Increased circulating CD31+/CD42b-EMPs in Perthes disease and inhibit HUVECs angiogenesis via endothelial dysfunction.

AIMS: Endothelial microparticles (EMPs) are extracellular vesicles secreted by endothelial cells. The purpose of this research is to explore that the clinical significance and roles in angiogenesis and endothelial dysfunction of circulating microparticles in Perthes disease. MAIN METHODS: We collected platelet-poor plasma (PPP) from patients and controls, then microparticles (MPs) were extracted. Flow cytometry was performed to calculate the concentrations of CD31+/CD42b-, CD62E+ and CD31+/CD42b+ MPs. ELISA was performed to detect the expression level of biomarkers of endothelial dysfunction and inflammatory factors in plasma. In vitro experiments to evaluate the effect of circulating MPs and EMPs derived from IL-6-stimulated human umbilical vein endothelial cells (HUVECs) on angiogenesis and endothelial dysfunction. KEY FINDINGS: Our results revealed that the CD31+/CD42b- EMPs were significantly higher in Perthes disease group than in the control group. The Perthes-MPs being taken up by HUVECs promoted endothelial cell apoptosis, endothelial dysfunction and inhibited angiogenesis in vitro. Moreover, the level of IL-6 in plasma significantly increased in patients with Perthes, which was tightly correlated with the elevated level of circulating CD31+/CD42b- EMPs. IL-6 promoted HUVECs to secrete CD31+/CD42b- MPs, and EMPs derived from high concentration IL-6-stimulated (100 and 1000 pg/mL) HUVECs promoted endothelial cell apoptosis, endothelial dysfunction and inhibited angiogenesis. SIGNIFICANCE: In summary, our study suggests that circulating EMPs in the phenotypic spectrum revealed unique phenotypes of endothelial dysfunction, showing close correlation with the secretion of IL-6. These circulating EMPs may give rise to endothelial cell apoptosis, endothelial dysfunction and angiogenesis in Perthes disease.

The role and underlying mechanisms of microRNA­214 in Legg­Calvé­Perthes disease.

Legg­Calvé­Perthes disease (LCPD) is a pediatric form of femoral head osteonecrosis with unknown etiology. MicroRNAs (miRs) have been revealed to serve an important role in LCPD. MiR­214 serves an important role in chondrogenesis. The aim of the present study was to investigate the potential role of miR­214 in LCPD and the underlying mechanisms. The expression levels of miR­214 and B­cell lymphoma 2 (Bcl­2)­associated X protein (Bax) in dexamethasone (DEX)­treated TC28 cells, and the femoral head cartilage tissues, serum and primary chondrocytes of patients with LCPD, and healthy individuals were determined via reverse transcription quantitative polymerase chain reaction and western blot analysis. A luciferase reporter assay was conducted to investigate the association between miR­214 and Bax, while cell viability was determined via an MTT assay, and flow cytometry was performed to investigate cell apoptosis. The results revealed that miR­214 was downregulated and Bax was upregulated in DEX­treated TC28 cells and tissues obtained from patients with LCPD. MiR­214 was demonstrated to directly target Bax and negatively regulate its expression. DEX administration significantly suppressed cell proliferation, promoted apoptosis and decreased the Bcl­2/Bax ratio in TC28 cells; overexpression of miR­214 induced opposing effects, which were reversed by Bax overexpression. In conclusion, the results indicated that miR­214 and Bax may be potential therapeutic targets for the future clinical treatment of LCPD.

Is VEGF under-expressed in Indian children with Perthes disease?

BACKGROUND: The role of vascular endothelial growth factor (VEGF) after ischaemic necrosis of the femoral head in Legg-Calve-Perthes disease (LCPD) has not been adequately studied in humans, especially in Indian population. Therefore, we aimed to evaluate the serum levels of VEGF-A in Indian children with various stages of LCPD and compare them with those of an age- and sex-matched control group of healthy children. METHODS: In this case-control study, we enrolled 42 children (below 14 years age) suffering from LCPD and 21 age- and sex-matched healthy controls. Patients were classified radiographically according to Waldenstrom's classification. Serum VEGF-A was estimated by sandwich enzyme-linked immunosorbent assay technique. The serum values were compared between the patient group and the control group, as well as between the Waldenstrom subgroups. Results were expressed as means with ranges or median with interquartile range. RESULTS: The mean age in the patient as well as the control group was 9 years (range 4-13 years). The median value (interquartile range) of serum VEGF-A was 162.5 pg/ml (673.75 pg/ml) in the patient group and 652 pg/ml (190.5 pg/ml) in the control group (p = 0.013). When compared between lower Waldenstrom stages (initial stage + stage of fragmentation) and higher Waldenstrom stages (re-ossification stage + stage of healing), the mean values of serum VEGF-A were 464.7 pg/ml (range 0-2211 pg/ml) and 301.1 pg/ml (range 0-1910 pg/ml), respectively (p = 0.305). CONCLUSIONS: VEGF is under-expressed in Indian children suffering from LCPD. As VEGF acts as a key regulator of endochondral ossification, our finding may open new therapeutic approaches to the disease. Also, serum VEGF may act as a valuable marker for the follow-up of the disease. Our study also provides baseline data about serum VEGF-A levels in Indian cohort of LCPD patients. Future multi-centre studies are warranted with a larger sample size to fully appreciate the patho-physiological changes in VEGF occurring in LCPD.

Alterations of serum osteocalcin levels in patients with Legg-Calvé-Perthes.

BACKGROUND: Legg-Calvé-Perthes disease (LCPD) is a paediatric form of osteonecrosis that ultimately heals but will cause femoral head and acetabular deformities. The purpose of this study was to investigate the early serum measurement of osteocalcin in children with femoral head necrosis compared with that of healthy children. METHODS: 20 patients with LCPD (4 girls and 16 boys) and 20 healthy volunteers (5 girls and 15 boys) were enrolled. All patients did not have additional treatment. Blood samples were obtained from all patients preoperatively in the morning. All cases had hip radiographs. The serum levels of osteocalcin comparisons between the LCPD patients and the healthy volunteers were performed using Wilcoxon signed-rank test. The Spearman rank correlation was used to assess correlation between LCPD grade and serum osteocalcin levels. Significance was set at p = 0.05. RESULTS: The 20 patients with LCPD (72.75 ± 24.92 ng/ml) had significantly higher serum osteocalcin levels compared with the healthy group (16.80 ± 4.04 ng/ml) (p<0.01). Serum osteocalcin levels of different LCPD grades were significantly different (Spearman's p = 0.540, p = 0.014). CONCLUSIONS: We observed a significant correlation between serum osteocalcin levels and LCPD. These results may be meaningful in clinical practice and to future studies.

Neutrophil to lymphocyte ratio may be a predictive marker of poor prognosis in Legg-Calvé-Perthes disease.

BACKGROUND: Legg-Calvé-Perthes disease (LCPD) is the idiopathic avascular necrosis of the femoral head in childhood. The pathologic changes seen in the femoral head are likely a result of vascular factors. Blood neutrophil to lymphocyte (N/L) ratio is a simple marker of subclinical inflammation. This study aims to to analyse the predictive ability of N/L ratio for the prognosis in LCPD patients. METHODS: Patients who had been diagnosed as LCPD from 2008 to 2014 were investigated retrospectively and 40 LCPD patients (33 male and 7 female) and 25 healthy age and sex-matched children (controls) were included in the study. LCPD patients were divided into 2 groups according to expected prognosis (good prognosis expected Herring A and B patients as Group I and poor prognosis expected Herring B-C and C patients as Group II) and healthy children (control) were included in Group III. All the patients' hematological markers were analysed. RESULTS: Mean age was 7.1 ± 2.0 years in group I (4.9-12 years), 8.3 ± 2.2 years (4-12.5 years) in group II and 7.8 ± 1.3 years (6-12 years) in group III. Mean values for Groups I, II and III for neutrophil to lymphocyte (N/L) ratio were 1.13 ± 0.65, 1.75 ± 0.95, 1.08 ± 0.37, respectively. The mean neutrophil to lymphocyte (N/L) ratio of Group II was higher than the other 2 groups (p = 0.003). CONCLUSIONS: N/L ratio may give us information about the natural course of LCPD and may be used as independent predictor of prognosis in patients with LCPD.

Comparative study of serum proteomes in Legg-Calve-Perthes disease.

BACKGROUND: Legg-Calve-Perthes Disease (LCPD) is an idiopathic osteonecrosis of the developing femoral head complicated by pain and disability of the hip joint. To date, the pathological mechanisms of LCPD are not well-known. This study screened the changes in serum protein expression in patients with LCPD. METHODS: Age- and sex-matched serum samples from 10 control subjects and 10 patients with LCPD were compared using the isobaric tags for relative and absolute quantification (iTRAQ) technique. Gene ontology analyses, KEGG pathway and functional network analyses were performed. Proteins of interest with large differences in expression, S100-A8, alpha-1-acid glycoprotein 1, haptoglobin and apolipoprotein E, were compared by western blotting. RESULTS: The disease/control ratios showed 26 proteins were significantly differentially expressed (all p < 0.05). Including higher abundances of complement factor H (1.44), complement C4-B (1.45), isocitrate dehydrogenase [NAD] subunit alpha (2.7) alpha-1-acid glycoprotein 1 (1.87), heptoglobin (1.53) and Ig lambda-2 chain C regions (1.46), and lower levels of apolipoprotein E (0.50), apolipoprotein F (0.60), apolipoprotein C-III (0.69), S100-A8 (0.73), S100-A9 (0.75) and prothrombin (0.77) in LCPD than in controls. The alpha-1-acid glycoprotein 1 and haptoglobin increases, and apolipoprotein E and S100-A8 decreases were confirmed by western blot. KEGG pathway analysis revealed these proteins were related to the complement and coagulation cascades, Staphylococcus aureus infection, PPAR signaling, fat digestion and absorption, and vitamin digestion and absorption. Functional network analysis suggested that the proteins were involved in lipid regulation. CONCLUSIONS: The complement and coagulation cascades, and abnormal lipid metabolism may be involved in the pathogenesis of LCPD.

Effects of circulating endothelial progenitor cells, serum vascular endothelial growth factor and hypogammaglobulinemia in Perthes disease.

OBJECTIVE: The aim of this study was to investigate Legg-Calvé-Perthes disease (PD) pathogenesis by comparing absolute circulating endothelial progenitor cell (EPC) counts, serum levels of vascular endothelial growth factor-A (VEGF-A) and immunoglobulins between PD patients and controls. METHODS: The study included 28 PD cases (mean age: 8 ± 3.8) and 25 healthy age-matched control subjects. EPC, serum VEGF-A and immunoglobulin levels were measured in peripheral blood samples. Comparisons and correlation analysis were performed. RESULTS: In the PD group, 17 subjects were in the fragmentation stage and 11 in the healing stage. Four patients had bilateral disease and 14 had hypogammaglobulinemia. Median EPC count of the PD group was 80 and was significantly higher than those of the control group (p=0.011). No significant difference was determined in serum VEGF-A levels (p=0.354). EPC count were inversely correlated with serum IgG levels of the PD group (r=0.403, p=0.03). Absolute EPC count was also significantly higher in the fragmentation stage than in the healing stage and were also greater in bilaterally affected than in unilaterally affected patients. Circulating EPC count was correlated to the serum VEGF-A levels in patients with fragmentation stage of PD (r=0.605, p=0.01) and in those with hypogammaglobulinemia (r=0.599, p=0.001). CONCLUSION: High EPC count at the fragmentation stage of PD and relatively higher counts in bilateral disease suggest that EPC may be a valuable marker in the diagnosis and follow-up of PD. Additional studies are needed to explain the strong correlation between EPC and serum VEGF-A level in the fragmentation stage and in the presence of hypogammaglobulinemia.

Thrombomodulin and GFC levels in Legg-Calve-Perthes disease.

Legg-Calve-Perthes disease (LCPD) is a self-limited microvascular disorder leading to the occlusion of the femoral blood supply, which results in bone necrosis. Endothelial injury and hemostatic alterations may play a role in the microvascular compromise and decreased blood flow, which occur during the course of LCPD. Global fibrinolytic capacity (GFC) is a novel assay reflecting the overall fibrinolysis response resulting from the dynamic interactions of numerous stimulatory and inhibitory fibrinolytic molecules. Circulating soluble thrombomodulin (TM) reflects endothelial activation and/or injury. It is a cofactor in the clinically important protein C natural anticoagulant system. Beyond the coagulation pathway it is shown to have effects on biological events, especially inflammation. The aim of this study was to determine GFC and TM levels in LCPD patients. The study included 77 children in two groups. Group I consisted of 42 patients with LCPD and Group II (control) comprised 35 healthy children. Median (interquartile ratios) GFC and TM levels were significantly higher in the LCPD patients (Group I) (p<0.0001 and p=0.049, respectively). Circulating high levels of soluble TM may be associated with ongoing endothelial injury or ongoing inflammation during the disease course. Along with increased overall fibrinolytic response, increased TM may be a compensatory reaction to thrombosis. Further investigations are needed to elucidate the endothelial, anticoagulant, and fibrinolytic kinetics associated with the microvascular compromise and self-limiting nature of LCPD.

The role of protein C deficiency in the etiology of Perthes disease.

BACKGROUND: Recent reports support the hypothesis that thrombophilia plays an important role in the pathogenesis of Perthes disease (PD) and the objective of this report is to show evidence of the role of protein C deficiency in the etiology of PD, based on a meta-analysis of current scientific literature. MATERIAL AND METHODS: Studies were selected in all languages over the last twenty years (1986 to 2006) in MEDLINE, LILACS and EMBASE data bases. The inclusion criteria involved controlled studies, those that presented protein C as a continuous variable, and studies conducted in children with Perthes disease. The fixed effect model for continuous data was used; differences between groups were assessed by the t-test, Z-test and Cochran Q test for independent data and the level of significance was p < 0.05. RESULTS: The selected studies involved 175 patients and 193 control subjects. The selected studies were shown to be heterogeneous, but there were no statistically significant differences in protein C levels between groups. CONCLUSION: The authors' findings were unable to support the hypothesis that protein C deficiency is associated with Perthes disease and that it may play an important role in the ethiopathogenesis of avascular necrosis of the femoral head in childhood.

Thrombophilia and Legg-Calvé-Perthes disease: is it a causative factor and does it affect the severity of the disease?

Coagulation parameters were studied in a population of 118 children with Perthes disease in order to determine the possible role of thrombophilia as a causative factor for the disease and to determine if thrombophilia could affect its course. We found 27 children presenting one or more coagulation disorders. The statistical analysis concurs with previous findings of a relationship between Legg-Calvé-Perthes disease and an increased liability to thrombosis; however, no significant effect of thrombophilia on the severity of the disease could be demonstrated.

Enhanced tissue factor pathway inhibitor response as a defense mechanism against ongoing local microvascular events of Legg-Calve-Perthes disease.

The precise pathogenetic basis of Legg-Calve-Perthes disease (LCPD) is currently unknown. Hemostatic abnormalities, i.e., hypercoagulability and/or hypofibrinolysis, were proposed in the genesis of the LCPD. Deficiency of tissue factor pathway inhibitor (TFPI), a critical natural anticoagulant molecule, may lead to a prothrombotic state in a wide variety of conditions. The aim of this study is to assess the circulating TFPI pool in the LCPD. Group I consisted of 44 patients with LCPD and group II comprised 38 healthy children. Median (IQR) TPFI concentration was significantly higher in the group I (p < .0001). Enhanced TFPI response could be regarded as a compensatory defense mechanism against ongoing local microvascular events of occlusion and revascularization of LCPD. TFPI molecule may be an important link between the crossroads of the LCPD genesis and pathogenetic microvascular changes in the disease course. Further investigations are needed to shed light on the endothelial anticoagulant kinetics, the unique microvascular compromise, and the self-limiting nature of the disease.

Legg-perthes disease and heritable thrombophilia.

The etiology of Perthes' disease is unclear. Recent reports have suggested that inheritable thrombophilic disorders may be one of its pathogenetic causes. The G20210A prothrombin gene, factor V Leiden, and MTHFR C677T mutations have been identified as predisposing genetic factors for thrombosis. Ninety children diagnosed with Perthes' disease were studied. A family history of thrombosis and any other personal thromboembolic events were researched. PCR and endonuclease digestion were used to analyze factor V Leiden, prothrombin G20210A, and MTHFR C677T. Two hundred healthy donors were included as a control group. No patient had a family or personal history of early thrombotic events. Four children with Perthes' disease (4.4%) were heterozygous for G20210A polymorphism compared with controls (odds ratio: 2.07; 95% confidence interval: 0.40-8.46). No association between factor V Leiden and Perthes' disease was observed. Three patients (3.33%) were heterozygous for factor V Leiden (odds ratio: 1.36; 95% confidence interval: 0.32-5.84). The prevalence of different genotypes of C677T MTHFR did not show statistical differences compared with controls. Eleven patients were homozygous for this polymorphism (odds ratio: 1.02; 95% confidence interval: 0.42-2.44). This study does not support the screening of this group of polymorphism in patients with Perthes' disease.

Evaluation of anticoagulant system in Turkish children with Perthes disease.

BACKGROUND: Perthes Disease (PD) is generally a self-limiting disease of childhood but it causes severe pain and may lead to deformity of the femoral head. Intravascular thrombosis seems to form the main mechanism in the pathogenesis of the disease. The aim of this study was to determine hereditary thrombotic risk factors in Turkish children with PD. METHODS: In 46 Perthes patients (35 male, 11 female), family history of thrombotic events was investigated, Protein C (PC), free-Protein S (f-PS), antithrombin (AT) activities, fibrinogen level, and resistance to activated Protein C (APC) were measured. The results were compared with a healthy control group of 79 children matched by age and sex. The relationship between the severity of disease and coagulation system abnormalities was evaluated. RESULTS: While the mean PC and AT activities were significantly lower in the patients than those of the controls, the proportions of patients with low AT activity, resistance to APC, and a history of hereditary thrombophilia were significantly higher than those of the controls. No difference was observed in coagulation system disorders relative to severity of the disease and bilateral or unilateral disease involvement. CONCLUSIONS: This study shows that a possible association between PD and inherited hypercoagulability. Determination of thrombotic risk factors in these patients may bring a new approach to the treatment. Most importantly, this may be a stimulant to take precautions for other thrombotic events, which patients may face later in life.

[Congenital disorders of hemostasis in children with Perthes disease]. / Wrodzone zaburzenia ukladu hemostazy u dzieci z choroba Perthesa.

The level of selected parameters of the coagulation system and fibrynolysis (prothrombin time, partial thromboplastin time, fibrinogen level, albumin C system, V Leiden factor and III antithrombin level) in 25 children who had been treated with Perthes disease was evaluated. In three children prolonged prothrombin time occurred; in one child anomalous protein C system was noted. The remaining parameters were normal in all children. The investigation results reveal that congenital disturbances of the haemostasis system were not the cause of Perthes disease in 24 children.

Legg-Calve-Perthes disease and thrombophilia.

BACKGROUND: Thrombophilia has previously been identified as a potential etiologic factor in Legg-Calve-Perthes disease. We prospectively studied the association between Legg-Calve-Perthes disease and coagulation abnormalities by comparing seventy-two children who had the disease with 197 healthy controls. METHODS: A nonselected, consecutive series of seventy-two patients with Legg-Calve-Perthes disease (mean age [and standard deviation], 6.6 +/- 2.6 years) was studied in their order of referral and compared with 197 healthy controls (mean age, 7.6 +/- 5.1 years). Assays were done for factor-V Leiden, prothrombin G20210A, methylenetetrahydrofolate reductase C677T, and plasminogen activator inhibitor-1 4G/5G gene mutations. Levels of anticardiolipin antibodies immunoglobulin G and M (IgG and IgM), homocysteine, protein C, protein S, antithrombin III, and plasminogen activator inhibitor-1 were also measured. RESULTS: The factor-V Leiden mutation was more common in the patients (eight of seventy-two) than in the controls (seven of 197) (chi-square = 5.7, p = 0.017). After we controlled for the false-discovery rate, the case-control difference remained significant (p = 0.017). The odds ratio for the development of Legg-Calve-Perthes disease in the presence of the factor-V Leiden mutation was 3.39 with a 95% confidence interval of 1.18 to 9.73. A high level of anticardiolipin antibodies (IgG and/or IgM) was found in nineteen of the seventy-two patients compared with twenty-two of the 197 controls (chi-square = 9.5, p = 0.002). After we controlled for the false-discovery rate, the case-control difference remained significant (p = 0.002). The odds ratio of patients with Legg-Calve-Perthes disease having one or more abnormalities in factor V, anticardiolipin antibody IgG, or anticardiolipin antibody IgM as opposed to normal values for all three variables was 3.29 (95% confidence interval, 1.73 to 6.24; p = 0.0003). CONCLUSIONS: Two thrombophilic risk factors, the factor-V Leiden mutation and anticardiolipin antibodies, are associated with Legg-Calve-Perthes disease, an association that may reflect causality. LEVEL OF EVIDENCE: Prognostic study, Level II-1 (retrospective study). See Instructions to Authors for a complete description of levels of evidence.

Endocrine profile and physical stature of children with Perthes disease.

Children with Perthes disease have been thought to be of smaller stature than their peers. No reproducible evidence exists to demonstrate an endocrinopathy as the cause. This study reviewed 139 children with Perthes disease. Height and weight were recorded and compared with standardized growth charts. A blood sample was analyzed for thyroid stimulating hormone (TSH), thyroxin (T4), and insulin-like growth factor 1 (IGF1). Two urine samples were collected at 8 am and 8 pm within 24 hours. From these a cortisol/creatinine ratio was calculated. The results were compared with those from 40 healthy matched controls. No significant differences were found between the study or control children for levels of TSH, T4, IGF1, or cortisol. Review of the height and weight results revealed that the study children were of normal body habitus compared with the general pediatric population and do not undergo a period of growth acceleration following the active stages of the disease.

Thrombotic and fibrinolytic alterations in the aseptic necrosis of femoral head.

Recent reports seem to support the role of the thrombophilia and decreased fibrinolysis in the aetiopathogenesis of aseptic necrosis of bone. In the present study, haemostatic disturbances were analysed in adults (n = 49) and patients in childhood (Perthes disease) (n = 47) with aseptic necrosis of the femoral head. Fibrinolytic parameters (in vitro clot lysis, plasminogen, plasmatic plasminogen activator inhibitor-1 activity, D-dimer) along with lipoprotein (a) [Lp(a)] and fibrinogen were measured. von Willebrand factor, platelet activation and some thrombophilic factors (activated protein C resistance and factor V Leiden mutation, protein C, protein S activity) were also determined. Impaired fibrinolysis, an increased Lp(a) level along with slow clot lysis and increased platelet activation were found in adult cases. We detected five cases of factor V Leiden mutations (one heterozygotic and four homozygotic) among patients with Perthes disease. The clinical course of the heterozygous case was similar to the usual form of Perthes disease. The most severe form of Perthes disease has been observed in homozygous factor V Leiden mutation cases. The mutation of factor V Leiden per se probably does not induce the development of aseptic necrosis of bone tissue in childhood, but it does play a role in its acceleration. Homozygous factor V Leiden mutation definitely runs a more severe course. On the other hand, in adult cases, the disturbances of haemostasis, impaired fibrinolysis, elevated Lp(a) level, increased platelet activation and slight elevation of fibrinogen might have clinical relevance. Further studies should focus on proving the role of the haemostatic alterations in the pathogenesis of severe forms of aseptic bone necrosis. The use of antithrombotic drugs in order to slow the process of aseptic necrosis also has to be addressed in future surveys.

Prospective reevaluation of the association between thrombotic diathesis and legg-perthes disease.

BACKGROUND: Legg-Perthes disease is associated with ischemia of the capital femoral epiphysis in children. Thrombophilia has been implicated as a potential cause of the condition, and screening of patients with Legg-Perthes disease for thrombophilia has been recommended. We analyzed the value of screening for inherited thrombophilia in patients with Legg-Perthes disease by examining the association between Legg-Perthes disease and abnormalities in the thrombotic pathway. METHODS: A random series of consecutive patients with Legg-Perthes disease were prospectively enrolled in this study. Assays for the detection of factor-V Leiden mutation and the plasma concentrations of protein C, protein S, antithrombin III, and lipoprotein (a) were performed on plasma samples from children with Legg-Perthes disease, and the results were compared with those for pooled plasma from normal controls. Plasma concentrations below the 95% midrange of the control values were classified as protein deficiencies. The estimated population frequency of each coagulation abnormality was compared with the proportion of the study group with the corresponding abnormality. RESULTS: The proportion of abnormalities observed in the study group did not differ from the estimated population frequency for protein C, protein S, antithrombin III, or factor-V Leiden mutation. A lipoprotein (a) level of >30 mg/dL (>1.07 micro mol/L) was found in 16% of the study group. CONCLUSIONS: Our data do not suggest that thrombotic diatheses due to deficiency of protein C, protein S, or antithrombin III or due to factor-V Leiden mutation are major causes of Legg-Perthes disease. The elevated levels of lipoprotein (a) in children with Legg-Perthes disease suggest that they may be at risk for atherosclerosis as adults.