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Lipoprotein(a) serum levels in patients with Paget’s disease and primary hyperparathyroidism
32S
Molecular genetics of Paget’s disease: Abstracts
Bone Vol. 24, No. 5, Supplement May 1999:31S–33S
Family History and Heritability of Paget’s Disease: A Multicenter Study F. SLEE,1 L. HOCKING,1 S. I. HASLAM,1 M. HOOPER,2 R. DARGIE,3 G. A. NICHOLSON,4 and S. H. RALSTON1 1 Department of Medicine and Therapeutics, University of Aberdeen, Aberdeen, UK 2 Concorde Hospital, Sydney, Sydney, Australia 3 University of Glasgow, Glasgow, UK 4 University of Melbourne, Melbourne, Australia Genetic factors are important in the pathogenesis of Paget’s disease, although estimates of the frequency with which the disease is inherited vary widely. Here, we report the results of a multicenter study on family history and heritability of Paget’s disease. A standard questionnaire asking for details of family history was administered to pagetic patients attending specialist out-patient clinics in Aberdeen (n 5 35), Glasgow (n 5 35), Melbourne (n 5 28), and Sydney (n 5 115). Calculation of heritability (recurrent risk in any relative) was made by administering the questionnaire to spouses and nonpagetic controls. Results are shown in Table 1.
Glasgow
Sydney
Aberdeen
Melbourne
14%
23%
25%
53%
●●●
●●●
14.5
Acknowledgments: This work was funded by the NARPD. Address for correspondence and reprints: Dr. Andrew Mee, Musculoskeletal Research Group, University School of Medicine, Manchester Royal Infirmary, Manchester M13 9WL, UK. PII S8756-3282(99)00041-100
Table 1. Questionnaire results
Family history Heritability
samples from each patient, to identify the expression of novel upregulated genes in the pagetic bone. After three rounds of subtraction, the results from southern blotting showed that sequences had been isolated from the affected bone that were not present in the uninvolved bone samples. In addition, other poly-A PCR products from pagetic patients (both affected and unaffected sites) were generated. Southern blotting confirmed that the cDNA sequences from the final subtraction hybridized only to cDNA derived from affected sites. The subtracted cDNA has been used to screen a pagetic bone cDNA library (a generous gift from Professor Paul T. Sharpe), from which 42 positive plaques have been isolated and are currently being sequenced. The results so far demonstrate that we have successfully isolated gene sequences that are only found to be expressed in affected pagetic bone biopsies and not in uninvolved sites. Sequence analysis of the positive clones derived from the pagetic cDNA library should yield valuable information to aid further investigations into the etiopathology of Paget’s disease.
4.9
All 26% 9.9
There was a wide variation between centers in frequency of a positive family history and in heritability, with values ranging between 14% and 53% for family history and 4.9 to infinity for heritability. Segregation analysis in kindreds with a positive family history showed an autosomal-dominant pattern of inheritance with high penetrance by age 65 years (55% offspring affected, 45% unaffected). The likelihood of disease transmission was similar for affected males (52%) and females (47%) and gender distribution of affected offspring was similar (53% of male offspring affected vs. 58% female). This study shows evidence of heterogeneity between centers in the frequency with which pagetic patients have a positive family history and in heritability estimates. These differences could either be due to a high frequency of pagetic individuals in population founders or selection bias due to more frequent referral of familial cases to certain clinics. Whatever the underlying reasons, overall heritability estimates for Paget’s were high, emphasizing the importance of genetic factors in disease etiology. Unlike a previous study, however, we were unable to confirm that there was an excess of maternal transmission in familial cases. Address for correspondence and reprints: Professor Stuart H. Ralston, Department of Medicine and Therapeutics, University of Aberdeen Medical School, Forestermill, Aberdeen AB25 2ZD, UK. PII S8756-3282(99)00040-X 00
Isolation of Novel Genes in Paget’s Disease by Subtractive Hybridization J. A. DIXON, J. A. HOYLAND, E. B. MAWER, P. L. SELBY, and A. P. MEE Musculoskeletal Research Group, University School of Medicine, Manchester Royal Infirmary, Manchester, UK
Pamidronate in Acquired Immune Deficiency Syndrome A. E. POSTLETHWAITE, M. A. LAND, G. M. A. PALAMIERI, L. PIFER, and J. INGLES University of Tennessee at Memphis and Baptist Memorial Hospital, Memphis, TN, USA There is evidence indicating a viral role in the etiology of Paget’s disease (PD). The increased bone destruction in PD has been attributed to overactive osteoclasts and available data suggest that there is upregulation of cytokines in PD bone multinucleated cells. Pamidronate (Pam), by inhibiting osteoclasts, is highly effective in this disease. We reported that Pam corrected AIDS cachexia and normalized serum cytokines in a patient with acquired immune deficiency syndrome (AIDS).1 We have now determined by ELISA, the spontaneous production of macrophage cytokines interleukin (IL)-6, IL-8, IL-10, transforming growth factor b1 (TGF-b1), and viral p24 antigen (prior stimulation with phytohemagglutinin 1 IL-2 for 48 h) by 106 cultured peripheral blood mononuclear cells (PBMC) before and at 1, 4, 12, and 24 weeks after a single dose of Pam, 60 mg intravenously to three additional AIDS patients, one with cachexia. The latter gained 12.7 kg in 12 weeks without signs of edema. The production of TGF-b1 by PBMCs, which suppresses HIV expression and replication in monocytes/macrophages, progressively rose in all patients by 188%–365% by 24 weeks after Pam treatment. In contrast, during the same period, PBMC cytokines progressively declined: IL-6 by 96%–90%; IL-8 by 99%–96%; IL-10 by 90%–35%; and p24 antigen by 97%– 81%. CD4 counts did not show a definitive pattern. We conclude that: (1) Pam may correct AIDS cachexia in at least some patients; (2) Pam may selectively bind to HIV-infected macrophages modulating cytokine production; and (3) these data strengthen the current opinion that macrophages play a major role in AIDS.
Reference 1. Carbone, L. D., Land, M. A., Stentz, F. B., and Palmieri, G. M. A. Elevated cytokine levels in a patient with AIDS and hypercalcemia: Effects of therapy with pamidronate. Clin Infec Dis 21:699; 1995. Address for correspondence and reprints: Dr. A. E. Postlethwaite, University of Tennessee at Memphis, 958 Court Ave., Memphis, TN 38163. PII S8756-3282(99)00042-3
The etiology of Paget’s disease of bone remains unclear. Several studies have demonstrated the presence of paramyxoviruses using a wide variety of techniques and, more recently, genetic linkage analysis has identified a locus for the disorder on chromosome 18q. We have previously shown that interleukin-6, c-fos, and Bcl-2 are all upregulated in pagetic bone. To determine other genes that are specifically upregulated in Paget’s disease, we have developed a novel subtractive hybridization technique. Bone biopsies were examined from five patients with unilateral Paget’s disease of the pelvis. Samples were also taken from the unaffected hemipelvis of each patient and RNA was extracted from each of the biopsies. A novel poly-A reverse transcriptase-polymerase chain reaction (RT-PCR) technique was then used to amplify cDNA from the RNA samples. Subtractive hybridization was then successfully performed using the affected and unaffected
Lipoprotein(a) Serum Levels in Patients With Paget’s Disease and Primary Hyperparathyroidism S. ADAMI and V. BRAGA University of Verona, COC Valeggio, Verona, Italy Lipoprotein a [Lp(a)] is a low-density lipoprotein (LDL)-like particle displaying strong atherothrombotic properties. Lp(a) is composed based on the covalent association of the unique and carbohydrate-rich apolipoprotein(a) [apo(a)] to
Bone Vol. 24, No. 5, Supplement May 1999:31S–33S
Molecular genetics of Paget’s disease: Abstracts
33S
Table 1. Various lipid parameters in patients with Paget’s disease
Subjects
No.
Age
LP(a) (mg/dL)
CHOL (mg/dL)
TGL (mg/dL)
HDL (mg/dL)
BALP (U/L)
Premenopause Postmenopause Men Paget’s disease PHPT
19 46 19 22 29
25.63 6 4.57 69.39 6 8.46 20.12 6 7.51 68.81 6 9.54 66.47 6 8.14
13.38 6 15.18 10.68 6 11.03 10.54 6 16.96 26.34 6 11.43a 22.24 6 27.01b
174.42 6 33.15 235.77 6 44.11 171.89 6 53.16 216.36 6 25.69 207.92 6 41.52
72.36 6 32.50 161.38 6 128.90 126.52 6 100.88 173.59 6 88.09 130.07 6 56.59
52.10 6 11.44 45.95 6 14.04 42.78 6 8.04 41.77 6 16.30 50.82 6 13.99
13.51 6 5.11 12.81 6 3.66 65.29 6 43.02 26.43 6 15.22c
KEY: BALP, bone alkaline phosphatase; CHOL, cholinesterase; HDL, high-density lipoprotein; LP(a), lipoprotein(a); PHPT, hyperparathyroidism; TGL, triglycerides. a p , 0.001; bp , 0.02; cp , 0.01 (vs. controls).
apolipoprotein-B100 (apoB100), the main protein moiety of LDLs. Although physical and chemical properties of Lp(a) are similar to LDL, Lp(a) displays distinct metabolism and functions. More than 90% of the concentration of Lp(a) in plasma is under genetic regulation. Apo(a) is a member of a family of Kringlecontaining proteins, including prothrombin, urokinase, and macrophage stimulating factor, and it also shares a high degree of sequence identity with plasminogen activator. Despite strong genetic regulation, Lp(a) levels may be altered by the acute phase response. The sequence of the apo(a) gene contains several interleukin-6 (IL-6)-responsive elements, potential targets for acute phase mediators. Table 1 lists the mean values (6SD) of bone alkaline phosphatase (BALP), Lp(a) and other lipid parameters in patients with Paget’s disease and primary hyperparathyroidism (PHPT), and in control subjects (healthy pre- and postmenopausal women, and men).
Control subjects were comparable to the pagetic and PHPT patients for age, serum total and HDL cholesterol, and triglycerides. BALP and Lp(a) were two to four times higher in pagetic and in PHPT patients than in control subjects. Elevated IL-6 activity has been found in high turnover bone diseases and plasminogen activator is an osteoblast-responsive element to parathyroid hormone and other cytokines. They are both somewhat related to Lp(a) metabolism. The clinical meaning of our observation of high levels of serum Lp(a) in patients with Paget’s disease and PHPT is unclear, but it might open a new horizon in the evaluation and pathophysiology of skeletal diseases. Address for correspondence and reprints: Dr. S. Adami, University of Verona, COC Valeggio, Verona, Italy. PII S8756-3282(99)00043-5
Molecular genetics of Paget’s disease: Abstracts
Bone Vol. 24, No. 5, Supplement May 1999:31S–33S
Family History and Heritability of Paget’s Disease: A Multicenter Study F. SLEE,1 L. HOCKING,1 S. I. HASLAM,1 M. HOOPER,2 R. DARGIE,3 G. A. NICHOLSON,4 and S. H. RALSTON1 1 Department of Medicine and Therapeutics, University of Aberdeen, Aberdeen, UK 2 Concorde Hospital, Sydney, Sydney, Australia 3 University of Glasgow, Glasgow, UK 4 University of Melbourne, Melbourne, Australia Genetic factors are important in the pathogenesis of Paget’s disease, although estimates of the frequency with which the disease is inherited vary widely. Here, we report the results of a multicenter study on family history and heritability of Paget’s disease. A standard questionnaire asking for details of family history was administered to pagetic patients attending specialist out-patient clinics in Aberdeen (n 5 35), Glasgow (n 5 35), Melbourne (n 5 28), and Sydney (n 5 115). Calculation of heritability (recurrent risk in any relative) was made by administering the questionnaire to spouses and nonpagetic controls. Results are shown in Table 1.
Glasgow
Sydney
Aberdeen
Melbourne
14%
23%
25%
53%
●●●
●●●
14.5
Acknowledgments: This work was funded by the NARPD. Address for correspondence and reprints: Dr. Andrew Mee, Musculoskeletal Research Group, University School of Medicine, Manchester Royal Infirmary, Manchester M13 9WL, UK. PII S8756-3282(99)00041-100
Table 1. Questionnaire results
Family history Heritability
samples from each patient, to identify the expression of novel upregulated genes in the pagetic bone. After three rounds of subtraction, the results from southern blotting showed that sequences had been isolated from the affected bone that were not present in the uninvolved bone samples. In addition, other poly-A PCR products from pagetic patients (both affected and unaffected sites) were generated. Southern blotting confirmed that the cDNA sequences from the final subtraction hybridized only to cDNA derived from affected sites. The subtracted cDNA has been used to screen a pagetic bone cDNA library (a generous gift from Professor Paul T. Sharpe), from which 42 positive plaques have been isolated and are currently being sequenced. The results so far demonstrate that we have successfully isolated gene sequences that are only found to be expressed in affected pagetic bone biopsies and not in uninvolved sites. Sequence analysis of the positive clones derived from the pagetic cDNA library should yield valuable information to aid further investigations into the etiopathology of Paget’s disease.
4.9
All 26% 9.9
There was a wide variation between centers in frequency of a positive family history and in heritability, with values ranging between 14% and 53% for family history and 4.9 to infinity for heritability. Segregation analysis in kindreds with a positive family history showed an autosomal-dominant pattern of inheritance with high penetrance by age 65 years (55% offspring affected, 45% unaffected). The likelihood of disease transmission was similar for affected males (52%) and females (47%) and gender distribution of affected offspring was similar (53% of male offspring affected vs. 58% female). This study shows evidence of heterogeneity between centers in the frequency with which pagetic patients have a positive family history and in heritability estimates. These differences could either be due to a high frequency of pagetic individuals in population founders or selection bias due to more frequent referral of familial cases to certain clinics. Whatever the underlying reasons, overall heritability estimates for Paget’s were high, emphasizing the importance of genetic factors in disease etiology. Unlike a previous study, however, we were unable to confirm that there was an excess of maternal transmission in familial cases. Address for correspondence and reprints: Professor Stuart H. Ralston, Department of Medicine and Therapeutics, University of Aberdeen Medical School, Forestermill, Aberdeen AB25 2ZD, UK. PII S8756-3282(99)00040-X 00
Isolation of Novel Genes in Paget’s Disease by Subtractive Hybridization J. A. DIXON, J. A. HOYLAND, E. B. MAWER, P. L. SELBY, and A. P. MEE Musculoskeletal Research Group, University School of Medicine, Manchester Royal Infirmary, Manchester, UK
Pamidronate in Acquired Immune Deficiency Syndrome A. E. POSTLETHWAITE, M. A. LAND, G. M. A. PALAMIERI, L. PIFER, and J. INGLES University of Tennessee at Memphis and Baptist Memorial Hospital, Memphis, TN, USA There is evidence indicating a viral role in the etiology of Paget’s disease (PD). The increased bone destruction in PD has been attributed to overactive osteoclasts and available data suggest that there is upregulation of cytokines in PD bone multinucleated cells. Pamidronate (Pam), by inhibiting osteoclasts, is highly effective in this disease. We reported that Pam corrected AIDS cachexia and normalized serum cytokines in a patient with acquired immune deficiency syndrome (AIDS).1 We have now determined by ELISA, the spontaneous production of macrophage cytokines interleukin (IL)-6, IL-8, IL-10, transforming growth factor b1 (TGF-b1), and viral p24 antigen (prior stimulation with phytohemagglutinin 1 IL-2 for 48 h) by 106 cultured peripheral blood mononuclear cells (PBMC) before and at 1, 4, 12, and 24 weeks after a single dose of Pam, 60 mg intravenously to three additional AIDS patients, one with cachexia. The latter gained 12.7 kg in 12 weeks without signs of edema. The production of TGF-b1 by PBMCs, which suppresses HIV expression and replication in monocytes/macrophages, progressively rose in all patients by 188%–365% by 24 weeks after Pam treatment. In contrast, during the same period, PBMC cytokines progressively declined: IL-6 by 96%–90%; IL-8 by 99%–96%; IL-10 by 90%–35%; and p24 antigen by 97%– 81%. CD4 counts did not show a definitive pattern. We conclude that: (1) Pam may correct AIDS cachexia in at least some patients; (2) Pam may selectively bind to HIV-infected macrophages modulating cytokine production; and (3) these data strengthen the current opinion that macrophages play a major role in AIDS.
Reference 1. Carbone, L. D., Land, M. A., Stentz, F. B., and Palmieri, G. M. A. Elevated cytokine levels in a patient with AIDS and hypercalcemia: Effects of therapy with pamidronate. Clin Infec Dis 21:699; 1995. Address for correspondence and reprints: Dr. A. E. Postlethwaite, University of Tennessee at Memphis, 958 Court Ave., Memphis, TN 38163. PII S8756-3282(99)00042-3
The etiology of Paget’s disease of bone remains unclear. Several studies have demonstrated the presence of paramyxoviruses using a wide variety of techniques and, more recently, genetic linkage analysis has identified a locus for the disorder on chromosome 18q. We have previously shown that interleukin-6, c-fos, and Bcl-2 are all upregulated in pagetic bone. To determine other genes that are specifically upregulated in Paget’s disease, we have developed a novel subtractive hybridization technique. Bone biopsies were examined from five patients with unilateral Paget’s disease of the pelvis. Samples were also taken from the unaffected hemipelvis of each patient and RNA was extracted from each of the biopsies. A novel poly-A reverse transcriptase-polymerase chain reaction (RT-PCR) technique was then used to amplify cDNA from the RNA samples. Subtractive hybridization was then successfully performed using the affected and unaffected
Lipoprotein(a) Serum Levels in Patients With Paget’s Disease and Primary Hyperparathyroidism S. ADAMI and V. BRAGA University of Verona, COC Valeggio, Verona, Italy Lipoprotein a [Lp(a)] is a low-density lipoprotein (LDL)-like particle displaying strong atherothrombotic properties. Lp(a) is composed based on the covalent association of the unique and carbohydrate-rich apolipoprotein(a) [apo(a)] to
Bone Vol. 24, No. 5, Supplement May 1999:31S–33S
Molecular genetics of Paget’s disease: Abstracts
33S
Table 1. Various lipid parameters in patients with Paget’s disease
Subjects
No.
Age
LP(a) (mg/dL)
CHOL (mg/dL)
TGL (mg/dL)
HDL (mg/dL)
BALP (U/L)
Premenopause Postmenopause Men Paget’s disease PHPT
19 46 19 22 29
25.63 6 4.57 69.39 6 8.46 20.12 6 7.51 68.81 6 9.54 66.47 6 8.14
13.38 6 15.18 10.68 6 11.03 10.54 6 16.96 26.34 6 11.43a 22.24 6 27.01b
174.42 6 33.15 235.77 6 44.11 171.89 6 53.16 216.36 6 25.69 207.92 6 41.52
72.36 6 32.50 161.38 6 128.90 126.52 6 100.88 173.59 6 88.09 130.07 6 56.59
52.10 6 11.44 45.95 6 14.04 42.78 6 8.04 41.77 6 16.30 50.82 6 13.99
13.51 6 5.11 12.81 6 3.66 65.29 6 43.02 26.43 6 15.22c
KEY: BALP, bone alkaline phosphatase; CHOL, cholinesterase; HDL, high-density lipoprotein; LP(a), lipoprotein(a); PHPT, hyperparathyroidism; TGL, triglycerides. a p , 0.001; bp , 0.02; cp , 0.01 (vs. controls).
apolipoprotein-B100 (apoB100), the main protein moiety of LDLs. Although physical and chemical properties of Lp(a) are similar to LDL, Lp(a) displays distinct metabolism and functions. More than 90% of the concentration of Lp(a) in plasma is under genetic regulation. Apo(a) is a member of a family of Kringlecontaining proteins, including prothrombin, urokinase, and macrophage stimulating factor, and it also shares a high degree of sequence identity with plasminogen activator. Despite strong genetic regulation, Lp(a) levels may be altered by the acute phase response. The sequence of the apo(a) gene contains several interleukin-6 (IL-6)-responsive elements, potential targets for acute phase mediators. Table 1 lists the mean values (6SD) of bone alkaline phosphatase (BALP), Lp(a) and other lipid parameters in patients with Paget’s disease and primary hyperparathyroidism (PHPT), and in control subjects (healthy pre- and postmenopausal women, and men).
Control subjects were comparable to the pagetic and PHPT patients for age, serum total and HDL cholesterol, and triglycerides. BALP and Lp(a) were two to four times higher in pagetic and in PHPT patients than in control subjects. Elevated IL-6 activity has been found in high turnover bone diseases and plasminogen activator is an osteoblast-responsive element to parathyroid hormone and other cytokines. They are both somewhat related to Lp(a) metabolism. The clinical meaning of our observation of high levels of serum Lp(a) in patients with Paget’s disease and PHPT is unclear, but it might open a new horizon in the evaluation and pathophysiology of skeletal diseases. Address for correspondence and reprints: Dr. S. Adami, University of Verona, COC Valeggio, Verona, Italy. PII S8756-3282(99)00043-5