In this study, two new, previously undescribed variants in the ALPL gene have been identified in two 15-year-old male patients, leading to the genetic screening of the patients' relatives for better understanding and personalized management for affected families. Both mutations have already been introduced into the VarSome database with references NM_000478:c.17T > C for the L6S variant and NM_001369805.2:c.498_500del for the T167del variant.
In silico predictions concluded that T167del is probably pathogenic while L6S does not present a clear consensus. The results of the alignment analysis indicate that the Met1, Leu6 and Leu8 amino acids for L6S variant and 165TTTR168 amino acids for T167del variant are highly conserved across all species included in this analysis (Figure S1, Supplemental Material). These results besides 3D modeling (Fig. 3B and 3C) suggest that these residues may play a major role in the protein function. We suggest that Met1, Leu6 and Leu8 amino acids in L6S variant could be involved in the cellular localization of the protein agreeing with the results obtained by Silvent et al. 32. For T167del variant T167 could play an important role as a stabilizer of the active site, since we found that it has a structural function providing stability to E332, a direct Mg2+ ligand that is part of the catalytic center.
Other mutations have been identified in the same positions as L6S variant (c.17T > A; L6*) 33 and T167del variant (c.500C > T; T167M) 34–36. For the first one, this variant is considered pathogenic; however, the effect of the mutation is not comparable to our identified variant since c.17T > A gives rise to a nonsense mutation resulting in a 6 amino acid peptide33. For the second one, it has been associated with a severe pathogenic phenotype of HPP being described in patients with severe childhood HPP 34, patients with non-lethal perinatal HPP35 and adult HPP36. Based on our results and previous scientific literature, T167del variant has been classified as pathogenic.
The results of the in vitro TNSALP activity are consistent with the results observed at the clinical level. P1 presented significantly higher serum ALP activity (73 and 42 UI/L) than P2 (45 and 38 UI/L) in concordance with the in vitro results where a substantial decrease in TNSALP activity was observed in T167del compared to L6S (Fig. 4C). The suspicion that both variants might be pathogenic is reinforced by the determination of their negative dominance over the WT monomer (Fig. 4D). In both cases, TNSALP activity was lower than that obtained in the control group. The fact that the L6S allele presents a moderate dominance over the WT allele is consistent with the serum ALP activity of P1 being close to the reference range. Furthermore, it has been described that mutations with higher DNE are usually found in the crown domain, homodimeric interphase, or the area of the active center37. This agrees with the results obtained for T167del (located close to the active centre of the protein) which showed more severe DNE.
Our results showed retention in the efflux of protein to the membrane both in homozygosity and in heterozygosity due to the drastic reduction both in the amount of protein in the membrane and in the number of cells expressing TNSALP. In this context, the use of flow cytometry is a very useful tool to obtain relevant explanations about TNSALP cellular localization. In the case of the L6S variant, the same mutation has been described in gap junction protein beta 1, producing its accumulation at the intracellular level 38. We have observed a decrease not only in the percentage of cells that express TNSALP but also in the amount of protein in the membrane. Thus the amino acid L6 could play an important role in the process of exporting TNSALP to the cell membrane which could explain the low antigenic density of TNSALP although the percentage of cells expressing this protein is higher. In the case of the T167del variant, the underlying mechanisms in the alteration of the cellular localization of the protein are unknown to date. However, due to the pathogenicity of the mutation, we suggest that the mutated protein could be taken to the proteasome for its complete degradation, which would explain the low activity and antigenic density of both homozygous and heterozygous.
Regarding clinical manifestations, in Family 1 there is a clear vitamin D deficiency in patients affected by L6S variant. Vitamin D insufficiency is a highly prevalent in HPP patients and should be treated with vitamin D supplements to avoid secondary hyperparathyroidism39 as observed in II-2 and P1. I-2 had tooth loss at an early age and a series of symptoms different from the pathognomonic symptoms of HPP such as digestive abnormalities (chronic diarrhea, esophagitis and diverticulitis) or inflammatory diseases (arteritis and polymyalgia rheumatica). II-2 presented chronic gastritis as a digestive symptom and giant cell tumor and hyperparathyroidism while II-3 suffered from neurological disorders such as fibromyalgia, immune disorders such as psoriatic arthritis and neutrophilia, skeletal disorders such as pelvic dysmetria and spondyloarthrosis with dyscarthrosis and HBP (Table 3). P1 inherited some of these manifestations such as digestive disorders, vitamin D deficiency and HBP at an early age. P1 also showed slightly elevated levels of parathyroid hormone (Table 1). As for Family 1, both carriers of new T167del variant from Family 2 showed vitamin D insufficiency. II-1 presented early tooth loss consistent with a phenotype of odontohypophosphatasia (Table 3) while P2 suffered from inverted psoriasis and Crohn's disease inherited from II-2 (Table 1). The in vitro results of T167del variant besides to the immune disturbances, suggest that this HPP-related genotype could contribute to a worse prognosis of the comorbidities. In this context, some symptoms such as fibromyalgia36,40–42, digestive affections43 or HBP42 have been slightly linked to HPP; however, these complications as a symptom related to HPP have not been explored in depth to date. Some studies suggest that digestive alterations may be associated with disturbances in the immune system44,45. This hypothesis is reinforced by the clinical manifestations of P2 who has been diagnosed with Crohn's disease and inverse psoriasis. All these symptoms could be a consequence of the role of TNSALP beyond bone metabolism. Accordingly, it has been described that TNSALP has an anti-inflammatory role, increasing the activity of this protein in the blood during episodes of late-onset sepsis in neonates46. TNSALP has ectonucleotidase capacity, hydrolyzing extracellular ATP or LPS that act on Toll-like receptor 4 which is responsible for the activation of the innate immune system by macrophages releasing proinflammatories cytokines as interleukins 6 and 847. These molecules are also degraded by intestinal alkaline phosphatase, and it has been described as a protein involved in the development of inflammatory bowel disease48. Furthermore, TNSALP has been reported to be expressed in phagocytes49, neutrophils50 and T lymphocytes. A study in mice showed that TNSALP is required for the complete stimulation of T lymphocytes and T-cell-dependent colitis8. Given the role of TNSALP in modulating inflammation and the immune response, a deficiency in TNSALP activity may lead to dysregulation of the immune system and the development of inflammatory diseases. In this line some clinical trials where TNSALP is being used as a treatment for acute kidney injury associated with sepsis have been tested51. Based on these findings, the symptomatology observed in in patients I-2 and II-2 of Family 1 with high IL6 levels (7 > pg/mL) and in the symptomatology of II-3 of Family 1 and P2 of Family 2 (Tables 1 and 3) could be explained by low TNSALP activity.
The computational 3D modeling and alignment linked to clinical results and functional analyses, suggest that L6S variant could be classified as likely pathogenic associated with a mild HPP phenotype. Regarding T167del variant, all the data collected suggest the classification of this variant as likely pathogenic with a moderate phenotype.
These results highlight the importance of establishing HPP as a systemic pathology, not only related to bone mineralization disturbances. Currently, digestive and autoimmune disorders are considered as independent processes in HPP patients; however, our findings reveal that these processes could be related to ALP deficiency. Despite this, the development of these comorbidities remains unknown, so more studies are needed in this area to understand the mechanisms of pathogenicity of the HPP-related comorbidities. On the other hand, although it is difficult to establish a gene-phenotypic relationship of each variant described in HPP due to the participation of several external factors that enhance phenotypic variability, it is important to identify and characterize new variants that serve as a starting point for future research and patient management.