Bone impairment in phenylketonuria is characterized by circulating osteoclast precursors and activated T cell increase.

Ilaria Roato,Riccardo Ferracini,Ludovica Fiore,Francesco Porta,Davide Garelli,Alessandro Mussa,Marco Spada,Lucia D'Amico

doi:10.1371/journal.pone.0014167

Ilaria Roato, Riccardo Ferracini + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0014167

Copy DOI

Abstract

BackgroundPhenylketonuria (PKU) is a rare inborn error of metabolism often complicated by a progressive bone impairment of uncertain etiology, as documented by both ionizing and non- ionizing techniques.MethodologyPeripheral blood mononuclear cell (PBMC) cultures were performed to study osteoclastogenesis, in the presence or absence of recombinant human monocyte-colony stimulating factor (M-CSF) and receptor activator of NFκB ligand (RANKL). Flow cytometry was utilized to analyze osteoclast precursors (OCPs) and T cell phenotype. Tumour necrosis factor α (TNF-α), RANKL and osteoprotegerin (OPG) were quantified in cell culture supernatants by ELISA. The effects of RANKFc and anti-TNF-α antibodies were also investigated to determine their ability to inhibit osteoclastogenesis. In addition, bone conditions and phenylalanine levels in PKU patients were clinically evaluated.Principal FindingsSeveral in vitro studies in PKU patients' cells identified a potential mechanism of bone formation inhibition commonly associated with this disorder. First, PKU patients disclosed an increased osteoclastogenesis compared to healthy controls, both in unstimulated and M-CSF/RANKL stimulated PBMC cultures. OCPs and the measured RANKL/OPG ratio were higher in PKU patients compared to healthy controls. The addition of specific antagonist RANKFc caused osteoclastogenesis inhibition, whereas anti-TNF-α failed to have this effect. Among PBMCs isolated from PKU patients, activated T cells, expressing CD69, CD25 and RANKL were identified. Confirmatory in vivo studies support this proposed model. These in vivo studies included the analysis of osteoclastogenesis in PKU patients, which demonstrated an inverse relation to bone condition assessed by phalangeal Quantitative Ultrasound (QUS). This was also directly related to non-compliance to therapeutic diet reflected by hyperphenylalaninemia.ConclusionsOur results indicate that PKU spontaneous osteoclastogenesis depends on the circulating OCP increase and the activation of T cells. Osteoclastogenesis correlates with clinical parameters, suggesting its value as a diagnostic tool for an early assessment of an increased bone resorption in PKU patients.

Highlights

Phenylketonuria (PKU; OMIM 261600) is an inborn error of amino acid metabolism resulting from deficiency of phenylalanine hydroxylase, the key enzyme for phenylalanine metabolism [1]
Our results indicate that PKU spontaneous osteoclastogenesis depends on the circulating OC precursors (OCPs) increase and the activation of T cells
Osteoclastogenesis correlates with clinical parameters, suggesting its value as a diagnostic tool for an early assessment of an increased bone resorption in PKU patients

Summary

Introduction

Phenylketonuria (PKU; OMIM 261600) is an inborn error of amino acid metabolism resulting from deficiency of phenylalanine hydroxylase, the key enzyme for phenylalanine metabolism [1]. Despite the recommendation of life-long adherence to treatment, poor compliance to dietary prescriptions is common during adolescence [3], as the risk of mental retardation due to hyperphenylalaninemia was historically thought to be insignificant at this age. This laxity of dietary restriction, has been related to systemic complications of PKU in adulthood, as extensively reported recently [4]. Phenylketonuria (PKU) is a rare inborn error of metabolism often complicated by a progressive bone impairment of uncertain etiology, as documented by both ionizing and non- ionizing techniques

Objectives

Methods

Results

Discussion

Conclusion