**3. Molecular process of mineralization and the role of TNAP in mineralization**

Biomineralization in hard tissues including bone occurs in a two-step process [34]. Hypertrophic chondrocytes, osteoblasts, and odontocytes in the bone and dental tissues bud matrix vesicles (MVs) from the cell membrane [2, 35]. MVs are 50–200 nm in diameter and are enclosed by a membrane. MVs are a type of extracellular vesicles; however, the difference between MVs and exosomes, which are secreted by cells in the nonmineralized condition, is unclear [36]. TNAP is one of the most abundant proteins on the membrane of an MV [34]. The other proteins that are abundant in MVs are annexins A2, A5, and A6, Ca2+-ATPase, nucleotide pyrophosphatase phosphodiesterase 1 (NPP1), Pit-1 (a sodium-phosphate cotransporter), and PHOSPHO1, all of which have important roles in mineralization [9, 34]. Biologically, mineralization is defined as the deposition of hydroxyapatite (Ca10(PO<sup>4</sup> )6 (OH)<sup>2</sup> ) crystals among the collagen fibers. If this process is insufficient, extracellular spaces are not mineralized, which leads to the formation of an abnormal soft tissue called osteoid tissue. In the first step of the mineralization, hydroxyapatite is formed in an MV. The membrane lipids of the MV provide a source of phosphate; of these lipids, phosphatidylcholine and phosphatidylethanolamine are hydrolyzed by phospholipase C (PLC), yielding phosphocholine (PCho) and phosphoethanolamine (PEA), respectively [37]. Subsequently, PCho and PEA are hydrolyzed by PHOSPHO1, a cytosolic phosphatase abundant in MVs [38]. The phosphate transporter, Pit-1, provides another source of phosphate. On the other hand, calcium is incorporated into MVs via an annexin calcium channel, which consists of annexins A2, A5, and A6 [34, 35]. When the concentration of calcium phosphate rises beyond the solubility of calcium phosphate, hydroxyapatite crystal formation begins. Subsequently, hydroxyapatite crystals penetrate the MV membrane and elongate in the extracellular space [34, 35]. For the elongation of hydroxyapatite, calcium and phosphate should be provided by the extracellular space. Although calcium ions may be abundant in this milieu, phosphate is provided mainly by the TNAP on the MV membrane, which hydrolyzes PPi to yield inorganic phosphate (Pi) [2, 8, 34]. This hydrolysis by TNAP has dual roles; it supplies a source of phosphate for hydroxyapatite formation and degrades an inhibitor of hydroxyapatite formation (PPi). Ultimately, formed hydroxyapatite crystals deposit among collagen fibers, and mineralization is complete (**Figure 1**). Although the crown domain of TNAP can bind collagen and is suggested to have a role in hydroxyapatite deposition, it has not been elucidated whether TNAP plays a direct role in hydroxyapatite deposition.

**4. Clinical features of HPP including laboratory tests**

**Figure 1.** Mineralization process focusing on the matrix vesicle.

HPP is classified into six forms depending on the onset age and the clinical severity (**Table 2**): perinatal (lethal) form, perinatal benign form, infantile form, childhood form, adult form, and odontohypophosphatasia [3]. The perinatal form occurs in utero and exhibits the most severe manifestations. Patients are stillborn or die during the early postnatal period. They show hypomineralization of the cranial bone and shortened and deformed limbs during gestation, which are easily revealed by ultrasonic examination. The hypomineralization of bones causes a membranous cranium and early craniosynostosis as well as musculoskeletal disorder after birth. The ribs are also hypomineralized, leading to respiratory failure after birth, which often requires respiratory aid. Failure of respiratory management often causes respiratory infections, which are the main cause of death. Epileptic seizures sometimes occur due to a deficit of PLP in neuronal cells, since PLP needs TNAP to enter neuronal cells. A deficit of PLP in neuronal cells causes a decrease in the inhibitory neurotransmitter GABA, leading to epileptic seizures. The perinatal benign form is a recently reported form [42]. Although the symptoms are recognized in gestation, prognosis is good and nonlethal. The infantile form occurs before 6 months of age and also shows severe manifestations. Patients display rickets and deformity of ribs and limbs, and fail to thrive. They also exhibit respiratory failure due to hypomineralization of the ribs, which requires respiratory aid. Recent progress in respiratory management elongates their lifespan. In addition, they often show hypercalcemia and hypercalciuria, leading to nephrocalcinosis. The childhood form shows manifestations after 6 months of age, whose symptoms are milder and not life-threatening. Patients show deformity of limbs, delayed walking, waddling gait, and muscle weakness. Craniosynostosis and

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Extracellular PPi is formed by NPP1 on the MV membrane by hydrolysis of ATP and also provided by a membrane transporter of PPi, ANKH (the human homolog of ANK, the mouse progressive ankylosis gene product). Therefore, mineralization is regulated by the balance of the activities of these three molecules: TNAP, NPP1, and ANKH [9, 39, 40], Experiments using mice with knockout of these three genes showed that loss of activity of NPP1 or ANKH leads to hypercalcification (ectopic calcification of aorta and/or vertebrae and joints), whereas that of TNAP causes hypomineralization [41].

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**Figure 1.** Mineralization process focusing on the matrix vesicle.

**3. Molecular process of mineralization and the role of TNAP in** 

Biomineralization in hard tissues including bone occurs in a two-step process [34]. Hypertrophic chondrocytes, osteoblasts, and odontocytes in the bone and dental tissues bud matrix vesicles (MVs) from the cell membrane [2, 35]. MVs are 50–200 nm in diameter and are enclosed by a membrane. MVs are a type of extracellular vesicles; however, the difference between MVs and exosomes, which are secreted by cells in the nonmineralized condition, is unclear [36]. TNAP is one of the most abundant proteins on the membrane of an MV [34]. The other proteins that are abundant in MVs are annexins A2, A5, and A6, Ca2+-ATPase, nucleotide pyrophosphatase phosphodiesterase 1 (NPP1), Pit-1 (a sodium-phosphate cotransporter), and PHOSPHO1, all of which have important roles in mineralization [9, 34].

Biologically, mineralization is defined as the deposition of hydroxyapatite (Ca10(PO<sup>4</sup>

TNAP plays a direct role in hydroxyapatite deposition.

of TNAP causes hypomineralization [41].

crystals among the collagen fibers. If this process is insufficient, extracellular spaces are not mineralized, which leads to the formation of an abnormal soft tissue called osteoid tissue. In the first step of the mineralization, hydroxyapatite is formed in an MV. The membrane lipids of the MV provide a source of phosphate; of these lipids, phosphatidylcholine and phosphatidylethanolamine are hydrolyzed by phospholipase C (PLC), yielding phosphocholine (PCho) and phosphoethanolamine (PEA), respectively [37]. Subsequently, PCho and PEA are hydrolyzed by PHOSPHO1, a cytosolic phosphatase abundant in MVs [38]. The phosphate transporter, Pit-1, provides another source of phosphate. On the other hand, calcium is incorporated into MVs via an annexin calcium channel, which consists of annexins A2, A5, and A6 [34, 35]. When the concentration of calcium phosphate rises beyond the solubility of calcium phosphate, hydroxyapatite crystal formation begins. Subsequently, hydroxyapatite crystals penetrate the MV membrane and elongate in the extracellular space [34, 35]. For the elongation of hydroxyapatite, calcium and phosphate should be provided by the extracellular space. Although calcium ions may be abundant in this milieu, phosphate is provided mainly by the TNAP on the MV membrane, which hydrolyzes PPi to yield inorganic phosphate (Pi) [2, 8, 34]. This hydrolysis by TNAP has dual roles; it supplies a source of phosphate for hydroxyapatite formation and degrades an inhibitor of hydroxyapatite formation (PPi). Ultimately, formed hydroxyapatite crystals deposit among collagen fibers, and mineralization is complete (**Figure 1**). Although the crown domain of TNAP can bind collagen and is suggested to have a role in hydroxyapatite deposition, it has not been elucidated whether

Extracellular PPi is formed by NPP1 on the MV membrane by hydrolysis of ATP and also provided by a membrane transporter of PPi, ANKH (the human homolog of ANK, the mouse progressive ankylosis gene product). Therefore, mineralization is regulated by the balance of the activities of these three molecules: TNAP, NPP1, and ANKH [9, 39, 40], Experiments using mice with knockout of these three genes showed that loss of activity of NPP1 or ANKH leads to hypercalcification (ectopic calcification of aorta and/or vertebrae and joints), whereas that

)6 (OH)<sup>2</sup> )

**mineralization**

104 Pathophysiology - Altered Physiological States

#### **4. Clinical features of HPP including laboratory tests**

HPP is classified into six forms depending on the onset age and the clinical severity (**Table 2**): perinatal (lethal) form, perinatal benign form, infantile form, childhood form, adult form, and odontohypophosphatasia [3]. The perinatal form occurs in utero and exhibits the most severe manifestations. Patients are stillborn or die during the early postnatal period. They show hypomineralization of the cranial bone and shortened and deformed limbs during gestation, which are easily revealed by ultrasonic examination. The hypomineralization of bones causes a membranous cranium and early craniosynostosis as well as musculoskeletal disorder after birth. The ribs are also hypomineralized, leading to respiratory failure after birth, which often requires respiratory aid. Failure of respiratory management often causes respiratory infections, which are the main cause of death. Epileptic seizures sometimes occur due to a deficit of PLP in neuronal cells, since PLP needs TNAP to enter neuronal cells. A deficit of PLP in neuronal cells causes a decrease in the inhibitory neurotransmitter GABA, leading to epileptic seizures. The perinatal benign form is a recently reported form [42]. Although the symptoms are recognized in gestation, prognosis is good and nonlethal. The infantile form occurs before 6 months of age and also shows severe manifestations. Patients display rickets and deformity of ribs and limbs, and fail to thrive. They also exhibit respiratory failure due to hypomineralization of the ribs, which requires respiratory aid. Recent progress in respiratory management elongates their lifespan. In addition, they often show hypercalcemia and hypercalciuria, leading to nephrocalcinosis. The childhood form shows manifestations after 6 months of age, whose symptoms are milder and not life-threatening. Patients show deformity of limbs, delayed walking, waddling gait, and muscle weakness. Craniosynostosis and


General

 Failure to thrive Poor feeding Weakness Skeletal

 Hypomineralization Rickets/osteomalacia Short, deformed limbs Membranous cranium Craniosynostosis Deformed ribs Skeletal pain Short statue Muscular

Muscle weakness

Neuronal

 Irritability Respiratory

Renal

Dental

Respiratory failure

Nephrocalcinosis

 Dental caries Blood examination Reduced serum ALP

Urinalysis

Elevated plasma Ca2+

 Elevated urine PEA Elevated urine Ca2+

Premature loss of deciduous teeth

Elevated plasma PPi, PLP and PEA

**Table 3.** Signs and symptoms of HPP.

Different presentation of symptoms is exhibited depend on the forms.

Gait disturbances; delayed walking, waddling gait

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Epileptic seizures (pyridoxine dependent)

**Table 2.** Clinical features of hypophosphatasia.

high intracranial pressure sometimes occur. These patients also show premature loss of deciduous teeth due to failure of cementum formation [43]. Radiologically, childhood form patients exhibit a characteristic tongue-like radiolucent projection from the rachitic growth plate into the metaphysis due to a focal bone defect at the ends of long bones [1, 3]. The adult form occurs during middle age. Although the natural history of the adult form has not been well characterized, patients sometimes have a history of rickets and/ or premature loss of deciduous teeth [44]. In the adult form, osteomalacia develops with pain associated with often recurring metatarsal stress fractures. In some patients, calcium pyrophosphate dehydrate crystals are deposited on articular cartilage due to an increase in concentrations of endogenous PPi [1]. Odontohypophosphatasia manifests only dental symptoms such as premature loss of deciduous teeth without skeletal symptoms due to rickets or osteomalacia.


Different presentation of symptoms is exhibited depend on the forms.

**Table 3.** Signs and symptoms of HPP.

high intracranial pressure sometimes occur. These patients also show premature loss of deciduous teeth due to failure of cementum formation [43]. Radiologically, childhood form patients exhibit a characteristic tongue-like radiolucent projection from the rachitic growth plate into the metaphysis due to a focal bone defect at the ends of long bones [1, 3]. The adult form occurs during middle age. Although the natural history of the adult form has not been well characterized, patients sometimes have a history of rickets and/ or premature loss of deciduous teeth [44]. In the adult form, osteomalacia develops with pain associated with often recurring metatarsal stress fractures. In some patients, calcium pyrophosphate dehydrate crystals are deposited on articular cartilage due to an increase in concentrations of endogenous PPi [1]. Odontohypophosphatasia manifests only dental symptoms such as premature loss of deciduous teeth without skeletal symptoms

**Onset Symptoms Prognosis**

Membranous cranium Respiratory failure Epileptic seizures

Respiratory failure

Failure to thrive Epileptic seizures

teeth

teeth

teeth

Stress fractures

Dental caries

Premature loss of deciduous

Musculoskeletal weakness Premature loss of deciduous

Rickets Benign

Benign

due to rickets or osteomalacia.

**Form Inheritance** 

106 Pathophysiology - Altered Physiological States

**pattern**

Childhood AR or AD After 6 months

AR: autosomal recessive, AD: autosomal dominant.

**Table 2.** Clinical features of hypophosphatasia.

Perinatal AR In utero Deformity of extremities Lethal

Perinatal benign AR or AD In utero Rickets Benign Infantile AR After birth Rickets, Craniosynostosis Mostly lethal Before 6 months

of age

of age

Adult AR or AD Middle age Osteomalacia Benign

Odontohypophosphatasia AR or AD Premature loss of deciduous

A common histopathological feature of HPP is hypomineralization of bone and teeth [1]. Extracellular hydroxyapatite crystals are reduced, although mineralization occurs within the MV, because PHOSPHO1 acts in the MV. Elongation of hydroxyapatite is impaired. Osteoid tissues are increased in bone, which contains nonmineralized extracellular matrix, and they cause rickets or osteomalacia [45].

are determined. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and high-resolution melting curve analysis (HRM) methods used to be employed for this purpose, but direct nucleotide sequencing may be the most effective current method of analysis. Once the mutation of the proband is determined, the inheritance can be pursued by testing the parents' DNA, which makes it possible to give a genetic counseling, because the inheritance pattern of HPP is basically Mendelian inheritance [54, 55]. However, as mentioned above, the same mutation can result in different phenotypes in some families. In addition, a rare case of paternal uniparental isodisomy has been reported [56]. Once a genetic diagnosis is established, enzymatic activity and mineralization activity can be evaluated [57]. An expression plasmid containing the mutant cDNA is transfected into U2OS cells, which are osteoblast-like cells that lack ALP activity. The cells are then cultured for an appropriate period, and enzymatic activity is estimated. For the mineralization assay, the transfected cells are cultured in a mineralization medium that contains β-glycerophosphate as an artificial substrate for TNAP, with or without ascorbic acid. After about 5 days of culture, mineralization is estimated by Alizarin

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Prenatal diagnosis by ALP enzymatic assay or by immunological detection using amniotic fluid and chorionic villus has been reported, but their diagnostic value is low [3] because of contamination of fetal intestinal ALP and maternal ALP. HPP can be diagnosed using ultrasonography and radiography during the second trimester, but the differential diagnosis is complicated. DNA-based diagnosis using chorionic villus is accurate if information about the nucleotide sequences within the family has been obtained [54, 58]. However, prediction of the prognosis of the disease is not easy, because of the fact that the same mutations can cause different phenotypes even in the same family. In addition, ethical considerations including genetic counseling are very important when prenatal genetic diagnosis is performed [54].

To date, a total of 335 mutations in the *ALPL* gene have been reported [6]. The TNAP gene mutations' databases (http://www.sesep.uvsq.fr/03\_hypo\_mutations.php) of the University of Versailles-Saint Quentin en Yvelines provide up-to-date information regarding mutations [55]. Almost all of these mutations are located within the exons, although some mutations are in the promoter region, exon-intron boundaries and introns. In addition, over 70% of the mutations are missense mutations, 11% are small deletions, 6% are splicing mutations, 5% are nonsense mutations, 3% are small insertions, and 3% are large deletions [6]. Only one regulatory mutation has been reported [59]. Many of the patients are compound heterozygotes. Generally, the interaction between the mutant alleles determines the phenotypes of the patients. Residual activities of mutant TNAPs influence the enzymatic activity and the mineralizing activity of the compound heterozygotes. However, the relationships of genotype and phenotype are rather complicated, and the phenotypes are not always estimated

Red S staining [57].

**5.4. Prenatal diagnosis**

**6. Mutations in the** *ALPL* **gene**

For all forms, a characteristic laboratory finding is low serum alkaline phosphatase activity, in which the bone isozyme is reduced [1]. In addition, the natural enzyme substrates, plasma PPi and PLP are elevated. Urine PEA is also elevated, although it is doubtful whether this compound is a natural substrate of TNAP. However, because urine PEA is easy to evaluate by using high-performance liquid chromatography (HPLC), the measurement of PEA is widely used for the diagnosis [2]. The combination of low ALP activity with elevated PPi or PEA is strong evidence for HPP. In some milder cases, however, an increase in PEA is not shown, and, in some cases, PEA is slightly elevated in carriers [46]. Signs and symptoms of HPP are summarized in **Table 3**.
