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Hypophosphatasia and the Extracellular Metabolism of Inorganic Pyrophosphate: Clinical and Laboratory Aspects: Part I a

c

Alison M. Caswell , Michael P. Whyte & R. Graham G. Russell

b

a

Department of Biochemistry and Molecular Biology, University of Leeds, LS2 9JT, Leeds, United Kingdom b

Department of Human Metabolism and Clinical Biochemistry, Medical School, University of Sheffield, Beech Hill Rd., Sheffield, S Yorks, UK S10 2RX c

Department of Medicine, The Jewish Hospital of St Louis, 216 S. Kingshighway, St Louis, MO, 63110 Published online: 03 May 2015.

To cite this article: Alison M. Caswell, Michael P. Whyte & R. Graham G. Russell (1991) Hypophosphatasia and the Extracellular Metabolism of Inorganic Pyrophosphate: Clinical and Laboratory Aspects: Part I, Critical Reviews in Clinical Laboratory Sciences, 28:3, 175-194 To link to this article: http://dx.doi.org/10.3109/10408369109106862

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Hypophosphatasia and the Extracellular Metabolism of Inorganic Pyrophosphate: Clinical and Laboratory Aspects Alison M. Caswell, Michael P. Whyte, and R. Graham G. Russell

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ABSTRACT Hypophosphatasia is a rare inherited disorder in which the activity of the boneiliverkidney or tissue nonspecific form of alkaline phosphatase (ALP) is reduced. The clinical expression of the disease is highly variable, but in early life the severity tends to reflect the age of onset. Accordingly, the disease is often classified into perinatal, infantile, and childhood forms. Hypophosphatasia also occurs in adults. Some exhibit symptoms in adulthood for the first time, but others have a history of the disease in early life with an intervening symptom-free period. Defective mineralization of bones and teeth is the predominant clinical feature of all forms of the disease. Biochemically, the reduction in ALP activity is associated with alterations in the extracellular metabolism of various phosphorylated compounds, including inorganic pyrophosphate (PPi), phosphoethanolamine, and pyridoxal 5‘-phosphate. Of these, PPi may have an especially important role in the development of the mineralization defect. Accordingly, the extracellular metabolism of PPi and its possible role in the regulation of mineralization will be discussed.

Key Words: hypophoshatasia, alkaline phosphatase, mineralization, inorganic pyrophosphate.

1. INTRODUCTION The human alkaline phosphatases (EC 3. I .3.1) are the products of at least four genes, but there is little definitive information about the function of these isoenzymes.’ Recently, and tissue nonspecific5 the genes that encode the placental ,* placental-like,3 inte~tinal,~ isoenzymes of alkaline phosphatase (ALP) have been cloned and sequenced. Hypophosphatasia is an inborn error of metabolism characterized by a lack of activity of tissue nonspecific ALP (TNSALP). This condition results in defective mineralization of bones and teeth, and in severe forms of hypophosphatasia various systemic symptoms also occur. Rathbun coined the term “hypophosphatasia” in 1948 when he described a boy in whom a lack of ALP activity in serum and tissues was associated with rickets and other systemic problems.6 However, patients with a similar clinical history had been described previously and are now considered to have had hypophosphatasia.’ The clinical expression of hypophosphatasia is highly variable, but there is a general relationship between the age of onset of symptoms and its severity and prognosis. Age at

A. M. Caswell, B.Sc., Ph.D., Department of Biochemistry and Molecular Biology, University of Leeds, Leeds, United Kingdom LS2 9JT. R. G. G. Russell, B.A., M.B. Ch.B., Ph.D., D.M., Department of Human Metabolism and Clinical Biochemistry, Medical School, University of Sheffield, Beech Hill Rd., Sheffield, S. Yorks, U.K. S10 2RX. M. P. Whyte, M.D., Department of Medicine, The Jewish Hospital of St. Louis. 216 S. Kingshiphwav. St. Louis. MO 631 10.

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presentation is therefore the usual basis of classification. Infantile (onset before six months of age), juvenile or childhood (onset after six months), and adult forms of hypophosphatasia were defined by Fraser in 1957.* Subsequently, the first group was divided into perinatal (symptoms developing in utero and present at birth) and infantile (symptoms developing between birth and six months) forms by Currarino and co-worker~.~ This review will utilize the latter classification when describing clinical, radiological, and laboratory findings of hypophosphatasia. The rather variable relationship between age of onset and severity and prognosis of the disease has, however, led to the development of alternative classifications. Taillard and co-workers, for example, differentiated between subjects with skeletal disease alone, and subjects with skeletal disease together with predominantly respiratory or neurological symptoms. l o These alternative classifications are not widely used. Lack of TNSALP activity results in the abnormal extracellular metabolism of several phosphorylated compounds including phosphoethanolamine (PEA), inorganic pyrophosphate (PPi) and pyridoxal 5'-phosphate. (PLP) (see below). Accumulation of these metabolites extracellulady may be involved in the pathogenesis of hypophosphatasia. In particular, increased extracellular concentrations of PPi may prevent normal skeletal mineralization and promote the formation of calcium pyrophosphate dihydrate (CPPD) crystals in articular cartilage. In view of the potential importance of PPi in hypophosphatasia, the latter part of this review will discuss the extracellular metabolism of PPi in cartilage and bone, and the role of PPi in mineralization.

II. CLINICAL AND RADIOLOGICAL FEATURES A. Perinatal Hypophosphatasia Perinatal hypophosphatasia is the severest form of the disease and is a lethal condition. Polyhydramnios is a frequent complication during the later stages of gestation. ''-I5 This form of hypophosphatasia can be detected early in utero.I6.l9 Both aborted fetuses and term newborns are grossly abnormal. The weight of term newborns may be norma17~9*'7~20 or red~ced,~.'.~' whereas aborted fetuses are usually small for gestational age.16*'8.22 The head is frequently soft and globular because of a generalized lack of bone mineral .9,11.18.23-25Widened or bulging fontanels have been noted o c ~ a s i o n a l l y , ~ ~ ~ ~ * ~ ~ but are evident only when bone structure is sufficiently defined. The long bones are almost always ~ h o r t e n e d , ~ . ~and . ' ~this ' ~ may ~ , ~ account ~ for the multiple skin folds that are sometimes present. 1423 Bowing of the long bones is frequently ~ b ~ e r v e d , ~ and. ~there ~ . may ~ ~ be . ~ ~ ~ ~ ~ other deformities.12.2'Mobility of the knees and elbows can be r e s t r i ~ t e d . ' ~ . 'The ~ . ' ~thoracic cage is often narrowed or ~ m a 1 1 , and " ~the ~ ribs ~ ~may ~ ~be~deformed."'25 ~ ~ A rachitic rosary has been observed in some ~ u b j e c t s , ' ~as* ~has ~ . widening ~~ of the w r i ~ t s , ~which . ~ ~ .is~ ~ another characteristic sign of rickets. Hepatic and/or splenic enlargement occurs ~ccasionally.~~~~~~~ Many affected newborns die within a few minutes of birth,9.'1.13t17.20.29 and those who survive for a few days tend to develop complications rapidly. Respiratory distress is very common and is the usual cause of death.7.9."-'3.17-20.29.3',32 Some subjects remain continuously dependent on a respirator until death. 12*25.26Convulsions are also relatively ~ o m m o n , ~ . ' ~ . ~ ' . ~ ~ and other complications include vomiting, feeding difficulties, irritability, hypotonia, thrombocytopenia, CNS hemorrhage, bradycardia, and renal f a i l ~ r e . ~ ~ ' ~ ~ ~ ~ . ~ ' . ~ ~ Skeletal radiographs (Figure 1) always reveal extensive and severe hypomineralization.7-9,14,17*18*23.27 The skull (Figure 2) is usually profoundly affected and may appear cornpletely unmineralized. 1 1 ~ 1 4 ~ 1 8 ~ 2Widened 0~31 sutures and fontanels have been observed oc~asionally,~ but only in those subjects whose bone structure is sufficiently defined. Shortening of the long bones is often apparent on radiograph^,^.'^.^' as is bowing9.*' and other d e f ~ r m i t i e s . ~ Fractures .~,~' are also relatively c ~ r n m o n . ~ . ' Bony spurs at the ends of the

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FIGURE 1. Perinatal hypophosphatasia. This radiograph of a stillborn with perinatal hypophosphatasia reveals profound skeletal hypomineralization, a finding that enables the condition to be readily distinguished from other congenital bone disorders. (From Whyte, M. P . , The Metabolic Basis of Inherited Disease, 6th ed., Scriver, C. R . , Beaudet, A. L., Sly, W. S . , and Valle, D . , Eds., McGrawHill, New York, 1989, 2843. With permission.)

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FIGURE 2. Perinatal hypophosphatasia. The skull of this new born boy with perinatal hypophosphatasia shows characteristic marked hypomineralization. Ossification is apparent in only a few areas: in the frontal bone (arrow), at the base, and in the occiput. (From Whyte, M. P., Endocrinol. Merab. Clin. North Am., 19, 133, 1990. With permission.)

long bones occur in a few subjects and are thought to be characteristic of hypopho~phatasia.~~ Mineralization is defective throughout the long bones in the perinatal form of the disease, but changes in the metaphyseal regions can be especially p r o n o u n ~ e d . Secondary ~ ~ * ~ ~ ~ centers ~ of ossification are usually poorly minerali~ed.~ In one subject, the spine was relatively well mineralized compared with the remainder of the skeleton,22but this does not appear to be a common finding. There is a report of ~ c o l i o s i s and , ~ ~ a few reports of “missing” or unossified ~ e r t e b r a e . ~ *Deformities ’ ~ ~ * ~ or fractures of the ribs are sometimes p r e ~ e n t , ~ ~ ‘ ~ ~ ~ ~ and the costochondral junction may appear ~ i d e n e d . ~ Postmortem findings in the skeleton will be described later, but abnormalities of nonosseous tissues are also frequently detected. Pulmonary atelectasis is a common finding,9.17,30*31 and pulmonary hypoplasia and hemorrhages have also been o b ~ e r v e d . ~ . ~ ~ , ~ ~ . ~ ~ Brain edema and hemorrhages have been noted o c c a ~ i o n a l l y , ’ ~as* ~has ~ , ~increased ~ hematopoietic activity in the liver and the spleen. 7.12.30 Nephrocalcinosis has been documented in some subject^,'*^'^*^ but in others the kidneys were No parathyroid glands were located in one ~ u b j e c t , and ’ ~ only one was identified in a However, normal parathyroid glands were found in two other subjects.’ Several congenital abnormalities were

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noted in an abortus described by Whyte and co-workers,26 including cleft lip and palate and absence of the diaphragm, but the karyotype was normal. It is difficult to define whether some of the changes in nonosseous tissues are directly attributable to the lack of ALP activity or are secondary to the skeletal abnormalities. For example, the defective rib cage, poor development of marrow spaces, and abnormal calcium and phosphate homeostasis could cause respiratory problems, altered hematopoiesis, and nephrocalcinosis, respectively. B. Infantile Hypophosphatasia Pregnancy and delivery are usually normal, but transient episodes of respiratory distress occur occasionally at birth,6,24.34,35 and hypotonia was noted in one newborn.36Short limbs,24 bony d e f ~ r m i t i e s , ~ . ~and ~ . widened ~ ~ . ~ ’ cranial sutures and fontanel^^^-^' are sometimes evident at birth. Most affected infants, however, are symptom-free for up to 6 months and then typically develop vomiting, failure to thrive, and In addition, there may be c o n v u I s i o n ~ , ~episodes * ~ ~ * ~of ~ .fever,7,9.36.40 ~~ respiratory anore~ia,’.~~.~~ c~nstipation,~,’.~~ and general irritability.’ At the time of onset of symptoms, subjects are usually small for their age,43-46 and some give evidence of retarded motor d e ~ e l o p r n e n t . ~ . ~ ~ . ~ ~ Affected infants may be mentally n017nal’~~~ or retarded.24Mental retardation can be secondary to craniostenosis (see below). On physical examination, skeletal changes predominate. The limbs are often ~ h o r t , ~ ~ . ~ ~ 4 ~ ~ and exhibit b ~ w i n g , ” . ~ ~or. ~valgus ’ d e f ~ r m i t i e s . ~Dimples ~.~’ are sometimes present over the long bones, and may be a characteristic feature of h y p o p h o ~ p h a t a s i a .Various ~~’~~~~~~~ deformities of the thorax have been r e p ~ r t e d , ’ . and ~ ~ .a~ rachitic ~ rosary is often f o ~ n d . ~ . ’ . ~ ’ , ~ , ~ ~ Widening of the wrists has also been noted occa~ionaIIy.~~’~.~’ Kyphosis occurs in a few s u b j e ~ t s . ~ ~ . “Rarer O ~ ~clinical ’ findings include anemia,9*36,47 and hepatic and splenic enlargement.40As in subjects with the perinatal form of the disease, extramedullary hematopoiesis may occur because of disrupted development of the bone marrow spaces, secondary to the skeletal changes. Poor nutrition may also contribute to the development of anemia. Inguinal herniae occur in some ~ u b j e c t s ,possibly ~ . ~ ~ resulting from muscle weakness, and various eye changes have been reported including blue sclerae, proptosis, papilledema, shallow orbits, failure of upward gaze, and pathological lid reetTa~tion.~~,’~.~’.~“ Some of these changes may be secondary to craniostenosis (see below). Renal compromise sometimes develops3“ and is probably attributable to nephrocalcinosis resulting from altered calcium and phosphate homeostasis secondary to the mineralization defect. Although nephrocalcinosis has been observed relatively frequently at autopsy (see below), it is seldom detectable on conventional radiographs. 24.45 The radiological appearance of the skeleton varies, but there is frequently generalized ~ s t e o p e n i a . ~ Bone ~ . ~ ~age . ~may ~ . ~be normal5’ or dec~eased.~? The long bones often appear ~ h o T t e n e d ~and . ~ ~exhibit * ~ ~ ,f ~r ~a c t u r e ~b, o~ ~ i~ n~ g~ ,~~or~, other ~ ~ .deformities ~~ .’ Bony spurs over the ends of the long bones have been reported, but are rare.24In some subjects, defective mineralization seems to be restricted to the metaphyseal regions of the long bones, and the diaphyseal regions are relatively well ossified. 34.35.47 In others, mineralization of the diaphyseal regions is either generally5’ or focally d e c r e a ~ e d . ~The ~ , ~metaphyseal ’ regions also exhibit variable mineralization with, again, either a general d e c r e a ~ e , ” . or ~ ~localized ~~~.~~ decreases that appear as areas of radiolucency .24.w*40,41Radiolucent defects may project into the metaphyses (tongues of radiolucency) and change with time.24The quality of the surrounding mineral can influence their radiological definition. Projections of growth plate cartilage into the osseous regions of the metaphyses are frequently observed on histological study (see below), and probably account for the “tongues of radiolucency”. The borders of the metaphyseal regions have been variously described as ill-defined ,47,51 f r a ~ e d , ~or ~,~’

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irregular.7.50 Mineralization of the epiphyseal regions may also be d e c r e a ~ e d , ~but, . ~ again, ~.~~ the degree of the reduction is variable. The calcified zone of the growth plate has been reported to be or even absent,52and its borders ill-defined or i r r e g ~ l a r .Conversely, ~~,~~ uncalcified regions of the growth plate appear to be ~ i d e n e d . ~ , ~ ~ , ~ ~ Thoracic changes include o ~ t e o p e n i a ,rib ~ ~ fracture^,^^.^^.^ shortening and irregularities of the rib^,^^,^' and widening of the costochondral junction (apparently attributable to an increase in the amount of cartilage) .6*7,24,38,47 Decreased mineralization of the spine38,a,5L and p e l v i ~ has ~ ~been ~ ~reported. ~ ~ ’ Sc01iosis~~ and flattening or wedging of the have been observed occasionally. The skull is often poorly mineralized and, as in the perinatal form of the disease, can feel soft.7.35.47 In a few subjects, the base of the skull appears to be relatively better m i n e r a l i ~ e d .Widened ~ . ~ ~ fontanels and sutures are frequently detected on both p h y ~ i ~ a 1 ~ and~ r. a~ d~i ,o ~l o- g~i ~~ a l ~ examination, * ~ ~ * ~ ~ ~ ~ ~especially ~~ during the first 6 months of life, but may be a clinical artifact resulting from poor mineralization of the calvarium. Postmortem findings in two 3-month-old infants support this view. In both, the sutures were of normal or decreased width, but were surrounded by wide bands of unmineralized ~ s t e o i d . ~Interestingly, .~* some subjects subsequently develop true craniostenosis, because of premature closure of the sutures. ’0.40s3 This suggests that functional craniostenosis develops during the first 6 months of life in these infants. The early detection and correction of craniostenosis are important in order to prevent brain damage, etc. Sty and ~ o - w o r k e r s ~ ~ have recommended the use of scintigraphy for the detection of craniostenosis in hypophosphatasia subjects. The course of infantile hypophosphatasja is variable, but about 50% of affected subjects die in infancy, usually from respiratory infections.25.38.39*43 A progressive deterioration in skeletal mineralization and increasing deformity have been observed in some subjects prior to death,9.36+’ but BCthenod and c o - ~ o r k e r reported s~~ improving skeletal mineralization in a subject who died as a result of convulsions. Surviving subjects may exhibit clinical and radiological i m p r o ~ e m e n t . ~However, ~ , ~ ~ . ~ retardation ~ of and of motor d e ~ e l o p m e n t ~may . ~ ~persist, . ~ ~ craniostenosis can d e ~ e l o p ,and ~ ~ new . ~ ~ problems such as the premature shedding of primary teeth9,10.24 may become manifest. Postmortem findings in nonosseous tissues have been reported for some subjects. Nephrocalcinosis has been observed fairly frequently, and was associated with varying degrees of inflammation and tissue degene~ation.~.~.’~.~~.~~.~~.~~ Hyaline or amorphous deposits, which did not contain calcium or phosphate, have sometimes been detected in the renal tubules or medulla, and were associated with a low-grade inflammatory r e s p o n ~ e . ~ *One * ~ .subject ~~ with nephrocalcinosis also exhibited calcification of the gastric m u ~ o s a Other . ~ ~ autopsy findings include pulmonary atelectasis with varying degrees of inflammatory ~ e s p o n s e , ~ - ~ ~ - ~ ~ cerebral ischemia54or edema,25 and in one subject hepatic lipid ac~umulation.~~ Although it is probable that abnormalities of the rib cage contribute to the development of respiratory problems and lung damage, it has been suggested that the lack of ALP activity may directly cause a defect in the biosynthesis of s ~ r f a c t a n t . ~ ~ . ~ ~ . ~ ~

C. Childhood Hypophosphatasia Childhood hypophosphatasia is milder than the aforementioned forms of the disease, but is more variable in its clinical expression. Some subjects exhibit dental changes a10ne,~~-~’ a condition that has been termed “odontohypophosphatasia” .55 In others, dental problems are associated with slight skeletal changes that are only apparent on radiograph^,^'.^^-^ and/ or merely cause growth retardation .59.61 More severely affected subjects exhibit premature loss of teeth and extensive skeletal d i s t u r b a n c e ~ , ~ . ~ and ~ . ”sometimes ~~ systemic sympt o m ~ . Very ~ ~ occasionally, - ~ ~ skeletal changes occur in the absence of any abnormality of the primary d e r ~ t i t i o n . ~ . ~ ~

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Affected subjects frequently present with dental problems, usually the premature shedding of primary teeth, i.e., before 5 years of age.58-61.70 Teeth “fall out” with minimal or no and there is seldom evidence of inflammatory periodontal d i ~ e a s e . ’ ~ Their ~~~~’’~~~ roots may be only partially resorbed, or even completely ~ n ~ e ~ ~ r b e dIncisors . ~ ~ are . ~ ~ . ~ ~ ~ ~ usually shed first. Sometimes no further loss of teeth occurs,57~58*66~73.74 but it may continue and can result in the premature loss of the entire primary d e n t i t i ~ n . ~ ~Remaining ,~’.~,~~ deciduous teeth may become c a r i ~ u s . ~The ~ . secondary ~ ~ . ~ ~ dentition develops normally in some subjects,56 but in others there are problems, including retarded eruption, excessive mobility, enamel hypoplasia, and caries.61.69.77 On radiographs, the remaining primary teeth sometimes appear normal ,55.61 but enlarged pulp chambers and root canals are frequently apparent.57.58.71-73 Decreased mineralization of dentine has been detected occasionally.68 Interestingly, both r e d u ~ e d and ~ ~ e. n~ h~a n ~ e d ~ ’ . ~ ~ root resorption have been observed. The development of the secondary dentition may appear norma1,57~66.71.78 but other subjects exhibit abnormalities such as enlarged pulp chambers and root canals and enamel h y p ~ p l a s i a . ~ ’ . ~ ’ . ~ ~ , ~ ~ One of the most common findings on dental radiographs is an apparent loss of alveolar bone. This osteopenia in the jaw is usually generalized and does not normally appear to be more pronounced in regions from which teeth have been ~ h e d . ~ ~It may . ~ be ~ attributable . ~ , ~ ~ . ~ ~ to the reduction in mechanical forces on the alveolar bone that results from the defective dentition. In some subjects, radiolucent areas in the alveolar bone are also a ~ p a r e n tand ~~.~~ indicate variable mineralization. As noted above, skeletal and systemic abnormalities do not always occur and, when present, are of variable severity. Subjects with skeletal involvement frequently exhibit growth retardation24*33.39.62,71 and characteristic rachitic changes such as widening of the ~ r i ~ t ~ ~ , ~ ~ , ~ and a rachitic rosary.7*33,62.67.7‘ Various skeletal deformities have been reported, including bowing and angulations of the long genu ~ a l g ~ m , and ~ . ~flat ~ feet.35,81 , ~ ~ , ~ ~ Spinal deformities have been observed oc ~asionally,and ~ ~ in one subject there was progressive kyphosis.82 Dimples, especially over the ends of the long bones of the lower limbs, may be present and appear to persist during d e ~ e l o p m e n t . ~Both ~ . ~skeletal , ~ ~ deformities and dimples were evident at birth in some subject^.^'.^^ Other physical abnormalities sometimes occur, including retarded motor development,24,58.59*62-75difficulties with running and ~ a l k i n g , ~ and ~ . pain ~ , ~in, the ~ ~e x t r e m i t i e ~ . ~ These ~ , ~ ~abnormalities .~~,~~ may be due to the skeletal disease and deformity, but their severity is not always well correlated with the extent of skeletal involvement evident on physical examination. Opthalmological abnormalities, including blue sclerae, proptosis and papilledema, have been observed occasionally .24.68,69,71 These may be secondary to the development of craniostenosis,as in infantile hypophosphatasia. Systemic symptoms are rare in childhood hypophosphatasia. However, a few subjects have had convulsions, respiratory infections, fevers, feeding problems, failure to thrive, vomiting, constipation, and irritability during the first year of life.9.35,67-69.83 The symptoms occurred episodically and resolved spontaneously. These individuals show that the distinction between infantile and childhood hypophosphatasia is somewhat artificial and imprecise. Muscle hypotonia has been observed in a few s u b j e ~ t s , ~and ’ . ~ three others reportedly exhibited a nonprogressive myopathy , although only mild changes were observed in muscle biopsy samples from two of these latter ~hildren.’~ Such problems may contribute to the locomotor difficulties described above. There are a few reports of renal problems in childhood hypophosphatasia, but this finding may be coincidental in some subjects. Two subjects, aged 5 and 8 years, died as a result of renal failure, and in the latter case nephrocalcinosis and glomerulonephritis were observed at autopsy .24.69 Another subject developed nephrotic syndrome. 77 One subject who transiently exhibited systemic symptoms during the first year of life concurrently developed renal problems that subsequently resolved spontane~ usly. ~ ~ Other rare clinical findings include abdominal distention65and enlargement of the liver and the ~ p l e e n . ~ . ~ ~

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The variable nature of childhood hypophosphatasia is also evident on skeletal radiographs. Some subjects, usually the more severely affected, exhibit generalized o ~ t e o p e n i a . ~ ~ ~ ~ ~ ~ ~ . ~ ~ . ’ Skeletal maturation, indicated by radiological assessment of the apparent bone age, may be norma157~75~78 or delayed,59.@’.x2 but is not well correlated with the extent of other skeletal changes. In severe cases, mineralization appears to be defective throughout the long bones, whereas in milder cases abnormalities are confined to the metaphyseal regions.65,68.80 Reported changes in the diaphyseal regions include irregular areas of osteopenia and thinning.24Metaphyseal abnormalities vary, probably because of the variable extent of the mineralization defect. Changes include o s t e ~ p e n i a , ~ ~ cupping .’~ and fraying,7*57.8’ banding,48.67*s’ and irregular radiolucent areas (Figure 3) .9*s7*63.66,81Widening of the growth plate may be a p ~ a r e n t , ~ . ~ ~ and the calcified zone may be poorly or irregularly m i n e r a l i ~ e d , ~with ~ . ~ill-defined ~.~~ or frayed border^.^'.'^ These changes are characteristic of rickets. Radiographs of the long bones may also reveal deformities such as b o ~ i n g . ~ Kozlowski . ~ ~ . ~ ~ and , ~ ~c o - w o r k e ~ s ~ ~ observed a S-like deformity of the tibia in two subjects and regarded this as a feature of childhood hypophosphatasia. In some subjects, spurs at the ends of the long bones were noted, which seemed to Unlike in other forms of the disease, pathological fractures have only been detected occasionally in childhood hypoph~sphatasia.’.~~ Rachitic changes are sometimes apparent on radiographs of the ribs, where there is widening of the anterior end^^^.^^.^^,^' and cupping and and of the wrists, where there is widening of the e p i p h y ~ e s . There ~~.~~ are reports of hypomineralization of the spine62.x5and p e l ~ i s , ’ ~and , ~ vertebral ~ deformities have been observed o c c a ~ i o n a l l y . ~ ~ ~ ~ ~ ~ ~ ~ or d e c r e a ~ e d . ~ ~ . ~ ~ Mineralization of the bones of the hands and feet may be Variable reductions in mineralization sometimes cause the bones to appear banded.67 Radiographs of the skull may reveal osteopenia that can be general,” p a t ~ h y , or ~.~~ especially pronounced in certain regions, e.g., around the anterior f ~ n t a n e l . Localized ~~,~~ decreases in mineral may account for the apparent persistence of open fontanels and sutures in some However, as in the infantile form of the disease, premature closure of the sutures frequently occurs and causes true craniosteno~is.~.~~-~~.~~ This is one of the most common complications of childhood hypophosphatasia. The long-term clinical course of childhood hypophosphatasia appears to be variable, but there is a lack of detailed follow-up. The majority of affected children seem to improve ~ ~ * ~ ~ ~ ~ there ’ ~ ~ ~are reports of subjects who remained clinically and r a d i o l ~ g i c a l l y . ~ However, clinically and radiologically ~ n c h a n g e dor , ~even ~ d e t e r i ~ r a t e d ,at~ ~ least . ~ ~in the short-term. A few individuals with infantile or childhood hypophosphatasia have been studied in young adulthood. All had improved clinically and radiologically and were symptom-free at that time, despite the persistence of biochemical abnormalities indicative of hypophosphatasia.24.63*87 In some subjects, spontaneous resolution of skeletal deformities had o ~ c u r r e d . ~ ~ . ~ ~ Despite the apparent improvement, however, most still had evidence of skeletal disease. In particular, nearly all exhibited skeletal abnormalities on radiographs, such as coarsening of the trabeculae and scalloping and erosions of the osseous surfaces,87and in one individual generalized osteopenia was apparent.63Iliac crest biopsies of five of these subjects all revealed o~teomalacia,~~ and approximately half of those studied had failed to reach normal adult height,24.63*88 suggesting continued growth retardation. These findings may account for the apparent recurrence of the disease later in adult life (see below).

D. Adult Hypophosphatasia Adult hypophosphatasia is also rather variable in clinical expression. In this review, only individuals with skeletal symptoms in adulthood will be described as having adult hypophosphatasia; the term “odontohypophosphatasia” will be used to describe subjects with dental manifestations alone. However, there are difficulties, particularly in relation to defining age of onset because symptoms may develop insidiously.

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FIGURE 3. Childhood hypophosphatasia. Characteristic tongues of radiolucency (arrows) project from the growth plates into the distal metaphysis of the left femur and into the proximal metaphysis of the left tibia.

A significant proportion of individuals with adult hypophosphatasia (40 to 50%) give a history of symptoms of the disease in infancy and childhood. Subsequently, there is usually a period without symptoms, except perhaps for dental problems, but skeletal problems then develop in a d ~ l t h o o d . ~ The ~ * *length ~ - ~ ~of the symptom-free period varies and is not always well correlated with the severity of the disease in either early life or adulthood. The mechanisms underlying the clinical improvement and subsequent relapse are not well understood,

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but the studies described above would suggest persistence of biochemical and radiologically detectable skeletal abnormalities in such individuals. Moreover, in some subjects, skeletal problems appear to persist or even develop during adolescence.92-94 These findings suggest that many cases of adult hypophosphatasia should be classified as a recurrence of the childhood or even infantile forms of the disease. However, it is difficult to define the proportion of cases in this category because symptoms of the disease in early life may be unknown or be poorly recalled by subjects with adult hypophosphatasia.” Equally, because of a lack of long-term studies, it is difficult to define the proportion of those with hypophosphatasia in infancy and childhood who subsequently develop the adult form of the disease. As in children, dental problems are frequently the first manifestation of hypophosphatasia in adults. Eberle and c o - w o r k e r ~described ~~ a small number of adults who exhibited dental abnormalities in the absence of any clinical or radiological evidence of bone disease. However, the subsequent development of skeletal disease is more usual. Various dental problems have been reported, including enamel h y p ~ p l a s i a ~ . ”and . ~ ~severe caries that resulted in dental clearance prior to the age of 30.76*B9.97-1w One subject gave a history of teeth “crumbling away” before the age of 30.96In a few subjects, teeth have to be extracted because of excessive mobility .96*99 As indicated above, skeletal abnormalities usually develop after the dental problems, but the precise age of onset is often difficult to define. Initial manifestations of skeletal involvement include pathological fracture^,^^.'^' poor healing of fracture^,^^^^^^^^^ back pain,”,1o4 and a~-thralgias*~*~~~ or frank a r t h r i t i ~ . ’The ~ * ~latter ~ ~ problem may be attributable to pathological calcification in the joints (see below). Subjects are often of short ~ t a t ~ r e ~ ~ . ~ ~ , ~ ~ . and, as noted above, this may result from growth retardation during childhood. However, there are also reports of adults who exhibited considerable loss of height from fractures and deformity over several year^.^^,^ Unfortunately, the relative lack of long-term studies of adults with hypophosphatasia precludes an assessment of the frequency of this height loss. Physical examination sometimes reveals tender sites over the long bones or rib^.^^,^^,^^^Fractures are frequently detected in these regions. Various skeletal deformities have been observed, but bowing of the long bones appears to be the most common abnormality.76.B9.94.w.103 This and/or poor healing of fractures can result in shortening of the legs.76.94-’w Reported thoracic changes include symetrical or asymetrical depressions of the ribs ,89*109 protrusion of the sternum,89and wideningB9and beading of the costochondral junction.Io3 S ~ o l i o s i slumbar , ~ ~ l o r d o ~ i s , and ~ ~ thoracic ~ ~ ~ ~kyphosisIw ’~ have been observed occasionally. Skeletal deformities may contribute to the development of pain and/or physical disability, and can appear to Restricted mobility of the pine,^^^^ hip^^.'^^ and wrists,IM and joint crepitation^^.^^-'^ have been observed on physical examination. Neuromuscular function has been reported to be normal in a few subject^,^^^^^^^^'^ but one subject has been described who developed a peripheral motor neuropathy .93 Symptoms were partially relieved by surgical decompression of the spine, but it is unclear whether spinal deformity wholly accounted for the neuropathy. The skull is sometimes small, indicative of premature closure of the sutures in i n f a n ~ y .Eye ~ . ~changes ~ such as pallor of the optic discs and proptosisS9*” are present occasionally, and may imply previous elevation of intracranial pressure. Radiographs, can show evidence of both defective skeletal mineralization and pathological calcification. There is frequently generalized ~ ~ t e ~ p e n iwhich a ,may ~ be ~ quan~ ~ ~ ~ ~ ~ . titated by bone densit~metry.%*~’ However, in a small number of subjects, radiographs only reveal pathological calcification, and there is no evidence of ~ s t e o p e n i a . ~ ~ ~ ~ * ’ ~ ~ . ” ~ Skeletal deformities are often apparent on radiographs. Bowing of the long bones is relatively common,76~98*109~’13 and there is sometimes periosteal bone formation along the cortices that appears to thicken the In two subjects, the ends of some of the long bones were reportedly misshapen, and this was attributed to a failure of r e s ~ r p t i o n . ~ ~ , ~ ~

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Spurs over the long bones were observed in one subject who developed skeletal deformities during childhood.” The ribs may be poorly m i n e r a l i ~ e d ’ ~andtor ~ , ’ ~ widened at their anterior end^.^^.^^^ Spinal abnormalities are detected fairly frequently and include s c o l i ~ s i s , ~ ~ . ’ ~ l ~ r d o s i s ,d’ ~e f ~ r m i t y ~ . ~ or ~ .c’o~m . “p~r e ~ s i o nof~the ~ ~vertebrae, ~~~ degenerative changes,97 and formation of spurs and o ~ t e o p h y t e s . ~Mineralization ~ ~ ~ ~ ~ ’ ~ ~ of ~ ’the ~ spine is often subnormal, but the extent of the decrease varies.99~’03~’06*fo8 In the pelvis, undermineralization and coarse trabeculae have been observed occasionally on radiograph^.^^.^^."' The hands may exhibit variable hypomineralization,108and more frequently a decrease in the width of the metacarpal The skull is sometimes small or m i s ~ h a p e n ,probably ~ ~ , ~ ~ as a result of premature closure of the sutures in infancy, and there may be patchy hypomineralization .76.89.97 Pathological calcification may be e x t e n s i ~ e ’ ~ . ~or~ .restricted ~ ~ . ’ ~ ~to one or two ~ i t e s . ~ ~ . In ~ the . ~ spine, ~ . ~ calcification ~ ~ . ” ~ of the intervertebral discs (Figure 4)94.97.107 and of the ligaments1w.114 has been observed. Joints in which articular calcification commonly occurs include knees,92.96*97.106.’07 pubic s y m p h y s i ~ , ~ ~ . ”metacarpophalangeal *%*~~~ ,94.96.106.107 proximal and distal interphalangeal of the and w r i ~ t . ~In~ the * I ~knees, calcification may be present in both meniscal and articular cartilage,” or be restricted to the menisci.99 Periarticular calcifications have also been observed in several I * and there are reports of calcification of the triangular ligament of the and of the tendons around the knee. ‘07 Various arthritic changes have also been observed radiologically,94~96~106~112~114 and are frequently but not always associated with the presence of calcification (see below). The major skeletal symptoms of adult hypophosphatasia are pain, fractures, and arthntis. The explanation for the bone and joint pains can vary, and the precise cause is not always evident. Bone pain is often attributable to fracture^,^^.^^^^^.^^' and subjects sometimes complain of discomfort in an affected bone for several months before a pathological or stress fracture is apparent on r a d i ~ g r a p h s . ~I4 ~Back ~ ~ ~pain ~~~ . ’ common and can be asis ~fairly sociated with various spinal abnormalities. Spinal changes detected in subjects complaining of back pain include osteopenia and compression of several vertebrae,Ios mild compression of a few vertebrae and calcification of intervertebral “degeneration” of vertebrae,w marked osteophyte formation,96and ‘‘mild osteomalacia” .Io4 Joint pains are highly variable with respect to frequency of occurrence, duration, seventy, and location.92,94,99,“2.’14 Pain may be associated with crystal deposition and/or osteoarthritis. Fractures and pseudofractures are a common skeletal problem in adult hypophosphatasia. As the disease evolves there appears to be both an increased tendency for fractures to occur, with or without and a marked slowing in fracture healing.89~’02.103-111.’13 Interestingly, subjects with pathological and/or poorly healed fractures sometimes give a history of earlier traumatic fractures that healed n ~ r m a l l y . ~ ~However, . l ~ l - ~ ~even ~ in early adulthood, these subjects may have been more fracture-prone. Sequential radiographs sometimes demonstrate progression of pseudofractures. For example, a series of radiographs from two subjects with multiple pseudofractures revealed several that started in the cortex and then slowly extended across the bone. Other incomplete fractures apparently remained stable for years (Figure 5 ) , although some ultimately progressed to completion. Healing was noted to have occurred only when a fracture had first progressed to c ~ m p l e t i o n . ~ Bone “ ~ ~scintigraphy frequently, but not always, reveals increased uptake of tracer at sites of fract u r e ~ . ~ ~However, ~ ~ ~radiographs ~ ~ ~ ~may ~ reveal ~ ~ m’ i ~ ~~i m’ aor’l ~a~b, ~n ~ r m a l *callus ~.~~ formation several months after a break has occurred, thereby demonstrating that healing is retarded. Poor healing of fractures in adult hypophosphatasia is an important cause of physical disability and can result in subjects becoming w h e e l c h a i r - b o ~ n d . ~ ~ ~ ~ ~ ~ ~ Arthritis is relatively common in adults with hypophosphatasia, but it is not always clear whether it is attributable to hypophosphatasia in a particular individual. As noted above,

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FIGURE 4. Pathological calcification in adult hypophosphatasia. A radiograph of the spine of a female aged 56 years showing calcification (arrows) of the nucleus pulposus of several intervertebral discs. Calcific deposits of this type may comprise CPPD (see below). The vertebrae are severely osteopenic.

joint problems, ranging from vague a r t h r a l g i a ~ ’to~ ~recurrent bouts of inflammatory polyarthriti~,’~’ are the first symptom of hypophosphatasia in a significant number of adult subjects. A few subjects reportedly exhibit arthritic problems a l ~ n e , ’ ~ ~but * ’ will ~ ’ possibly develop fractures later, since some other subjects with skeletal disease initially manifested only joint problems.89.92.96.w Calcification of articular and fibrocartilage and sometimes of periarticular tissues may be evident on radiographs of affected joints,w~92~96~10s~’07.1’z but its relationship to the arthritic changes is difficult to define because there is considerable variation in the latter. Symptoms in joints where there is evidence of calcification range from pain, stiffness, and crepitation^^^.^^^^^^'^' to inflammatory episodes. The latter were described as acute, mono-

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FIGURE 5. Pseudofractures in adult hypophosphatasia. Sequential radiographs of the proximal left femur of a female subject showing a pseudofracture in the lateral aspect of the cortical bone that remained unhealed for at least 10 years. Left, subject aged 52 years; right, subject aged 56 years.

articular, and self-limiting in two affected s ~ b j e c t s ,but ~ ~in~ another '~ individual there was multiple joint involvement and persistence of mild inflammation between attacks. lo' Similarly, the extent of joint damage evident on physical or radiological examination is variable. Reported abnormalities include crepitation^,^^.^^^'^^ restricted mobility and contractures,90*wJ"fraying and erosion of the joint surfaces,'12destruction of the metacarpophalangeal and radioscaphoid and Heberden's nodes. '06 Calcification of cartilage an& or of periarticular tissues and arthritis are not always associated in adult hypophosphatasia, as there are reports of individuals who exhibit only a r t h T i t i ~ , ~ ~ ,or ~ c~a*l c~i ~f i~~ a' ~t i ~o n, ~ "I l o~ . ~ ~ , in an affected joint. However, arthritis that occurs in the absence of calcification may not be attributable to hypophosphatasia, and subjects with calcification alone may subsequently develop athritis. The highly variable nature of adult hypophosphatasia makes it difficult to assess a given subject's prognosis. The redevelopment of symptoms in adults with a history of infantile or childhood h y p o p h o ~ p h a t a s i a , ~and ~ ~ ~ evidence ~-~~ that biochemical abnormalities persist throughout the symptom-free imply a relationship between aging and the onset or reappearance and the subsequent progression of the disease in adults. The apparent increase with age in both the frequency of fractures, with or without t r a ~ m a , ~ ~ , 'and ~ ' . 'problems '~ of fracture healing,89~'02~'03~'''~''3 also implies that age-dependent factors influence the pathogenesis of adult hypophosphatasia. However, it would appear that this relationship is not a simple one because the majority of subjects reported in the literature developed skeletal or joint problems between the third and sixth decades of life and few, if any, first developed symptoms after the age of 60. A possible explanation for the apparent association of adult hypophosphatasia with aging is that age-related skeletal changes are superimposed on abnormalities that result from the lack of TNSALP activity, and the combined effect of these two processes causes the increased susceptibility to fractures, etc. This appears to be supported by the fact that more than 75% of adult hypophosphatasia subjects reported in the literature were women, and women are more prone to develop osteopenia in later life than men. Moreover, the other forms of hypophosphatasia do not appear to be influenced by gender.

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Progression of the disease may result in loss of height97.99and the development or worsening of skeletal d e f o r m i t i e ~However, .~~ “remissions” have also been reported, as in one subject who spontaneously improved on a number of occasions with healing of fractures, etc. yet subsequently relapsed months or years later. Clinical changes in this subject were correlated with changes in serum ALP activity, but the cause of the biochemical and clinical remissions was not identified. lo’ There are a few reports of subjects who developed renal stones,1m*1’5 but this association may be a coincidence or result from inappropriate medical therapy (see below). One subject died of renal failure at the age of 23, but had exhibited symptoms of hypophosphatasia in infancy and was never entirely sympt~m-free.~~

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111. HISTOLOGY A. Skeletal Histology 1. Perinatal and infantile Hypophosphaiasia Skeletal histological findings are similar in perinatal and infantile hypophosphatasia, and these forms of the disease will be considered together. It should be noted, however, that the majoricy of samples examined from subjects with infantile hypophosphatasia have been obtained postmortem. The abnormalities are most severe in fatal cases of infantile hypophosphatasia, and milder in affected survivors. Both endochondral and membranous ossification are greatly disrupted in the perinatal and severe infantile forms of the disease. At sites of endochondral ossification, there are usually abnormalities in both the cartilaginous and the osseous regions (Plate 1*). However, changes were reportedly restricted to the osseous regions in samples from three fetuses aged less than 30 weeks gestation. 19,22,116 Cartilage changes may result in part from a disruption of the normal progression of cartilage to bone because of the failure of mineralization. The hypertrophic and calcified zones of the cartilaginous growth plate are usually severely affected, whereas the resting and proliferative zones may be relatively or only moderately widened and d i ~ r u p t e d . ~l6. ~ ~ . ’ Mineralization is profoundly d e c r e a ~ e d ~ - ~ ,I6 ~and, ~ , ~consequently, ~ * ~ ~ * ~ ~ . the ’ calcified zone sometimes appears irregular, ill defined, or even absent .7.43 Conversely, the hypertrophic zone may be dramatically ~ i d e n e d , ~ . ’and ~ . ”the ~ columnar arrangement of the chondrocytes is frequently distorted as in other forms of r i c k e t ~ . ~ . ~ ~Areas . ~ ~ .of~unmineralized ~ . ~ ~ . ~ ~ cartilage are often present adjacent to the osseous regions of the metaphysis, and may be or completely surrounded by contiguous with the growth plate (projection~),~~~.”.’~,~~*~~.’~~ l6 osseous material. In the osseous regions, trabecular abnormalities are usually present in both the metaphyses and the diaphyses. However, occasionally only the metaphyseal regions are affected, possibly reflecting a milder form of the disease or recent worsening of the mineralization d e f e ~ t . ~ ~ . ” ~ Trabecular abnormalities include ~ i d e n i n g ; ~ . distortion, ’ ~ . ~ ~ * ~sometimes ~ caused by infrac’ ~ - ’ ~mineralization . ~ ~ ~ ~ ~ with a consequent abundance of tions or callus f o r m a t i ~ n ; ’ ~ ~ ~reduced OSte~id;9,15.19.22.43.48and foci of uncalcified cartilage. 11.18.19.38.43.1 16 There is also evidence of a failure of mineralization at sites of membranous ossification. For example, histological examination of the skull has revealed either a total absence of mineral’.” or, interestingly, the presence of only one or two small calcified plaque^.^.^ Calcium could not be detected elsewhere in the ~ s t e o i d .Although ~.~ there is an apparent abundance of unmineralized osteoid because of the lack of mineral, the gross architecture Excess unmineralized osteoid has of the skull has been reported to be relatively nor~nal.~.’~ also been observed in subperiosteal regions of the long bones and in the rib^.^.^.'^.^' With respect to the cellular elements, lack of information about bone and cartilage from 7~1432354,1

* Color plates for this article appear after page number 194

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normal infants renders it somewhat difficult to interpret findings in pennatal and infantile hypophosphatasia. Moreover, for growth plate cartilage, the problem of the interpretation of both cell numbers and cell morphology in these forms of the disease is exacerbated by the disruption of the progression of cartilage to bone, and the morphology of the chondrocytes appears to vary between subjects. In samples of growth plate cartilage from five individuals with perinatallinfantile hypophosphatasia, the chondrocytes were considered to be morphologically normal in all regions except the hypertrophic zone, where they were smaller and contained less glycogen.’I6 However, in tissue from two other subjects with the infantile form of the disease, the cells were described as “swollen and ~acuolated”.~’There may also be regional variations in the rate of degeneration of the chondrocytes in severe forms of hypophosphatasia, since in samples from five affected subjects, breakdown of cells appeared to be accelerated in all regions except the lower hypertrophic zone, where it was retarded.’I6 Moreover, in one study, chondrocytes were observed in foci of cartilage deep within the osseous region^,'^ which again suggests that the cells do not degenerate normally in the final stages of the differentiation of growth plate cartilage. Osteoblasts of normal morphology and in appropriate numbers have been observed in most studies of bone from various sites from subjects with severe forms of hypophosphatasia,6~’1~20~43.1’7 but in a few samples of cranial and metaphyseal bone their number and activity were reduced. 10.47.1 18.1‘9 Findings in two other studies imply that osteobtast number and activity are selectively reduced in osseous regions adjacent to abnormal cartilage projection~.~.” Studies of osteoclast number and their apparent activity in severe forms of hypophosphatasia have yielded extremely variable results. ’ There was evidence of active resorption in some samples,’6.20but in others remodeling appeared to be abnormal.54 There is very little information about osteocyte number and morphology in severe forms of hypophosphatasia, but available data suggest that these are both normal .6*20.11y Matrix vesicles are extracellular structures that are derived from the plasma membrane of chondrocytes and are believed to promote initial mineral formation at sites of endochondral ossification.120In two studies of subjects with severe hypophosphatasia, a normal distribution of matrix vesicles was observed in growth plate cartilage, and some were reportedly present in adjacent regions of unmineralized osteoid. ‘16.1l7 However, few matrix vesicles contained hydroxyapatite crystal^,'^^*"^ although some free crystals were detected in the cartilage and bone matrices. Such crystals appeared to be developing abnormally. Aggregates of crystals were rare and tended to be Development of both marrow cavities and capillaries has also not been well studied in severe forms of hypophosphatasia and, again, results are inconsistent, probably because of variable disruption of cartilage and bone structure. Marrow cavities were of normal width in one bone sample,38but were narrow in several other s p e c i r n e n ~and , ~ ~in~at~ least ~ ~ ~one of these narrowing was attributable to the presence of uncalcified cartilage in the marrow spaces.3yActive marrow formation has sometimes been observed.I ’ There was evidence of ingrowth of capillaries in the chondroosseous regions in some s a m p l e ~ , ’but ~ . ~in~others the process appeared to be d i s r ~ p t e d . ~ . ’ ~ .15,20*43947s4

2. Childhood Hypophosphatasia Skeletal histology is rarely reported for this form of hypophosphatasia. Understandably, autopsy studies have not been carried out, and biopsies of bone tissue have seldom been performed. A specimen obtained from a subject with only mild skeletal disease contained excess unmineralized osteoid, but was otherwise normal with respect to the structure of the growth plate cartilage and bone, remodeling activity, the rate of mineralization (measured by labeling in vivo with tetracycline), and the number and morphology of osteoblasts, osteoclasts, and matrix vesicles. ‘I7 Bone specimens obtained from two more severely affected children contained relatively larger amounts of unmineralized osteoid, and dynamic mea-

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surements of the rate of mineralization based upon the uptake of tetracycline also gave evidence of a reduction in bone formation.68.'I 7 In addition, these samples contained woven bone, and, in one,68areas of cartilage remained in the osseous regions adjacent to the growth plate cartilage. In the other sample, many "active" appearing osteoblasts were present in regions of cortical bone, but only a few "inactive" appearing osteoblasts were noted on the trabecular bone surfaces. 'I7

3. Adult Hypophosphatasia Skeletal histological findings vary markedly, which is not unexpected in view of the variable clinical seventy of this form of the disease. For example, biopsy samples obtained from adults with odontohypophosphatasia were only slightly abnormal,' l 7 whereas samples obtained postmortem from a severely affected adult who died at the age of 23 were grossly abnormal.92 Indeed, the abnormalities noted in the samples from this latter individual resembled those observed in the perinatal or infantile forms of the disease, especially at sites of endochondral ossification where there was evidence of a persistent disturbance in the transition of cartilage to bone, despite the subject's age. Growth plate cartilage was present in these samples, but the calcified zones were irregular or even absent, implying a failure of mineralization. Adjacent osseous regions contained areas of cartilage, either as isolated units or contiguous with the growth plate cartilage. Osseous regions also contained abundant unmineralized osteoid. Findings in bone samples from the majority of subjects with adult hypophosphatasia typically lie between these extremes. Trabecular bone volume is frequently norma1,94.96~107~17 but an increase has been observed o c ~ a s i o n a l l y . The ~ ~ ~most ' ~ common finding is an excessive amount of unmineralized osteoid (Plate 2 ) , consistent with a defect in mineralization, but the relative osteoid volume is increased to a variable degree. Reported values range from a few percent to as much as 75%.94~96~99~100~107 The magnitude of the increase reflects, to some extent, the clinical severity of the disease. For example, Fallon and c o - w o r k e r ~ ~ ' ~ observed that the relative osteoid volume was much greater in samples from subjects with skeletal problems than in samples from subjects with odontohypophosphatasia. The increase in the relative osteoid volume may be due to increases in osteoid seam width and/or in the Tetracycline labeling also reveals a mineralization defect area of osteoid of variable seventy. Findings range from minimal incorporation of to a relatively normal banding pattern. '07 The proportion of osteoid seams exhibiting no tetracycline uptake is often indicating that a significant proportion of the osteoid is not being actively mineralized. There is only limited information about the rate of overall bone turnover in adults with hypophosphatasia. Whyte and c o - ~ o r k e r sdid ~ ~not ~'~ detect woven bone or peritrabecular fibrous tissue in samples from three subjects. However, in specimens from other subjects, these workers96 observed considerable peritrabecular fibrosis, consistent with accelerated remodeling. With respect to the cellular elements of bone in adult hypophosphatasia, there is particular interest in osteoblasts because both their number and activity are frequently r e d u ~ e d . ~ ~ - % . ' ~ This could account for the mineralization defect and contribute to the reduction in serum ALP activity in this form of the disease, since approximately 45% of serum ALP activity in adults is bone (osteoblast) derived. 12' Indeed, a cellular dysfunction causing a decrease in the number of osteoblasts was considered as a possible cause of adult hypophosphatasia, '00 However, later studies have suggested that this is not the usual cause of adult hypophosphatasia, since the reduction in ALP activity is not normally found to be restricted to bone (see below). Moreover, similar cellular changes have been observed in the bone of adults with advanced osteomalacia,'22.123 which suggests that such abnormalities may be secondary to defective mineralization per se. There is also at least one report of a normal number of

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osteoblasts in a sample from an adult with hypophosphatasiaio’ and, despite a reduction in the number of active osteoblasts in some samples, those that are present may be morphologically The number of osteoclasts may be normal ,96.99.1n1 but is sometimes increased96 or decreased in adult hypophosphatasia. loo There is similar variability in the areas of both the osteoclastic resorbing surface94~96*1n7 and the inactive resorbing surface.96 An appropriate number of osteocytes of normal morphology was observed in one Matrix vesicles have not been detected in biopsy samples from the iliac crest from either subjects with adult hypophosphatasia or normal adult^.^^."^ There is one report of histological findings in a sample obtained from the site of an unhealed femoral fracture.In2The femoral cortex was vascular and thin, and the fracture callus contained considerable amounts of fibrous tissue and small trabeculae that did not appear to be maturing, remodeling or mineralizing normally. Histology of articular cartilage and synovium was studied postmortem in the severely affected adult described by Eade and c o - w o r k e r ~Deposits .~~ of CPPD and calcium oxalate crystals were identified in both tissues.

B. Dental Histology 1. Perinatal Hypophosphatasia

Detailed postmortem dental findings have been reported for one subject with perinatal hypophosphatasia. Cementum was almost totally absent, and there was little evidence of cementoblast activity. Dentine was also severely and variably reduced, and only thin layers of predentine and odontoblasts were present. Numbers of odontoblasts and the thickness of the predentine layer appeared to vary in parallel, suggesting that the reduction in the amount of predentine was attributable to decreased synthesis. In addition, there was no evidence of mineralization of predentine. Enamel was present on the tips of some cusps, but the total amount was markedly reduced. The periodontal spaces were wide, and the development of the periodontal membrane appeared to be abnormal. 2. Childhood Hypophosphatasia Several prematurely shed teeth have been examined histologically and almost always the most notable finding was total absence of or a profound reduction in cementum (Figure 6).55.58s9,71.72.77 Normal cementoblasts and precementum were present in some samples , 5 8 but one other sample contained only acellular c e m e n t ~ r nThere . ~ ~ is some evidence that, in the absence of cementum, the periodontal ligament is attached directly to the surface of the root dentine.59.77Root resorption often appears to be severely restricted and its distribution abnormal ,55.57.s8,78 but extensive resorption has been observed occasionally .61 Osteodentine has sometimes been detected in the apical regions of the root^;^^,^^,^^ its formation may be promoted by injury to the roots resulting from the excessive mobility of the teeth.59Dentine is also frequently abnormal. Changes include decreased t h i ~ k n e s s ~or* .variable ~ mineraliati ion,^^ increases in the size and decreases in the number of dentinal t ~ b u l e s , ~and ~~~’ increases in the amounts of predentine5*and interglobular dentine.5s.57,58 Enamel, by contrast, is usually relatively n~rmaI,’~~@-’ although scalloping of the dentinal/enamel junction was observed in one sample.55The pulp chamber is sometimes of normal size,78but is frequently enlarged.31,59m.72*77 The pulp may be abnormal and reported changes include fibrosis ,72 i n f l a m m a t i ~ n , and ~ ~ , ~death.78 ~ Secondary dentine or osteodentine can be present on the surface of the pulp In some teeth, the pulp chamber is lined with odontoblasts of normal r n ~ r p h o l o g y ,but ~ ~ there , ~ ~ is at least one report of a reduction in the number of odontoblasts. 58

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A

B

FIGURE 6. Aplasia of cementum in hypophosphatasia. (A) A decalcified section of a maxillary incisor from a child with X-linked hypophosphatemic rickets. This is essentially normal and shows the presence of cementum (arrows) between the dentine of the tooth root and the periodontal ligament. (B) A similar section of an incisor from a subject with childhood hypophosphatasia. This shows an absence of cementum. Part of the root surface is covered with plaque rather than the periodontal ligament.

IV. BIOCHEMICAL STUDIES

A. Alkaline Phosphatase 7. Activity in Serum All forms of hypophosphatasia, except pseudohypophosphatasia (see below) are characterized by low serum ALP activity,9~12~L5~23~24~3s~40~"~61~64~76~94~96~97~1 I ' but the magnitude of the reduction is not always correlated with the seventy of the clinical symptom^.^' There are also reports of subjects with infantile and childhood hypophosphatasia who exhibited considerable clinical improvement despite persistence of low serum ALP a ~ t i ~ i t y . An ~ ~ . ~ ~ , ~ ~ , inverse correlation between serum ALP activity and the clinical seventy of the disease over time has been described occasionally in single cases of adult hypophosphatasia, most notably in a subject reported by Eisenberg and Pimstone,108who exhibited alternating periods of remission and exacerbation of clinical symptoms. Similarly, Wendling and c o - w o r k e ~ s ~ ~ observed an inverse relationship between serum ALP activity and clinical symptoms in two adults with markedly different disease severity, but stated that this was somewhat unusual. Some lack of correlation between clinical seventy of hypophosphatasia and the magnitude of the reduction in serum ALP activity is not completely unexpected, because serum ALP is derived from many tissues, whereas the skeletal abnormalities probably result mainly from the lack of ALP activity in bone and cartilage.

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ALP activity in the serum of subjects with hypophosphatasemia can fluctuate somewhat, and a single measurement may not always reveal hypophosphatasemia. In adults with hypophosphatasia, transient increases in serum ALP activity, which sometimes result in values in the normal range, have been noted following fractures or bone grafting,’o’.’03.’m1” and during episodes of liver disease.10z.124 It is interesting that serum ALP activity also tends to increase in normal individuals in these ~ituations,’~’which implies that the mechanisms controlling TNSALP biosynthesis are operative in at least some subjects with hypophosphatasia. Marked variations in serum ALP activity have also been observed in children with hypopho~phatasia~~ and may be related to growth. In addition, there are reports of subjects with infantile hypophosphatasia in whom serum ALP activity was normal at birth and only decreased after several weeks or months.10.’26It was suggested that placental ALP was present in the circulation of these subjects during the perinatal period.’26 Finally, there is the report of an unusual adult with hypophosphatasia who exhibited marked and apparently spontaneous fluctuations in serum ALP activity over several years. ’08 Isoenzyme analysis consistently reveals a reduction in the activity of TNSALP in serum, but the relative amounts of this enzyme originating from liver and bone may differ between the various clinical forms of hypophosphatasia. It has been reported that bone-derived ALP activity is markedly reduced and liver-derived ALP activity is undetectable in sera from the majority of subjects with infantile or childhood hypophosphatasia. 10.6531,126.127 However, the findings are reversed in sera from the majority of subjects with the adult form of the di~ease.~’.’~’.’~’.’~~ Interestingly, sera from two adults who had previously exhibited childhood hypophosphatasia with subsequent resolution of symptoms gave the same pattern as described in children.Iz7This suggests that the differences between the various clinical forms of the disease may not simply be age related. Normal or slightly increased activity of intestinal ALP (IALP) has been detected in some sera from subjects with hypoph~sphatasia,~~’~~.’~~ but little or no activity of IALP has been found in other^.^^.'*^ However, lack of this isoenzyme of ALP is not necessarily attributable to hypophosphatasia, because there is significant activity of IALP in the circulation of only approximately 25% of normal individuals. There have been a few studies of the properties of serum ALP in hypophosphatasia. Mixing of sera from affected and normal individuals suggests that the reduction in ALP activity in hypophosphatasia is not attributable to the presence of an inhibitor or to the There are conflicting reports of the effect of the addition absence of an of divalent cations in vitro or in vivo. Birtwell and c o - w o r k e r ~ observed ’~~ a twofold increase in ALP activity on adding magnesium in virro to the serum of an adult with hypophosphatasia, and Eisenberg and PimstoneIo8 observed that intravenous infusion of magnesium resulted in a transient increase in serum ALP activity in their subject during a period of exacerbation of the disease, but not during a period of clinical remission. However, in a study of a different subject, magnesium was without effect when administered in vitro or in vivo, and zinc was also without effect when added in vitro.‘O0 These divergent findings may indicate that there are different molecular defects of TNSALP in individual subjects. The occurrence of variants of TNSALP with altered biochemical or physical properties is also implied by a study in which a form of ALP with some characteristics of TNSALP, but with increased sensitivity to inhibition by L-phenylalanine, was detected in the serum of a subject with childhood hypophosphatasia.a Furthermore, bone-derived ALP in the serum of a subject with infantile hypophosphatasia was reported to migrate abnormally slowly on electrophoresis.’26 These abnormal enzymes partially resemble IALP, and it has been suggested that IALP is induced in certain tissues to compensate for the lack of TNSALP.28 However, if these variants are indeed products of the gene for IALP, they must be modified differently from normal IALP after translation, e.g., in the pattern of glycosylation. There was evidence for the induction of normal IALP in a subject with childhood hypophosphatasia

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Critical Reviews in Clinical Laboratory Sciences

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aged 18 months in whom there was an increase in serum IALP activity, but the tissue(s) of origin was not identified.Iz7 With respect to other enzyme activities that have been attributed to ALP, there have been relatively few studies using serum from subjects with hypophosphatasia. However, a reduction in serum inorganic pyrophosphatase activity has been observed in some subjects with childhood and adult hypophosphatasia. Io6.I3I 2. Activity in Leukocytes ALP activity in leukocytes is usually reduced or absent in subjects with perinatal or infantile h y p o p h o s p h a t a ~ i a but , ~ in ~ ~subjects ~ ~ ~ ~with ~ ~ the ~ ~ childhood ~ ~ ~ ~ ~ or adult forms of the disease it may be no~a164.75.94.~05.108 or redUced.60.61.74,83.94.102.103.109 Interestingly, histochemical measurements in leukocytes from a subject with adult hypophosphatasia revealed good ALP activity in approximately 50% of the cells, but no ALP activity in the remaining cells.'08 There are a few reports of isoenzyme analysis in leukocytes with reduced ALP activity. TNSALP alone was present in leukocytes from one subject,13* but a mixture of TNSALP and IALP was present in leukocytes from another. 133 This implies that leukocytes from some subjects with hypophosphatasia express additional forms of ALP, possibly induced to compensate for the lack of TNSALP activity. However, there are also conflicting results with respect to which isoenzyme is present in leukocytes from normal individuals,'34-'37and, although leukocytes usually express only TNSALP, a different isoenzyme may be present in leukocytes from a small minority of the population. Thus, subjects with hypophosphatasia who express normal ALP activity in leukocytes may be members of this latter group, Clearly, there needs to be further characterization of ALP in leukocytes with both normal and reduced activity from subjects with hypophosphatasia.

3. Activity in Tissues Measurements of ALP activity in tissues have been performed mainly in subjects with perinatal and infantile hypophosphatasia because of the availability of material postmortem, but findings appear to be similar in all forms of the disease. Bone ALP activity, measured biochemically or histochemically, is almost always r e d ~ ~ e d . " O , ~ ~ ALP - ' ~activity ~ . ~ ~is~ , ~ ~ ~ , ~ ~ ~ particularly low in bone from subjects with perinatal h y p o p h o s p h a t a ~ i a , which ~~~~~*~~~~~~ correlates with the clinical severity of this form of the disease. In adults, the magnitude of the reduction in ALP activity is more variable, but, again, there is some correlation with the seventy of the symptoms. For example, ALP activity was very low in bone from a severely affected adult,9zwhereas it was almost normal in bone from a subject who exhibited arthritis but no skeletal problems.107In addition, Fallon and co-workers'17reported a negative correlation between the severity of osteomalacia, assessed by the relative osteoid volume, and ALP activity in samples of bone from a fairly large group of adults with hypophosphatasia. The distribution of ALP activity in trabecular bone has been studied, but interpretation of the findings is hampered by the small number of reports. ALP activity was present only on limited areas of the trabecular bone surface in individuals with adult hypophosphatasia, 117,139and in a subject with childhood hypophosphatasia. 'I7 In the latter, ALP activity appeared to be associated with sites that were lined with active osteoblasts. I I7 However, no ALP activity was detected on osteoblasts in bone from a few subjects with adult hypophosphatasia, or in autopsy material from subjects with infantile hypophosphatasia (Plate 3).'17 Matrix vesicles in bone samples from the latter subjects were also devoid of ALP activity. Markedly reduced ALP activity has been detected in growth plate cartilage in perinatal and infantile h y p o p h o s p h a t a ~ i a ~ and . ~ ~ in ~ ~ clinically ~.~~ severe adult hypopho~phatasia.~' ALP activity in the liver, measured biochemically or histochemically , is also usually severely r e d ~ ~ e d . ~ ~ . In ~ ~the. kidneys, ~ ~ ~ ~biochemical ~ ~ ~ ~ ~measurements ~ ~ ~ ' ~ ~frequently . ' ~ ~

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PLATE 1. Histology of bone and growth plate cartilage in infantile hypophosphatasia.Rachtic changes, characterized by disorganization of the columnar pattern of proliferating chondrocytes (arrows), are present in this autopsy specimen of a growth plate. Defective skeletal mineralizationis also evident from the marked accumulation of unmineralized osteoid (red stain) that covers the mineralized bone (green stain) of the primary spongiosa. (Goldner stain, magnification x 100.)

Hypophosphatasia and the extracellular metabolism of inorganic pyrophosphate: clinical and laboratory aspects.

Hypophosphatasia is a rare inherited disorder in which the activity of the bone/liver/kidney or tissue nonspecific form of alkaline phosphatase (ALP) ...
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