	4 (36)		21.5 (2.6-174)	36 (8-65)
Controls	59	1 (2)	0.0016/0.004			98 (95-100)
Factor-V Leiden
Patients	13	2 (15)			15 (0-35)
Controls	64	0 (0)	0.027/0.008			100
High plasminogen activator inhibitor activity (>21.1 U/mL)
Patients	13	3 (23)		4.7 (1.1-20.7)	23 (0-46)
Controls	61	3 (5)	0.063/0.008			95 (90-100)
eNOS T-786C allele (no. of alleles)
Patients	22	9 (41)		2.3 (1.0-5.0)	41 (20-61)
Controls	44	8 (18)	0.047†/0.016			82 (70-93)
Comparison of secondary multifocal osteonecrosis and secondary unifocal osteonecrosis
High lipoprotein (a) (=35 mg/dL)
Multifocal osteonecrosis	13	1 (8)		0.2 (0.03-1.4)	8 (0-22)
Unifocal osteonecrosis	45	17 (38)	0.046/0.008		38 (24-52)
Low free protein S (<66%)
Multifocal osteonecrosis	11	4 (36)		5.2 (1.4-20.0)	36 (8-65)
Unifocal osteonecrosis	43	3 (7)	0.026/0.004		7 (0-14)

The values are given as the Fisher p value/Benjamini-Hochberg adjusted p value⁴³.Chi square.

Patients

Orthopaedic surgeons referred patients from seven states in the Midwest. The findings of a thorough history and physical examination, anteroposterior and frog-leg lateral radiographs, and magnetic resonance imaging scans⁹ of the hips, knees, shoulders, ankles, and other joints (Table III) were used to verify the diagnosis of osteonecrosis.

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TABLE III Anatomic Sites and Number of Affected Joints in Thirteen Patients with Multifocal Idiopathic Osteonecrosis and in Thirteen Patients with Multifocal Secondary Osteonecrosis

Anatomic Sites	Hip	Knee	Shoulder	Ankle	Other
Idiopathic multifocal osteonecrosis
Patients (n = 13)	13 (100%)	11 (85%)	6 (46%)	4 (31%)	4 (31%)
Joints involved (n = 71)	25 (35%)	19 (27%)	10 (14%)	7 (10%)	10 (14%)
Secondary multifocal osteonecrosis
Patients (n = 13)	11 (85%)	12 (92%)	7 (54%)	3 (23%)	2 (15%)
Joints involved (n = 65)	22 (34%)	21 (32%)	11 (17%)	5 (8%)	6 (9%)

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TABLE III Anatomic Sites and Number of Affected Joints in Thirteen Patients with Multifocal Idiopathic Osteonecrosis and in Thirteen Patients with Multifocal Secondary Osteonecrosis

Anatomic Sites	Hip	Knee	Shoulder	Ankle	Other
Idiopathic multifocal osteonecrosis
Patients (n = 13)	13 (100%)	11 (85%)	6 (46%)	4 (31%)	4 (31%)
Joints involved (n = 71)	25 (35%)	19 (27%)	10 (14%)	7 (10%)	10 (14%)
Secondary multifocal osteonecrosis
Patients (n = 13)	11 (85%)	12 (92%)	7 (54%)	3 (23%)	2 (15%)
Joints involved (n = 65)	22 (34%)	21 (32%)	11 (17%)	5 (8%)	6 (9%)

Normal Control Subjects

For comparison of polymerase chain reaction and serologic measures of thrombophilia and hypofibrinolysis, the normal controls included forty-four healthy adult men (seventeen were hospital personnel and twenty-seven were being evaluated for hyperlipidemia), and fifty-seven healthy adult women (twenty-three were hospital personnel and thirty-four were being evaluated for hyperlipidemia). From this group of 101 healthy adult controls, we matched four normal controls to each patient with idiopathic multifocal osteonecrosis by race, sex, and age (fifty-two controls) and five normal controls to each patient with secondary multifocal osteonecrosis (sixty-five controls) (Table I). We targeted our age matches to five years or less. Of these 101 healthy adult controls, seventy-six had previously been compared¹⁰ with patients with unifocal osteonecrosis.

For comparison of the eNOS T-786C polymorphism, the normal controls included thirty-two healthy adult men (seventeen were hospital personnel and fifteen were being evaluated for hyperlipidemia) and forty healthy adult women (twenty-three were hospital personnel and seventeen were being evaluated for hyperlipidemia). We matched (by race, sex, and age) nine normal controls to each of six patients with idiopathic multifocal osteonecrosis who had measurement of the eNOS T-786C polymorphism (fifty-four controls) (Fig. 1), and two normal controls to each of eleven patients with secondary multifocal osteonecrosis (twenty-two controls) (Fig. 1). We targeted our age matches to five years or less.

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Fig. 1: The top panel shows eNOS T-786C distribution and T-786C allele frequency in six patients with idiopathic multifocal osteonecrosis (ON) compared with fifty-four race, sex, and age-matched normal controls. The bottom panel shows eNOS T-786C distribution and T-786C allele frequency in eleven patients with secondary multifocal osteonecrosis compared with twenty-two race, sex, and age-matched normal controls.

Patient Controls with Osteonecrosis of a Single Anatomic Site (Unifocal Osteonecrosis of One or Both Hips)

A cohort of 198 patients (116 men and eighty-two women) referred to our center with osteonecrosis of a single anatomic site (unifocal) involving one or both hips, which included 100 patients who had idiopathic osteonecrosis and ninety-eight who had secondary osteonecrosis, was used to provide control subjects with unifocal osteonecrosis. The thirteen patients with idiopathic multifocal osteonecrosis were matched three to one by race, age, and sex with thirty-nine patients with idiopathic unifocal osteonecrosis, and the thirteen patients with secondary multifocal osteonecrosis were matched four to one by race, age, and sex with fifty-two patients with secondary unifocal osteonecrosis (Table I). We targeted our age matches to five years or less (Table I). Of the ninety-one unifocal osteonecrosis controls, fifty-five came from our previous report of 133 patients with unifocal osteonecrosis¹⁰.

Laboratory Methods

Blood was processed as previously described^14,36. We studied mutations associated with thrombophilia (G1691A factor-V Leiden, G20210A prothrombin, C677T-methylenetetrahydrofolate reductase gene [MTHFR], and platelet glycoprotein PL A1A2 polymorphism)^14,36-40, with nitric oxide production (eNOS T-786C)⁴¹, and with hypofibrinolysis (plasminogen activator inhibitor-1 4G/4G³⁶). Serologic measures of thrombophilia^{14,36,37,39,42} included protein C, total and free protein S, antithrombin III, lupus anticoagulant, homocysteine, anticardiolipin antibodies immunoglobulin G and M (IgG and IgM), and factors VIII and XI. Measures of hypofibrinolysis^{14,36,37,39,42} included plasminogen activator inhibitor activity and lipoprotein (a).

Protein C, total and free protein S, and antithrombin III levels below the 5th percentile for normal population controls were considered abnormal³⁷. Homocysteine, anticardiolipin antibodies IgG and IgM, factor-VIII, and factor-XI levels at or more than the 95th percentile for normal population controls were considered abnormal^37,39. Plasma plasminogen activator inhibitor activity and lipoprotein-(a) levels at or more than the 95th percentile for normal population controls were considered abnormal³⁷.

Tests of factors VIII and XI were not available to us until November 1999; five of the thirteen patients with idiopathic multifocal osteonecrosis and eight of the thirteen patients with multifocal secondary osteonecrosis were seen before these assays were available to us.

Statistical Analyses

Categorical comparisons were assessed with use of chi-square tests or Fisher exact tests when at least one cell size was <5 (Fig. 1), and with Mantel-Haenszel tests.

Risk ratios with 95% confidence intervals were calculated (Table II). In the 2 × 2 table, patients with idiopathic or secondary osteonecrosis were compared with normal controls, and patients with secondary multifocal osteonecrosis were compared with those with secondary unifocal osteonecrosis (Table II); proportions (sensitivity and specificity) were reported with 95% confidence intervals which were done by asymptotic normality estimations (Table II).

Because of multiple comparisons of polymerase chain reaction (six comparisons) and serologic (twelve) measures of thrombophilia and hypofibrinolysis between patients with multifocal osteonecrosis and controls, and between patients with multifocal osteonecrosis and patients with unifocal osteonecrosis, we made a Benjamini-Hochberg correction⁴³ in the critical p value. In Table II, we display Fisher p values and the adjusted p value (Bp) required for significance after doing the Benjamini-Hochberg correction⁴³.

To assess the associations of steroids and alcohol with free protein S, factor-V Leiden, and the eNOS T-786C polymorphism allele frequency, three stepwise logistic regressions were carried out. The dependent variables were, separately, free protein S (low or not low), the factor-V Leiden mutation (present or not present), and the eNOS T-786C mutation (homozygosity, heterozygosity, or wild-type normal homozygosity), respectively, and explanatory variables included age, race, sex, group (secondary multifocal osteonecrosis or normal control), steroids, and alcohol use.

Limited by the small numbers of patients with multifocal osteonecrosis, the study was not powered a priori to examine a few well-defined hypotheses, but rather was exploratory.

Results

Introduction | Materials and Methods | Results | Discussion | References

Of 224 patients with osteonecrosis who were referred to our center, twenty-six (11.6%) had multifocal osteonecrosis categorized as involvement of three or more anatomic sites^1,2 (Table III). The thirteen patients with idiopathic multifocal osteonecrosis had seventy-one joints affected, and the thirteen with secondary multifocal osteonecrosis had sixty-five joints affected. The joints most commonly affected were hips, knees, and shoulders. Three of the thirteen patients with idiopathic multifocal osteonecrosis and four of the thirteen patients with secondary multifocal osteonecrosis smoked one pack of cigarettes or more per day.

Of the thirteen patients with idiopathic multifocal osteonecrosis, four were men, nine were women, and all were white (Table I). They had a mean age (and standard deviation) of 48 ± 6 years. All thirteen patients were successfully matched with patients with idiopathic unifocal osteonecrosis and with normal controls by race and sex. For age, the matches of patients who had multifocal idiopathic osteonecrosis with fifty-two normal controls were all in the target range (an age difference of five years or less); two (5%) of thirty-nine matched patients with idiopathic unifocal osteonecrosis had an age difference of more than five years.

The thirteen patients with secondary multifocal osteonecrosis included six men and seven women; twelve of them were white and one was black (Table I). They had a mean age of 44 ± 15 years. All thirteen patients with secondary multifocal osteonecrosis were successfully matched with fifty-two patients with unifocal secondary osteonecrosis and were matched with sixty-five normal controls by race and sex. For fourteen (27%) of the fifty-two matched patients with secondary unifocal osteonecrosis, the age match was more than five years (Table I). For ten (15%) of the sixty-five matched controls, the age match was more than five years.

Multifocal Osteonecrosis Compared with Unifocal Osteonecrosis

A comparison of the thirteen patients with idiopathic multifocal osteonecrosis and the thirty-nine race, sex, and age-matched patients with idiopathic unifocal osteonecrosis demonstrated no difference (p > 0.5) in the total numbers of abnormalities in five polymerase chain reaction or twelve serologic measures of thrombophilia-hypofibrinolysis, or in the eNOS T-786C polymorphism (data not shown). The eNOS genotype did not differ between the patients with idiopathic multifocal osteonecrosis and the eighteen with idiopathic unifocal osteonecrosis (p = 0.45) (data not shown).

In a comparison of the thirteen patients with secondary multifocal osteonecrosis and fifty-two patients with secondary unifocal osteonecrosis matched for race, sex, and age, there were no differences (p > 0.5) in the total numbers of abnormalities in five polymerase chain reaction or in twelve serologic measures of thrombophilia-hypofibrinolysis. The eNOS genotype did not differ between the eleven patients with secondary multifocal osteonecrosis and the twenty-two with secondary unifocal osteonecrosis (p = 0.23) (data not shown).

For individual polymerase chain reaction and serologic measures, thirteen patients with secondary multifocal osteonecrosis (compared with forty-five patients with secondary unifocal osteonecrosis who had a lipoprotein-(a) measure) were less likely to have a high lipoprotein-(a) level (=35 mg/dL) (risk ratio = 0.2; 95% confidence interval, 0.03 to 1.4) (Table II). There was a higher frequency of a low level of free protein S (<66%) in patients with secondary multifocal osteonecrosis than in patients with secondary unifocal osteonecrosis (risk ratio = 5.2; 95% confidence interval, 1.4 to 20.0) (Table II).

Idiopathic Multifocal Osteonecrosis Compared with Normal Controls

Two (15%; 95% confidence interval, 0% to 35%) of thirteen patients with idiopathic multifocal osteonecrosis had a high level of homocysteine (>13.5 µmol/L) compared with none of fifty-one normal controls (p = 0.039) (Table II). After the Benjamini-Hochberg correction⁴³, for this homocysteine difference to be significant, the p value would have to be =0.004 (Table II). The frequency of a high level (>150%) of factor VIII was greater in patients with idiopathic multifocal osteonecrosis (risk ratio = 3.4; 95% confidence interval, 1.3 to 9.1) (Table II). In the thirteen patients with idiopathic multifocal osteonecrosis, the eNOS T-786C polymorphism could be studied in six, and five (83%; 95% confidence interval, 54% to 100%) of the six patients were heterozygous and one (17%; 95% confidence interval, 0% to 46%) was homozygous. In comparison, twenty-three (43%; 95% confidence interval, 29% to 56%) of fifty-four race, sex, and age-matched normal controls were heterozygous and one (1.9%; 95% confidence interval, 0% to 5%) was homozygous (Fisher exact test, p = 0.012) (Fig. 1). The T-786C allele was present in 58% (95% confidence interval, 30% to 86%) of the twelve alleles in these six patients with idiopathic multifocal osteonecrosis compared with 23% (95% confidence interval, 15% to 31%) of the 108 alleles in the fifty-four normal controls (risk ratio = 2.5; 95% confidence interval, 1.4 to 4.5; p = 0.015, Bp = 0.008) (Table II, Fig. 1).

The thirteen patients with idiopathic multifocal osteonecrosis were more likely to have higher frequencies of gene mutations than were the fifty-two normal controls, particularly for two abnormalities among the five genes (factor-V Leiden, prothrombin, MTHFR, plasminogen activator inhibitor-1, and PL A1A2) studied. Two gene mutations were present in 42% (95% confidence interval, 14% to 70%) of the twelve patients with idiopathic multifocal osteonecrosis compared with 7% (95% confidence interval, 0% to 14%) in the normal controls (Mantel-Haenszel chi-square = 4.91, p = 0.027). Complete data on the five genes were not available for one patient.

In the comparison of the patients with idiopathic multifocal osteonecrosis and the normal controls, a high homocysteine level was seen in 15% of the patients (sensitivity, 15%) and in none of the normal controls (specificity, 100%) (Table II). A high level of factor VIII had a sensitivity of 50% (95% confidence interval, 15% to 85%) in the patients and a specificity of 85% (95% confidence interval, 75% to 95%) in the controls (Table II). The eNOS T-786C polymorphic allele had a sensitivity of 58% (95% confidence interval, 30% to 86%) in the patients and a specificity of 77% (95% confidence interval, 69% to 85%) in the controls (Table II). Overall, specificity was moderate to high, and sensitivity was low to moderate.

Secondary Multifocal Osteonecrosis Compared with Normal Controls

Four (36%; 95% confidence interval, 8% to 65%) of eleven patients with secondary multifocal osteonecrosis had a low level of free protein S compared with one (2%; 95% confidence interval, 0% to 5%) of fifty-nine normal controls (risk ratio = 21.5; 95% confidence interval, 2.6 to 174; p = 0.0016, Bp = 0.004) (Table II). Of the thirteen patients with secondary multifocal osteonecrosis, two (15%; 95% confidence interval, 0% to 35%) were heterozygous for the factor-V Leiden mutation compared with none of the sixty-four normal controls (p = 0.027, Bp = 0.008) (Table II). Three (23%; 95% confidence interval, 0% to 46%) of thirteen patients with secondary multifocal osteonecrosis had high (>21.1 U/mL) plasminogen activator inhibitor activity compared with three (5%; 95% confidence interval, 0% to 10%) of sixty-one normal controls (risk ratio = 4.7; 95% confidence interval, 1.1 to 20.7; p = 0.063, Bp = 0.008) (Table II).

The eNOS polymorphisms did not differ between the eleven patients with secondary multifocal osteonecrosis in whom they were measured and the twenty-two race, sex, and age-matched normal controls (Fisher p = 0.084) (Fig. 1). The T-786C allele was present in 41% (95% confidence interval, 20% to 61%) of the twenty-two alleles in these eleven patients with secondary multifocal osteonecrosis compared with 18% (95% confidence interval, 7% to 30%) of the forty-four alleles in the twenty-two normal controls (risk ratio = 2.3; 95% confidence interval, 1.0 to 5.0; p = 0.047, Bp = 0.016) (Table II, Fig. 1).

For the comparison of secondary multifocal osteonecrosis with the normal controls, a low level of free protein S, the factor-V Leiden, high plasminogen activator inhibitor activity, and eNOS T-786C allele polymorphism had low-to-moderate sensitivity of 36%, 15%, 23%, and 41%, respectively, in the patients and, overall, high specificity of 98%, 100%, 95%, and 82%, respectively, in the controls (Table II).

By stepwise logistic regression, in secondary osteonecrosis, neither steroid use nor alcohol were independently related to a low level of free protein S, factor-V Leiden, or eNOS polymorphism, which differed between the secondary osteonecrosis group and the normal controls (Table II).

Discussion

Introduction | Materials and Methods | Results | Discussion | References

Only approximately 3% of patients with osteonecrosis have multifocal involvement². Perhaps reflecting our referral patterns, twenty-six (11.6%) of 224 patients with osteonecrosis referred to our center had multifocal osteonecrosis. Multifocal osteonecrosis is usually thought to be secondary to corticosteroid use^2-4 or alcoholism^5,44. We were surprised that half of our twenty-six patients with multifocal osteonecrosis were diagnosed as having idiopathic disease, and that only ten (38%) of the twenty-six patients previously had long-term exposure to high-dose corticosteroids.

Rather than assume that most multifocal osteonecrosis is secondary to steroid use or alcoholism, we suggest that studies be done of thrombophilia, hypofibrinolysis, and the eNOS T-786C polymorphism associated with reduced nitric oxide production. Within this frame of reference, nine of 101 patients with multifocal osteonecrosis in a previous multicenter study² had no history of corticosteroid therapy and four of the nine patients had thrombophilia or hypofibrinolysis (one had familial protein-S deficiency, two had a high level of plasminogen activator inhibitor activity, and one had a factor-V Leiden mutation)².

In the current study, the total numbers of thrombophilic-hypofibrinolytic coagulation abnormalities did not differ between the thirteen patients with idiopathic multifocal osteonecrosis and the thirty-nine race, sex, and age-matched patients with idiopathic unifocal osteonecrosis or between the thirteen patients with secondary multifocal osteonecrosis and the fifty-two race, sex, and age-matched patients with secondary unifocal osteonecrosis. This suggests that the overall coagulation burden does not explain why some patients have multifocal and some have unifocal osteonecrosis develop.

The higher frequency of a low level of protein S, perhaps the most thrombophilic of the inherited thrombophilias⁷, seen in secondary multifocal osteonecrosis compared with the frequency seen in secondary unifocal osteonecrosis (36% compared with 7%, p = 0.026), suggests that there might be a risk for secondary multifocal osteonecrosis when high-dose steroids are given to patients with familial protein-S deficiency.

In the current study, to correct for the possibility that some of the multiple comparisons might be significant by chance, we controlled for the false discovery rate with the Benjamini-Hochberg method⁴³. Use of this correction⁴³ sets a higher hurdle for declaring significance, which was reached only by secondary multifocal osteonecrosis patient-control differences in low free protein-S levels. Despite limitations imposed by the small sample size, associated with the relative rarity of multifocal osteonecrosis², our exploratory study suggested that familial thrombophilia was associated with both idiopathic multifocal osteonecrosis (hyperhomocystinemia and a high level of factor VIII) and secondary multifocal osteonecrosis (factor-V Leiden and an increased frequency of protein-S deficiency), as was the eNOS T-786C mutation.

In our present study, thirteen (50%) of the twenty-six patients had idiopathic multifocal osteonecrosis, and these thirteen patients were more likely than race, sex, and age-matched normal controls to have thrombophilia (homocystinemia [15% compared with 0%, p = 0.039] and high factor VIII [risk ratio = 3.4; 95% confidence interval, 1.3 to 9.1]). Familial thrombophilias (low antithrombin III⁶ and familial protein-S deficiency⁷) have previously been associated with idiopathic multifocal osteonecrosis.

In the present study, high levels of homocysteine (>13.5 µmol/L) were more likely to occur in patients with idiopathic multifocal osteonecrosis than in normal controls, an observation previously noted in patients with predominantly unifocal osteonecrosis^45-47.

High levels of factor VIII^48-52 were more common in patients with idiopathic multifocal osteonecrosis than in normal controls (risk ratio = 3.4; 95% confidence interval, 1.3 to 9.1). This is a new finding, but it is consistent with the associations of familial thrombophilias with osteonecrosis^{2,6,7,45-47,53}.

The G1691A factor-V Leiden mutation⁵³ was more common in patients with secondary multifocal osteonecrosis than in normal controls (15% compared with 0%, p = 0.027). Bjorkman et al.¹⁹ reported a higher prevalence of the factor-V Leiden mutation in patients with idiopathic (but not secondary) osteonecrosis compared with normal controls in the Swedish population. The higher frequency of the thrombophilic factor-V Leiden mutation in patients with osteonecrosis compared with controls in our study and in the study by Bjorkman et al.¹⁹ further suggests an association of this heritable thrombophilia with osteonecrosis.

The associations of thrombophilia and hypofibrinolysis with osteonecrosis are important because this provides an opportunity for anticoagulation with enoxaparin with a goal of stopping the progression of unifocal osteonecrosis⁹. Anticoagulation^9,42,54 with enoxaparin^55-60 for the same duration as that used in deep vein thrombosis in the leg⁵⁷ has been reported⁹ to stop the progression of idiopathic Ficat⁶¹ stage-I and stage-II unifocal osteonecrosis of the femoral head in patients with thrombophilia-hypofibrinolysis, decreasing the frequency of total hip replacement⁹. In our prospective enoxaparin treatment study⁹, sixteen patients (twenty-five hips) with one or more thrombophilic-hypofibrinolytic disorders and Ficat stage-I or II osteonecrosis of one or both hips were selected. Enoxaparin (60 mg/day) was given for the first twelve weeks of the study. Sixteen patients were initially followed for two years or more⁹, with extension of our yearly follow-up to an average of six years⁶². Maintenance of hips at Ficat stages I and II compared with progression to stage III or IV or total hip replacement was the primary significant study outcome^9,62. On the basis of an intent-to-treat analysis, seventeen (68%) of twenty-five hips remained at Ficat stage I or II at an average follow-up of six years⁶². Compared with untreated historical control hips (approximately 20% had two-year survival)^63-65, the survival of seventeen (68%) of twenty-five hips at the time of the six-year follow-up suggests that the original twelve-week thromboprophylaxis with enoxaparin produced lasting benefit in idiopathic osteonecrosis in patients with heritable thrombophilia and hypofibrinolysis⁶². Enoxaparin seemingly prevents the progression of Ficat stages-I and II idiopathic unifocal osteonecrosis of the hip in patients with heritable thrombophilia-hypofibrinolysis, causing a decrease in the prevalence of total hip replacement^9,62.

Consistent with the report by Koo et al.³¹, and with our recent report on idiopathic unifocal osteonecrosis³², patients with idiopathic multifocal osteonecrosis were more likely than controls to have the eNOS T-786C polymorphism associated with reduced nitric oxide production. Koo et al.³¹ reported that the frequency of the 27-base pair repeat polymorphism in intron 4, a noncoding part of the eNOS gene, was higher in Koreans with idiopathic osteonecrosis of the hip than in controls (9% compared with 2.4%, p = 0.03). Since this 4a polymorphism is associated with reduced synthesis of eNOS, those authors concluded that a carrier state of 4a allele in intron 4 might be a genetic risk factor for osteonecrosis, and it could provide insight into the protective role of nitric oxide in the pathogenesis of osteonecrosis. Koo et al.³¹ determined that the frequency of the intron-4 polymorphism did not differ in patients with osteonecrosis of the hip secondary to steroid use or alcoholism compared with controls³¹. We recently reported³² that eNOS T-786C homozygosity was present in eleven (20%) of the fifty-four patients with predominantly unifocal idiopathic osteonecrosis compared with one (2%) of the fifty-one race, sex, and age-matched normal controls, heterozygosity was present in twenty-eight (52%) of fifty-four patients compared with seventeen (33%) of fifty-one normal controls, and wild-type normal was seen in fifteen (28%) of fifty-four patients compared with thirty-three (65%) of fifty-one normal controls (p = 0.0001). The eNOS T-786C mutant allele frequency in idiopathic osteonecrosis (46%; fifty of 108) was higher than in race, sex, and age-matched normal controls (19%; nineteen of 102) (p < 0.0001). Logistic regression revealed that, in the fifty-four patients with idiopathic osteonecrosis, the eNOS T-786C mutant allele was independently associated with idiopathic osteonecrosis (p < 0.0001; odds ratio = 5.86; 95% confidence interval, 2.63 to 13.1)³².

The finding of an association of the T-786C mutation with idiopathic unifocal and multifocal osteonecrosis opens the possibility of pharmacologic intervention with agents that increase nitric oxide production³², such as L-arginine. L-arginine enhances nitric oxide synthesis through eNOS and, through nitric oxide, improves endothelial dilatation, decreases platelet aggregation, and reverses endothelial dysfunction^66-70. We previously reported that the development and severity of Buerger disease in cigarette smokers who were homozygous or heterozygous for stromelysin-1 5A/6A and eNOS T-786C mutations are related to a gene-environment vasospastic interaction with reduced nitric oxide-mediated vasodilatation⁷¹. Increasing nitric oxide production by L-arginine appears to have therapeutic benefit in patients with the eNOS T-786C mutation and Buerger disease⁷¹. There is, to our knowledge, no data yet on the therapeutic effect of L-arginine in patients with idiopathic osteonecrosis associated with eNOS T-786C homozygosity³².

The overall coagulation burden, as assessed in the current report, does not explain why some patients have multifocal and some have unifocal osteonecrosis develop. Multifocal and unifocal osteonecrosis are similarly associated with thrombophilia, hypofibrinolysis, and with the eNOS T-786C polymorphism, which are potentially treatable^9,32 pathophysiologic conditions. The associations of both multifocal and unifocal³² idiopathic osteonecrosis with the eNOS T-786C polymorphism also open novel treatment possibilities with pharmacologic agents that increase nitric oxide production^66-70.

Limited by the small numbers of patients with multifocal osteonecrosis—approximately 3% of all patients with osteonecrosis²—our exploratory study suggested that both idiopathic multifocal osteonecrosis (hyperhomocystinemia and a high level of factor VIII) and secondary multifocal osteonecrosis (factor-V Leiden and increased frequency of protein-S deficiency) were associated with familial thrombophilia and with the eNOS T-786C polymorphism, all potentially treatable^9,32 pathophysiologic conditions. Our current study was not powered a priori to examine a few well-defined hypotheses but rather was exploratory. Image Not Available

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Topics

thrombophilia ; polymorphism ; nitric oxide synthase ; osteonecrosis

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Charles J. Glueck, Richard A. Freiberg, Swapna Boppana, Ping Wang; Thrombophilia, Hypofibrinolysis, the eNOS T-786C Polymorphism, and Multifocal Osteonecrosis. The Journal of Bone & Joint Surgery. 2008 Oct;90(10):2220-2229.

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