Clinical severity in Lesch-Nyhan disease: the role of residual enzyme and compensatory pathways

Abstract

Mutations in the HPRT1 gene, which encodes the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGprt), cause Lesch-Nyhan disease (LND) and more mildly affected Lesch-Nyhan variants. Prior studies have suggested a strong correlation between residual hypoxanthine recycling activity and disease severity. However, the relevance of guanine recycling and compensatory changes in the de novo synthesis of purines has received little attention. In the current studies, fibroblast cultures were established for 21 healthy controls and 36 patients with a broad spectrum of disease severity related to HGprt deficiency. We assessed hypoxanthine recycling, guanine recycling, steady-state purine pools, and de novo purine synthesis. There was a strong correlation between disease severity and either hypoxanthine or guanine recycling. Intracellular purines were normal in the HGprt-deficient fibroblasts, but purine wasting was evident as increased purine metabolites excreted from the cells. The normal intracellular purines in the HGprt-deficient fibroblasts were likely due in part to a compensatory increase in purine synthesis, as demonstrated by a significant increase in purinosomes. However, the increase in purine synthesis did not appear to correlate with disease severity. These results refine our understanding of the potential sources of phenotypic heterogeneity in LND and its variants.

Keywords: Genotype–phenotype correlation; Inherited metabolic disease; Purine metabolism; Purinosome.

Figure 1

Enzyme activity and clinical severity. The enzyme activities for Hprt and Gprt are presented as box-whisker plots. The middle horizontal line in each box shows the median. The upper and lower limits of box define the upper and lower quartiles of the data. The whiskers span the entire data range. Each dot represents a single case, which was determined in quadruplicate.

Figure 2

Correlation of Hprt and Gprt activities. Each case was determined in quadruplicate and shown as a separate symbol. The groups are normal controls (squares), LND (triangles), HND (diamonds), and HRH (circles). The line shows the empirically-derived correlation of Hprt to Gprt by linear regression.

Figure 3

Purinosomes in fibroblasts. (A) Diffuse fluorescence in the control fibroblast cells transfected with FGAMS-GFP. (B) Representative images of purinosomes formed in LND fibroblast cells. FGAMS-GFP is used as a purinosome marker. Representative images of diffuse fluorescence in the control cells transfected with FGAMS-GFP (A) were clearly distinct from those of the HGprt-deficient patients (B)

Figure 4

Quantification of purinosomes in fibroblasts from controls and HGprt deficient patients. Five LND subjects (PB, DP, CC, TS, and TH), four HND subjects (JGS, BT, LW, and DD), three HRH subjects (RB, LG, and BF), and six controls (MG, CH2, JS, SK, AR, and AK) were evaluated. The ratio of cells containing purinosome is presented as box-whisker plots for the controls and each patient subgroup. Purinosomes were quantified by calculating the ratio of cells forming fluorescent cytoplasmic clustering over cells expressing diffuse fluorescent staining. The middle horizontal line in each box shows the median, and the quartiles of each clinical group are shown as the upper and lower limits of box.