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. 2012 Jan 27;287(5):2997-3008.
doi: 10.1074/jbc.M111.317701. Epub 2011 Dec 7.

Genotype-phenotype correlations in Lesch-Nyhan disease: moving beyond the gene

Rong Fu  1 , H A Jinnah
Affiliations

Genotype-phenotype correlations in Lesch-Nyhan disease: moving beyond the gene

Rong Fu et al. J Biol Chem. .

Abstract

Lesch-Nyhan disease and its attenuated variants are caused by mutations in the HPRT1 gene, which encodes the purine recycling enzyme hypoxanthine-guanine phosphoribosyltransferase. The mutations are heterogeneous, with more than 400 different mutations already documented. Prior efforts to correlate variations in the clinical phenotype with different mutations have suggested that milder phenotypes typically are associated with mutants that permit some residual enzyme function, whereas the most severe phenotype is associated with null mutants. However, multiple exceptions to this concept have been reported. In the current studies 44 HPRT1 mutations associated with a wide spectrum of clinical phenotypes were reconstructed by site-directed mutagenesis, the mutant enzymes were expressed in vitro and purified, and their kinetic properties were examined toward their substrates hypoxanthine, guanine, and phosphoribosylpyrophosphate. The results provide strong evidence for a correlation between disease severity and residual catalytic activity of the enzyme (k(cat)) toward each of its substrates as well as several mechanisms that result in exceptions to this correlation. There was no correlation between disease severity and the affinity of the enzyme for its substrates (K(m)). These studies provide a valuable model for understanding general principles of genotype-phenotype correlations in human disease, as the mechanisms involved are applicable to many other disorders.

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Figures

FIGURE 1.
FIGURE 1.
Michaelis-Menten plots for human HGprt. Each data point was determined in triplicate, and the symbols reflect average ± S.D. Panels A and C show the Hprt reaction, whereas panels B and D show the Gprt reaction. Panels A and B were plotted at varied purine concentration with PRPP fixed at 1 mm, whereas panels C and D were plotted at varied PRPP concentration with fixed purine at 200 μm.
FIGURE 2.
FIGURE 2.
Kinetic properties of HGprt according to clinical subgroup. The kinetic parameters for Hprt or Gprt were normalized to that of the native enzyme and are presented as box-whisker plots. The middle horizontal line in each box shows the median. The upper and lower limits of the box define the upper and lower quartiles of the data. The whiskers span the entire data range. The clinical groups were compared using the Kruskal-Wallis H-statistic, which revealed significant differences for Hprt with either hypoxanthine or PRPP as fixed substrates and for Gprt with either guanine or PRPP as fixed substrates. There also were significant differences between the clinical subgroups and combined purine recycling (Hprt + Gprt). There were no significant differences among the clinical groups for Km parameters for any substrate for either the Hprt or Gprt reaction.
FIGURE 3.
FIGURE 3.
Correlation of Hprt and Gprt reactions. Panel A and B compares kcat values for Hprt and Gprt, whereas panels C and D compares Km values for purine and PRPP. The latter are shown in a log scale due to the large variation in absolute values. The normal enzyme is shown as a solid circle in each panel. The mutants are shown as open circles (LND), open squares (HND), or open triangles (HRH). The dotted line shows the predicted relation of normal Hprt to Gprt across different enzyme levels, and the solid line shows the empirically derived correlation of Hprt to Gprt by linear regression. Some mutants with skewed loss of activity toward one or the other reaction were evident (arrows). There were good correlations between kcat values for Hprt and Gprt with fixed PRPP and for kcat values for PRPP with fixed hypoxanthine or guanine. There also were correlations between Km values with fixed PRPP or for PRPP with fixed hypoxanthine or guanine, although these correlations were driven largely by a small number of mutants with very high Km values.
FIGURE 4.
FIGURE 4.
Stability of HGprt. Residual enzyme activity was monitored for 84 h at 37 °C. Most of the mutants were tested with guanine as substrate because of the superior UV absorbance changes associated with conversion of guanine to GMP. Two mutants (G71V and D194E) were tested with hypoxanthine because of poor activity toward guanine. Panel A shows mutants associated with the LND phenotype, panel B shows mutants associated with the HND phenotype, and panel C shows mutants associated with the HRH phenotype.
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