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Novel ALPL genetic alteration associated with an odontohypophosphatasia phenotype.

ABSTRACT: Hypophosphatasia (HPP) is an inherited disorder of mineral metabolism caused by mutations in ALPL, encoding tissue non-specific alkaline phosphatase (TNAP). Here, we report the molecular findings from monozygotic twins, clinically diagnosed with tooth-specific odontohypophosphatasia (odonto-HPP). Sequencing of ALPL identified two genetic alterations in the probands, including a heterozygous missense mutation c.454C>T, leading to change of arginine 152 to cysteine (p.R152C), and a novel heterozygous gene deletion c.1318_1320delAAC, leading to the loss of an asparagine residue at codon 440 (p.N440del). Clinical identification of low serum TNAP activity, dental abnormalities, and pedigree data strongly suggests a genotype-phenotype correlation between p.N440del and odonto-HPP in this family. Computational analysis of the p.N440del protein structure revealed an alteration in the tertiary structure affecting the collagen-binding site (loop 422-452), which could potentially impair the mineralization process. Nevertheless, the probands (compound heterozygous: p.[N440del];[R152C]) feature early-onset and severe odonto-HPP phenotype, whereas the father (p.[N440del];[=]) has only moderate symptoms, suggesting p.R152C may contribute or predispose to a more severe dental phenotype in combination with the deletion. These results assist in defining the genotype-phenotype associations for odonto-HPP, and further identify the collagen-binding site as a region of potential structural importance for TNAP function in the biomineralization.

SUBMITTER: Martins L

PROVIDER: S-EPMC3872001 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Novel ALPL genetic alteration associated with an odontohypophosphatasia phenotype.

Martins Luciane L Rodrigues Thaisângela L TL Ribeiro Mariana Martins MM Saito Miki Taketomi MT Giorgetti Ana Paula Oliveira AP Casati Márcio Z MZ Sallum Enilson A EA Foster Brian L BL Somerman Martha J MJ Nociti Francisco H FH

Bone 20130619 2

Hypophosphatasia (HPP) is an inherited disorder of mineral metabolism caused by mutations in ALPL, encoding tissue non-specific alkaline phosphatase (TNAP). Here, we report the molecular findings from monozygotic twins, clinically diagnosed with tooth-specific odontohypophosphatasia (odonto-HPP). Sequencing of ALPL identified two genetic alterations in the probands, including a heterozygous missense mutation c.454C>T, leading to change of arginine 152 to cysteine (p.R152C), and a novel heterozyg ...[more]

PMID: 23791648

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Project description:Hypophosphatasia (HP) is a rare genetic disease caused by mutation in the alkaline phosphatase, liver/bone/kidney (ALPL) gene with highly variable clinical manifestations. Efforts have been made to collect cases with novel mutations and to examine how a missense mutation affects ALPL protein function, which remains difficult to predict. The present study investigated the underlying mechanism of ALPL dysfunction in a patient diagnosed with HP. Bidirectional sequencing of the ALPL gene was conducted in a 5‑year‑old Chinese girl preliminary diagnosed with childhood HP. Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping v2 (PolyPhen‑2) tools were used to forecast the impact of the mutation on protein function. Site‑directed mutagenesis was performed and transfected into cells to verify the role of the specific mutation. Furthermore, the mechanism of the impact was investigated via all‑atom molecular dynamics (MD) simulation. The patient demonstrated a compound heterozygote with two missense mutations in the ALPL gene, p.Trp29Arg and p.Ile395Val. Trp29 and Ile395 were determined to be 'tolerable' by SIFT, whereas they were 'possibly damaging' by PolyPhen‑2 in terms of conservation. Additionally, HEK293 cells were transfected with plasmids expressing wild type and/or mutated ALPL. Only 4.1% of ALP activity remained when Trp29 was substituted by Arg, whereas 19.1, 33.7, 50.1 and 7.6% ALP activity remained in cells expressing p.Ile395Val, wild type+p.Trp29Arg, wild type+p.Ile395Val and p.Trp29Arg+p.Ile395Val substitutions, respectively. All‑atom MD simulation demonstrated that the N‑terminal helix of mutated ALPL, where Trp29 is located, separated from the main body of the protein after 30 nsec, and moved freely. These results demonstrated that p.Trp29Arg, as a novel missense mutation in the ALPL gene, reduced the enzymatic activity of ALPL. This effect may be associated with an uncontrolled N‑terminal helix. These results provide novel information about the genetic basis of HP, and may facilitate the development of future therapies.

Project description:It is generally accepted that human cancers derive from a mutated single cell. However, the genetic steps characterizing various stages of progression remain unclear. Studying a unique case of metastatic melanoma, we observed that cell lines derived from metachronous metastases arising over a decade retained a central core of genetic stability in spite of divergent phenotypes. In the present study we expanded our previous observations comparing these autologous cell lines of clonal derivation with heterologous ones and correlated array Comparative Genomic Hybridization (aCGH) with gene expression profiling to determine their relative contribution to the dynamics of disease progression. aCGH and gene expression profiling were performed on autologous cell lines and heterologous melanoma cell lines originated from other patients. A striking correlation existed between total extent of genetic imbalances, global transcriptional patterns and cellular phenotypes; they did not follow a strict temporal progression but stemmed independently at various time points from a central core of genetic stability best explained according to the cancer stem cell hypothesis; although their contribution was intertwined, genomic imbalances detectable by aCGH contributed only 25% of the transcriptional traits determining autologous tumors distinctiveness. Our study provides important insights about the dynamics of cancer progression and supports the development of targeted anti-cancer therapies against stable genetic factors determining the individuality of each patientâs disease that are maintained throughout the end stage of disease. Keywords: genetic modification design PBMC from a female donor were Ficoll gradient separated and used throughout the study as reference.. Validation of array CGH accuracy was done by obtaining six additional PBMC from female donors and six PBMC from male donors to confirm stability of gene representation in autosomes and sex-determined imbalances within the X and Y chromosome regions. Five melanoma cell lines were generated from distinct cutaneous melanoma metastases that progressively appeared in patient 888. Other melanoma cell lines were generated from cutaneous melanoma metastases from other patients. The melanoma cell line A375 was purchased from the American Type Culture Collection. For each cell line arrayCGh has been performed.

Dataset Information

Novel ALPL genetic alteration associated with an odontohypophosphatasia phenotype.

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Novel ALPL genetic alteration associated with an odontohypophosphatasia phenotype.

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