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Characterization of Genetic Variants of Uncertain Significance for the ALPL Gene in Patients With Adult Hypophosphatasia.

ABSTRACT: Hypophosphatasia (HPP) a rare disease caused by mutations in the ALPL gene encoding for the tissue-nonspecific alkaline phosphatase protein (TNSALP), has been identified as a potentially under-diagnosed condition worldwide which may have higher prevalence than currently established. This is largely due to the overlapping of its symptomatology with that of other more frequent pathologies. Although HPP is usually associated with deficient bone mineralization, the high genetic variability of ALPL results in high clinical heterogeneity, which makes it difficult to establish a specific HPP symptomatology. In the present study, three variants of ALPL gene with uncertain significance and no previously described (p.Del Glu23_Lys24, p.Pro292Leu and p.His379Asn) were identified in heterozygosis in patients diagnosed with HPP. These variants were characterized at phenotypic, functional and structural levels. All genetic variants showed significantly lower in vitro ALP activity than the wild-type (WT) genotype (p-value <0.001). Structurally, p.His379Asn variant resulted in the loss of two Zn²⁺ binding sites in the protein dimer which may greatly affect ALP activity. In summary, we identified three novel ALPL gene mutations associated with adult HPP. The correct identification and characterization of new variants and the subsequent study of their phenotype will allow the establishment of genotype-phenotype relationships that facilitate the management of the disease as well as making it possible to individualize treatment for each specific patient. This would allow the therapeutic approach to HPP to be personalized according to the unique genetic characteristics and clinical manifestations of each patient.

SUBMITTER: Sanabria-de la Torre R

PROVIDER: S-EPMC9047899 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Characterization of Genetic Variants of Uncertain Significance for the <i>ALPL</i> Gene in Patients With Adult Hypophosphatasia.

Sanabria-de la Torre Raquel R Martínez-Heredia Luis L González-Salvatierra Sheila S Andújar-Vera Francisco F Iglesias-Baena Iván I Villa-Suárez Juan Miguel JM Contreras-Bolívar Victoria V Corbacho-Soto Mario M Martínez-Navajas Gonzalo G Real Pedro J PJ García-Fontana Cristina C Muñoz-Torres Manuel M García-Fontana Beatriz B

Frontiers in endocrinology 20220414

Hypophosphatasia (HPP) a rare disease caused by mutations in the <i>ALPL</i> gene encoding for the tissue-nonspecific alkaline phosphatase protein (TNSALP), has been identified as a potentially under-diagnosed condition worldwide which may have higher prevalence than currently established. This is largely due to the overlapping of its symptomatology with that of other more frequent pathologies. Although HPP is usually associated with deficient bone mineralization, the high genetic variability of ...[more]

PMID: 35498405

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Project description:IntroductionHypophosphatasia (HPP) is an inborn metabolic error caused by mutations in the ALPL gene encoding tissue non-specific alkaline phosphatase (TNSALP) and leading to decreased alkaline phosphatase (ALP) activity. Although the main characteristic of this disease is bone involvement, it presents a great genetic and clinical variability, which makes it a systemic disease.MethodsPatients were recruited based on biochemical assessments. Diagnosis was made by measuring serum ALP and pyridoxal 5-phosphate levels and finally by Sanger sequencing of the ALPL gene from peripheral blood mononuclear cells. Characterization of the new variants was performed by transfection of the variants into HEK293T cells, where ALP activity and cellular localization were measured by flow cytometry. The dominant negative effect was analyzed by co-transfection of each variant with the wild-type gene, measuring ALP activity and analyzing cellular localization by flow cytometry.ResultsTwo previously undescribed variants were found in the ALPL gene: leucine 6 to serine missense mutation (c.17T>C, L6S) affecting the signal peptide and threonine 167 deletion (c.498_500delCAC, T167del) affecting the vicinity of the active site. These mutations lead mainly to non-pathognomonic symptoms of HPP. Structural prediction and modeling tools indicated the affected residues as critical residues with important roles in protein structure and function. In vitro results demonstrated low TNSALP activity and a dominant negative effect in both mutations. The results of the characterization of these variants suggest that the pleiotropic role of TNSALP could be involved in the systemic effects observed in these patients highlighting digestive and autoimmune disorders associated with TNSALP dysfunction.ConclusionsThe two new mutations have been classified as pathogenic. At the clinical level, this study suggests that both mutations not only lead to pathognomonic symptoms of the disease, but may also play a role at the systemic level.

Project description:BackgroundHypophosphatasia (HPP) is a rare hereditary disorder characterized by defective bone and tooth mineralization caused by mutations in the alkaline phosphatase (ALPL) gene encoding tissue-nonspecific alkaline phosphatase (TNSALP). Here we performed clinical and molecular studies on 5 HPP children to investigate the pathogenic mechanisms of the ALPL gene variants.MethodsClinical and genetic analyses were performed on 5 HPP children, and the loci where ALPL variants were identified. Plasmids containing the relevant loci were constructed. The molecular and cellular mechanisms of the pathogenic ALPL variants were investigated by cellular immunofluorescence, enzyme activity assay, and protein expression assay.ResultsA total of 6 ALPL variants were identified in 5 HPP children: proband 1: c.346G>A (p.A116T); proband 2: c.346G>A (p.A116T)/deletions from c.1097 to c.1099 CCT (p.T366_S367deli) compound heterozygous variant; proband 3: insertion of G from c.1014 to c.1015 (p.H338fs)/c.1446C>A (p.H482Q) compound heterozygous variant; proband 4: c.920C>T (p.P307L); and proband 5: c.883A>G (p.M295V). Twenty-four hours after the HEK-293T was transfected with different variant plasmids, its alkaline phosphatase activity and enzyme protein content were reduced compared with the wild type, and there were differences among different variants. Except for 1014-G-1015+C1446A, the degree of reduction in enzyme activity was negatively correlated with the severity of clinical manifestations. Immunofluorescence revealed that the variants (especially c.883A>G and c.920C>T) caused a decrease in alkaline phosphatase expression in the cellular membrane.ConclusionsIn total, 3 novel variants were identified in these 5 HPP children, the discovery of which will enrich the human ALPL gene mutation database. Different variants in the ALPL gene can downregulate the activity of TNSALP enzyme (and thus affect its function) by affecting protein expression and translational modifications. The same variant may cause clinical manifestations of different severities in different individuals due to the presence of dominant negative effects, alterations in noncoding sequences, blind area of intron regulatory region sequencing, and variations in environmental and individual factors. The molecular mechanisms via which the ALPL gene exerts its expression effect in vivo are highly variable and warrant further investigation.

Dataset Information

Characterization of Genetic Variants of Uncertain Significance for the ALPL Gene in Patients With Adult Hypophosphatasia.

Publications

Characterization of Genetic Variants of Uncertain Significance for the <i>ALPL</i> Gene in Patients With Adult Hypophosphatasia.

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