Two novel mutations in the ALPL gene of unrelated Chinese children with Hypophosphatasia: case reports and literature review

Hypophosphatasia (HPP) is a rare hereditary disorder caused by loss of function mutations in the ALPL gene that encodes the Tissue Nonspecific Alkaline Phosphatase (TNSALP), one of the alkaline phosphatase (ALP) family members [1‐3]. TNSALP is predominantly expressed in the liver, skeleton, kidney and teeth [4]. Its specific function is to cleave the extracellular substrates including inorganic pyrophosphate (PPi), pyridoxal-5-phosphate (PLP) and phosphoethanolamine (PEA) [5]. The deficiency of TNSALP causes the extracellular accumulation of PPi, a potent inhibitor of mineralization, resulting in the defective teeth and bones.

HPP is clinically characterized by decreased level of serum ALP activity and defective skeletal mineralization. It is subdivided into six forms depending on the age at diagnosis: perinatal lethal, prenatal benign, infantile, childhood, adult, and odonto HPP [6]. The patients with lethal perinatal form show markedly impaired mineralization in utero, whereas the patients with infantile form mostly present respiratory complications, widespread demineralization and rachitic symptoms during the first 6 months of life, both of which are defined as severe forms of HPP [6]. The childhood form of HPP displays milder symptoms between 6 months and 18 years with premature loss of primary teeth, delayed walking, short stature and bone deformities. As one of the mildest forms of HPP, odonto HPP is characterized by premature exfoliation of primary and/or severe dental caries without abnormalities of the skeletal system.

Both autosomal dominant and recessive transmission have been shown in HPP. In general, the more severe the HPP is, the more often it could be recessive inheritance. For instance, lethal perinatal form and most infantile forms of HPP are recessively inherited, whereas less severe forms, including perinatal benign, childhood, odonto and adult forms of HPP, show both dominant and recessive inheritance [7]. It has been reported that severe HPP has lower prevalence (1/300,000) than that of less severe forms of HPP (1/6370) [8]. Further, more severe forms of the HPP present lower serum AP activity levels. The studies all indicated that the severity of the HPP is correlated with the activity level of ALP, which is encoded by the ALPL gene [9].

The diagnosis of HPP is based on low level of serum ALP activity and genetic testing of the ALPL gene mutations. To date, a total of 390 disease-causing mutations in the ALPL gene have been reported, most of which are missense mutations (70.3%) (http://​www.​sesep.​uvsq.​fr/​03_​hypo_​mutations.​php). It has been suggested that large variety of mutations results in variable deficiency of ALP activity and distinct clinical phenotypes [6, 10]. Although genotype-phenotype correlation has been observed, it needs more clinical and genetic data to support. Particularly, there are few pediatric HPP patients reported in the Chinese population.

The ALPL gene encoding TNSALP was located on chromosome 1p34–36. HPP is caused by loss of function mutations in the ALPL gene. The first case of HPP was reported in 1948 and the ALPL gene mutation was first reported in 1988 [1, 21]. HPP presents a heterogeneous phenotype ranging from life threatening to asymptomatic presentation. In general, the pattern of inheritance in HPP is autosomal recessive. However, HPP may also be transmitted by autosomal dominant and incomplete penetrance of dominant transmission [22]. The severe forms of HPP usually transmit by autosomal recessive, while milder forms of HPP more often present with autosomal dominant with variable expressivity. These heterogeneous clinical features and inheritance patterns result in the difficulty of timely diagnosis and challenging of genetic counselling [23, 24].

In the present study we investigated four Chinese children affected by three forms of HPP, which were lethal infantile, childhood and odonto HPP. All patients follow autosomal recessive inheritance pattern. They presented clinical symptoms early at 1 day old (patient 1) or at 1 year old (patient 2, 3 and 4). All four patients had low serum ALP activity. As reported, patients with perinatal or infantile HPP have high morbidity and mortality in the first 5 years of life [7]. Patient 1 was a typical infantile form of HPP who presented respiratory distress, nearly undetectable ALP activity and general skeletal hypomineralization. He died of respiratory failure at 3 months old, therefore was diagnosed as lethal infantile form of HPP. To the best we know, this was the first case of lethal infantile HPP reported in the Chinese population. Recent researches revealed that a rapidly worsening clinical course often occurs in prenatal [25] or infantile HPP mainly due to respiratory compromise [7]. However, further investigation is need to clarify the mechanism.

It has been reported that diagnostic delay is common due to limited awareness of HPP [26]. Patient 2 and 3 were Childhood HPP who presented premature loss of deciduous teeth and muscle weakness at 1 year old. Patient 3 also had joint swelling and bone pain starting at 1 year old, however, he was confirmed with HPP at 8 years old. Patient 4, although his early loss of deciduous teeth started at 1 year old, was diagnosed at 4 years old. These observation demonstrated that some HPP cases may not be recognized well and managed timely.

Molecular diagnosis provides great advantage to confirm the diagnosis of HPP. HPP is caused by a loss of function mutation in the ALPL gene encoding TNSALP. It has been reported that few mutations may be frequent in particular populations [23]. For instance, c.1559delT and p.F327 L are two common mutations in the Japanese population, whereas c.571G > A (p.E191 K) is identified in half of European patients with moderate HPP [27, 28]. To elucidate the mutational characteristics of the ALPL gene in the Chinese population, we reviewed all reported Chinese HPP cases in the literature. We found that the most mutations in Chinese HPP patients were missense variants located in exon 5 in the ALPL gene. No frequent mutations were recognized (Fig. 3). In the present study, six ALPL mutations, including five missense mutations and one splicing mutation, were identified. All of these mutations were first reported in the Chinese population.

It is considered that the variety of ALPL mutations results in highly variable clinical expressivity, resulting in difficulty to assess the severity of a novel mutation in the ALPL gene [15]. The severity of HPP symptoms was correlated with the level of ALP activity affected by the ALPL mutation [29]. Severe HPP often exhibit very low level of ALP activity, whereas mild HPP usually retain most ALP activity. Severe form manifests early, whereas mild form may be diagnosed in adulthood [30]. Patient 1 was a homozygote of c.359G > C which has not been previously described in the literature. However, typical clinical features, very low level of serum ALP activity, overall hypomineralization and homozygote status all were consistent with severe infantile form of HPP, indicating the variant c.359G > C seems to be at a crucial position of ALP protein. Further investigation revealed that the variant c.359G > C (p.G120A) is located in the homodimer interface, a crucial site of secondary structure in the TNSALP and highly conserved throughout many species. Thus, the novel mutation c.359G > C was strongly indicated to be disease-causing and related to severe infantile form of HPP. Further functional study of the mutation c.359G > C is needed.

Patient 2, 3 and 4 were compound heterozygote in the ALPL gene. Patient 2, the childhood HPP, had been identified with two known pathogenic variants, c.212G > A (p.R71H) at exon 4 and c.571G > A (p.E191K) at exon 6 [31, 32]. As another childhood HPP, Patient 3 carried the identical c.571G > A (p.E191K) variant of Patient 2 and another pathogenic c.203C > T (p.T68 M) variant at exon 4 which was also reported previously [33]. Interestingly, the variant c.571G > A (p.E191 K) in Patient 2 and 3, was the common mutation reported in European population with moderate HPP. Patient 4 was the mildest odonto HPP in the present study. He demonstrated c.979 T > C (p.G120A) and c.1017dupG (p. H340AfsX3). The c.979 T > C (p.G120A) variant was pathogenic reported previously [34], whereas the novel variant c.1017dupG (p.H340AfsX3) was predicted to result in a translation frameshift and premature protein termination (p. H340AfsX3).

Some limitations exist in the present study. First, the number of HPP patients is not big enough to reveal the phenotype–genotype correlations. Second, PLP, the best markers of HPP, was not detected in the present study since the method is still not available in our center. Future study will be focused on collecting more data to reveal the correlation between phenotype and genotype of HPP in the Chinese population.

Currently enzyme replacement therapy with asfotase alfa (Strensiq™, USA) is currently the only approved treatment for HPP. Asfotase alfa can restore normal ALP levels and prevented skeletal and dental manifestations of HPP. Unfortunately it has been approved in many countries but not in China. None of the patients in the present study have received asfotase alfa for treatment.

In the present study, we described the clinical and genetic characteristics of HPP in four unrelated Chinese pedigrees who were affected with infantile, childhood and odonto forms of HPP. All patients followed autosomal recessive inheritance pattern. Six mutations in the ALPL gene were identified including four known mutations and two novel mutations. The novel missense mutation (c.359G > C) caused the decrease of ALP activity and was related to lethal infantile form of HPP. The study expanded knowledge about the characteristics of HPP.