Ex Vivo Osteoclastogenesis from Peripheral Blood Mononuclear Cells Is Unchanged in Adults with Phenylketonuria, Regardless of Dietary Compliance

Abstract

Pathogenic variants in the phenylalanine hydroxylase gene can result in phenylalanine (Phe) accumulation leading to phenylketonuria (PKU; OMIM #261600), a metabolic disease diagnosed in newborn screening. Early treatment with a Phe-restricted diet prevents severe mental retardation. Next to several other health complaints, patients with PKU present with low bone mineral density (BMD) more often than the general population. The etiology of the phenotype is not yet fully understood, and current research focuses on improving special medical foods and changes in osteoclasts (OC) and osteoblasts. Analysis of osteoclastic and oxidative stress control gene expression next to the simple number of OC developing from peripheral blood mononucleated cells (PBMCs) in association with dietary compliance and BMD was therefore part of our analysis. PBMCs were obtained from 17 adults with PKU and 17 age- and sex-matched controls on the same day. PBMCs were differentiated into osteoclasts (OC, Trap-positive multi-nucleated cells (≥3 nuclei)) for 14 days by adding human macrophage colony-stimulating factor (MCSF) and receptor activator of NF-κB Ligand (RANKL). Subsequently, quantitative real-time PCR was performed on OC function and oxidative stress control. Data on dietary compliance during the previous 12 months and 5 years and BMD were collected. PBMCs from adults with PKU and controls were differentiated into comparable numbers of OC (PKU: 53 [17-87] vs. controls: 39 [19-52], p = 0.381) without differences in mRNA expression of genes related to OC function and oxidative stress control. Dietary compliance in short-term and mid-term was not associated with OC number or mRNA expression, but CTSK negatively correlated with BMD T-Score in the hips of adults with PKU (Spearman r = -0.518, p = 0.040). Osteoclastogenesis was not changed in adult patients with PKU, nor were most mRNA expressions of OC marker genes or those of oxidative stress control. However, 44% of patients presented with BMD below -1 in their hips, and the OC of these tended to express higher CTSK (above -1: 0.2 [0.2-0.8] vs. below -1: 0.9 [0.6-3.4], p = 0.055). Thus, alternative regulatory mechanisms of OC activity may play a role in the development of low BMD in patients with PKU.

Keywords: aromatic amino acids; bone turnover; congenital metabolic disease; dietary compliance; osteoclasts; oxidative stress response; phenylalanine.

Figure 1

Cell count and relative expression of osteoclast specific and antioxidative genes in osteoclasts differentiated from PBMCs of patients with phenylketonuria (PKU) and controls (Co). Numbers of tartrate-resistant acid phosphatase (Trap)-positive multi-nucleated cells after 14 days of differentiation (A) and exemplary images (40× magnification (B)) of Trap-positive cells of a patient with PKU (1) and a control (2); white arrows mark representatives of osteoclasts (Trap-positive, ≥3 nuclei) at different stages of development. Relative mRNA expression of Osteoclast-associated receptor (OSCAR, (C)), Nuclear factor of activated T cells 1 (NFATc1, (D), BTB domain and CNC homolog 1 (BACH1, (E)) and Cathepsin K (CTSK, (F)). Dotted lines indicate patient–control pairs. Analyzed using paired Student’s t-test and paired Wilcoxon test.