A Japanese single-center experience of the ecacy and safety of enzyme replacement therapy in childhood-onset hypophosphatasia

Hypophosphatasia (HPP) is a rare inherited metabolic disorder caused by mutations in the ALPL gene, which encodes tissue nonspecic alkaline phosphatase. The phenotype of HPP is widely diverse from the perinatal severe form to the adult mild form. The former represents the most severe form and was earlier associated with high mortality due to impaired development of the lungs and severe hypomineralization of the bones. Enzyme replacement therapy (ERT) using asfotase alfa was approved in 2015 in Japan for treating patients with HPP and has improved their pulmonary function and life prognosis. There are several practical and ethical challenges related to using orphan drugs for a rare disorder in a publicly funded healthcare system. Sharing experiences about their application is essential towards formulating guidelines to assist clinicians with decisions about their initiation and withdrawal. We report the details of ERT experience in ten cases of childhood-onset HPP in nine families from January 2015 to November 2019 (median [interquartile range] age 11.0 years [7.6–12.5] years; 60% male). This is the largest study of a single-center cohort describing the clinical course of HPP patients treated with ERT in Asia. One case of perinatal lethal form of HPP, two cases of perinatal benign form, six cases of childhood form, and one case of odontohypophophatasia were observed. The most common symptom at onset was bone abnormalities. All patients had low serum ALP levels as compared to the age-matched reference range before the commencement of ERT. All HPP patients responded to ERT without serious adverse effects. Genetic analysis showed that eight out of ten patients had compound heterozygosity; two patients had only one heterozygous variant. In this study, two patients had a heterozygous variant of ALPL and responded to ERT, although the variants did not have the dominant-negative effect. ERT only ALPL dominant-negative effect. Early diagnosis abnormalities low alkaline phosphatase levels prognosis.

compared with age-speci c normal values [12]. If one or more primary symptoms are seen on clinical examination and a low serum ALP level is observed, HPP should be suspected, and gene testing should be performed for de nitive diagnosis. ALPL gene analysis was performed with informed consent. Ten children (4 girls and 6 boys) of 9 families were included. The demographic characteristics, clinical features, laboratory investigations, and genetic analysis of all patients are summarized in Table 1. The reference values for serum PLP, PL, and PLP/PL were determined based on a study published by Akiyama et al [13].
Short stature was de ned as a height of − 2 SD below the mean for a respective age as speci ed by the 2000 growth survey on BMI for age references for Japanese children (cross-sectional national survey data by the Japanese Ministry of Health, Labour and Welfare, and the Ministry of Education, Culture, Sports, Science and Technology) [14,15]. ERT using asfotase alfa was administered subcutaneously three times a week at a dose of 2 mg/kg or six times a week at a dose of 1 mg/kg, and the maximum volume of a single injection was 1 mL (40 or 100 mg/mL concentration). This was an observational retrospective patient report that did not involve any research-based patient intervention. All diagnostic procedures and treatment protocols were based on established protocols intended to diagnose and treat patients and adhered to the principles of the Declaration of Helsinki 1975, as revised in 2000. The assay of pyridoxal phosphate and pyridoxal was approved by the Research Ethics Board at Okayama University Hospital (Approval No. 1603-012). The patient experienced pain in his lower legs at 2 years of age. At 4 years, he had pain in the upper arms and exhibited premature loss of three deciduous teeth. The serum ALP level was 197 IU/L (reference value for this age: 430-1,200 IU/L). Genetic testing con rmed that he had compound heterozygosity for c.613G > A (p.Ala205Thr) and c.1559delT (p.Leu520fs) of the ALPL gene. We initiated ERT, and there was consequent improvement in his symptoms of pain in the lower extremities and gradual decline in the rate of premature loss of deciduous teeth. The patient's height increased from 103.7 cm (− 0.8 SD below the mean for his age group as per the 2000 growth survey) [14] to 116.5 cm (− 0.5 SD) 2 years after the initial presentation.
Case 2: 12-year-old female patient The patient had poor weight gain, and her radiographs showed rachitic changes at the ends of the bones of the trunk at 6 months of age. At 1 year of age, she was suspected to have HPP as her serum ALP levels were in the range of 260-380 IU/L (reference value for this age: 395-1,289 IU/L), but she had no other symptoms. Thereafter, her height had been − 1.0 to − 2.5 SD below the mean for her age group as per the 2000 growth survey [14]. At 8 years of age, she experienced neck pain and lumbago. There were no abnormalities on imaging tests, and HPP was once again suspected. Genetic analysis revealed a heterozygous variant of the ALPL gene, c.984_986delCTT (p.Phe327del). Her father similarly had the same variant and was asymptomatic; therefore, this variant was not thought to be the cause of her symptoms. The patient was rehabilitated and treated with anti-in ammatory drugs. At the age of 10 years, she could not attend school on account of her inability to walk, carry her bag by herself, or play with her friends because of pain in her extremities (Additional File 1). She was referred to our hospital to commence ERT as the serum PLP level was elevated at 164.4 nmol/L (reference value: 14.5-57.3 nmol/L). The pain in her extremities gradually decreased after the introduction of ERT. Approximately 3-4 months after the initiation of ERT, she was able to join an athletic festival without the support of any anti-in ammatory drugs. Six months after ERT initiation, she did not have any di culty in attending school (Additional File 1).

Case 3: 3-year-old male patient
The patient lost ve deciduous teeth prematurely with roots intact, starting from the rst year of his life. He visited a dentist who suspected HPP. He was then referred to our hospital for further investigation and treatment. The serum ALP level was 79 IU/L (reference value for this age: 395-1,339 IU/L). The ALPL variants of c.526G > A (p.Ala176Thr) and c.920C > T (p.Pro307Leu) were detected. We started treatment with ERT and noticed a decline in the rate of premature loss of deciduous teeth. His height increased from 73.8 cm (− 2.0 SD) to 86.9 cm (− 1.8 SD) in 1 year and 7 months after ERT initiation.
Case 4: 12-year-old male patient At 9 years of age, the patient complained of pain in the right knee and hip joint and was referred to an orthopedic surgeon. No abnormalities were noted on radiography, and the patient was monitored. Over the next several months, he developed pain in his chest and extremities and was referred to our hospital for a detailed investigation. Blood examination revealed a low serum ALP level of 421 IU/L (reference value for this age: 470-1,500 IU/L). Genetic testing for the ALPL gene exhibited a heterozygous variant of c.1559delT (p.Leu520fs). Based on our experience in treating case 2 of this series, an HPP patient with a heterozygous variant and reactive to ERT, we likewise commenced ERT in case 4 at the age of 11 years. The patient's pain gradually subsided after the introduction of ERT.
Case 5: 12-year-old male patient At the age of 7 years, the patient had a short stature at − 2.0 SD as per the 2000 growth survey [14]. A general practitioner performed a blood examination; the serum ALP level was 338 IU/L (reference value for this age: 450-1,250 IU/L). He was referred to our hospital at the age of 9 years for further examination and treatment. His serum ALP level continued to remain low, and he had developed bilateral ankle pain, which prompted us to perform further examinations. ALPL variants c.382G > A (p.Val128Met) and c.529G > A (p.Ala177Thr) were found, and treatment with ERT was initiated. His height increased from 126.6 cm (− 1.9 SD) to 142.0 cm (− 1.4 SD) in 2 years and 3 months, and his pain was gradually alleviated after the introduction of ERT.

Case 6: 18-year-old female patient
This patient, the older sister of patient 5 of this series, experienced left knee pain at the age of 10 years and was treated surgically after a diagnosis of meniscus injury, but the knee pain persisted. She was also diagnosed with scoliosis at 13 years of age, and when she turned 15, her younger brother (Case 5) was diagnosed with HPP. Consequently, she was suspected to have the same disease. Blood examination revealed a low serum ALP level of 140 IU/L (reference value for this age: 120 − 570 IU/L). Genetic testing showed the same ALPL variants as her brother, which con rmed the diagnosis of HPP. At 16 years of age, she was started on ERT. Her knee pain gradually improved, and she experienced considerably less fatigue in her daily life after the initiation of ERT.
Case 7: 5-year-old male patient At 4 years of age, the patient lost a deciduous tooth with the root intact. He was taken to a general practitioner, and his serum ALP level was found to be 116 IU/L (reference value for this age: 430-1,200 IU/L). Thereafter, he was referred to our hospital for further examination and treatment. A genetic test con rmed ALPL variants; c.572A > G (p.Glu 191Gly) and c.1559delT (p.Leu520fs), and the diagnosis of HPP was con rmed. After the initiation of ERT, he did not experience any further premature loss of deciduous teeth.  [14] and periodontal disease were noted. At the age of 8 years, he was referred to our hospital for surgical correction of the difference in the length of his lower legs. When the patient was 15 years old, asfotase alfa was approved in Japan. Therefore, we performed a genetic test before treatment to con rm HPP; ALPL gene variants: c.979T > C (p.Phe327Leu) and c1559delT (p.Leu520fs) were found. Hence, ERT was commenced. After the initiation of ERT, his 6-minute walk test results improved from 420 m to 560 m, marking a clinical improvement in performance capacity.
Case 9: 9-year-old female patient Fetal ultrasound at 29 weeks of age revealed bilateral femoral deformities. A low serum ALP level (96 IU/L; reference value for this age is 530-1,610 IU/L) at birth and presence of ALPL variants, namely c.568_570delAAC (p.Asn190del) and c.979T > C (p.Phe327Leu), con rmed the diagnosis of HPP. She subsequently had a short stature of less than − 2.0 SD. ERT was started at the age of 4 years for short stature. She experienced gradual alleviation in fatigue after the initiation of ERT.
Case 10: 2-year-old female patient The patient had prominently shorter limbs than normal since the fetal period and was born at 38 weeks and 3 days of gestation. She had severe respiratory failure immediately after birth and was therefore intubated. Blood test results after birth revealed a low serum ALP level of 8 IU/L (reference value for this age: 530-1,610 IU/L). Consequently, she was clinically diagnosed as having HPP and was started on ERT and vitamin B6 supplements on day 0. A genetic test later con rmed ALPL variants, c.1333T > C (p.Ser445Pro) and c.1559delT (p.Leu520fs), and a diagnosis of HPP. The patient has sensorineural hearing loss that is gradually improving. She needs respiratory support during the night, but the respirator can be taken off during the daytime. She can pull herself up at the age of 2 years. Although she still has severe short stature, her height increased from 35.0 cm (− 6.5 SD) to 72.3 cm (− 4.8 SD) in 2 years and 4 months after ERT initiation.
To summarize, our case series reports the details of ERT experience in ten HPP patients of 9 families from January 2015 to November 2019 (median [interquartile range] age 11.0 years [7.6-12.5] years; 60 % male patients) ( Table 2). There was one case of perinatal lethal form of HPP, two cases of perinatal benign form, six cases of childhood form, and one case of odontohypophophatasia. The most common symptom at onset was bone abnormalities (four out of ten patients). The most common symptom during the clinical course of the disease was also bone abnormalities (eight out of ten patients). Two patients (cases 8 and 9) had calci cation in the kidneys bilaterally. All ten patients had low serum ALP levels as compared with the age-matched reference range before ERT.  ※ The height of patient 9 was measured at the age of 6 years 3 months before restarting ERT for a second time.
Genetic analysis showed that eight out of ten patients had compound heterozygosity, and two patients (cases 2 and 4) had only one heterozygous variant. Five out of ten patients had the c.1559delT (p.Leu520fs) variant, which is common in Japan [6]. In contrast, only two patients had the c.979T > C (Phe310Leu) variant, which is also common in Japan [6].
All patients showed improvements in clinical symptoms after ERT such as increase in height, pain alleviation, cessation of premature deciduous tooth loss, better performance in the 6-minute walk test, or improvement in respiratory insu ciency. All ve patients (cases 1, 2, 4, 5, and 6) who experienced pain in extremities had alleviation of pain after ERT initiation. Two patients (cases 3 and 7) with premature loss of deciduous teeth, with the roots intact, did not lose teeth prematurely after initiation of ERT. Clinical improvements in patients with the prenatal benign form (cases 8 and 9) were increased distance in the 6minute walk test and alleviation of fatigue. Case 10 had a severe short stature with a height of 72.3 cm (− 4.8 SD) at 2 years and 4 months of age even after the initiation of ERT (her height was − 6.5 SD at birth). There was improvement in respiratory insu ciency, and motor, mental, and auditory development progressed gradually. Changes in biochemical data after the initiation of ERT showed a tendency for PLP and PEA to decrease and PL to increase. These changes are consistent with the pathophysiology of the disease.
In three patients (cases 5, 6, and 9), ERT was either discontinued temporarily or the total dose was reduced owing to injection site reaction or fever. Patient 5 resumed an adequate dose of ERT 6 months after dose reduction. Patient 6 continues to receive ERT twice a week, instead of three times a week owing to the pain with ERT injection. Patient 9 resumed ERT for the rst time 1 year and 10 months after stopping ERT.

Discussion
This is the largest single-center cohort study in Asia describing the clinical course of patients with HPP treated by ERT. In particular, we have elucidated the e cacy of ERT in childhood-onset HPP. However, there were some limitations in our study. First, effectiveness of ERT in reducing pain was assessed based on the subjective responses of the patients or their parents. Second, our study group was a single-center cohort. Nevertheless, we believed that presenting the clinical courses of HPP patients after ERT would bene t clinicians treating patients with HPP.
Although the e cacy of ERT in the perinatal and infantile form of HPP has been demonstrated in a Japanese clinical trial, the number of patients with childhood and adult form of HPP was comparatively lower [11]. There are currently no adequate biomarkers available to evaluate the effectiveness of ERT, which necessitates the close monitoring of improvement in clinical ndings of each case [16]. In our study, clinical ndings of the patients such as pain, short height, premature loss of teeth, poor performance in the 6-minute walk test, or respiratory insu ciency were alleviated after initiating ERT. Conversely, clinical improvement of signs (6-minute walk test or the level of fatigue) in patients with the prenatal benign form (cases 8 and 9) seemed less obvious than in patients with the childhood form of HPP. The reduced effectiveness of ERT can be attributed to the differences in disease forms or the late start of treatment.
Further studies are needed to explore the improvement of clinical signs in different forms of HPP after ERT.

Case 10
with perinatal lethal HPP continued to have severe short stature even though the growth rate improved considerably after the initiation of ERT (from − 6.5 SD to − 4.8 SD). We could not draw a comparison between the clinical signs before and after the initiation of ERT with respect to case 10 as ERT was initiated immediately after birth. The natural prognosis of perinatal lethal HPP is poor, with death occurring within 1 year [17]. Therefore, we can say that ERT improved the patient's prognosis and quality of life.
HPP is usually inherited in an autosomal recessive manner; however, an autosomal dominant genetic form has been also recognized. In general, the autosomal recessive form is prevalent in more severe phenotypes, while the autosomal dominant form is prevalent in milder phenotypes. In our study, two patients (cases 2 and 4) deserve additional attention. Even though they only had a heterozygous variant that was reported as an autosomal recessive form of HPP, c.1559delT (p.Leu520fs) and c.984_986delCTT (p.Phe327del) in cases 2 and 4, respectively, their symptoms were relieved through ERT. Case 2 was very responsive to ERT (Additional File 1). In general, dominant inheritance is caused by a dominant-negative effect (DNE), gain of function, or haploinsu ciency.
Owing to the allosteric properties of TNSALP, dominant HPP is believed to result from DNE [18]. Although the homozygous variant of c.1559delT (p.Leu520fs) or c.984_986delCTT (p.Phe327del) has quite a low enzymatic activity, a heterozygous variant of either c.1559delT (p.Leu520fs) or c.984_986delCTT (p.Phe327del) does not show DNE in vitro study [19]. Conversely, it is known that patients with HPP, who have a heterozygous variant of the autosomal recessive genetic form of HPP without DNE, have HPP symptoms [20]. In a Japanese clinical trial, a heterozygous c.1559delT (p.Leu520fs) variant caused the patient to exhibit symptoms of the adult form of HPP, and symptoms such as myalgia and arthralgia were improved through ERT [11]. Recent reports suggest heterogeneity of the genotype-phenotype correlation and other genetic or environmental factors modulating HPP phenotype [10,17]. A study reported that ERT was initiated in a patient with odontohypophosphatasia who had a heterozygous missense variant, c.1183A > T (p.Ile395Phe), in the ALPL gene [21]. However, in this report, ERT did not demonstrate signi cant e cacy. To the best of our knowledge, our study is the rst report that shows the e cacy of ERT in patients with the childhood form of HPP and having a heterozygous variant of the ALPL gene.
Although genetic analysis is recommended to diagnose HPP patients, a detailed patient history and clinical examination are the crucial rst steps. There might be patients with heterozygous ALPL variants like case 2 who respond to ERT. We should comprehensively consider the indications for starting ERT in patients with HPP and closely monitor their clinical improvement while evaluating the safety of ERT for each patient.

Conclusion
We reported the largest single-center cohort in Asia describing the clinical course of patients with HPP, treated with ERT. Every patient bene ted from ERT. A noteworthy observation was that even if patients have HPP symptoms with only heterozygous variants, ERT can still be a valuable therapeutic option. We will continue to conduct future studies with long-term follow-up periods to investigate the e cacy of ERT in patients with HPP.
Abbreviations ERT, enzyme replacement therapy; HPP, hypophosphatasia; PEA, phosphoethanolamine; PLP, pyridoxal 5' phosphate; TNSALP, tissue-nonspeci c alkaline phosphatase; SD, standard deviation Declarations Ethics approval and consent to participate: This is an observational retrospective patient report that did not involve any research-based patient intervention. All diagnostic procedures and treatment protocols were based on established protocols intended to diagnose and treat the patients. No aspect of the case series is in contradiction with the Helsinki Declaration of 1975, as revised in 2000. The assay of pyridoxal phosphate and pyridoxal was approved by the Research Ethics Board at Okayama University Hospital (Approval No. 1603-012). Written informed consent for publication of the data and for performing measurements of PLP and PL were obtained from the patients or patient's parents (in case of patients below 18 years of age).
Consent for publication: Written informed consent for publication of the data, measurements of PLP and PL, and the video record of patient 2 was obtained from the patient's parents.
Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.