New biomarkers for early diagnosis of Lesch-Nyhan disease revealed by metabolic analysis on a large cohort of patients

Abstract

BackgroundLesch-Nyhan disease is a rare X-linked neurodevelopemental metabolic disorder caused by a wide variety of mutations in the HPRT1 gene leading to a deficiency of the purine recycling enzyme hypoxanthine-guanine phosphoribosyltransferase (HGprt). The residual HGprt activity correlates with the various phenotypes of Lesch-Nyhan (LN) patients and in particular with the different degree of neurobehavioral disturbances. The prevalence of this disease is considered to be underestimated due to large heterogeneity of its clinical symptoms and the difficulty of diagnosing of the less severe forms of the disease. We therefore searched for metabolic changes that would facilitate an early diagnosis and give potential clues on the disease pathogenesis and potential therapeutic approaches.MethodsLesch-Nyhan patients were diagnosed using HGprt enzymatic assay in red blood cells and identification of the causal HPRT1 gene mutations. These patients were subsequently classified into the three main phenotypic subgroups ranging from patients with only hyperuricemia to individuals presenting motor dysfunction, cognitive disability and self-injurious behavior. Metabolites from the three classes of patients were analyzed and quantified by High Performance Ionic Chromatography and biomarkers of HGprt deficiency were then validated by statistical analyses.ResultsA cohort of 139 patients, from 112 families, diagnosed using HGprt enzymatic assay in red blood cells, was studied. 98 displayed LN full phenotype (86 families) and 41 (26 families) had attenuated clinical phenotypes. Genotype/phenotype correlations show that LN full phenotype was correlated to genetic alterations resulting in null enzyme function, while variant phenotypes are often associated with missense mutations allowing some residual HGprt activity. Analysis of metabolites extracted from red blood cells from 56 LN patients revealed strong variations specific to HGprt deficiency for six metabolites (AICAR mono- and tri-phosphate, nicotinamide, nicotinic acid, ATP and Succinyl-AMP) as compared to controls including hyperuricemic patients without HGprt deficiency.ConclusionsA highly significant correlation between six metabolites and the HGprt deficiency was established, each of them providing an easily measurable marker of the disease. Their combination strongly increases the probability of an early and reliable diagnosis for HGprt deficiency.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-014-0219-0) contains supplementary material, which is available to authorized users.

Disruption of Nucleotide Homeostasis by the Antiproliferative Drug 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside Monophosphate (AICAR)

“…Strikingly, AICAR is accumulated in body fluids of patients suffering purine metabolism diseases, including hypoxanthine-guanine phosphoribosyltransferase-deficient patients (15,26). It should be stressed that the levels of AICAR accumulated by these patients (15) are way below those resulting in synthetic lethality in yeast cells (this work). However, should AICAR have similar effects in human cells than the ones observed in yeast, it could contribute to the phenotypic outcome of these complex developmental genetic diseases.…”

“…However, should AICAR have similar effects in human cells than the ones observed in yeast, it could contribute to the phenotypic outcome of these complex developmental genetic diseases. Accordingly, we recently found that, in addition to AICAR accumulation, NTP levels were significantly lower in erythrocytes from a large cohort of hypoxanthine-guanine phosphoribosyltransferase-deficient patients (15).…”

“…15. Peaks were identified by their retention time, as well as by co-injection with standards and/or their UV spectrum signature.…”

MicroRNAs

“…However one of the major limitations associated with this classification has been the heterogeneity of HPRT-deficient phenotypes which, along with the varying degrees of severity, can lead to misclassification and diagnostic uncertainty (Ceballos-Picot et al, 2015;Fu, Ceballos-Picot, et al, 2014). Consequently, there have been multiple attempts to develop new biomarkers that will facilitate diagnostic and also allow a more refined classification of the clinical phenotypes of LND (Ceballos-Picot et al, 2015). Circulating miRNAs are currently used as biomarkers not only in cancer but also in neurological diseases.…”

Metabolomics and proteomics identify the toxic form and the associated cellular binding targets of the anti-proliferative drug AICAR

“…accumulate when its riboside precursor AICAR is provided to the cell, but also in several metabolic diseases. Indeed, ZMP as well as ZDP and ZTP were found at very high concentrations (ZTP reaching even higher levels than ATP) in red blood cells of an AICAR transformylase IMP-cyclohydrolase-deficient patient lacking the enzyme that catalyzes the last two steps of the purine biosynthesis pathway (25) and was the best marker for hypoxanthine-guanine phospho-ribosyl-transferase deficiency in a large cohort of Lesch-Nyhan syndrome patients (26). The precise role of ZMP in the etiology of these diseases is unclear but should be addressed in connection with the fact that treating eukaryotic cells with AICAR at high concentration is toxic.…”