In 1948, J.C. Rathbun, a Canadian pediatrician, coined the term hypophosphatasia when he described an infant boy who developed and then died from severe rickets, weight loss, and seizures, yet whose ALP activity in serum, bone, and other tissues was paradoxically subnormal.44 Several historical reviews mention case reports published earlier that probably depicted this condition.45,46 In 1953, premature loss of deciduous teeth was noted to be a second major clinical feature of hypophosphatasia.47 About 350 patients have been described in the medical literature.
Discoveries of elevated levels endogenously of three phosphocompounds (Fig. 207-1) clarified the metabolic basis for hypophosphatasia and the physiological role of TNSALP. In 1955, increased urinary concentrations of phosphoethanolamine (PEA)48,49 provided a useful biochemical marker for the disorder. In 1965 and 1971, high levels of PPi were noted in urine50 and in blood,38 respectively, suggesting a mechanism for the defective mineralization of hard tissues. In 1985, elevated plasma levels of pyridoxal 5′-phosphate (PLP) were found—an observation that indicated an ectoenzyme action for TNSALP51 (see below). In 1988, the structure of the TNSALP gene was characterized12 and the first molecular defect was discovered in hypophosphatasia.52 .
Natural substrates for TNSALP. Three phosphocompounds appear to be natural substrates for TNSALP, because each accumulates endogenously in hypophosphatasia: inorganic pyrophosphate (PPi), phosphoethanolamine (PEA), and pyridoxal 5′-phosphate (PLP).
Hypophosphatasia occurs throughout the world. However, the disorder is especially prevalent in inbred Mennonite families from Manitoba, Canada, where 1 in 2500 newborns manifests severe disease and approximately 1 in 25 individuals is a carrier.53 The incidence of severe forms in Toronto, Canada was estimated in 1957 to be 1 per 100,000 live births.46
Despite the presence of relatively high levels of TNSALP in bone, liver, kidney, and adrenal tissue in healthy individuals (and at least some TNSALP throughout the body), the clinical impact of hypophosphatasia predominantly disturbs the skeleton and dentition. However, the severity of expression is remarkably variable and ranges from death in utero to mere problems with dentition in adult life.46,54-57 In fact, some individuals who demonstrate characteristic biochemical abnormalities may never become symptomatic.55,56 Although hypophosphatasia generally breeds true within sibships, significantly variable clinical expression can occur in this setting as well.55,56,58,59 Because the genetic basis for hypophosphatasia is being uncovered (see later),15 there is promise for a molecular nosology in the future. Nevertheless, the current classification of patients for prognostication, recurrence risk estimates, and so on remains a clinical one. Several schemes have been proposed that attempt to deal with the disorder's remarkably variable expression.46,54 Six clinical forms constitute a useful separation. The age at which lesions in bone are discovered distinguish the perinatal (lethal), infantile, childhood, and adult forms.46,57 Patients who have only dental manifestations are regarded as having odontohypophosphatasia. An especially rare variant called pseudohypophosphatasia resembles infantile hypophosphatasia, except that serum ALP activity is not subnormal in the clinical laboratory (discussed below). The prognoses for these six forms of hypophosphatasia generally reflect the severity of the skeletal disease which, in turn, corresponds with the age at presentation. Usually, the younger a patient becomes symptomatic, the more severe the disorder.33,43,46 Although this nosology is useful, there is considerable variability within each clinical form and no clear-cut separations between them.
Perinatal (Lethal) Hypophosphatasia.
Perinatal hypophosphatasia is the most severe form. It is expressed in utero and can result in stillbirth. The pregnancy may be complicated by polyhydramnios. Caput membranaceum and limbs that are shortened and deformed from profound skeletal hypomineralization are noted at birth. Unusual osteochondral spurs may protrude through the skin from the midportion of the forearms and legs.60,61 Some affected neonates live a few days, but then suffer increasing respiratory compromise from rachitic defects in the chest and from hypoplastic lungs.62 Clinical findings also include failure to gain weight and often a high-pitched cry, irritability, periodic apnea with cyanosis and bradycardia, unexplained fever, myelophthisic anemia (perhaps from encroachment on the marrow space by excess osteoid), intracranial hemorrhage, and seizures.54,57 Very rarely there is prolonged survival.63
Radiographic study of the skeleton enables perinatal hypophosphatasia to be distinguished from even the most severe types of osteogenesis imperfecta and other forms of congenital dwarfism. Indeed, the radiographic changes may be considered diagnostic.60,61 Nevertheless, the findings can be diverse and there is marked patient-to-patient variability.61 In some cases, the skeleton appears to be almost completely unmineralized (Fig. 207-2). In others, there is marked bony undermineralization and severe rachitic changes including irregular extensions of radiolucency into the metaphyses together with poorly ossified epiphyses. Fractures are often present. The individual membranous bones of the cranium may show calcification only at their center portions, so that the areas of unossified skull give the illusion that the cranial sutures are widely separated. However, these sutures can be functionally closed.60 The teeth are poorly formed.61 Other unusual features include parts of (or entire) vertebrae that appear to be missing and bony spurs that protrude laterally from the midshaft of the ulnae and fibulae.64
Perinatal hypophosphatasia. Profound hypomineralization is obvious (an umbilical clip [arrow] is more dense than the skeleton). The ends of the long bones in the upper limbs show severe rachitic change. Reproduced with permission from Whyte MP. Hypophosphatasia: Nature's window on alkaline phosphatase function in man, in Bilezikian J, Raisz L, Rodan G (eds.): Principles of Bone Biology. San Diego, Academic Press, p. 951, 1996.
Infantile hypophosphatasia presents before 6 months of age.46 Postnatal development often seems normal until the onset of poor feeding, inadequate weight gain, and clinical signs of rickets. The cranial sutures feel wide, but the ossification defects in the skull can cause a “functional” craniosynostosis. There may be raised intracranial pressure, with bulging of the anterior fontanel, papilledema, proptosis, mild hypertelorism, and brachycephaly. True premature fusion of the cranial sutures may occur if the patient survives infancy.60 Blue sclerae have been reported.65 A flail chest from rachitic deformity or rib fractures may predispose the infant to pneumonia. Hypercalcemia and hypercalciuria are common and can cause recurrent vomiting, nephrocalcinosis, and renal compromise.46,66,67
The radiographic features of infantile hypophosphatasia are characteristic and resemble those of the perinatal form, although they are less severe.60 In some newly diagnosed patients, there is a seemingly abrupt transition from a normal-appearing diaphysis to a poorly calcified metaphysis. This finding is of interest because it suggests that a pathophysiological change suddenly occurred.46 Sequential radiographic studies may disclose not only the persistent defective skeletal mineralization typical of rickets, but gradual demineralization of osseous tissue as well.67 Skeletal scintigraphy can help demonstrate functional closure of cranial sutures, because these structures exhibit decreased tracer uptake although they appear “widened” on conventional radiography.68
Childhood hypophosphatasia is also highly variable in its clinical expression.46,62,69 Premature loss of deciduous teeth (i.e., earlier than 5 years of age) occurs with only minimal tooth root resorption because of aplasia, hypoplasia, or dysplasia of dental cementum.70,71 Less likely, destruction of cementum has been caused by periodontal infection.72 The lower incisors are typically lost first, and occasionally nearly the entire dentition is exfoliated. Dental radiography may show enlarged pulp chambers and root canals (“shell teeth”). Alveolar bone attrition, especially in the anterior mandible, can occur from lack of mechanical stimulation because defects in cementum prevent periodontal ligaments from properly connecting the teeth to the jaw.73 The prognosis for the permanent dentition is generally better.74
In childhood hypophosphatasia, rickets often causes short stature and is associated with delayed walking and a characteristic waddling gait.46,66 Rachitic deformities include beading of the costochondral junctions; either bowed legs or knock-knees; enlargement of the wrists, knees, and ankles from flared metaphyses; and, occasionally, a brachycephalic skull. Patients may complain of pain and stiffness, as well as isolated episodes of joint discomfort and swelling. They can have significant muscle weakness in their extremities (especially the thighs) consistent with a nonprogressive myopathy.75
Radiography of the metaphyses of major long bones usually reveals characteristic focal bony defects—“tongues” of radiolucency that project from growth plates into metaphyses (Fig. 207-3). This feature, if present, distinguishes hypophosphatasia from other forms of rickets and metaphyseal dysplasias.60 Epiphyseal centers of ossification may be well preserved. Functional craniosynostosis can occur in affected infants and young children despite widely “open” fontanels that are an illusion caused by hypomineralized areas of calvarium. Later, true premature bony fusion of cranial sutures may cause raised intracranial pressure, proptosis, and cerebral damage. The skull can then have a “beaten-copper” appearance.
Childhood hypophosphatasia. Posteroanterior view of the knee of a 5-year-old boy with hypophosphatasia shows growth plates that are not greatly widened, but defective endochondral bone formation is revealed by irregular radiolucencies (arrows) that project into the metaphyses. This finding is characteristic of the childhood form of hypophosphatasia.
Adult hypophosphatasia usually presents during middle age.55,56 Not infrequently, however, patients recount a history of rickets and premature loss of deciduous teeth followed by relatively good health. Subsequently, osteomalacia manifests with pain in the feet due to recurrent, poorly healing metatarsal stress fractures and discomfort in the thighs or hips due to femoral pseudofractures (Fig. 207-4). Early loss or extraction of the adult dentition is common.55,56,76 Calcium pyrophosphate dihydrate (CPPD) deposition, occasionally with attacks of arthritis (pseudogout), troubles some patients. Others may suffer from a PPi arthropathy. Apparently, these complications are due to increased endogenous levels of PPi (see below).56,77 Affected individuals may also be somewhat predisposed to develop primary hyperparathyroidism (personal observation). Screening studies often reveal symptomatic or asymptomatic family members.55,56 In some kindreds with hypophosphatasemia, there is periarticular calcium phosphate deposition that manifests clinically as “calcific periarthritis” and with ossification of ligaments (syndesmophytes) resembling spinal hyperostosis (Forestier disease).78,79
Adult hypophosphatasia. The femur of this middle-aged woman has a pseudofracture (Looser zone) that has been unhealed for several years (arrow). These cortical bone defects characteristically form on the lateral side of the femur in adult hypophosphatasia, rather than medially as in most other forms of osteomalacia.
Radiographic study may show pseudofractures (Looser zones), a hallmark of osteomalacia. Inexplicably, these defects occur most often in the lateral cortices of the proximal femora, rather than medially as in most other types of osteomalacia.80 There may also be osteopenia, chondrocalcinosis, features of pyrophosphate arthropathy, and perhaps calcific periarthritis.56,78,79
Odontohypophosphatasia is present when the only clinical abnormality is dental disease with radiographic and/or bone biopsy studies showing no evidence of rickets or osteomalacia. Odontohypophosphatasia may explain some cases of “early-onset periodontitis,”81 although hereditary leukocyte abnormalities and other disorders usually account for this condition.
Pseudohypophosphatasia is a particularly interesting, but especially rare, form of hypophosphatasia. The disorder was convincingly documented in two infants.82,83 In this unusual hypophosphatasia variant, the clinical, radiographic, and biochemical findings are like those of patients who have infantile hypophosphatasia, except serum ALP activity is consistently normal or increased in assays performed in the clinical laboratory.82,83 The enzymatic defect seems to involve a mutant TNSALP that either retains or has enhanced catalytic activity under the nonphysiological conditions of routine ALP assay procedures, but has diminished activity endogenously. As a consequence, PEA, PPi, and PLP accumulate (see below).84,85
Some reports of pseudohypophosphatasia are not convincing86-88 and seemingly describe individuals with hypophosphatasia for whom there had been transient normalization of serum ALP activity during fracture, illness, and so on, or, more likely, misinterpretation of reference ranges for serum ALP activity and/or overemphasis on the significance of a slightly elevated urinary PEA level (see below).
Hypophosphatasia can be diagnosed with confidence when a consistent clinical history, physical findings, and radiographic changes occur with serum ALP activity that is clearly and consistently subnormal. In general, the more severe the disease the lower the serum ALP activity appropriate for age (Fig. 207-5). Even patients with odontohypophosphatasia are distinguishable from healthy individuals by their hypophosphatasemia. In the perinatal and infantile forms, hypophosphatasemia is detectable in umbilical cord blood.66,89 In fact, in types of rickets or osteomalacia other than hypophosphatasia, serum ALP activity is typically increased.30 Nevertheless, a variety of diagnostic pitfalls must be avoided. First, blood must be collected correctly.90 Chelation of Mg2+ or Zn2+ by EDTA and other means will destroy ALP activity.1 Second, levels of serum ALP activity must be interpreted knowing that control values vary significantly depending on age and sex; for example, infants and children have considerably higher levels (due to a relative abundance of the bone isoform of TNSALP) compared to adults. Serum ALP activity is especially high during the growth spurt of adolescence, which occurs earlier in girls than in boys.1 Because the reference ranges cited by many clinical laboratories are, unfortunately, appropriate only for adults, some infants or children with hypophosphatasia are mistakenly judged to have normal enzyme activity, or perhaps pseudohypophosphatasia, when the higher pediatric reference range for ALP is not provided. Third, hypophosphatasemia may occur in hypothyroidism, starvation, scurvy, severe anemia, celiac disease, Wilson disease, hypomagnesemia, or Zn2+ deficiency and with exposure to certain drugs (glucocorticoids, chemotherapy, clofibrate, intoxication levels of vitamin D, or milk-alkali syndrome), as well as with radioactive heavy metal poisoning or massive transfusion of blood or plasma.90,91 However, each of these clinical situations should be readily apparent. Rarely, newborns with severe osteogenesis imperfecta can have low serum ALP activity.92 Finally, a few case reports of hypophosphatasia describe transient increases in serum ALP activity (probably the bone isoform of TNSALP) after fracture or orthopedic surgery.55 Theoretically, conditions that increase circulating activity of any type of ALP (e.g., pregnancy, liver disease) could mask a biochemical diagnosis of hypophosphatasia.66,93 Accordingly, in puzzling cases, documentation that serum ALP activity is low on more than one occasion during clinical stability seems advisable. Quantitation of ALP isoenzyme levels or TNSALP isoforms in serum may also be helpful. Assay of PEA, PPi, and PLP levels are especially important in uncertain situations.94
Serum ALP activity in hypophosphatasia. ALP activity in serum in normal children and normal adults (▴) and in 52 patients (•, ○) from 47 families with the various clinical forms of hypophosphatasia. Note the logarithmic scale. All assays were performed at the Metabolic Research Unit, Shriners Hospital for Children, St. Louis, MO.
In contrast to most types of rickets or osteomalacia, neither calcium nor Pi levels in serum are low in hypophosphatasia. In fact, hypercalciuria and hypercalcemia occur frequently in the infantile form of the disease.45,54,57 In childhood hypophosphatasia, severely affected patients can have hypercalciuria but without hypercalcemia. The pathogenesis of the calcium disturbance may involve defective uptake of mineral by a poorly developing skeleton. Circulating levels of the bioactive forms of vitamin D (25-hydroxyvitamin D and 1,25-dihydroxyvitamin D) and parathyroid hormone (PTH) are usually unremarkable.95,96 Several patients, however, reportedly had elevated serum PTH levels, but renal compromise from hypercalcemia with retention of immunoreactive PTH fragments may have been the explanation in the severe cases. Conversely, low circulating levels of PTH, possibly reflecting an abnormality in the Ca2+-PTH feedback system, have also been described.97
Patients with the childhood and adult forms of hypophosphatasia have serum Pi levels that are above the mean value for controls, and approximately 50 percent of patients are distinctly hyperphosphatemic. Enhanced renal reclamation of Pi (increased tubular maximum for Pi/glomerular filtration rate [TmP/GFR]) accounts for this finding.98 Conversely, especially rare hypophosphatasemic patients who are hypophosphatemic from renal Pi wasting have been reported.99,100
Routine Biochemical Studies.
Other standard laboratory tests, including serum parameters of liver or muscle function (e.g., bilirubin, aspartate aminotransferase, lactate dehydrogenase, creatine kinase, and aldolase), are typically unremarkable in all forms of hypophosphatasia. Increased levels of proline in blood and urine have been reported in a few patients, but the significance of this observation is not known.101 Acid phosphatase activity in serum is generally normal,102 but tartrate-resistant enzyme of the mononuclear/phagocyte type (band 5), possibly of osteoclast origin, has been elevated for more than a decade in the blood of one affected woman.103
Increased urinary phosphoethanolaminuria (PEA) levels support a diagnosis of hypophosphatasia,104 but the finding is not pathognomonic. Elevated PEA can occur in a variety of other disorders, including several metabolic bone diseases.105 Ideally, a 24-h urine collection is assayed and PEA excretion is “normalized” to creatinine content prior to interpretation. It is important for diagnosing mild cases of hypophosphatasia to recognize that PEA levels are conditioned by age, depend on diet, follow a circadian rhythm, and have been reported to be normal in several mildly affected individuals.54,106 The following reference ranges (micromoles of PEA per gram of urine creatinine) have been published: less than 15 years, 83 to 222; 15 to 30 years, 42 to 146; 31 to 41 years, 38 to 155; and over 45 years, 48 to 93.105
An increased plasma level of PLP is the most sensitive and specific TNSALP substrate marker for hypophosphatasia51,84,107 (Fig. 207-6). Even patients with odontohypophosphatasia manifest this finding.51 However, to exclude low-level false positive values, vitamin supplements must not be taken for 1 week before testing.107 In general, the more severely affected the patient, the greater the elevation in the plasma PLP level. Nevertheless, overlap occurs between the clinical subtypes. Assay of plasma PLP levels after oral challenge with pyridoxine hydrochloride distinguishes patients especially well, and is helpful for identifying Canadian Mennonite carriers of severe hypophosphatasia.108
Plasma PLP levels in hypophosphatasia. PLP levels in plasma in the clinical forms of hypophosphatasia (hatched area is the normal range for children and adults). Elevated levels are present in all 71 patients (representing 60 families). In general, the plasma PLP level reflects the disease severity. Note the logarithmic scale with some overlap between the clinical forms. (Assays performed courtesy of Dr. Stephen P. Coburn, Fort Wayne State Developmental Center, Fort Wayne, IN.)
Urinary PPi levels are increased in most hypophosphatasia patients,94 but are occasionally unremarkable in mildly affected individuals.39 Nevertheless, quantitation of urinary PPi levels has been reported to be a sensitive means for carrier detection.109 Unfortunately, assay of PPi remains a research technique.
Radiographic studies of the skeleton are diagnostic in perinatal hypophosphatasia (Fig. 207-2). In the infantile and childhood forms, characteristic abnormalities are usually demonstrated (Figs. 207-3 and 207-4) (see above). Bone scanning retains its utility for identifying fractures and may help to diagnose craniosynostosis.68
Abnormalities are observed primarily in the hard tissues. In severe cases, hypoplastic lungs have been found and extramedullary hematopoiesis is occasionally noted in the liver.29,69
In growth plates, rachitic changes are described29,69,110 where there is disruption of the normal columnar arrangement of chondrocytes, zones of provisional calcification are widened, and areas near degenerating cartilage cells fail to calcify. However, the cellular sources of the bone isoform of TNSALP (chondrocytes and osteoblasts), as well as their matrix vesicles, are present, although with reduced levels of TNSALP activity.29,69
In all but the mildest cases (odontohypophosphatasia),55 nondecalcified sections of bone reveal evidence of defective mineralization of the skeleton.29,69,110 However, features of secondary hyperparathyroidism, which occur from hypocalcemia in most other types of rickets or osteomalacia, are generally absent. Unmineralized matrix accumulates because osteoid does not calcify properly. Cranial “sutures” that seem to be widened are not fibrous tissue, but the result of this pathologic process.46 Impaired skeletal mineralization is confirmed when brief courses of a tetracycline are given orally prior to bone biopsy; fluorescence microscopy will fail to show characteristic fluorescent bands on bone surfaces where calcification should normally be occurring at “mineralization fronts.” Some questionable cases of pseudohypophosphatasia (with normal serum ALP activity, skeletal symptoms, dental caries, elevated urinary PEA levels, and excessive amounts of osteoid in bone specimens) lack this important information from tetracycline labeling.111 The severity of the mineralization defect in hypophosphatasia generally reflects the clinical outcome.69 In lethal cases, even the bony structures of the middle ear can be poorly ossified.112 Woven bone, a finding that can reflect either bone repair or defective skeletal formation, may be present.69
Unless histochemical studies of ALP activity are performed, the histopathologic changes of hypophosphatasia in the skeleton cannot be distinguished from most other forms of rickets or osteomalacia.69 The numbers and morphology of osteoblasts and osteoclasts, as well as the appearance of unmineralized osteoid, vary from patient to patient. The level of ALP activity in boneÂtissue correlates inversely with the degree of osteoid accumulation.69
Electron microscopy of bone from cases of perinatal hypophosphatasia has shown normal distribution of proteoglycan granules, collagen fibers, and matrix vesicles in the extracellular space.29,69 Matrix vesicles are deficient in ALP activity yet contain hydroxyapatite crystals.110 However, in the osteoid, only isolated or tiny groups of hydroxyapatite crystals (calcospherites), frequently not associated with matrix vesicles, have been observed.29,61,110 The significance of this observation is discussed later.
Premature loss of deciduous teeth occurs in a variety of diseases (including toxicities, metabolic errors, and malignancies).72 In hypophosphatasia, this complication is due to aplasia, hypoplasia, or dysplasia of cementum, despite the presence of cells that look like cementoblasts (Fig. 207-7).71,74,113 The cementum may be afibrillar.114 The magnitude of the defect varies from tooth to tooth, but generally reflects the severity of the skeletal disease. Incisors are the most vulnerable. Big pulp chambers suggest retarded dentinogenesis. Dentin tubules may be enlarged although reduced in number. The excessive width of predentin, increased amounts of interglobular dentin, and impaired calcification of cementum are analogous to the osteoidosis observed in bone. Conflicting reports concern whether the enamel is directly affected.72,113 The histopathologic changes found in the permanent teeth seem similar, but more mild, compared to those in the deciduous teeth.74,114 Desiccated teeth exfoliated years earlier may still be useful for microscopic examination.115
Dental histopathology in hypophosphatasia. A, Decalcified section of part of the root of a maxillary incisor from a child with X-linked hypophosphatemic rickets shows primary cementum (delineated by arrows) at the tooth surface. B, In hypophosphatasia, cementum is absent. Magnification ×150. PL = periodontal ligament; PQ = plaque; D = dentin.
Biochemical and Genetic Defect.
Early on, autopsy studies of perinatal and infantile cases elucidated the enzymatic defect causing hypophosphatasia, but they also pointed to its etiology. Profound deficiency of ALP activity was documented in liver, bone, and kidney, yet ALP activity was not diminished in intestine or in placenta (fetal trophoblast).116,117 This observation was consistent with results emerging from amino acid sequence analysis of ALPs purified from healthy human tissues,3,33 and indicated a defect that selectively diminished the catalytic activity of all of the secondary isoforms of the TNSALP isoenzyme family.
Investigation of the cardinal biochemical feature of hypophosphatasia, hypophosphatasemia, supports the autopsy studies. There is deficient activity of both the liver and the bone isoform of TNSALP in serum.24 However, the hypophosphatasemia does not seem to be due to enhanced clearance of TNSALP from the circulation.55,118 The bone isoform of TNSALP (enriched in plasma from patients with Paget bone disease) and purified placental ALP have unremarkable circulating half-lives when given intravenously to severely affected infants with hypophosphatasia during attempted enzyme-replacement therapy (see below).67 Furthermore, coincubation experiments with mixtures of serum as well as cell coculture and heterokaryon studies using fibroblasts from severely affected patients have excluded the presence of an inhibitor or the absence of an activator of TNSALP.46,55,77,119 Instead, the hypophosphatasemia of hypophosphatasia appears to reflect failure of especially liver and bone tissue to contribute adequate amounts of TNSALP activity into the circulation. Leukocyte ALP activity, first noted to be absent in an adult with hypophosphatasia,120 is a mixture of the TNSALP and the placental ALP isoenzymes and therefore can also be subnormal in any clinical form of the disease except perhaps pseudohypophosphatasia.69 During pregnancy in hypophosphatasia, low levels of leukocyte ALP activity may correct due to increased placental ALP isoenzyme activity.121
In hypophosphatasia tissue specimens, preliminary observations using a polyclonal antibody to the liver isoform of TNSALP suggested the presence of normal amounts of enzyme protein.122,123 However, a monoclonal antibody-based immunoassay demonstrated low levels of bone and liver TNSALP isoforms in the serum of patients with all clinical forms of hypophosphatasia except pseudohypophosphatasia.124 This immunoassay measured dimeric TNSALP.124 Accordingly, disruption of the immunoreactivity, and perhaps the tertiary structure of TNSALP, may occur after its release from cell surfaces in hypophosphatasia patients.124
In infants with hypophosphatasia, some ALP activity is detectable by sensitive methods in liver, bone, and kidney tissue and in skin fibroblasts in culture.125,126 The ALP in the fibroblasts has different physicochemical properties when compared with the enzyme in normal cells.125 In one case of infantile hypophosphatasia, catalytic inhibition and isoelectric focusing studies suggested that the residual ALP activity was intestinal ALP,117 perhaps reflecting compensatory expression of an intestinal ALP gene. Indeed, studies of homogenates of small bowel mucosa from a family with a clinically mild childhood/adult form of hypophosphatasia,127 and autopsy tissue from severely affected patients,126 showed increased amounts of intestinal ALP. However, fibroblasts in culture from severely affected patients seem to produce low ALP activity with physicochemical properties that are TNSALP-like.125 Nevertheless, the physicochemical and immunologic properties differ from patient to patient.128 Accordingly, the effect of TNSALP gene mutations (see below) on circulating, as well as tissue, ALP requires further investigation.
Autopsy studies of children or adults with hypophosphatasia have not been reported. However, these nonlethal subtypes also seem to feature globally diminished TNSALP activity within tissues. TNSALP isoform activity can be deficient in serum,24 circulating granulocytes,69 bone,69 and cultivated skin fibroblasts.129 TNSALP isoform immunoreactivity in serum is also reduced in these patients.124
The first evidence that hypophosphatasia was a heritable disorder came when affected sibs were reported in 1950.130 Early on, family studies of severe disease in infants or children indicated autosomal recessive inheritance. The parents of such patients often had low or low-normal levels of serum ALP activity, and PEA was detectable in their urine.45,46 Furthermore, consanguinity was reported in some kindreds.
The inheritance pattern for the milder forms of hypophosphatasia is, however, less clear. In some reports, childhood and adult hypophosphatasia, as well as odontohypophosphatasia, are regarded as autosomal recessive conditions.131-133 Indeed, vertical transmission of clinically apparent disease seems to be unusual.55,56 Nevertheless, multigenerational occurrence of clinical and biochemical abnormalities of hypophosphatasia suggests that mild disease can be transmitted as an autosomal dominant trait.55,56,74,134-136 Family studies rarely show mildly affected individuals with severe disease in their offspring.55,136,137
Identification of carriers for hypophosphatasia is difficult, necessitating quantitation of several biochemical markers, including urinary PPi.138 Pyridoxine loading followed by assay of plasma PLP levels is especially helpful in heterozygote detection, particularly among the Mennonite population in Canada.108
Chromosomal defects have rarely been reported in hypophosphatasia. A common D/D translocation was found in 1970 in one adult patient, but it was not present in other affected family members, and was therefore presumed coincidental.77
Phenylketonuria was described in one infant with hypophosphatasemia, phosphoethanolaminuria, and generalized skeletal demineralization.139 Morquio syndrome together with hypophosphatasia has occurred in a Canadian Hutterite kindred.140 These patients, however, appear to reflect the coincidence of two autosomal recessive conditions.140
In 1984, a preliminary report using skin fibroblast heterokaryons deficient in ALP activity from patients representing 10 families with perinatal or infantile hypophosphatasia described absence of complementation.119 This observation indicated a molecular defect involving one gene locus.119
In 1987, genetic linkage of the Rh blood group in six inbred Mennonite kindreds from Manitoba, Canada, provided evidence that the “candidate” TNSALP gene was involved in severe hypophosphatasia in this population.141
In 1988, characterization of the gene encoding the TNSALP 12 isoenzyme provided the background for significant advances in our understanding of the genetic basis for hypophosphatasia.12,142 That same year, a missense mutation of the TNSALP gene was discovered in an infant with perinatal hypophosphatasia born to second cousins in Nova Scotia.52 The patient was homozygous and both parents carriers of a single base-transition that caused a threonine-for-alanine substitution at amino acid position 162. Site-directed mutagenesis and transfection analysis of the patient's TNSALP gene defect confirmed that the mutation diminished the enzyme's catalytic activity. Three-dimensional structure information concerning Escherichia coli ALP by x-ray crystallography16 suggested that the base change compromised the spatial relationship of metal ligands to an important arginine residue at the catalytic pocket.50 Later, this defect was shown to impair transport of the mutated enzyme leading to its intracellular aggregation.143
In 1992, sequence analysis of the TNSALP cDNAs of four additional unrelated patients with perinatal or infantile hypophosphatasia revealed a different missense mutation in each of the eight TNSALP alleles examined.142 Screening of 50 unrelated patients with all clinical forms of hypophosphatasia disclosed 23 individuals with one of these defects, but in whom the nature of the other TNSALP allele was not known. Of interest, however, two sibs with typical childhood hypophosphatasia and one unrelated elderly woman with classic adult hypophosphatasia were found to be compound heterozygotes for the identical TNSALP missense mutations. This observation showed that childhood and adult hypophosphatasia can be the same disorder and can be transmitted as an autosomal recessive trait.142 Each of these 9 TNSALP missense mutations altered an amino acid residue that is shared in mammalian TNSALPs.15 Indeed, several of these amino acid residues are conserved even among bacteria. The three-dimensional structure of E. coli ALP 15,16 suggests that some of the base substitutions would disturb metal ligand-binding in the mature enzyme, but how the other mutations are deleterious was not fully understood.15
In 1993, homozygosity for a tenth TNSALP missense mutation was found to account for severe hypophosphatasia prevalent in Canadian Mennonites, presumably explained by a founder effect and inbreeding.53
In 1998, 18 TNSALP gene mutations were reported in 26 distinct chromosomes carrying a possible mutation in the TNSALP gene in 13 European families with the perinatal, infantile, or childhood form of hypophosphatasia; 15 were novel, and most were missense mutations.144 In this study, 24 of 26 alleles had a mutation responsible for severe hypophosphatasia. In two patients, only one mutation was found, which was consistent with a possible dominant effect and a functional role for polymorphisms in the TNSALP gene.144,145 Computer-assisted modeling of the mutated proteins did not show any obvious modification in the predicted enzyme structure except for mutation R206W that could abolish a turn between two β-sheets.144 To date, 58 different defects have been reported in the TNSALP gene in hypophosphatasia patients,145a including missense, nonsense, donor splice-site, and frame-shift deletions.52,53,63,142-151 All principal clinical forms of hypophosphatasia (perhaps also including pseudohypophosphatasia) can involve TNSALP mutations. Molecular studies from Japan suggest that even odontohypophosphatasia can be inherited as an autosomal recessive trait.152 Genetic diagnosis of hypophosphatasia requires extensive analysis of the TNSALP gene worldwide in outbred populations.142,144,145
Especially rare cases of hypophosphatasia, however, may be due to a regulatory defect in the biosynthesis of TNSALP. In one boy with the infantile form, a series of IV infusions of pooled normal plasma in attempted enzyme replacement therapy was followed by a 4-month correction of hypophosphatasemia due to skeletal synthesis of the bone isoform of TNSALP.153 Remarkable transient remineralization of osseous tissue occurred during this time. The observation could not be attributed to the infused ALP, which had a circulating half-life of just several days.153
Regulation of TNSALP biosynthesis may affect disease expression in other ways. Patients with the childhood form of hypophosphatasia usually have higher absolute levels of ALP activity than adult-onset cases (Fig. 207-5). Physiological decreases in individual serum (skeletal) ALP levels during the adult years possibly engender clinical reexpression of the condition. However, the degree of hypophosphatasemia (relative to the serum ALP level that is appropriate for age) is similar in affected children and adults and perhaps helps to explain the “overlap” in defining these two clinical forms of hypophosphatasia.
Recent transfection studies indicate that some TNSALP gene mutations inactivate the enzyme and lead to its intracellular accumulation.143,144,152 Other defects may diminish expression of the mutated allele or mRNA stability.63
Perinatal (lethal) hypophosphatasia is almost always a fatal condition. Rarely, prolonged survival occurs.63 Infantile hypophosphatasia has an unpredictable outcome when first diagnosed. In some patients, there is progressive skeletal deterioration;67 in others, there is significant spontaneous improvement.154 Sequential radiographic studies are critical for prognostication. Approximately 50 percent of these patients die from respiratory compromise and pneumonia that follow worsening skeletal disease in the chest.46 The prognosis seems to improve after infancy. Indeed, a preliminary report from Canada suggests that in their patient population, the adult stature of survivors of infantile hypophosphatasia may be normal (although I am aware of significant exceptions in the United States). Childhood hypophosphatasia may also spontaneously improve during adolescence,46 but recurrence of symptoms in adult life is possible, if not likely.46,55,155 Adult hypophosphatasia causes chronic orthopedic problems after the onset of skeletal symptomatology.46,55,80,155 Worsening osteomalacia, leading to osteopenia and fractures, can occur in affected women at menopause, and was not prevented by estrogen replacement therapy in two patients (personal observation).
There is no established medical therapy for hypophosphatasia, although a variety of treatments have been studied.46,55,153,156,157 Assessment of any regimen is made difficult by the uncertain clinical course of many affected individuals, some of whom improve spontaneously on radiographic study.
Traditional therapies for rickets and osteomalacia (vitamin D and mineral supplements) should be avoided, unless clear-cut deficiencies are documented, because circulating levels of calcium, Pi, and the vitamin D metabolites are not low. Indeed, in infantile cases, excess vitamin D could augment intestinal absorption of calcium without enhancing skeletal formation and thus cause or exacerbate hypercalcemia and hypercalciuria. However, complete restriction of vitamin D intake or exposure to sunshine should be guarded against, because superimposed vitamin D-deficiency rickets has occurred.96 Hypercalcemia in infantile hypophosphatasia can be improved by lowering dietary calcium intake and/or with glucocorticoid therapy,60,67 but progressive skeletal demineralization may follow.67,155,156,158 Synthetic calcitonin treatment to control hypercalcemia and to block mineral loss may be beneficial.159
In theory, therapy with agents that could stimulate TNSALP biosynthesis or enhance its activity might be helpful for hypophosphatasia. Administration of cortisone to a few patients with severe disease was reportedly followed by periods of normalization of serum ALP activity and radiographic improvement,46,156,160 but this has not been a consistent finding.46 Brief treatments with zinc, magnesium, and an active fragment of PTH to stimulate ALP activity or synthesis have been unsuccessful.55,153 In other metabolic bone diseases, sodium fluoride enhances osteoblast function and increases the activity of the bone form of TNSALP in serum.161 However, an excessive amount of fluoride can itself impair skeletal mineralization and bone quality, and fluoride has not been rigorously tested in hypophosphatasia.
If extracellular accumulation of PPi is a key pathogenetic factor in hypophosphatasia (see below), reduction in endogenous PPi levels might enable skeletal mineralization to proceed normally.33,39 In 1968, an attempt to achieve this outcome using oral Pi supplementation to promote renal PPi excretion reportedly met with some radiographic success.162 However, in subsequent studies, plasma PPi levels were found to be essentially unchanged by this treatment. In fact, increased urinary PPi levels after Pi is administered orally may merely reflect enhanced renal PPi synthesis.33,39 This therapeutic approach has been repeated, but its efficacy has not been confirmed.50,116
Enzyme replacement therapy for hypophosphatasia has been attempted by IV infusion of several types of ALP given to patients with the infantile form of the disease. The results generally have been disappointing. Serum from an individual with Paget bone disease given to one affected infant was associated with some radiographic improvement.67,163 However, subsequent trials of this therapy for four patients showed no significant clinical or radiographic benefit, and their disease proved fatal.156 Weekly intravenous infusions of fresh plasma were followed by clinical and some radiographic improvement in one patient.164 Also, infusions of plasma from several normal individuals, which had been frozen and then pooled, were followed by transient correction of hypophosphatasemia and marked temporary clinical, radiographic, and histologic improvement in one severely affected boy (see above).153 However, it may be that he had a unique defect in TNSALP gene regulation or TNSALP biosynthesis. Indeed, a subsequent trial of pooled plasma infusions in a different patient did not reproduce this response.126 Recently, after a brief report that suggested that IV administration of ALP purified from liver improved the histologic appearance of bone and decreased urinary PEA levels in one patient,165 a vigorous therapeutic attempt was conducted with IV infusion of purified placental ALP. In a study of pregnant women who were carriers for hypophosphatasia, placental ALP was shown to be catalytically active toward PEA, PPi, and PLP.166 Infusions of placental ALP caused hyperphosphatasemia, but resulted in only modest decrements of plasma PLP and urinary PEA concentrations, and no change in urinary PPi levels. Furthermore, there was no clinical or radiographic improvement for lethal disease.167 These cumulative observations may reflect the fact that the amount of ALP in the body is much greater than levels achieved in the circulation by these treatments. Alternatively, they are consistent with a requirement for ALP to be present on cell surfaces, particularly in the skeleton, to act therapeutically.167 In this regard, it is notable that the extreme skeletal disease characterizing perinatal hypophosphatasia occurs in utero in an environment that is not protective. Preliminary findings in the TNSALP gene knock-out mouse,168 and in one patient with infantile hypophosphatasia,169 indicate that bone marrow transplantation could be beneficial.
Infants and young children with hypophosphatasia should be followed carefully for increased intracranial pressure from either “functional” or “true” premature craniosynostosis. As discussed, functional synostosis (that may require craniotomy) can occur despite the radiographic illusion of widely open fontanels.60
Fractures in children do mend, although delayed healing after femoral osteotomy with casting has been reported.170 In adult patients, pseudofractures may remain unchanged for years, but will not heal unless they progress to completion, or are treated orthopedically.55 Use of intramedullary rods rather than load-sparing devices, such as plates, seems best for the prophylactic or acute surgical management of femoral fractures and pseudofractures.80 For recurrent metatarsal stress fractures, ankle-foot orthoses are useful.
Expert dental care is especially important for affected children. Severely involved dentition can impair nutrition, and efforts to preserve teeth in position or use of complete or partial dentures may be necessary.70,71 One study indicates that proliferation of bacteria on the tooth surface, perhaps related to deficiency of TNSALP activity in leukocytes, may contribute to loss of dentition.114
Symptoms from CPPD or calcium phosphate crystal deposition may respond to nonsteroidal anti-inflammatory medication.78
Assay of ALP activity in amniotic fluid is not helpful.171 At 14 to 18 weeks of gestation, most is intestinal ALP excreted from the fetus.172 Measurement of α-fetoprotein in amniotic fluid, however, can help to differentiate anencephaly from severe hypophosphatasia.
The perinatal (lethal) form of hypophosphatasia has been diagnosed in utero.79 During the first trimester, chorionic villus samples from 15 pregnancies were assessed utilizing a monoclonal antibody-based assay specific for TNSALP.173,174 A precisely timed and carefully prepared specimen is required.173,175 RFLP analysis, using a chorionic villus sample, was used successfully for a Canadian Mennonite176 and for a Japanese family.177 During the second trimester, perinatal hypophosphatasia has been diagnosed with ultrasonography (with attention to the limbs as well as the skull),178 radiographic study of the fetus, and assay of ALP activity in amniotic fluid cells by an experienced laboratory.179 An ultrasound study, however, was judged to be normal at 16 to 19 weeks of gestation in 3 cases of perinatal hypophosphatasia in which radiographic study at 38 weeks of gestation showed absence of a fetal skeleton.180,181 Combined use of radiologic techniques, including serial ultrasonography, seems to be best. The utility of assaying cord blood ALP is untested. TNSALP gene mutation information has been used successfully to evaluate pregnancies at risk for lethal disease.144,182,183
Mild forms of hypophosphatasia have not been diagnosed prenatally. I am aware of two such children who reportedly had normal ultrasonography in early pregnancy. Conversely, severe bowing of the lower extremities detected by ultrasound, suggestive of a potentially lethal form of skeletal dysplasia, occurred in four pregnancies in three families in whom the deformity corrected spontaneously postnatally and the clinical phenotype otherwise was childhood hypophosphatasia.184,185
Mouse Model for Hypophosphatasia.
In 1995 and 1997, two independent laboratories reported use of homologous recombination to inactivate the equivalent of the TNSALP gene in mice.186,187
Although there were some minor phenotypic differences, perhaps explained by the background strains,188 the animals developed skeletal disease that resembled a form of rickets and had endogenous accumulation of PEA, PPi, and PLP.188 Homozygous knock-out mice had unremarkable skeletons at birth, suggesting in utero protection or redundant or back-up mechanisms for skeletal development.186-188 A striking feature was seizures apparently due to low extracellular and tissue levels of PL leading to decreased gamma-aminobutyric acid in the brain.186 Parenteral administration of vitamin B6 was necessary to keep the animals alive. Subsequent investigation showed that the mice were an excellent model for infantile hypophosphaturia with postnatal development of defective skeletal mineralization, endogenous accumulation of PEA, PPi, and PLP and, interestingly, a disturbance in epiphyseal and physeal chondrocyte development.188