Clinical and Genetic Characteristics of Pediatric Patients with Hypophosphatasia in the Russian Population

(1) Hypophosphatasia (HPP) is a rare inherited disease caused by mutations (pathogenic variants) in the ALPL gene which encodes tissue-nonspecific alkaline phosphatase (TNSALP). HPP is characterized by impaired bone mineral metabolism due to the low enzymatic activity of TNSALP. Knowledge about the structure of the gene and the features and functions of various ALPL gene variants, taking into account population specificity, gives an understanding of the hereditary nature of the disease, and contributes to the diagnosis, prevention, and treatment of the disease. The purpose of the study was to describe the spectrum and analyze the functional features of the ALPL gene variants, considering various HPP subtypes and clinical symptoms in Russian children. (2) From 2014–2021, the study included the blood samples obtained from 1612 patients with reduced alkaline phosphatase activity. The patients underwent an examination with an assessment of their clinical symptoms and biochemical levels of TNSALP. DNA was isolated from dried blood spots (DBSs) or blood from the patients to search for mutations in the exons of the ALPL gene using Sanger sequencing. The PCR products were sequenced using a reagent BigDye Terminator 3.1 kit (Applied Biosystems). Statistical analysis was performed using the GraphPad Prism 8.01 software. (3) The most common clinical symptoms in Russian patients with HPP and two of its variants (n = 22) were bone disorders (75%), hypomyotonia (50%), and respiratory failure (50%). The heterozygous carriage of the causal variants of the ALPL gene was detected in 225 patients. A total of 2 variants were found in 27 patients. In this group (n = 27), we identified 28 unique variants of the ALPL gene, of which 75.0% were missense, 17.9% were frameshift, 3.6% were splicing variants, and 3.6% were duplications. A total of 39.3% (11/28) of the variants were pathogenic, with two variants being probably pathogenic, and 15 variants had unknown clinical significance (VUS). Among the VUS group, 28.6% of the variants (7/28) were discovered by us for the first time. The most common variants were c.571G > A (p.Glu191Lys) and c.1171del (Arg391Valfs*12), with frequencies of 48.2% (13/28) and 11% (3/28), respectively. It was found that the frequency of nonsense variants of the ALPL gene was higher (p < 0.0001) in patients with the perinatal form compared to the infantile and childhood forms of HPP. Additionally, the number of homozygotes in patients with the perinatal form exceeded (p < 0.01) the frequencies of these genotypes in children with infantile and childhood forms of HPP. On the contrary, the frequencies of the compound-heterozygous and heterozygous genotypes were higher (p < 0.01) in patients with infantile childhood HPP than in perinatal HPP. In the perinatal form, residual TNSALP activity was lower (p < 0.0005) in comparison to the infantile and childhood (p < 0.05) forms of HPP. At the same time, patients with the heterozygous and compound-heterozygous genotypes (mainly missense variants) of the ALPL gene had greater residual activity (of the TNSALP protein) regarding those homozygous patients who were carriers of the nonsense variants (deletions and duplications) of the ALPL gene. Residual TNSALP activity was lower (p < 0.0001) in patients with pathogenic variants encoding the amino acids from the active site and the calcium and crown domains in comparison with the nonspecific region of the protein.


Introduction
Hypophosphatasia (HPP, OMIM: 146300, 241500, 241510) is a rare inherited mineral metabolism disorder caused by low enzymatic activity in the tissue nonspecific alkaline phosphatase (TNSALP) as a result of mutations (pathogenic variants) in the ALPL gene that is localized on chromosome 1p36.12 [1]. The prevalence of HPP is variously estimated at 1/100,000-1/900,000 liveborn [2]. The incidence of pathogenic variants in the ALPL gene in the Russian population is 4.2% [3]. HPP was first mentioned in 1948, and the first pathogenic variant of c. 711A > G (Ala162Thr) in the ALPL gene was detected in 1988 [4,5].
A high level of TNSALP expression is observed in the liver, kidneys, and bones [6]. TNSALP is involved in the dephosphorylation of pyridoxal-5-phosphate (PLP) and inorganic pyrophosphate (PPi) in these organs and tissues. A decrease in its serum activity leads to the accumulation of PPi mineralization inhibitors in tissues, which causes bone and tooth defects [7]. It has been demonstrated that a decrease in TNSALP activity and the concentration of its substrates correlate with the severity of the clinical symptoms of HPP, defining a heterogeneous phenotype of the disease [8]. However, the activity of TNSALP can decrease under the influence of drugs, the lack of zinc, magnesium (in celiac disease), achondroplasia, and anemia and lead to an increase in liver diseases and osteogenesis imperfecta [9].
HPP is usually classified into six clinical forms: perinatal, prenatal benign, infantile, childhood, odonto-HPP, and adult [10]. The most severe perinatal form of HPP manifests in utero or can be obviously diagnosed at birth based on profound skeletal hypomineralization, caput membraneceum, shorted and deformed limbs, and pyridoxine-dependent seizures. These patients have a very high risk of death in the first days of life due to the hypomineralization of the ribs, pulmonary hypoplasia, and asphyxia [11]. The infantile type is characterized by respiratory complications, total demineralization, skeletal deformities, and rickets, with symptoms present in the first six months of life [12]. The childhood form of HPP appears between 6 months and 18 years of age, with mineralization defects, bone deformities, premature loss of deciduous teeth, delayed walking, short stature, brain damage, delayed motor development and skill formation, and the progression of convulsions [13]. Odonto-HPP is typical in early isolated damage to the dentition, with the premature loss of deciduous teeth, impaired teeth growth and structure, and periodontal disease. Adult HPP expresses skeletal hypomineralization leading to fractures in the metatarsal bones and femur, with premature tooth loss, muscle and joint pain, arthropathy, pseudogout and enthesopathy, and neurological symptoms [12].
The clinical symptoms and severity of HPP, however, can vary considerably in different patients, even within the same form of the disease, which is associated with the heterogeneous pathogenic variants in the ALPL gene [14,15]. For this reason, the diagnosis of HPP involves measuring TNSALP activity levels in the serum according to age and gender and searching for mutations in the ALPL gene (NM_000478.6, 12 exons, and a length of about 70,000 bp) [16].
The missense variants of the ALPL gene can affect the level of expression, modification, folding, membrane transport, and dimerization of TNSALP, which is phenotypically indicated by different levels of its residual enzymatic activity [19]. At the same time, it has been established that the severity of HPP depends on the inheritance type. In mild forms, there is a functional loss of a single copy of the ALPL gene, which is reflected in the autosomal-dominant inheritance of the disease, with varying expression and incomplete penetrance. The perinatal and infantile forms are characterized by recessive inheritance [10]. Meanwhile, the pathogenic variants of the ALPL gene can exhibit a dominant-negative effect (DNE) due to the mutant monomer inhibiting the interaction between the wild-type monomers or due to the sequestering of the wild-type protein by the mutant monomer, preventing its transport to the membrane, thereby explaining the presence of the dominant forms of the disease [20]. Some results demonstrate that the in vitro residual enzymatic activity of the mutant protein and/or DNE value correlates with the clinical symptoms of HPP [21]. Study objective: to describe the spectrum and analyze the functional features of the variants of the ALPL gene with regard to different HPP subtypes and clinical symptoms in Russian children.

Variant Frequencies in the ALPL Gene at HPP
Compound heterozygotes were found in 27 of the patients with HPP. A total of 28 different genetic variants were found in this group of patients, including some previously undescribed in other populations (

Variant Frequencies in the ALPL Gene at HPP
Compound heterozygotes were found in 27 of the patients with HPP. A total of 28 different genetic variants were found in this group of patients, including some previously undescribed in other populations ( Table 2). The variants were classified according to the American College of Medical Genetics and Genomics (ACMG) guidelines (Richards et al., 2015). In bold are the variants that are not represented in the Global Variome shared LOVD ALPL database as of 1 August 2022 (VUS: a variant of uncertain (or unknown) significance).
The heterozygous carriage of the variants was detected in 225 of the total 1612 patients with decreased alkaline phosphatase activity. A total of 66 unique genetic variants were identified (Table 3).  In bold are variants that are not represented in the Global Variome shared LOVD ALPL database as of 1 August 2022 (VUS a variant of uncertain (or unknown) significance). All genetic variants are presented in Table S1 in the Supplementary Materials.

Analysis of the Variants Depending on Type and Localization in the ALPL Gene
Compound hererozygotes in the ALPL gene were detected in 27 of the individuals. The majority of them were missense (75.0%), with 17.9% frameshift and in frame, 3.6% splice variants, and 3.6% duplications ( Figure 5).  Table S1 in the Supplementary Materials.

Analysis of the Variants Depending on Type and Localization in the ALPL Gene
Compound hererozygotes in the ALPL gene were detected in 27 of the individuals. The majority of them were missense (75.0%), with 17.9% frameshift and in frame, 3.6% splice variants, and 3.6% duplications ( Figure 5). Interestingly, 50.0% of the variants (14/28) in the patients with two variants were localized in the 5th (6 variants), 10th (4 variants), and 12th (4 variants) exons of the ALPL gene ( Figure 6). Interestingly, 50.0% of the variants (14/28) in the patients with two variants were localized in the 5th (6 variants), 10th (4 variants), and 12th (4 variants) exons of the ALPL gene ( Figure 6).     Interestingly, the two variants (Ser368del and Glu455Asp) were localized in the active center of the enzyme. The Glu291Gln mutation was located in the calcium-binding domain, and mutations Tyr388Cys, Arg391ValfsTer12, Arg455His, and GGYTPRG386_392ATGGVST) were located in the corona domain. These mutations may also lead to decreased enzyme activity. Another mutation, Asp294Ala, located in the binding center of this domain leads to pathological changes [22]. There were also seven mutations, Val50AlafsTer20, Gly61Glu, Arg71Cys, Gln76His, Thr85Pro, Val459Met, and Val483Met, located in the homodimer interaction site. These mutations may cause the disruption of homodimer formation and, as a consequence, the disruption of protein incorporation into the cell membrane.

Analysis of ALPL Gene Variants Depending on the Form of HPP and Clinical Symptoms in Patients
We analyzed the genotypes of the patients with two pathogenic and VUS forms (n = 22) separately, according to their most severe clinical symptoms and the form of HPP (Table 4).  A total of three out of four patients with the perinatal form, according to the data in Table 4, were homozygotes (two in the missense and one in the nonsense variants) and had bone disorders and skull hypomineralization; one child had hypotonia and another had respiratory disorders. Of the eight patients with the infantile form, six were compound heterozygotes and two were heterozygotes of the missense and nonsense variants. This group of patients was predominated by skeletal deformities (87.5%), loss of teeth (75.0%), and neuropsychomotor developmental delay (75.0%). Of the ten patients with the pediatric form, seven were compound heterozygotes, two were splice heterozygotes, and one was nonsense heterozygote. Tooth loss (100.0%) and hypotonia (60.0%) prevailed in this group of patients. It is noteworthy that the homozygous patients, regarding missense variants or compound heterozygotes for nonsense variants, had severe clinical symptoms.
Further, we compared the frequencies of missense and nonsense variants depending on the form of HPP (Table 3, Figure 8). A total of three out of four patients with the perinatal form, according to the data in Table 4, were homozygotes (two in the missense and one in the nonsense variants) and had bone disorders and skull hypomineralization; one child had hypotonia and another had respiratory disorders. Of the eight patients with the infantile form, six were compound heterozygotes and two were heterozygotes of the missense and nonsense variants. This group of patients was predominated by skeletal deformities (87.5%), loss of teeth (75.0%), and neuropsychomotor developmental delay (75.0%). Of the ten patients with the pediatric form, seven were compound heterozygotes, two were splice heterozygotes, and one was nonsense heterozygote. Tooth loss (100.0%) and hypotonia (60.0%) prevailed in this group of patients. It is noteworthy that the homozygous patients, regarding missense variants or compound heterozygotes for nonsense variants, had severe clinical symptoms.

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Further, we compared the frequencies of missense and nonsense variants depending on the form of HPP (Table 3, Figure 8). A total of three out of four patients with the perinatal form, according to the data in Table 4, were homozygotes (two in the missense and one in the nonsense variants) and had bone disorders and skull hypomineralization; one child had hypotonia and another had respiratory disorders. Of the eight patients with the infantile form, six were compound heterozygotes and two were heterozygotes of the missense and nonsense variants. This group of patients was predominated by skeletal deformities (87.5%), loss of teeth (75.0%), and neuropsychomotor developmental delay (75.0%). Of the ten patients with the pediatric form, seven were compound heterozygotes, two were splice heterozygotes, and one was nonsense heterozygote. Tooth loss (100.0%) and hypotonia (60.0%) prevailed in this group of patients. It is noteworthy that the homozygous patients, regarding missense variants or compound heterozygotes for nonsense variants, had severe clinical symptoms.
Further, we compared the frequencies of missense and nonsense variants depending on the form of HPP (Table 3, Figure 8).
(60.0%) prevailed in this group of patients. It is noteworthy that the homozygous patients, regarding missense variants or compound heterozygotes for nonsense variants, had severe clinical symptoms.
Further, we compared the frequencies of missense and nonsense variants depending on the form of HPP (Table 3, Figure 8). As follows from the data presented in Figure 4, there is not a statistically significant (p > 0.05) predominance of the nonsense mutations of the ALPL gene in patients with the perinatal form compared with the infantile and childhood forms of HPP.
We also compared the frequencies of the homozygous and heterozygous variants of the ALPL gene in different forms of HPP (Table 3, Figure 9). As follows from the data presented in Figure 4, there is not a statistically significant (p > 0.05) predominance of the nonsense mutations of the ALPL gene in patients with the perinatal form compared with the infantile and childhood forms of HPP.
We also compared the frequencies of the homozygous and heterozygous variants of the ALPL gene in different forms of HPP (Table 3, Figure 9). The number of homozygotes with the perinatal form was significantly (p < 0.01) higher than the number of these genotypes in patients with the infantile and childhood forms of HPP. On the contrary, the frequency of the compound heterozygotes and heterozygotes was higher (p < 0.01) in patients with the infantile and childhood forms when compared to the perinatal form of HPP. The number of homozygotes with the perinatal form was significantly (p < 0.01) higher than the number of these genotypes in patients with the infantile and childhood forms of HPP. On the contrary, the frequency of the compound heterozygotes and heterozygotes was higher (p < 0.01) in patients with the infantile and childhood forms when compared to the perinatal form of HPP.

Analysis of TNSALP Activity Depending on the Form, Type of Variants, and the Genotype of the Patients
We analyzed the TNSALP activity, depending on the form of HPP (Table 3, Figures 10  and 11). The number of homozygotes with the perinatal form was significantly (p < 0.01) higher than the number of these genotypes in patients with the infantile and childhood forms of HPP. On the contrary, the frequency of the compound heterozygotes and heterozygotes was higher (p < 0.01) in patients with the infantile and childhood forms when compared to the perinatal form of HPP.

Analysis of TNSALP Activity Depending on the Form, Type of Variants, and the Genotype of the Patients
We analyzed the TNSALP activity, depending on the form of HPP (Table 3, Figures  10 and 11).  of normal TNSALP at this age. *Most patients with the childhood form were diagnosed at the age of 2-9 years. The middle limit of normal was taken from the study [23]. Figure 11. Residual activity of TNSALP variants relative to their wild-type, according to HPP subtype [21].
Based on the works of [21], we compared the theoretical residual activity of the variants in our group of patients according to its TNSALP domain localization (Figure 11).
The results presented in Figures 10 and 11 establish that residual TNSALP activity in the perinatal form was statistically significantly (p < 0.0005) lower than in the infantile and pediatric (p < 0.05) forms of HPP.
However, the theoretically calculated (the real data for these groups of patients are partially available), heterozygotes and compound heterozygotes, with variants in the ALPL gene (Table 4, Figure 12A), have higher residual protein activity compared to the homozygotes. We did not detect differences between the real age-related changes and the Figure 11. Residual activity of TNSALP variants relative to their wild-type, according to HPP subtype [21].
Based on the works of [21], we compared the theoretical residual activity of the variants in our group of patients according to its TNSALP domain localization (Figure 11).
The results presented in Figures 10 and 11 establish that residual TNSALP activity in the perinatal form was statistically significantly (p < 0.0005) lower than in the infantile and pediatric (p < 0.05) forms of HPP.
However, the theoretically calculated (the real data for these groups of patients are partially available), heterozygotes and compound heterozygotes, with variants in the ALPL gene (Table 4, Figure 12A), have higher residual protein activity compared to the homozygotes. We did not detect differences between the real age-related changes and the theoretically-calculated activity of the TNSALP for different genotypes ( Figure 12B).

Analysis of the Activity of the ALPL Gene Variants Depending on Their Domain Localization and Origin
We compared the activity of the ALPL gene variants in our group of patients depending on their domain localization ( Figure 13).

Analysis of the Activity of the ALPL Gene Variants Depending on Their Domain Localization and Origin
It was found that the activity of TNSALP for those variants encoding the amino acids in the "active center" and corona domain was statistically significantly (p < 0.05) lower than the activity in the variants at the homodimeric interface (nonspecific sites of the protein). This implies that these sites in the protein structure are crucial for the preservation of enzyme activity.
We compared the activity of the ALPL gene variants in our group of patients depending on their domain localization ( Figure 13). It was found that the activity of TNSALP for those variants encoding the amino acids in the "active center" and corona domain was statistically significantly (p < 0.05) lower than the activity in the variants at the homodimeric interface (nonspecific sites of the protein). This implies that these sites in the protein structure are crucial for the preservation of enzyme activity.

Discussion
In our study of Russian children with HPP, the most frequent symptoms were tooth loss (72.7%), skeletal deformities (63.6%), hypotonia (50.0%), and neuropsychomotor developmental delay (50%) (Figure 1). Whereas, in the European and American childhood populations (n = 121), the main symptoms of HPP were premature tooth loss (48.2%), bone disorders (44.2%), craniosynostosis (26.7%), and pain syndrome (19.3%) [24]. Among the four Russian children with perinatal HPP, the most frequent symptoms were bone deformities (75%), skull bone deformity (75%), skull hypomineralization (75%), hypotonia (50%), craniosynostosis (50%), pectus excavatum (50%), bone hypomineralization (50%), limb shortening (50%), and respiratory failure (50%) (Figure 2). Whereas, for Canadian and American children with perinatal HPP (n = 30), the most frequent symptoms were fractures (40.0%), skull abnormalities (40.0%), and general motor difficulties (23.3%) [25]. Notably, there are common clinical features for different populations, as well as specific ones. The most common symptoms in the eight Russian children with infantile HPP were skeletal deformities (87.5%), loss of teeth (75.0%), neuropsychomotor developmental delay (75.0%), and nutritional disorders (50.0%) (Figure 3). In contrast, in a group of Canadian and American children with infantile HPP (n = 102), the leading symptoms were loss of teeth (61.8%), cranial malformations (43.1%), and motor and mobility disorders (40.2%) [25]. Among the 10 Russian patients with childhood HPP, the most frequent symptoms were loss of teeth (100.0%), hypotonia (60.0%), skeletal deformity (40.0%), gait disorders (40.0%), muscle and joint pain (40.0%) motor retardation (40.0%), and fatigue (40.0%) (Figure 4). In Canadian and American children with childhood HPP (n = 87), the main symptoms were loss of teeth (70.1%), fractures (34.5%), and pain syndrome (43.7%) [25]. Note that those patients with more severe symptoms (skeletal deformities, loss of teeth, etc.) in the Russian, American, and European populations have a homozygous genotype or have nonsense variants (deletions or splice variants) in the genotype. In general, the low frequency of HPP detection in the Russian population is due to the fact that the main criterion for selecting patients was not the presence of specific clinical symptoms but a recorded low level of alkaline phosphatase, which could not be associated with HPP. The difference in the frequency of the clinical symptoms in Russian patients compared with foreign data is due to (1) a small sample of our patients with a confirmed diagnosis, for whom the detailed clinical data were available, (2) the retrospective analysis of clinical data from the medical record, and (3) a lack of uniform criteria for assessing some symptoms (for example, if a child did not complain of pain by their own accord, they could not be asked about it).
Additionally, we identified a total of 79 unique variants of the ALPL gene, characterized by residual enzymatic activity, from 252 Russian patients with HPP. Among them, patients with two pathogenic variants had 28 different variants. Of these, 75% of the patients had missense variants, 17.9% had frameshift deletions, 3.6% had duplications, and 3.6% had splicing variants. American and French researchers obtained similar frequencies for their patients. They found a total of 155 variants of the ALPL gene in 345 patients with HPP. Missense variants accounted for 146 (94.2%) patients, nonsense variants (variants with a broken chain) for five (3.2%), variants with a reading frame shift for two (1.3%), and deletions for two (1.3%) patients [23]. It should be noted that our group of patients was less characterized by missense variants, while the reading frameshift deletions were more representative. This is probably associated with the fact that our group included patients both with two variants and/or VUS variants and with a more severe form of HPP.
The c.571G > A(p.Glu191Lys) variant in the 6th exon in the ALPL gene was the most frequent among the compound-heterozygote and heterozygotes in the Russian population at 28.89% (73/252). According to the study by del Angel G. et al., the frequency of this c.571G > A/p.Glu191Lys variant is more than 10 −3 [23]. The c.571G > A/p.Glu191Lys variant is also the most common (with a frequency of up to 55%) in patients with HPP of a European origin [26], and the initial frequency of this variant was 0.08 in infants, children, and patients with odonto-HPP [6]. The c.571G > A/p.Glu191Lys variant has been demonstrated to reduce TNSALP enzymatic activity by up to 68% (207.1 ± 39.3 U/µg) when compared with wild-type enzyme activity (302.5 ± 102.2 U/µg) [26].
The other variants, such as c.1001G > A (p.Gly334Asp), both in our study and in the study by del Angel G. et al. [23], were detected only in patients with HPP at a carrier frequency of less than 1%, which corresponds to the frequency of this variant in Manitoba Mennonites [27]. Finally, it should be noted that some variants can be detected with a higher frequency in a particular population. For example, the frequency of the c.787T > C/p.Tyr263His variant in the Japanese population is 0.31-0.55, and in the gnomAD database, 0.1833 [28,29] of the p.Asp378Val and p.Leu520ArgfsX86 variants are most common in the USA [6] and Japan [30], respectively. The majority of the mutations in the ALPL gene were localized in the 5th, 7th, 10th, and 3rd exons [18].
In the study by del Angel G. et al. (2020), the pathogenic variants (reading frame shift, deletions, and splicing variants) exhibited close to zero residual enzymatic activity. An interesting fact is that, whereas low residual enzymatic activity can serve as a predictor of pathogenicity, the converse statement is incorrect. High residual activity values for the variants are not necessarily a predictor of their benignity. In calculating the activity within patients with HPP, it was found that extremely low in vitro values correlate with perinatal lethal or infantile HPP subtypes (Figure 7). At the same time, there was an overlap in activity between patients with HPP and healthy donors (≥50%) [21]. Consequently, the prognostic value of low TNSALP enzymatic activity is limited by extremely low values in the most severe HPP subtypes. Data obtained by gnomAD (version 2.1.1) [6] show that 844 out of 923 genetic variants are rare, with allele frequencies less than <10 −4 in the population, independent of the residual activity level, which is compatible with the rare nature of HPP, especially in its perinatal and infantile subtypes [15,31]. Meanwhile, variants with frequencies of more than 10 −3 have higher residual TNSALP activity [21]. This is related to evolutionary pressure, which results in variants with low activity occurring at low population frequencies. This conclusion is supported by the comparison of variant residual activity with clinical significance [32]. In addition, high values of residual enzymatic activity (>50% of WT values) can also be associated with the clinical subtype of HPP. Notably, pathogenic variants in the ALPL gene can have a dominant-negative effect (DNE) due to the inhibition of the interaction with the wild-type monomers by mutant monomers or as a result of the sequestration of the wild-type protein by the mutant monomer, preventing its transport to the membrane [33]. In our study, the residual enzymatic activity of TNSALP was most decreased for those variants associated with amino acid substitutions in the active center, corona domain, and the homodimeric interface.
Another study noted that pathogenic variants are localized at the calcium-binding site [34]. In our study, curiously, the residual activity of TNSALP at the homodimer interaction site covered a wide range. Consequently, the clinical significance of the variants in this domain may depend on the positional interactions and the specific amino acid changes involved. This conclusion is confirmed by comparing the residual activity of a variant to its clinical significance [32]. At the same time, high values of residual enzymatic activity (>50% of WT values) may also be associated with the clinical subtype of HPP. In addition, it is suggested that some alleles in patients with severe types of HPP may not reduce enzymatic activity but impair its transport and the localization of the TNSALP dimer in the extracellular domain or by impairing the creation of aggregates that are improperly cleaved in the proteasome [19,35].

Patients with Hypophosphatasia
Genetic testing was performed on 1612 patients, with decreased activity of alkaline phosphatase in the blood plasma from different cities and regions of the Russian Federation (Moscow, Saint Petersburg, Yekaterinburg, Surgut, Khabarovsk, Cheboksary, Krasnodar, etc.) from 2014-2021. The inclusion criteria for the patients were a detected low level of alkaline phosphatase, clinical symptoms, radiographic changes, and genetic variants in the ALPL gene. Heterozygous carriage of pathogenic and likely pathogenic variants in the ALPL gene was detected in 225 patients. Compound heterozygotes and homozygotes were found in 27 patients. The sex ratio of all subjects was approximately 3:1 (males to females). The average age of onset was 7 months (from 2 h after birth to 14 years old). The mean age of diagnosis was 2.43 years (2 h after birth to 19 years old).

Biochemical Studies
Patients included in this study were tested for screening diagnostic purposes. Fasting blood samples were collected from all patients to obtain serum. TNSALP activity in the serum was measured according to reference values, depending on age and sex using kinetic and colorimetric methods and an alkaline phosphatase kit (BioSystems S.A. biochemical analyzer) (Costa Brava, Barcelona Spain) [36]. The TNSALP concentration in the serum samples was calculated using the following formula: where ∆A/min is the average absorption difference per minute, ε is the molar absorption coefficient of 4-nitrophenol at 405 nm, which is 18450, the optical path (l) is 1 cm, the total reaction volume (Vt) is 1.02, the sample volume (Vs) is 0.02, and 1 U/L is 0.0166 µkat/L. The following factors were used to calculate enzyme activity.
∆A × 2764 = U/L or × 46.08 = µkat//L TNSALP values in patients with two variants in the ALPL gene in different clinical forms of HPP are presented in Table 1.

Molecular Genetic Analysis of a ALPL Gene
The search for mutations in the exons of the ALPL gene was performed using Sanger sequencing. DNA was isolated from dry blood spots or whole blood by phenol-chloroform extraction. Special primers were developed for each exon in the ALPL gene. PCR conditions were selected individually for each exon. The synthesized PCR products were purified with 5 m NH4Ac and 96% ethanol, and then 70% ethanol, dried at 60 • C and dissolved in 10 µL of deionized water. Further, the purified PCR products were sequenced using the reagent BigDye Terminator 3.1 kit (Applied Biosystems, Waltham, MA, USA). Then, capillary electrophoresis was performed in a genetic analyzer 3500xl (Applied Biosystems, Waltham, MA, USA). The obtained exon sequences were analyzed using the Sequence Scanner (version 2.0, Applied Biosystems, Waltham, MA, USA) software. The presence of the mutation was confirmed by visual inspection of the sequencing chromatograms.

Predicting Theoretical Activity of Patient Genotypes
Predicting theoretical TNSALP residual activity in Russian HPP patients (n = 252) depended on genotype (homozygotes, heterozygotes, and compound heterozygotes) and was calculated as a fraction of WT activity for each patient. The TNSALP residual activity for the homozygous patients, which had a variant with complete loss of function (nonsense, splice, or frameshift), was assumed to be null. The TNSALP residual activity for the heterozygotes was detected as 50% from the activity of the homozygotes. In patients with a single LoF variant, the TNSALP residual activity was 0.5, with the suggestion that the LoF allele would not express any protein. The TNSALP residual activity for the compound heterozygotes was calculated as the average activity of both alleles. If an allele had a dominant negative effect (DNE), the TNSALP activity was determined as "activity divided by four", or leaving only 25% from total TNSALP activity, with two copies of allele one [21].

Statistical Analysis
The statistical data were analyzed using GraphPad Prism 8.01 software. The Kolmogorov-Smirnov test was used to determine the type of data distribution. ANOVA, with posthoc (usually Tukey's honest significant difference, HSD) used to find the differences between several normally-distributed samples. When pairwise comparing data with a distribution other than normal, the Mann-Whitney U test was used [37].

Conclusions
From 2014-2021, we performed genetic testing on 1612 patients from various regions of the Russian Federation, with decreased alkaline phosphatase activity in their blood plasma. Among them, we identified 75 variants of the ALPL gene characterized by residual enzymatic activity in 252 Russian patients with HPP. A total of 75% of these patients had missense variants, and the others had nonsense variants. Among them, 71.4% (20/28) of the genetic variants were identified as being pathogenic, and eight variants (28.6%) were of uncertain (or unknown) significance (VUS). Among these variants, 25.0% (7/28: four pathogenic and three VUS) were absent from the database and were initially identified by us.
The high frequency of hypotension in children from the Russian population is notable in the clinical picture, whereas the other clinical symptoms were similar. Among the heterozygous patients, 60.6% (40 of 66) of the genetic variants were pathogenic, one variant (1.5%) was likely pathogenic or pathogenic, and 25 variants (37.9%) were VUS. A total of 18 variants (27.3%: 18/66) were not previously described in the LOVD and dbSNP databases and the literature and were first identified in this research. The frequencies of the nonsense variants and low TNSALP enzymatic activity were higher in patients with the perinatal form of HPP compared to the infantile and childhood forms. Residual activity was detected as a function of the HPP subtype, variant type, and protein domain localization. TNSALP enzymatic activity cannot be used to assess the severity of HPP, but in combination with clinical symptoms, it will contribute to a better understanding of the phenotypic spectrum of HPP.