Children Who Had Legg-Perthes Disease
A letter was mailed to 112 children who had been managed for Legg-Perthes disease at Children's Hospital in Cincinnati from 1977 to 1994 and for whom the current address was known. The letter invited the children to participate in this study; forty-four responded and form the basis of this report. The remaining sixty-eight did not respond. There was no selection bias with regard to sex, race, age at onset, or severity of the disease.
After the patient had fasted overnight, we measured fibrinolytic activity7,8,12; the levels of protein C, protein S, C4b-binding protein, antithrombin III11,17,21, lipids8, and lipoprotein(a)2; and the prothrombin time. A lipoprotein(a) level of 0.25 gram per liter or more was considered high. We recorded the age at which the Legg-Perthes disease was first diagnosed as well as any diseases or drugs that could produce secondary osteonecrosis7,10-12,24. We systematically sought evidence of current infection (which might have raised the level of C4b-binding protein with a decrease in the level of free protein S), malabsorption, obstructive jaundice, other liver disease, or antibiotic therapy that may have produced vitamin-K deficiency. A detailed family history was obtained.
Thirty-three of the children who had Legg-Perthes disease had a low level of protein C or S, a high level of lipoprotein(a), or a low level of stimulated fibrinolysis, and vigorous efforts were made to obtain samples from all of their living first-degree relatives and, when possible, second and third-degree relatives, to determine whether these disorders were familial.
Controls
Fibrinolytic activity and the levels of protein C and protein S were studied in thirty healthy children (twelve girls and eighteen boys) who served as controls. The mean age (and standard deviation) of the controls was 10.8 ± 4.9 years, which was comparable with that of the forty-four patients (10.1 ± 4.4 years) at the time of the study.
Measurement of Fibrinolytic Activity and Levels of Protein C, Protein S, and Antithrombin III
Basal and stimulated fibrinolytic activity was measured as previously reported7,8,10,12. After a twelve-hour-long fast, blood was drawn from the antecubital vein while the patient was seated, between the hours of 8:00 and 9:00 A.M. to minimize circadian influence on fibrinolytic activity8. The first three milliliters of blood was discarded, after which blood was collected in five-milliliter 0.13-molar sodium citrate Vacutainer tubes (Becton Dickson, Rutherford, New Jersey) and was immediately placed in wet ice. For measurement of tissue-plasminogen activator activity, blood was collected in five-milliliter Stabilyte tubes (American Diagnostica, Greenwich, Connecticut) containing an acidified citrate anticoagulant solution, which preserves the level of tissue-plasminogen activator activity. While the patient remained seated during a standard stimulus (ten minutes of venous occlusion at 100 millimeters of mercury [13.33 kilopascals] with a blood pressure cuff8,12), citrated blood was again collected as just described for the measurement of stimulated fibrinolysis. Within sixty minutes after collection, the blood samples were centrifuged at 2000 times gravity for twenty minutes at 4 degrees Celsius. Platelet-poor plasma was stored at -70 degrees Celsius until it was processed.
The concentrations of all factors in healthy individuals and the lower and upper limits of the concentrations (reference range) have been determined statistically from a normal, or so-called gaussian, distribution with use of the middle 80, 90, or 95 per cent of values.
Tissue-plasminogen activator activity, the major stimulator of fibrinolysis, was measured by chromogenic assay (Biopool Spectrolyse/Fibrin; Biopool, Umea, Sweden)8. The within-day coefficient of variation was 7.7 per cent and the between-day coefficient of variation was 15.2 per cent. The bottom-to-top decile reference range for stimulated tissue-plasminogen activator activity that included 80 per cent of the thirty controls was 2.33 to 9.57 international units per milliliter, which is comparable with that in adults (2.28 to 11.30 international units per milliliter)11. The reference range for stimulated tissue-plasminogen activator activity that included 90 per cent of the thirty controls was 2.19 to 17.03 international units per milliliter.
Plasminogen activator-inhibitor activity, the major inhibitor of fibrinolysis, was measured by chromogenic assay (Biopool Spectrolyse pL; Biopool)8. The within-day coefficient of variation was 5.6 per cent, and the between-day coefficient of variation was 7.7 per cent. The bottom-to-top decile reference range for basal plasminogen activator-inhibitor activity that included 80 per cent of the thirty controls was 5.5 to 19.6 units per milliliter, similar to that in adults (5.2 to 19.9 units per milliliter)12. The reference range for plasminogen activator-inhibitor activity that included 90 per cent of the thirty controls was 4.3 to 24.0 units per milliliter.
For the analysis of the levels of antithrombin III, protein C, and protein S, plasma samples from thirty-three healthy, normal adults were pooled and were used for standard dilution curves by making serial dilutions of the pooled normal plasma. The optical density reading for the undiluted pooled normal plasma is defined as 100 per cent of normal. The optical density readings of plasma levels of antithrombin III, protein C, and protein S in the thirty control children and the forty-four patients who had Legg-Perthes disease were read against these dilution curves to determine their concentrations, with the unit of measurement being the percentage of normal.
The protein-C level was measured by enzyme-linked immunosorbent assay (Asserachrom Protein C; Diagnostica Stago, distributed by American Bioproducts, Parsippany, New Jersey). The within-day coefficients of variation for low (53 per cent of normal) and normal (92 per cent of normal) protein-C plasma pools were 6.7 and 3.6 per cent, respectively, and the between-day coefficients of variation were 10.7 and 7.6 per cent, respectively. The bottom-to-top decile reference range for adults is 70 to 140 per cent of normal. The bottom-to-top decile reference range that included 80 per cent of the thirty controls was 70 to 106 per cent of normal, the reference range that included 90 per cent of the controls was 65 to 114 per cent of normal, and the reference range that included 95 per cent of the controls was 54 to 114 per cent of normal. Depending on the reference interval used, protein-C deficiency was identified when the protein-C level in a child who had Legg-Perthes disease was less than 70, 65, and 54 per cent of normal, respectively.
The protein-S level was measured by enzyme-linked immunosorbent assay (Asserchrom Protein S; American Bioproducts). The within-day coefficients of variation for low (42 per cent of normal) and normal (87 per cent of normal) protein-S plasma pools were 8.2 and 7.2 per cent, respectively, and the between-day coefficients of variation were 8.9 and 10.7 per cent, respectively. The bottom-to-top decile reference range for adults is 70 to 140 per cent of normal. The bottom-to-top decile reference range that included 80 per cent of the thirty controls was 76 to 134 per cent of normal, the reference range that included 90 per cent of the controls was 76 to 150 per cent of normal, and the reference range that included 95 per cent of the controls was 43 to 151 per cent of normal. Depending on the reference interval used, protein-S deficiency was identified when the protein-S level in a child who had Legg-Perthes disease was less than 76, 76, and 43 per cent of normal, respectively.
The level of antithrombin III was measured with use of chromogenic assay (Dade antithrombin III; Baxter Healthcare, Miami, Florida). The bottom-to-top decile reference range for adults is 80 to 120 per cent of normal. Antithrombin-III deficiency is recognized when the measurement is in the bottom decile for normal (less than 80 per cent of normal).
The level of C4b-binding protein was quantitated by latex immunoassay (Liatest C4b-BP; American Bioproducts). Free protein-S and protein-E activity assays were not performed.
We chose not to display post-occlusion values for fibrinogen, plasminogen, and a2-antiplasmin since they are primarily affected by hemoconcentration and are not released from the endothelium in response to increased vascular transmural pressure12.
Statistical Methods
Differences in the age at onset of the Legg-Perthes disease were assessed with chi-square analysis.
Children Who Had Legg-Perthes Disease
None of the forty-four children in the present investigation had been included in our previous study of Legg-Perthes disease11. All forty-four had characteristic clinical, radiographic, and physical findings of Legg-Perthes disease3,11,19,24,26,27. The osteonecrosis was not secondary to use of corticosteroids, sickle-cell disease, lupus erythematosus, or fracture of the hip7,10-12,24. All of the children had a normal prothrombin time and a normal level of C4b-binding protein.
With regard to the age at the onset of the disease, sex, race, or the percentage who had unilateral or bilateral disease, these forty-four children did not differ significantly (p =0.09) from the sixty-eight children with Legg-Perthes disease who did not respond to the invitation to participate in the study. Thus, there was no self-selection bias on the part of the forty-four participants.
Only eleven (25 per cent) of the forty-four children who had Legg-Perthes disease were entirely normal in that they had normal levels of lipoprotein(a), protein C, and protein S; normal stimulated tissue-plasminogen activator activity; and normal plasminogen activator-inhibitor activity. In the thirty-three families in which the proband had a low level of protein C or S, hypofibrinolysis, or a high level of lipoprotein(a) (thought to increase the risk for Legg-Perthes disease11), sixty-one children (including the thirty-three probands) were tested. Fifty-two (85 per cent) of them had a low level of protein C or S, hypofibrinolysis, or a high level of lipoprotein(a); thirty-three (63 per cent) of these fifty-two children had Legg-Perthes disease.
Family Studies
The thirty-three probands who had a low level of protein C or S, a high level of lipoprotein(a), or low stimulated tissue-plasminogen activator activity had ninety-eight living first-degree relatives, eighty (82 per cent) of whom were studied. Thirty-two second and third-degree relatives were also evaluated.
Deficiency in Antithrombotic Factors C and S
The forty-four probands and the 112 relatives who were studied all had normal levels of antithrombin III, fibrinogen, plasminogen, and a2-antiplasmin.
Although the mean age (and standard deviation) at the time of the diagnosis of Legg-Perthes disease was 5.8 ± 2.7 years (range, two to fourteen years), the patients were first seen by us at a mean age of 10.1 ± 4.4 years (range, two to twenty-four years), which is comparable with the mean age of the controls (10.8 ± 4.9 years). The protein-C level and the lower limit of the normal range increase with age in childhood1, being lowest at the ages of one to five years and more closely approximating adult values by the ages of eleven to sixteen years1. Of the nineteen children with Legg-Perthes disease whom we identified as having a low level of protein C, seven were more than eleven years old when they were studied and ten were six to ten years old (Table I). Although the protein-S level also increases with age in childhood, it does not differ significantly from that in adulthood1. Two of the four children who had a low level of protein S were sixteen years old at the time of the study, one was six years old, and one was five years old (Table II).
Of the forty-four children who had Legg-Perthes disease, twenty-three had thrombophilia (nineteen had a low level of protein C and four had a low level of protein S), seven had a high level of lipoprotein(a), and three had hypofibrinolysis (low stimulated tissue-plasminogen activator activity and a normal level of lipoprotein[a]) (Tables I, II, III through IV). At least one first-degree relative of eleven (C1 through C11) of the nineteen probands who had a low level of protein C also had a low level of protein C (Table I and Fig. 1). Consequently, previously undiagnosed familial protein-C deficiency was identified in these individuals. Conversely, both parents and at least one brother of five (C12 and C14 through C17) of the remaining eight probands had a normal level of protein C (Table I). None of these five probands had a sister.
Of the eight probands (C12 through C19) who had a low level of protein C and in whose families protein-C deficiency could not be demonstrated (Table I), four (C13, C14, C17, and C18) are of particular interest as they were very young (three years old or less) when the Legg-Perthes disease was first diagnosed.
Four probands had a low level of protein S (Table II). At least one relative of one (S1) of these four probands also had a low level of protein S (Table II and Fig. 1). Of the three probands (S2, S3, and S4) who had a low level of protein S and in whose families familial protein-S deficiency could not be demonstrated, two (S2 and S4) were very young (four years old or less) when the Legg-Perthes disease was first diagnosed.
High Levels of Lipoprotein(a) or Hypofibrinolysis, or Both
Seven children who had Legg-Perthes disease also had a high level of lipoprotein(a) (Table III); six (L1 through L5 and L7) had a familial high level of lipoprotein(a), with at least one first-degree relative also having a high level of lipoprotein(a) (Figs. 2, 3, and 4). Of the seven probands who had a high level of lipoprotein(a), six (L1, L2, and L4 through L7) also had hypofibrinolysis with a low level of stimulated tissue-plasminogen activator activity (Table III and Figs. 2, 3, and 4). At least one first-degree relative of three (L1, L4, and L5) of these six probands (Figs. 2, 3, and 4) also had a high level of lipoprotein(a) and low stimulated tissue-plasminogen activator activity, indicating a concurrent heritable high level of lipoprotein(a) and hypofibrinolysis. Six (L1 and L3 through L7) of the seven probands had normal basal plasminogen activator-inhibitor activity.
Three probands had a normal level of lipoprotein(a) and low stimulated tissue-plasminogen activator activity (Table III). One of these probands (H1), along with her father, also had high plasminogen activator-inhibitor activity, indicating hereditary hypofibrinolysis (Fig. 5).
Race, Sex, Age at Onset, and Nature of Legg-Perthes Disease
All forty-four of the children were white, boys predominated (75 per cent [thirty-three] of the patients), and most of the children had unilateral disease (Table IV). The onset of the Legg-Perthes disease for the eleven children who had familial protein-C deficiency was more likely to have been before the age of five years (nine of the eleven) than the onset for the eleven children who did not have a hypofibrinolytic or thrombophilic disorder (three of the eleven) (chi-square = 6.6; p = 0.01) (Table IV). The onset of the Legg-Perthes disease for all nineteen children who had protein-C deficiency was also more likely to have been before the age of five years (thirteen of the nineteen) than the onset for the eleven children who did not have a hypofibrinolytic or thrombophilic disorder (chi-square = 4.74; p = 0.03) (Table IV).
Familial Aggregation of Legg-Perthes Disease, Thrombophlebitis, Premature Myocardial Infarction, and Stroke
In two families (the kindreds of C3 and C5) of the eleven probands who had familial protein-C deficiency, Legg-Perthes disease had been diagnosed in a maternal uncle (Table I). In one of the eleven families, a first-degree relative had thrombophlebitis. In five of the eleven families, a member (four grandparents and one great aunt) had had a myocardial infarction or stroke before the age of sixty-five years.
At least one first-degree relative of two of the four probands who had protein-S deficiency had thrombophlebitis, and in two families at least one member had had a myocardial infarction or stroke before the age of sixty-five years.
A maternal uncle in one family (the kindred of L3) of the six probands who had a familial high level of lipoprotein(a) had Legg-Perthes disease (Fig. 3). Thrombophlebitis was diagnosed in at least one member in two of the families, and at least one member in five of the families had had a myocardial infarction or stroke before the age of sixty-five years.
One relative of one of the three probands who had hypofibrinolysis had Legg-Perthes disease and three other relatives had adult idiopathic osteonecrosis (two paternal aunts, one paternal uncle, and one maternal aunt were affected). At least one member in two of the families had had a myocardial infarction or stroke before the age of sixty-five years. In one of these two families, all eight brothers and sisters of the maternal grandfather of the proband had had a myocardial infarction before the age of sixty-five years.
Over-all, four members of the twenty-four families just mentioned had abnormalities of coagulation, which increase the risk of osteonecrosis; four families had a history of Legg-Perthes disease; and three members of one family had adult idiopathic osteonecrosis.
In aggregate, 58 per cent (sixty-seven) of the 116 adult members in families with familial hypofibrinolysis, thrombophilia, or a high level of lipoprotein(a) had had a myocardial infarction or stroke before the age of sixty-five years. This high rate was comparable with that (50 per cent9) in adult first-degree relatives of 432 hyperlipidemic patients at high risk for myocardial infarction and stroke.