Ada Medica et Biologica Vol. 49, No.l, 31-37, 2001

Pycnodysostosis with a Cathepsin K Mutation:

A Case Report

Hiroshi YAMAGIWA1, Mayumi ASAOKA2, Hisayoshi KATO2, Minoru SHIBATA3, and Naoto ENDO1

'Department of Orthopedic Surgery, "Rehabilitation, and 3Plastic Surgery, Niigata University School of Medicine, Niigata, Japan

Received October 16 2000 ; accepted January 22 2001

Summary.Pycnodysostosis (PKND) is a type of osteo- the distal phalanges, frequent fractures, and skull sclerosing bone disease. Recent studies have demon- deformities with delayed suture closure4'5'. The cathe- strated that several cathepsin K mutations can be psin K gene, which wa cloned originally from rabbit identified in PKND families. A fifty-three-year-old Japanese womandiagnosed with PKND with typical osteoclasts6', was highly expressed in osteoclasts. features suffered from the nonunion of the right tibial This molecule plays an important role in bone resorp- shaft. We did open reduction and performed a vascular- tion and remodeling. Cathepsin K knockout mice ized fibular graft using an external fixator. A low- show impaired osteoclastic bone resorption, which intensity pulsed ultrasound device was used three leads to osteopetrosis7'8). Recent studies have demon- months after surgery. Union of the tibia was completed strated several mutations in patients with about one year after surgery. Histomorphometric anal- PKND1'9-10'11'. In this paper, we present a clinical, ysis of the iliac bone revealed that the patient had low histomorphometric, and genomic study of a patient turnover bone with increased bone volume. Analysis of with the typical form of pycnodysostosis. Since the cathepsin K coding region from the genomic DNA parental consanguinity has been noted in more than of the patient and her family (consanguineous parents 30% of cases, we also analyzed her consanguineous and three sisters who were all of normal stature) revealed that the patient had a deletion of genomic parents and three sisters. A cathepsin K gene muta- DNA nucleotide 426 T in exon 5. Her parents and two tion was detected by direct DNA sequencing analysis. sisters had a heterozygous mutation, while one sister had a normal sequence. In summary, a mutation in the cathepsin K gene was identified, providing further CASE REPORT evidence that a deficiency in the activity of this enzyme causes PKND. A fifty-three-year-old Japanese womanwas refer- red to Niigata University Hospital with nonunion of Key words-cathepsin K, Pycnodysostosis, mutation, the right tibial shaft. Eight years prior to admission, histomorphometric analysis, nonunion. she had injured her tibia and had it operated on using a plate, because closed reduction and casting were not successful. The plate broke and fracture union INTRODUCTION did not ensue. Six and three years prior to admission, nonunion of the tibia was fixed each time using an Pycnodysostosis (PKND), an autosomal recessive intramedullary nail with iliac bone graft, resulting in sclerosing skeletal dysplasia, has recently been failure of the nail three years after re-fixation. She shown by a positional candidacy approach to result was referred to our hospital for investigation and from a deficient activity of the lysosomal cysteine treatment. protease cathepsin K1>2'3). The disease is character- Physical examination of the patient revealed that ized by dwarfism, osteosclerosis, acro-osteolysis of she was obese (133 cm tall and 53 kg in weight) (Fig. la). She had a saddle nose, hypoplasia of the face, and Correspondence: Naoto Endo, M.D., Ph.D., Department of Orth- an open cranial suture. She has used dental prosthesis opedic Surgery, Niigata University School of Medicine, 1-757 due to dental caries and periodontal disease following Asahimachi-dori, Niigata 951-8510, Japan. malformed teeth since the age of twenty. She appear-

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r n b sit ･ b c ･ R a d i o r a h r o f th e a ti e n t ･ a ･ P h o t o r a h s h o w i n s h o r t s t a t u e a d o e F i g . 1 . C li ni c al f e a t u e s p g p g y , g p

r o r o n a c r a n a s u t u r e s a r r o w s ･ e a n e o t e o f th e s k u ll s h o w i n g n o f u s i o n o f ei th e r s a g gi t a l o c l i l ( ) T h gl f h n l n a r r r d . R i o r a h d e m o n st r a ti n a c r o 0 S t e O l si s o f t h e h a n d s a r r o w s a d u m a n d ib l e i s o b t u s e ( a o w ) . a d g p g 1 y ( ) f h l i a n d s cl e r o s e s o f t h e f e m o r a l m i n u s v a ri a n t . e . R a d i o g r a p h ill u s t r a ti n g i n c r e a s e d b o n e d e n sit y o t e p e v s l l f . 1 m o a c r a l r a d i o r a h s h o w i n s o n d l o li s th e si s a t t h e L 5 e v e s h a ft d u e t o s t r e s s f r a c t u r e ( a r r o w s) . u b s g p g p y

( a r r o w ) .

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n x n x r o x x r w - r u r s i n clu di n th e st u d of s e r u m el e ct r ol t e a e t e r al fi at o O r t h 丘 O f o d , UK it h ili a c p o c e d e g y y ( , ) l r f n i n a n d b o n e a n d v a s c ul a ri z e d 丘b ul a r r a ft . I n t r a o e r a ti v e c o n c e n t r a ti o n s , r e n al f u n c ti o n , liv e u c t o , g p y , i n d c o u n t s of bl o o d c ell s w e r e w it hi n n o r m al li m it s . P a s t a bi o p s y s p e ci m e n f r o m t h e ili a c c r e st w a s o b t a e

m e di c al hi st o r y r e v e al e d t h a t t h e p ati e n t h a d b e e n f o r hi st o m o r p h o m et ri c st u d y . T h r e e m o nt h s aft e r

D i h i al f e a t u r e s si n c e a d o s u r e r a s e ci丘c all r o r a m m e d uls ed l o w - di a g n o s e d a s PKN w t t y p c - g y , p y p g , p ,

n h h d m u lti l e f r a c t u r e s i n b o t h f e m o r a i n t e n sit ult r a s o u n d d e vi c e S AFH S : S o n i c A c c ele r - l e s c e c e . S e a p y (

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ll h r f o r e cl o s e d r ed u c ti o n w a s a w a N w a s u s e d f o r 2 0 m i n . e r d a . Si x m o n t h s r e d . I n a c a s e s t e e y , J) p y , ,

r i n h i n t w a s a bl e t o b e a r e ff e cti v e a n d o e n r e d u cti o n w a s n o t n e c e s s a r . H e r aft e r t h e o e a t o t e a t e , p y p , p

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n h r i r s all of n o r m al u n til t h e r e m o v al . N i n e m o n t h s aft e r s u r e r t h e g u l n e O u S p a r e n t s a d t e e s st e , g y ,

m n h o r t l e b r a c e st a t u r e . Si x f e m al e a n d t w o m al e sibli n g s of h e r e x t e r n al 丘Ⅹ a t o r w a s r e o v e d , a d a s g

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r n n o m o v e m e n t w a s als o u n aff e c t e d . t e n d e r n e s s w a s o b s e v e d , a d C a s e R e p o r t : P y c n o d y s o st o si s 3 3

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l a t e r t h e n ail h a s b r k , , o e n a r r o w ･ c ･ E x t e r n al 丘x a ti o n w i th v ( ) a s c ul a ri z e d 肋 u l a r a n d ili a c b o n e g r a ft w a s rf r m p e o e d (f o u rt h o p e r a ti o l-) . d . O n e e a r a ft e r th e f o u r th o e r a ti o ll u n i o n h a n y p , s b e e a c h i e v e d .

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sit e t o b e s t a bl e .

R e s u lts R o e n t g e n o g r a p h ic 丘n d i n g s T h e b o n e l m v o u e ( BV / TV) a n d w all thi c k n e s s ( W . A r o n e t g e n o g r a m s h o w ed a n o b t u s e m a n dibl e T h) w e r e si g ni丘c a n tly i n c r e a s ed c o m p a r e d w it h a g e -

a n le a n d n o f u si o n f - 1 3 ) g o t h e c r a n i al s u t u r e s F i . 1 b c a n d s e x m ( g , ) , a t ch e d J a p a n e s e w o m e n . T h e o st e o id

- a c r o o st el y sis of t h e h a n d s Fi . 1 d i n c r e a s ed b o n e v ol u m e OS BS a n ( g ) , ( / ) d o s t e oid th ic k n e s s ( 0 . T h) w e r e d e n sit of t h e el v i s a n d s cl e r o sis of f e m o r al sh aft d e r y p c e a s e d . O s t e ob l a s t s w e r e r a r el o b r y s e v e d . T h e

d u e t o st r e s s fr a c t u r e F i . 1 e a n d s o n d l oli st h e sis e r o d e d s u rf ( g ) , p y a c e ( ES / BS) w a s i n c r e a s e d alt h o u g h n o

of 丘fth l u m b a r s i n e Fi . 1f . - p ( g ) o s t e o cl a s t s w e r e o b s e r v e d . A d o u b le l a b ele d s u rf a c e

A r o e n t e n o r a m of t h e ri h t l o w e r e x t r e m it w l g g g y a s s ig h tly d et e c t e d . T h e m i n e r al a p p o siti o n r a t e m a e a e s e c o n n a n s o w d t t h d ili g h ed t h e alig n m e n t of ( MAR) w a s n o r m al b u t b o n e f o r m ati o n r at e BFR , ( /

t h fr - e a m e n t s t o b e s atisf a ct o r F i . 2 a . A r o nt B g ( ) e S w a s a t a l o w e r le v el T a bl . 1 n y g ) ( e a d Fi g . 3) .

g e n o g r a m a t a d m is si o n s h o w ed n o n - u n i o n of t h e tibi a

a n d t h e b r e a k a e of t h e i n t r a m e ull r n g d a y ail ( Fi g . 2 b) , DNA s e ll e n C l n a n al sis - q g A p o s t o p e r ati v e r o e n t g e n o g r a m sh o w e d t h a t t h e y f r a c t u r e w a s fi x e w n d a s a e x t e r n al fi x a t o r ( Fi . 2 c) . A g M e th o ds f r a c t u r e c all u s a p p e a r ed a b o u t t e n m o n t h s a ft e r th e

o e r a ti o n n a d th e u ni o n of t h e tibi a w a s c o m l e t e d Bl o o m - p , p d s a p le s w e r e o b t ai n ed w it h i n f o r m ed c o n

a b o u t o n e e a r aft e r s u r e r F i . 2 . y g y ( g d) s e n t fr o m t h e p ati e n t a n d h e r f a m il y ( p a r e n t s a n d

t h r e e sis t e r s) . H e r f a m ily w a s n o t p h e n o t y p ic ally

a ff e c t ed w i h P t KND . G e n o m i c DNA w a s e x t r a c t e d Ⅲist o m o r p h o m e t ric a n aly sis f r o m t o p e rip h e r al bl o o d ･ E x o n s ト 8 o f t h e c a t h e p si n 1 4 ) K e n e w e r e a m lifi e d f r o m t h e e n o m i DN M e th o d s g p g c A of t e a t n n h PKND p i e t a d h e r f a m il y b y p ol y m e r a s e

A bi o p s y s p e ci m e n of th e ili a c c r e st w a s o b t ai n e d c h ai n r e a c ti o n PCR is ol a t ed a n d n ( ) , , s e q u e c e d vi a

d u ri n s u r e r f o r h n - g g y t e o n u ni o n t r e a t m e n t (l a b eli n g c y cl e s e q u e n ci n g w ith a n A B 1 3 77 S e q u e n c e r ( P e r k i n

s c h e d ul e 2 5 2 6 . I nf o r m n n - - - ) e d c o s e t w a s o b t ai n ed . El m e r C o r . N o r w alk o n n i p , , C e c t c u t , USA ) . PCR

T h e s p e ci m e n w a s 丘Ⅹ ed i n 70 e th a n ol r e st ai n e d ri m e r s w e r e d e si n ed f r o m t h e s e u e n f - % , p p g q c e o C a th e p

w it h V ill a n u e v a b o n e st ai n e m b ed d ed i n m e t h l m e th - si n K m RNA S 7 9 89 5 i n h , y ( t e G e n B a n k) a n d t h e 1 4 ) a c r l a t e a n d c u t t o a t hi c k n e s s ()f 5 m i c r o m r . y , e t e s g e n o m i c st r u c t u r e a s f olll o w s :

H i st o m o r p h o m et ri c a n aly s ュs W a s p e rf o r m e d a s p r e vi -

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' ' - m - - n n . s y st e S y t e m S u p p ly , N a g a o , a a H is t o m o r H C KIR 5 c t ct a t a a a t c t tt t 3 ( J p ) , g g g g g g A l ∴ 3 4 Ⅲ . Y AMAGIW e t a

m r h o m e t ri c a n al si s T a bl e 1 . R e s u lt s o f h i s t o o p y

* R e f e r e n c e r a n g e p a r a m e t e r A b b r e v i a ti o n V al u e ( M e a n ± S D )

B o n e v o l u m e( %) BV / TV 3 3 . 8 2 3 . 1 9 ± 4 . 3 7

1 . 2 2 0. T r a b e c ul a r t h i c k n e s( s m c m ) T b . T h 1 2 9 6. 3 3 0 ±

4 5 . 0 3 4 . 1 6 2 . 3 2 W a ll t hi c k n e s s( m c m ) W . T h ±

O s t e oi d v o l u m e( % ) O V / TV 0 . 2 0 . 3 2 ± 0 . 1 9

O V BV 0 . 5 1 . 4 8 ± 0 . 9 3 O s t e oi d V o l ノB o n e V o l (. %) /

1 2 . 1 4 . 6 4 O s t e o id s u rf a c e( % ) O S / BS 5 . 4 ±

O st e o id th i c k n e s s( m d m ) 0 . T h 5 . 2 1 0 . 3 4 ± 2 0. 5

E r o d e d s u r f a c e(%) ES / BS 1 2 . 3 4 . 0 9 ± 2 . 3 3

. 0 . 5 8 9 0 . 0 8 2 M i n e r a l a p p o s . r a t e( m c m / d a y) MAR 0 5 8 7 ±

B 0 . 7 3 D o u b l e l a b e l e d s u rf a c e/ B S( % ) d L S/ S LS BS 0 S i n gl e l a b e l e d s u rf a c e/ BS (%) S /

3 2 . 0 1 6 0 . 0 0 8 B o n e f o r m a ti o n r a t e( m m / m m / y e a r) BFR / BS 0 . 0 0 1 5 0 ±

‥ B o n e f o r m a ti o n r a t e(% / y e a) r BFR / B V 2 . 4 2 2 4 . 6 ± 1 3 . 3

* 1 1 1 9 8 7 . o n n o . : n r o s s o c : K T JJ p O t h p A , 6 1 1 0 8 1 1 0 9 ,

･ m i r ･ N o M i r h o t o r a h s o f t r a b e c u l a o f ili a c b o n e u n d e r li t a ･ a n d 8 u o r e s c e n t b ･ c o s c o F ig . 3 . c o p g p gh p y i n ri n l m a n i 丘c a ti o n : × 4 0 e r a c c n e a e s e e c e . a n u e v a o n e s a o a t t y li l b l i d t t d ( V ill b t , gi g )

' ' H C K 2 F 5 - c t a c a t t c t t c t g c a g g a t g - 3 R e s u lt s ' ' H C K 2 R 5 - ct t g tt g tt a t a tt g c tt c c t g-3

' ' H C K 3 F 5 - t a t g ct tt g t tt a g g t g g a t g a - 3 A n al y sI SOf t h e c at h e p si n K c o °i n g r e g l O n f r o m

' ' a a c a t t c - 3 e n o m i c DNA of t h e a ti e n t r e v e al e d a d el eti o n of H C K 3 R , 5 - t g g t a t a c tt g c c g g p

' ' - H C K 4 F 5 a c c a t a a a t t t c a 3 e n o m i c DNA n u cl e o tid e 4 26 T i n e x o n 5 Fi . 4 . , g g g g g g g - g ( g )

' ' - T his m u t a ti o n w a s cl o s el l o c a t e d a t t h e a c ti v e HCK 4 R , 5 c t g at tt t t g a c a g g a g t a a c a-3 y

' ' H K F 5 - t c a t t tt c c t tt - 3 c s t ei n e r e sid u le i n a m a t u r e e tid e r e s ulti n l n a C 5 , g g g g g g g g y p p , g

' ' - - - f r m hif t F 1 4 2 L a n d st o c o d o n L 1 6 0 Ⅹ r e dic t H C K 5 R , 5 c c t g t c c c a c a t a t g g g t a g 3 a e s ( ) p ( ) , p

' ' - - t 3 1 n r e m a t u r e t e r m i n a ti o n of t h e m a t u r e c t h e si n K H C K 6 F , 5 t c c a g c c a g g a a g a g a g t g g p a p

' ' ' - H C K 6 R 5 c a c tt c t t a c c tt t ct 3 o l e tid e . A n al sis o f h e r a r e n t s e n o m i c DNA , g g g g- p y p p y p g

' ' - - d e m o n s t r at e d t h a t t h e a r e n t s a n d t w o si st e r s w e r e Ii C K 7 F , 5 t a t t c t a g g t g t a t t at g at g 3 p

' ' H C K 7 R 5 - t tt c c c a tt a c ct tt tt 3 h et e r o z o u s f o r t h e d el e ti o n m u t a ti o n w h ile o n e , g g - y g ,

' '

- - c a c a i t r h a d a n o r m al s e u e n c e Fi . 4 . T h e s e r e s ult s HCK 8 F , 5 t t g g t t t g c t g g g g a g 3 s s e q ( g )

' '

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m e n t s w e r e s u c c e s sf ul . I n t h e f u t u r e , g e n e t r a n sf e r of - m o n L D e s n i c k R , G e lb B D : C h a r a c t e ri z a J , R i i D , J o l a st s o r a ti s s u e e n l c at h e p si n K i n t o p u ri丘e d o st e c g - ti o n o f n o v el c a th e p si n K m u t a ti o n s i n t h e p r o a n d

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n o s t o si s . G e n o m e R e s 6 l l : 1 0 5 0 1 0 5 5 1 9 9 6 . p y c o d y s ( ) - ,

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n D r . H i k i E . T a k a h a h i st o m o r o m e t r : S t a n d a rdi z a ti o n o f n o m e n cl a t u r e a s si s t a n c e o f M s . A k e m i lt o a d d e a - p y n i R r f h e A S B M R H i st o m o r s h i N ii a t a B o n e S ci e n c e I n s ti t u t e N ii a t a a a n f o r s m b o l s a n d u t s . e o t o t ( g , g , J p ) y , p -

h i s t o m o r p h o m e t ri c a n a l y si s a n d m a n u s c ri p t p r e p a r a ti o n . p h o m e t r y N o m e n cl a t u r e C o m m it t e e . I B o n e M i n e r 1 e a u o r s a s o a n r . e a z a w a o r w o r n e s : . T h t h l t h k M H i d k i A k f k i R 2 5 9 5- 6 1 0 , 9 8 7

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: . n a a n 6 1 1 0 8 ト 1 0 9 1 , 1 9 8 7 (i J e s e M e ta p ) b 8 5 1 : 4 2 5 4 3 1 2 0 0 0 . ( ) - , e 1 4 G l b B D , S h i G P H ell e r M W e r e m o w i c z S M r ) , , , o t o n 1 6 S a r n s e th si ri P H i r n r ) , tt O K , E y i g E J , F o st II M :

e s n c a m a n - C , D i k R J , C h HA : S t r u c t u r e a n d T p e t r a c y cli n e b a s e d s t u d y o f b o n e d y n a m i c s i n c h r o m o s o m a l a s s l g n m e n t O f th e h u m a l- C a t h e si n K c n o d s o i p p y s t o s s . C li n O r th o 7 4 : 3 0 ト 3 1 2 1 y p , 9 7 1 .

e n e . G e n o m i c s 4 1 2 : 2 5 8 2 g ( ) 6 2 1 9 9 7 . H - , 1 7 e c k m a n D , R a b P , M c a e r e o e ) J y y J C b ∫, F y JJ , R 1 5) F u i t a Y N a k a t a K Y a s u i N M a t s u i Y j , , , K a t a o k a E LR : A c c l r - - , , e e a ti o n o f tibi a l f r a c t u r e h e ali n g b y n o n

H i r o sh i m a K S h i a c : o v e m u a o II - , b R J , O h i T N l t ti SO f i n v a si v e l o w i n t e n i , s t y p u l s e d u lt r a s o u n d . I B o n e th e c a th e p si n K g e n e i n a ti e n t s w i th c n o d s o s o i n t S u 7 p p y y 7 6 A 1 : 2 6 3 4 1 9 9 4 . - J g ( ) - ,