**5. Genetic aspect of HPP**

#### **5.1. Inheritance of HPP**

HPP is an autosomal recessive inherited disease [1]. Carriers usually do not exhibit any manifestations. Sometimes, however, carriers show subnormal serum ALP activity and slightly higher urine PEA values [2, 46]. The penetrance differs among forms. In some milder cases, an autosomal dominant cases have been reported [47, 48], and the dominant negative effect accounts for some autosomal dominant cases [48]. In addition, different severity of the symptoms within the same family has been reported [49, 50], suggesting the involvement of epigenetic mechanisms.

#### **5.2. Prevalence of HPP**

The prevalence of HPP was estimated as 1 in 100,000 live births in the Toronto area in Canada, where the first case was found [51]. In Manitoba, Canada, the prevalence is higher in the Mennonite group, being 1 in 2500, according to a founder effect of a particular mutation [52]. In Europe, the prevalence of severe cases is estimated as 1 in 300,000 [53], whereas in Japan it is 1 in 450,000 for patients who have the c.1559delT allele [46]. This particular allele is a severe allele and is characteristic of Japanese families (46.8% of Japanese patients with HPP have this deletion allele) [46].

#### **5.3. Genetic diagnosis**

When HPP is suspected, collection of the family history and the making of a pedigree are important for genetic counseling [54]. Clinical diagnosis can be done by laboratory biochemical examinations and ultrasonic and radiographic findings. Definitive diagnosis is performed by genetic testing. Genomic DNA of the patient is amplified, and the nucleotide sequences 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 Red S staining [57].

#### **5.4. Prenatal diagnosis**

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

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

HPP is an autosomal recessive inherited disease [1]. Carriers usually do not exhibit any manifestations. Sometimes, however, carriers show subnormal serum ALP activity and slightly higher urine PEA values [2, 46]. The penetrance differs among forms. In some milder cases, an autosomal dominant cases have been reported [47, 48], and the dominant negative effect accounts for some autosomal dominant cases [48]. In addition, different severity of the symptoms within the same family has been reported [49, 50], suggesting the involvement of epi-

The prevalence of HPP was estimated as 1 in 100,000 live births in the Toronto area in Canada, where the first case was found [51]. In Manitoba, Canada, the prevalence is higher in the Mennonite group, being 1 in 2500, according to a founder effect of a particular mutation [52]. In Europe, the prevalence of severe cases is estimated as 1 in 300,000 [53], whereas in Japan it is 1 in 450,000 for patients who have the c.1559delT allele [46]. This particular allele is a severe allele and is characteristic of Japanese families (46.8% of Japanese patients with HPP have this

When HPP is suspected, collection of the family history and the making of a pedigree are important for genetic counseling [54]. Clinical diagnosis can be done by laboratory biochemical examinations and ultrasonic and radiographic findings. Definitive diagnosis is performed by genetic testing. Genomic DNA of the patient is amplified, and the nucleotide sequences

cause rickets or osteomalacia [45].

108 Pathophysiology - Altered Physiological States

summarized in **Table 3**.

**5.1. Inheritance of HPP**

genetic mechanisms.

**5.2. Prevalence of HPP**

deletion allele) [46].

**5.3. Genetic diagnosis**

**5. Genetic aspect of HPP**

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].

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

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 from the combination of the genotypes. Mutation sites are scattered throughout the gene, but there are some "hot spots." In Caucasian patients, p.E191K (a moderate allele with a dominant negative effect) and p.D378V (a severe allele) are frequent mutations [60, 61], whereas c.1559delT (pL520RfsX86; a severe allele) and p.F327L (a moderate allele) are frequent in Japanese patients [62, 63]. c.1559delT also has founder effects, and the frequency of c.1559delT is mentioned above [46, 62].

TNAP in a knockout mouse (*Akp2*−/−; *AKP2* is the mouse homolog of the *ALPL*) that is a good mimic of the perinatal form of HPP, showed elongation of life and improvements in bone and dental defects without respiratory failure [66, 67]. The clinical trial with the bioengineered TNAP (ENB-0040; asfotase alfa) was conducted with five perinatal and six infantile patients [7]. It was administered first as a single intravenous infusion of 2 mg/kg, which was then followed by subcutaneous injections three times per week at a dose of 1 mg/kg for 48 weeks. With the exception of one case who died of respiratory failure that was unrelated with asfotase alfa, the recruited patients showed improvements in rickets and respiratory failure [7]. Asfotase alfa (StrensiqST; Alexion Pharmaceuticals, Inc.) was approved in Japan, the EU, Canada, and the USA in that order in 2015 [2]. Asfotase alfa has dramatically changed the treatment of HPP [68]. Asfotase alfa is indicated for the treatment of patients with perinatal-, infantile- and juvenile-onset HPP [69], in which juvenile-onset HPP means almost the same as the childhood form. The current protocol of the recommended administration is subcutaneous injection six times a week at a dose of 1 mg/kg or three times a week at 2 mg/kg, and the maximal volume of injection is 1 ml [69]. The half-life of asfotase alfa is 5 days in the case of subcutaneous administration. The most common adverse reactions (≥ 10%) are injection site reactions, lipodystrophy, ectopic calcifications, and hypersensitivity reactions. Patients with HPP are at increased risk for developing ectopic calcifications, especially of the eye including the cornea and conjunctiva, and the kidneys (nephrocalcinosis). Although ectopic calcification of the blood vessels has not been reported, it is conceivable that long -term administration may cause medial artery calcification. Medial artery calcification or Mönckeberg-type calcification is often shown as a lethal complication in chronic kidney disease (CKD) patients [70]. In CKD patients, hyperphosphatemia triggers transformation of smooth muscle cells in the media into osteoblastic cells that express elevated TNAP, which then stimulates calcification in the medial artery by a mechanism similar to that of bone mineralization [71, 72]. Asfotase alfa is still not indicated for milder form HPP patients. In this regards, the natural history of the adult form has not been well elucidated [44], and more study is needed. Similarly, odontohypophosphatasia may be still underdiagnosed, because dentists usually do not evaluate the serum ALP value. There should be more investigation into the feasibility

Hypophosphatasia: A Systemic Skeletal Disorder Caused by Alkaline Phosphatase Deficiency

http://dx.doi.org/10.5772/intechopen.70597

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Although current ERT has drastically changed the treatment of HPP, many problems are indicated regarding asfotase alfa administration. First of all, two or three injections per week are needed for this ERT treatment, which burdens patients with injections and parents with administration fees. The interval between injections can be elongated by introducing some modifications into the enzyme preparation. Other possible therapies are bone marrow stemcell transplantation and/or combination therapy of such transplantation with ERT. Another possible trial is a trial of gene therapy. Using viral vectors, gene therapy was successfully used to treat knockout mice (*AKP2*−/−) [73, 74]. Since a viral vector containing *ALPL* cDNA that is injected into blood cannot maintain an effective concentration, gene therapy in combination with stem-cell transplantation (*ex vivo* gene therapy) may be more effective [75].

of using asfotase alfa for those milder forms.

**9. Future perspective**
