Bone and Mineral, 0169-6009/92/$05.00
19 (1992) 159-174 0 1992 Elsevier
159 Science Publishers B.V. All rights reserved.
BAM 00413
Canine distem er virus transcripts sequence from pagetic bone
M.T. Gordona, A.P. Meea, DC. Andersonb and P.T. Sharpea ‘Bone Disease Research Cetwe, Manchester,
University
UK and bChinese University
of Manchester,
Dept. OJ Cell and Structural
of Hong Kong, Prince of Wales Hospital,
Shatin, New Territories,
Biology,
Dept. of Medicine,
Hong Kong
(Received 7 February 1992) (Accepted 21 May 1992)
Summary Paget’s disease is a chronic disease of the skeleton which is believed to be caused by a persistent virus of the paramyxovirus family. There is still conflict as to the precise ‘dentity of the virus(es) involved in causing this disease. Our previous results using in situ hybridisation have implicated distemper as a possible cause of this disease. In order to confirm these results, we have reverse transcribed RNA from pagetic bone and have specifically amplified for distemper and measles sequences using the polymerase chain reaction (FCR) technique. We have found that 8/13 of the patients examined had distemper and l/10 cases had measles nucleic acids sequestered within their boa.: cells. One individual was found to have both viruses sequestered in his bone cells. Dideoxy sequencing of the distemper virus PCR products revealed 2% base pair changes in the nucleic acid sequences relative to the Onderstpoort strain of canine distemper. We can conclude that in Paget’s disease, canine distemper and possible other paramyxoviruses reside in bone cells and that the persistent nature of the virus may be due to mutations in the viral genome.
Key words: Paget’s disease; Canine distemper virus; Paramyxoviruses
Introduction
Paget’s disease of bone is a chronic disease of the skeleton associated with an increase in bone resorption dnd formation. It leads to a progressive d:struction, remodelling and deformity of the bone [l]. Characteristically, it can affect any Correspondence to: Dr P.T. Sharpe, Bone Disease Research Centre, University of Manchester, Dept. of Cell and Structural Biology, Stopford Building, Oxford Rd., Manchester Ml3 9PT, UK.
160
part of the skeleton and cause complications that include pressure on the surrounding nerves, fracture and in extreme cases, tumours. Geographically, its prevalence varies around the world, with a disproportionate incidence (up to 8%) having been found in the North West region of G:ieat Britain [2,3,4]. It is estimated that 50-100000 people in Great Britain develop symptoms by middle and old age. Paget’s disease also has a striking high incidence in North America, but in other regions of the world, such as Japan and Finland, the disease is rare [4]. Various studies have implicated a slow virus infection to be the cause of this disease. Electron microscopical [5,6], immunocytochemical [7,8] and in situ hybridisation studies [9,10] have concluded that paramyxoviruses are the most likely aetiological agents causing this disease. Those viruses implicated have included canine distemper virus [lo], measles [9], respiratory syncyctial virus [I 11and simian virus 5 [8] which have been detected in both osteoblasts [9,10] and osteoclasts [9,10]. As yet, the virus/es involved in Paget’s disease have not been definitively identified by DNA sequencing. We report here the use of the polymerase chain reaction (PCR), followed by cloning and sequencing to establish the presence of paramyxoviruses in pagetic bone. The results show that in some cases of Paget’s disease, canine distemper is the virus sequestered in bone cells.
Materials and Methods
All molecular biology reagents and enzymes were obtained from Promega UK or Boehringer Mannheim. Transiliac bone biopsies were obtained under local anaesthetic from 12 patients with Paget’s disease following approval from the Salford Area Health Authority. A specimen from the head of the femur was obtained from a further patient with Paget”sdisease who was undergoing hip replacement surgery. The specimens were immediately placed into liquid nitrogen and subsequently stored at - 70°C until they were used for RNA extraction. Eleven of these specimens had been cut longitudinally prior to freezing, with one half of the sample also being placed into formol-saline for in situ hybridisation studies as previously described [lo]. Non-pagetic control bone samples were obtained from a number of sources. Femoral heads or osteophytes were obtained from three individuals without Paget’s disease who were undergoing routine hip replacement surgery. A further six non-pagetic transiliac bone specimens were obtained from patients with a number of different disease states including hyperparathyroidism, mastiocytosis, osteomalacia, osteoporf,sis and normal fracture healing. Three specimens (two femoral heads and one osteoporotic biopsy) were used for RNA extraction. It was possible to process one of these femoral specimens for both RNA extraction and in situ hybridisation. The remaining six biopsies were only used for in situ hybridisation investigations.
161 RNA extraction from bone
Bone samples (
19 (1992) 159-174 0 1992 Elsevier
159 Science Publishers B.V. All rights reserved.
BAM 00413
Canine distem er virus transcripts sequence from pagetic bone
M.T. Gordona, A.P. Meea, DC. Andersonb and P.T. Sharpea ‘Bone Disease Research Cetwe, Manchester,
University
UK and bChinese University
of Manchester,
Dept. OJ Cell and Structural
of Hong Kong, Prince of Wales Hospital,
Shatin, New Territories,
Biology,
Dept. of Medicine,
Hong Kong
(Received 7 February 1992) (Accepted 21 May 1992)
Summary Paget’s disease is a chronic disease of the skeleton which is believed to be caused by a persistent virus of the paramyxovirus family. There is still conflict as to the precise ‘dentity of the virus(es) involved in causing this disease. Our previous results using in situ hybridisation have implicated distemper as a possible cause of this disease. In order to confirm these results, we have reverse transcribed RNA from pagetic bone and have specifically amplified for distemper and measles sequences using the polymerase chain reaction (FCR) technique. We have found that 8/13 of the patients examined had distemper and l/10 cases had measles nucleic acids sequestered within their boa.: cells. One individual was found to have both viruses sequestered in his bone cells. Dideoxy sequencing of the distemper virus PCR products revealed 2% base pair changes in the nucleic acid sequences relative to the Onderstpoort strain of canine distemper. We can conclude that in Paget’s disease, canine distemper and possible other paramyxoviruses reside in bone cells and that the persistent nature of the virus may be due to mutations in the viral genome.
Key words: Paget’s disease; Canine distemper virus; Paramyxoviruses
Introduction
Paget’s disease of bone is a chronic disease of the skeleton associated with an increase in bone resorption dnd formation. It leads to a progressive d:struction, remodelling and deformity of the bone [l]. Characteristically, it can affect any Correspondence to: Dr P.T. Sharpe, Bone Disease Research Centre, University of Manchester, Dept. of Cell and Structural Biology, Stopford Building, Oxford Rd., Manchester Ml3 9PT, UK.
160
part of the skeleton and cause complications that include pressure on the surrounding nerves, fracture and in extreme cases, tumours. Geographically, its prevalence varies around the world, with a disproportionate incidence (up to 8%) having been found in the North West region of G:ieat Britain [2,3,4]. It is estimated that 50-100000 people in Great Britain develop symptoms by middle and old age. Paget’s disease also has a striking high incidence in North America, but in other regions of the world, such as Japan and Finland, the disease is rare [4]. Various studies have implicated a slow virus infection to be the cause of this disease. Electron microscopical [5,6], immunocytochemical [7,8] and in situ hybridisation studies [9,10] have concluded that paramyxoviruses are the most likely aetiological agents causing this disease. Those viruses implicated have included canine distemper virus [lo], measles [9], respiratory syncyctial virus [I 11and simian virus 5 [8] which have been detected in both osteoblasts [9,10] and osteoclasts [9,10]. As yet, the virus/es involved in Paget’s disease have not been definitively identified by DNA sequencing. We report here the use of the polymerase chain reaction (PCR), followed by cloning and sequencing to establish the presence of paramyxoviruses in pagetic bone. The results show that in some cases of Paget’s disease, canine distemper is the virus sequestered in bone cells.
Materials and Methods
All molecular biology reagents and enzymes were obtained from Promega UK or Boehringer Mannheim. Transiliac bone biopsies were obtained under local anaesthetic from 12 patients with Paget’s disease following approval from the Salford Area Health Authority. A specimen from the head of the femur was obtained from a further patient with Paget”sdisease who was undergoing hip replacement surgery. The specimens were immediately placed into liquid nitrogen and subsequently stored at - 70°C until they were used for RNA extraction. Eleven of these specimens had been cut longitudinally prior to freezing, with one half of the sample also being placed into formol-saline for in situ hybridisation studies as previously described [lo]. Non-pagetic control bone samples were obtained from a number of sources. Femoral heads or osteophytes were obtained from three individuals without Paget’s disease who were undergoing routine hip replacement surgery. A further six non-pagetic transiliac bone specimens were obtained from patients with a number of different disease states including hyperparathyroidism, mastiocytosis, osteomalacia, osteoporf,sis and normal fracture healing. Three specimens (two femoral heads and one osteoporotic biopsy) were used for RNA extraction. It was possible to process one of these femoral specimens for both RNA extraction and in situ hybridisation. The remaining six biopsies were only used for in situ hybridisation investigations.
161 RNA extraction from bone
Bone samples (
