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A novel missense mutation in the ALPL gene causes dysfunction of the protein.

ABSTRACT: 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.

SUBMITTER: Chen B

PROVIDER: S-EPMC5482188 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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A novel missense mutation in the ALPL gene causes dysfunction of the protein.

Chen Bin B Li Lili L Ren Weitong W Yi Long L Wang Yaping Y Yan Fuhua F

Molecular medicine reports 20170531 1

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 conduct ...[more]

PMID: 28586049

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Project description:PurposeRetinitis pigmentosa (RP) is an inherited retinal disorder that results in the degeneration of photoreceptor cells, ultimately leading to severe visual impairment. We characterized a consanguineous family from Southern India wherein a 25 year old individual presented with night blindness since childhood. The purpose of this study was to identify the causative mutation for RP in this individual as well as characterize how the mutation may ultimately affect protein function.MethodsWe performed a complete ophthalmologic examination of the proband followed by exome sequencing. The likely causative mutation was identified and modeled in cultured cells, evaluating its expression, solubility (both with western blotting), subcellular distribution, (confocal microscopy), and testing whether this variant induced endoplasmic reticulum (ER) stress (quantitative PCR [qPCR] and western blotting).ResultsThe proband presented with generalized and parafoveal retinal pigmented epithelium (RPE) atrophy with bone spicule-like pigmentation in the midperiphery and arteriolar attenuation. Optical coherence tomography scans through the macula of both eyes showed atrophy of the outer retinal layers with loss of the ellipsoid zone, whereas the systemic examination of this individual was normal. The proband's parents and sibling were asymptomatic and had normal funduscopic examinations. We discovered a novel homozygous p.Pro388Ser mutation in the tubby-like protein 1 (TULP1) gene in the individual with RP. In cultured cells, the P388S mutation does not alter the subcellular distribution of TULP1 or induce ER stress when compared to wild-type TULP1, but instead significantly lowers protein stability as indicated with steady-state and cycloheximide-chase experiments.ConclusionsThese results add to the list of known mutations in TULP1 identified in individuals with RP and suggest a possible unique pathogenic mechanism in TULP1-induced RP, which may be shared among select mutations in TULP1.

Dataset Information

A novel missense mutation in the ALPL gene causes dysfunction of the protein.

Publications

A novel missense mutation in the ALPL gene causes dysfunction of the protein.

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