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Deletion of exon 3: a cause of hereditary vitamin D3 resistant rickets
680
Abstracts
0 5 . Deletion o f exon 3: a cause of hereditary vitamin D3 resistant rickets SM Farrow, FJ, Cockerill, AR Rut, JLH O ' R i o r d a n
Department of Medicine, University College London Medical School, Middlesex Hospital, London W I N 8AA H e r e d i t a r y v i t a m i n D 3 r e s i s t a n t rickets (HVDRR) is a n a u t o s o m a l recessive d i s o r d e r c h a r a c t e r i s e d b y severe rickets, hypocalcaemia and secondary hyperparathyroidism. A number of m u t a t i o n s h a v e b e e n i d e n t i f i e d w i t h i n the v i t a m i n D3 receptor g e n e w h i c h are associated with HVDRR a n d here we report the first case of exon s k i p p i n g in a patient with HVDRR. Skin fibroblasts f r o m this patient s h o w e d no detectable b i n d i n g of 1,25(OH)2D3 a n d t h e r e w a s n o i n d u c t i o n of 24-hydroxylase a c t i v i t y b y 1,25(OH)2D3. R N A isolated from these cells w a s reverse transcribed a n d the coding region w a s amplified b y PCR. D N A s e q u e n c i n g indicated that this deletion c o r r e s p o n d s to the second zinc finger (44 a m i n o acids) in the DNA b i n d i n g d o m a i n w h i c h is e n c o d e d b y exon 3. G e n o m i c DNA p r e p a r e d f r o m fibroblasts w a s a m p l i f i e d u s i n g p r i m e r s within the introns on the 5' a n d 3' of e x o n 3. Resulting PCR p r o d u c t s s h o w e d the presence of exon 3 in g e n o m i c DNA of both patient and control cells t h u s i n d i c a t i n g t h a t the disease w a s not associated with a m a j o r g e n e d e l e t i o n e n c o m p a s s i n g this exon. Therefore, the absence of exon 3 in m R N A is d u e to a b n o r m a l RNA processing in the n u c l e u s i n v o l v i n g e x o n s k i p p i n g . This m a y be d u e to splice site m u t a t i o n s at the e x o n - i n t r o n b o u n d a r i e s . These f i n d i n g s r e p r e s e n t a n o v e l c a u s e of h o r m o n e resistance in HVDRR.
0 6 . Identification and cloning of human P2U purlnoceptor present in osteoclastoma, bone and osteoblasts WB Bowler, JA G a l l a g h e r , G Bilbe*
Department of Human Anatomy and Cell Biology, The University of Liverpool, PO Box 147, Liverpool L69 3BX, and "Biotechnology, Ciba Geigy AG, CH-4022, Basel, Switzerland E x t r a c e l l u l a r ATV a c t i n g t h r o u g h p u r i n o c e p t o r s m a y be an i m p o r t a n t f a c t o r in t h e m o d u l a t i o n of b o n e t u r n o v e r . Classically, the p r e s e n c e of p u r i n o c e p t o r s w a s established by p h a r m a c o l o g i c a l s t u d y in w h i c h the elevation of intracellular c a l c i u m w a s m o n i t o r e d in response to agonist binding. In this s t u d y w e r e p o r t for the first time the expression of m R N A for the P2U p u r i n o c e p t o r s u b t y p e in h u m a n skeletal tissue including bone, o s t e o c l a s t o m a , p r i m a r y cultures of h u m a n osteoblast-like cells a n d t w o o s t e o s a r c o m a cell lines, Saos2 a n d Te85. Using d e g e n e r a t e a n d nested p r i m i n g PCR techniques, 230bp of the h u m a n P2U s e q u e n c e w e r e cloned from an osteoclastoma cDNA library in L g t l l . Following the recent publication of the complete h u m a n P2u receptor sequence, cloned from respiratory epithelia, specific p r i m e r s b a s e d on this sequence were designed a n d used to g e n e r a t e 1.2 Kb of c D N A f r o m osteoclastoma c o r r e s p o n d i n g to the o p e n r e a d i n g f r a m e of the h u m a n P2U receptor. PCR a n d S o u t h e r n b l o t t i n g w e r e u s e d to d e m o n s t r a t e the expression of P2U r e c e p t o r t r a n s c r i p t s in s a m p l e s of h u m a n bone, p r i m a r y cultures of h u m a n osteoblast-like cells, Saos2 cells and Te85 cells. The P2U p u r i n e r g i c r e c e p t o r m a y p r o v i d e a f u t u r e target for t h e r a p e u t i c i n t e r v e n t i o n in b o n e d i s e a s e s . F u r t h e r m o r e , p h a r m a c o l o g i c a l a n d m o l e c u l a r evidence points to the presence of o t h e r p u r i n o c e p t o r s in bone. In a d d i t i o n to ATP, potential agonists for these receptors include ADP, Ap4A a n d metabolites of the b i s p h o s p h o n a t e s .
0 7 . P T H / P T H r P receptor mRNA expression on forming but not resorbing surfaces in growing rat ulnae B Fermor, TM Skerry
University of Bristol BS2 8EJ The a n a b o l i c a n d catabolic actions of PTH in vioo p o s e m a n y questions. This e x p e r i m e n t w a s p e r f o r m e d to d e t e r m i n e w h i c h cells are c a p a b l e of e x p r e s s i n g the P T H / P T H r P receptor, a n d c o u l d t h e r e f o r e be t a r g e t s for the h o r m o n e . U s i n g in-situ h y b r i d i s a t i o n of 35S labelled sense a n d antisense riboprobes we h a v e i d e n t i f i e d P T H / P T H r P r e c e p t o r m R N A e x p r e s s i o n on f o r m i n g b u t n o t r e s o r b i n g s u r f a c e s in u n d e c a l c i f i e d serial sections of 6 w e e k old male rat ulnae.
B o n e Vol. 16, No. 6 June 1995:679~595
Forming surfaces were characterised by continuous f l u o r o c h r o m e i n c o r p o r a t i o n , presence of osteoblasts a n d osteoid, positive alkaline p h o s p h a t a s e s t a i n i n g b u t n o tartrate resistant acid p h o s p h a t a s e (TRAP) activity. R e s o r b i n g s u r f a c e s w e r e jagged, covered w i t h tightly a d h e r e n t cells with positive TRAP staining, but no osteoid, and had no fluorochrome incorporation. P T H / P T H r P receptor m R N A w a s expressed on all f o r m i n g surfaces, b u t not on a n y of the resorbing surfaces. Silver g r a i n s w e r e l o c a l i s e d to o s t e o b l a s t s w h i c h w e r e a c t i v e l y s y n t h e s i z i n g n e w b o n e m a t r i x a n d not to m o r e p e r i p h e r a l osteoblast p r e c u r s o r cells even those expressing h i g h levels of alkaline p h o s p h a t a s e . Expression on m a r r o w cells w a s minimal. Expression of m R N A m a y not a l w a y s be followed b y protein s y n t h e s i s , so t h e s e results d o not p r o v e c o n c l u s i v e l y that osteoblasts are the targets for IYFH. H o w e v e r , the absence of m R N A on cells on r e s o r b i n g surfaces does c o n f i r m that a n y effect of PTH on their actions.is indirect.
08. Mechanical stimulation of rat tail vertebrae induces i n d o m e t h a c l n - s e n s i t i v e e x p r e s s i o n of i n s u l i n - l l k e growth factor-1 m R N A in osteocytes J Lean, C Jagger, A Mackay, J C h o w , T C h a m b e r s
Department of Histopathology, St. George's Hospital Medical School, London We h a v e recently d e v e l o p e d an experimental m o d e l w h e r e b y a single 1 0 - m i n u t e e p i s o d e of m e c h a n i c a l s t i m u l a t i o n induces b o n e f o r m a t i o n in the 8th c a u d a l vertebra of 13-week old rats. We h a v e n o w u s e d this m o d e l for an in situ h y b r i d i z a t i o n analysis of c h a n g e s in gene expression. We f o u n d a n 8-10-fold increase in the p r o p o r t i o n of trabecular b o n e surfaces u p o n w h i c h transcripts for collagen type l, IGF-I a n d osteocalcin w e r e detectable w h i c h w a s maximal 72 h o u r s after loading. More striking, h o w e v e r , was intense hybridization for IGF-I, in osteocytes, p r e d o m i n a n t l y in the diaphyseal cortex, a n d in m e t a p h y s e a l trabeculae, within 6 h o u r s of loading. This w a s o b s e r v e d only in loaded bones, a n d became undetectable in trabecular osteocytes after 48 hours, a n d cortical osteocytes after 120 hours. Both the increase in b o n e formation a n d os,teocytic IGF-1 e x p r e s s i o n w e r e ' i n h i b i t e d b y a d m i n i s t r a t i o n of i n d o m e t h a c i n b e f o r e m e c h a n i c a l s t i m u l a t i o n . T h u s , IGF-1 e x p r e s s i o n b y o s t e o c y t e s a p p e a r s to d e p e n d on p r o s t a g l a n d i n p r o d u c t i o n . E x p r e s s i o n of IGF-1 b e f o r e the i n c r e a s e in t r a n s c r i p t i o n of m a t r i x p r o t e i n m R N A , a n d b e f o r e the t r a n s c r i p t i o n of IGF-1 m R N A in b o n e surface cells, represents p e r s u a s i v e e v i d e n c e for a role for osteocytes, a n d for IGF-1, in the osteogenic response of b o n e to mechanical stimulation.
0 9 . Evidence that programmed cell death contributes to the regulation of bone formation in adult human bone
N Arnott, JN Beresford
Bath Institute of Rheumatic Diseases, 1 Tr!mbridge, Bath BA1 2HD and Bath University O s t e o p o r o s i s is the m o s t c o m m o n metabolic b o n e disease. It occurs in l in 4 w o m e n after the m e n o p a u s e a n d 1 in 8 elderly m e n and is a f r e q u e n t c o m p l i c a t i o n of r h e u m a t o i d arthritis. Decreased b o n e f o r m a t i o n is a consistent finding in all forms of osteoporosis a n d there is evidence that this is d u e to a reduction in the n u m b e r of osteoblasts. It is generally a s s u m e d that the r e d u c t i o n in osteoblast n u m b e r s results f r o m an i m p a i r m e n t in the r e c r u i t m e n t a n d / o r p r o l i f e r a t i o n of their precursors. The majority of investigators h a v e therefore focussed their attention on the identification of factors that m i g h t increase the size of the o s t e o b l a s t p o p u l a t i o n . A n a l t e r n a t i v e h y p o t h e s i s is t h a t the r e d u c t i o n in osteoblast n u m b e r s is a c o n s e q u e n c e of p r e m a t u r e cell d e a t h , b y a p r o c e s s w h i c h , u n d e r n o r m a l circumstances, c o n t r i b u t e s to the m a i n t e n a n c e of tissue h o m e o s t a s i s a n d has been t e r m e d p r o g r a m m e d cell d e a t h (PCD). To investigate this p o s s i b i l i t y w e h a v e i m m u n o l o c a l i s e d in a d u l t h u m a n o s t e o p h y t e tissue the sites of expression of the p r o d u c t of the bcl-2 g e n e , w h i c h h a s b e e n i m p l i c a t e d in the r e g u l a t i o n of a p o p t o s i s , a m o r p h o l o g i c a l l y d e f i n e d p a t h w a y of PCD. 81xm cryostat sections w e r e reacted with the m o n o c l o n a l antibodies Bc1-124 a n d Bcl-100, w h i c h recognise distinct epitopes on the Bcl2 p r o t e i n , o r a n i s o t y p e c o n t r o l a n t i b o d y . Sites of
Abstracts
0 5 . Deletion o f exon 3: a cause of hereditary vitamin D3 resistant rickets SM Farrow, FJ, Cockerill, AR Rut, JLH O ' R i o r d a n
Department of Medicine, University College London Medical School, Middlesex Hospital, London W I N 8AA H e r e d i t a r y v i t a m i n D 3 r e s i s t a n t rickets (HVDRR) is a n a u t o s o m a l recessive d i s o r d e r c h a r a c t e r i s e d b y severe rickets, hypocalcaemia and secondary hyperparathyroidism. A number of m u t a t i o n s h a v e b e e n i d e n t i f i e d w i t h i n the v i t a m i n D3 receptor g e n e w h i c h are associated with HVDRR a n d here we report the first case of exon s k i p p i n g in a patient with HVDRR. Skin fibroblasts f r o m this patient s h o w e d no detectable b i n d i n g of 1,25(OH)2D3 a n d t h e r e w a s n o i n d u c t i o n of 24-hydroxylase a c t i v i t y b y 1,25(OH)2D3. R N A isolated from these cells w a s reverse transcribed a n d the coding region w a s amplified b y PCR. D N A s e q u e n c i n g indicated that this deletion c o r r e s p o n d s to the second zinc finger (44 a m i n o acids) in the DNA b i n d i n g d o m a i n w h i c h is e n c o d e d b y exon 3. G e n o m i c DNA p r e p a r e d f r o m fibroblasts w a s a m p l i f i e d u s i n g p r i m e r s within the introns on the 5' a n d 3' of e x o n 3. Resulting PCR p r o d u c t s s h o w e d the presence of exon 3 in g e n o m i c DNA of both patient and control cells t h u s i n d i c a t i n g t h a t the disease w a s not associated with a m a j o r g e n e d e l e t i o n e n c o m p a s s i n g this exon. Therefore, the absence of exon 3 in m R N A is d u e to a b n o r m a l RNA processing in the n u c l e u s i n v o l v i n g e x o n s k i p p i n g . This m a y be d u e to splice site m u t a t i o n s at the e x o n - i n t r o n b o u n d a r i e s . These f i n d i n g s r e p r e s e n t a n o v e l c a u s e of h o r m o n e resistance in HVDRR.
0 6 . Identification and cloning of human P2U purlnoceptor present in osteoclastoma, bone and osteoblasts WB Bowler, JA G a l l a g h e r , G Bilbe*
Department of Human Anatomy and Cell Biology, The University of Liverpool, PO Box 147, Liverpool L69 3BX, and "Biotechnology, Ciba Geigy AG, CH-4022, Basel, Switzerland E x t r a c e l l u l a r ATV a c t i n g t h r o u g h p u r i n o c e p t o r s m a y be an i m p o r t a n t f a c t o r in t h e m o d u l a t i o n of b o n e t u r n o v e r . Classically, the p r e s e n c e of p u r i n o c e p t o r s w a s established by p h a r m a c o l o g i c a l s t u d y in w h i c h the elevation of intracellular c a l c i u m w a s m o n i t o r e d in response to agonist binding. In this s t u d y w e r e p o r t for the first time the expression of m R N A for the P2U p u r i n o c e p t o r s u b t y p e in h u m a n skeletal tissue including bone, o s t e o c l a s t o m a , p r i m a r y cultures of h u m a n osteoblast-like cells a n d t w o o s t e o s a r c o m a cell lines, Saos2 a n d Te85. Using d e g e n e r a t e a n d nested p r i m i n g PCR techniques, 230bp of the h u m a n P2U s e q u e n c e w e r e cloned from an osteoclastoma cDNA library in L g t l l . Following the recent publication of the complete h u m a n P2u receptor sequence, cloned from respiratory epithelia, specific p r i m e r s b a s e d on this sequence were designed a n d used to g e n e r a t e 1.2 Kb of c D N A f r o m osteoclastoma c o r r e s p o n d i n g to the o p e n r e a d i n g f r a m e of the h u m a n P2U receptor. PCR a n d S o u t h e r n b l o t t i n g w e r e u s e d to d e m o n s t r a t e the expression of P2U r e c e p t o r t r a n s c r i p t s in s a m p l e s of h u m a n bone, p r i m a r y cultures of h u m a n osteoblast-like cells, Saos2 cells and Te85 cells. The P2U p u r i n e r g i c r e c e p t o r m a y p r o v i d e a f u t u r e target for t h e r a p e u t i c i n t e r v e n t i o n in b o n e d i s e a s e s . F u r t h e r m o r e , p h a r m a c o l o g i c a l a n d m o l e c u l a r evidence points to the presence of o t h e r p u r i n o c e p t o r s in bone. In a d d i t i o n to ATP, potential agonists for these receptors include ADP, Ap4A a n d metabolites of the b i s p h o s p h o n a t e s .
0 7 . P T H / P T H r P receptor mRNA expression on forming but not resorbing surfaces in growing rat ulnae B Fermor, TM Skerry
University of Bristol BS2 8EJ The a n a b o l i c a n d catabolic actions of PTH in vioo p o s e m a n y questions. This e x p e r i m e n t w a s p e r f o r m e d to d e t e r m i n e w h i c h cells are c a p a b l e of e x p r e s s i n g the P T H / P T H r P receptor, a n d c o u l d t h e r e f o r e be t a r g e t s for the h o r m o n e . U s i n g in-situ h y b r i d i s a t i o n of 35S labelled sense a n d antisense riboprobes we h a v e i d e n t i f i e d P T H / P T H r P r e c e p t o r m R N A e x p r e s s i o n on f o r m i n g b u t n o t r e s o r b i n g s u r f a c e s in u n d e c a l c i f i e d serial sections of 6 w e e k old male rat ulnae.
B o n e Vol. 16, No. 6 June 1995:679~595
Forming surfaces were characterised by continuous f l u o r o c h r o m e i n c o r p o r a t i o n , presence of osteoblasts a n d osteoid, positive alkaline p h o s p h a t a s e s t a i n i n g b u t n o tartrate resistant acid p h o s p h a t a s e (TRAP) activity. R e s o r b i n g s u r f a c e s w e r e jagged, covered w i t h tightly a d h e r e n t cells with positive TRAP staining, but no osteoid, and had no fluorochrome incorporation. P T H / P T H r P receptor m R N A w a s expressed on all f o r m i n g surfaces, b u t not on a n y of the resorbing surfaces. Silver g r a i n s w e r e l o c a l i s e d to o s t e o b l a s t s w h i c h w e r e a c t i v e l y s y n t h e s i z i n g n e w b o n e m a t r i x a n d not to m o r e p e r i p h e r a l osteoblast p r e c u r s o r cells even those expressing h i g h levels of alkaline p h o s p h a t a s e . Expression on m a r r o w cells w a s minimal. Expression of m R N A m a y not a l w a y s be followed b y protein s y n t h e s i s , so t h e s e results d o not p r o v e c o n c l u s i v e l y that osteoblasts are the targets for IYFH. H o w e v e r , the absence of m R N A on cells on r e s o r b i n g surfaces does c o n f i r m that a n y effect of PTH on their actions.is indirect.
08. Mechanical stimulation of rat tail vertebrae induces i n d o m e t h a c l n - s e n s i t i v e e x p r e s s i o n of i n s u l i n - l l k e growth factor-1 m R N A in osteocytes J Lean, C Jagger, A Mackay, J C h o w , T C h a m b e r s
Department of Histopathology, St. George's Hospital Medical School, London We h a v e recently d e v e l o p e d an experimental m o d e l w h e r e b y a single 1 0 - m i n u t e e p i s o d e of m e c h a n i c a l s t i m u l a t i o n induces b o n e f o r m a t i o n in the 8th c a u d a l vertebra of 13-week old rats. We h a v e n o w u s e d this m o d e l for an in situ h y b r i d i z a t i o n analysis of c h a n g e s in gene expression. We f o u n d a n 8-10-fold increase in the p r o p o r t i o n of trabecular b o n e surfaces u p o n w h i c h transcripts for collagen type l, IGF-I a n d osteocalcin w e r e detectable w h i c h w a s maximal 72 h o u r s after loading. More striking, h o w e v e r , was intense hybridization for IGF-I, in osteocytes, p r e d o m i n a n t l y in the diaphyseal cortex, a n d in m e t a p h y s e a l trabeculae, within 6 h o u r s of loading. This w a s o b s e r v e d only in loaded bones, a n d became undetectable in trabecular osteocytes after 48 hours, a n d cortical osteocytes after 120 hours. Both the increase in b o n e formation a n d os,teocytic IGF-1 e x p r e s s i o n w e r e ' i n h i b i t e d b y a d m i n i s t r a t i o n of i n d o m e t h a c i n b e f o r e m e c h a n i c a l s t i m u l a t i o n . T h u s , IGF-1 e x p r e s s i o n b y o s t e o c y t e s a p p e a r s to d e p e n d on p r o s t a g l a n d i n p r o d u c t i o n . E x p r e s s i o n of IGF-1 b e f o r e the i n c r e a s e in t r a n s c r i p t i o n of m a t r i x p r o t e i n m R N A , a n d b e f o r e the t r a n s c r i p t i o n of IGF-1 m R N A in b o n e surface cells, represents p e r s u a s i v e e v i d e n c e for a role for osteocytes, a n d for IGF-1, in the osteogenic response of b o n e to mechanical stimulation.
0 9 . Evidence that programmed cell death contributes to the regulation of bone formation in adult human bone
N Arnott, JN Beresford
Bath Institute of Rheumatic Diseases, 1 Tr!mbridge, Bath BA1 2HD and Bath University O s t e o p o r o s i s is the m o s t c o m m o n metabolic b o n e disease. It occurs in l in 4 w o m e n after the m e n o p a u s e a n d 1 in 8 elderly m e n and is a f r e q u e n t c o m p l i c a t i o n of r h e u m a t o i d arthritis. Decreased b o n e f o r m a t i o n is a consistent finding in all forms of osteoporosis a n d there is evidence that this is d u e to a reduction in the n u m b e r of osteoblasts. It is generally a s s u m e d that the r e d u c t i o n in osteoblast n u m b e r s results f r o m an i m p a i r m e n t in the r e c r u i t m e n t a n d / o r p r o l i f e r a t i o n of their precursors. The majority of investigators h a v e therefore focussed their attention on the identification of factors that m i g h t increase the size of the o s t e o b l a s t p o p u l a t i o n . A n a l t e r n a t i v e h y p o t h e s i s is t h a t the r e d u c t i o n in osteoblast n u m b e r s is a c o n s e q u e n c e of p r e m a t u r e cell d e a t h , b y a p r o c e s s w h i c h , u n d e r n o r m a l circumstances, c o n t r i b u t e s to the m a i n t e n a n c e of tissue h o m e o s t a s i s a n d has been t e r m e d p r o g r a m m e d cell d e a t h (PCD). To investigate this p o s s i b i l i t y w e h a v e i m m u n o l o c a l i s e d in a d u l t h u m a n o s t e o p h y t e tissue the sites of expression of the p r o d u c t of the bcl-2 g e n e , w h i c h h a s b e e n i m p l i c a t e d in the r e g u l a t i o n of a p o p t o s i s , a m o r p h o l o g i c a l l y d e f i n e d p a t h w a y of PCD. 81xm cryostat sections w e r e reacted with the m o n o c l o n a l antibodies Bc1-124 a n d Bcl-100, w h i c h recognise distinct epitopes on the Bcl2 p r o t e i n , o r a n i s o t y p e c o n t r o l a n t i b o d y . Sites of