NAD metabolism in HPRT-deficient mice.

The activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) is virtually absent in Lesch-Nyhan disease (LND), an X-linked genetic disorder characterized by uric acid accumulation and neurodevelopmental dysfunction. The biochemical basis for the neurological and behavioral abnormalities have not yet been completely explained. Prior studies of cells from affected patients have shown abnormalities of NAD metabolism. In the current studies, NAD metabolism was evaluated in HPRT gene knock-out mice. NAD content and the activities of the enzymes required for synthesis and breakdown of this coenzyme were investigated in blood, brain and liver of HPRT(-) and control mice. NAD concentration and enzyme activities were found to be significantly increased in liver, but not in brain or blood of the HPRT(-) mice. These results demonstrate that changes in NAD metabolism occur in response to HPRT deficiency depending on both species and tissue type.

Mendelian non-syndromic obesity.

Obesity is highly heritable and arises from the interplay of many genes and environmental factors. It can be defined as the result of prolonged imbalance between calorie intake and energy utilization. About 5% of cases of non-syndromic obesity are monogenic (Mendelian obesity). The amount of adipose tissue in the body is mainly regulated by leptin, a hormone produced by adipocytes, and Mendelian obesity is mainly caused by mutations that disrupt the leptin/melanocortin pathway. In this article, we summarize the genes involved in genetic obesity and the test we use for genetic analysis.

HPRTSardinia: a new point mutation causing HPRT deficiency without Lesch-Nyhan disease.

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency always causing hyperuricemia presents various degrees of neurological manifestations, the most severe which is Lesch-Nyhan syndrome. The HPRT gene is situated in the region Xq26-q27.2 and consists of 9 exons. At least 300 different mutations at different sites in the HPRT coding region from exon 1 to exon 9 have been identified. A new mutation in the HPRT gene has been determined in one patient with complete deficiency of erythrocyte activity, with hyperuricemia and gout but without Lesch-Nyhan disease. Analysis of cultured fibroblasts revealed minimal residual HPRT activity mainly when guanine was the substrate. Genomic DNA sequencing demonstrated patient's mother heterozygosity for the mutation and no mutation in her brother. The mutation consists in a C-->T transversion at cDNA base 463 (C463T) in exon 6, resulting in proline to serine substitution at codon 155 (P155S). This mutation had not been reported previously and has been designated HPRT(Sardinia). The mutation identified in this patient allows some expression of functional enzyme in nucleated cells such as fibroblasts, indicating that such cell type may add further information to conventional blood analysis. A multicentre survey gathering patients with variant neurological forms could contribute to understand the pathophysiology of the neurobehavioral symptoms of HPRT deficiency.

Biochemical and molecular study of mentally retarded patient with partial deficiency of hypoxanthine-guanine phosphoribosyltransferase.

Nucleotide metabolism was studied in erythrocytes of a mentally retarded child and family members. Partial hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency was found in the propositus and an asymptomatic maternal uncle. Studies in crude lysates demonstrated decreased apparent V(max) and slightly decreased apparent K(m) for hypoxanthine in both HPRT-deficient subjects. Genomic DNA analysis revealed a single nucleotide change with leucine-147 to phenylalanine substitution in both subjects; mother and grandmother were heterozygous carriers of the same defect. This new variant has been termed HPRT(Potenza). Increased erythrocyte concentration of NAD and rate of synthesis by intact erythrocytes were found in the patient; increased activities of nicotinic acid phosphoribosyltransferase (NAPRT) and NAD synthetase (NADs) were demonstrated in erythrocyte lysates, with normal apparent K(m) for their substrates and increased V(max). These alterations were not found in any member of the family, including the HPRT-deficient uncle. These findings show multiple derangement of nucleotide metabolism associated with partial HPRT deficiency. The enzyme alteration was presumably not the cause of neurological impairment since no neurological symptoms were found in the HPRT-deficient uncle, whereas they were present in the propositus' elder brother who had normal HPRT activity.

Simple non-radiochemical HPLC-linked method for screening for purine metabolism disorders using dried blood spot.

BACKGROUND: Pathologies associated with rare inherited disorders affecting purine metabolic pathways range from renal failure to neurological dysfunction and immunodeficiency. The disorders are usually diagnosed by measuring enzyme activities in hemolysates. A non-radiochemical HPLC-linked method is described for simultaneous determination of the activities of hypoxanthine-guanine phosphoribosyltransferase (HPRT: E.2.4.2.8.), adenine phosphoribosyltransferase (APRT: E.2.4.2.7.), adenosine deaminase (ADA: E.3.5.4.4.) and purine nucleoside phosphorylase (PNP: E.2.4.2.1.) in dried blood spots. METHOD: 7-mm-diameter blood spots stored at 4 degrees C or room temperature were transferred to an Eppendorf tube and eluted with 500-microl 0.1 mol/l Tris-HCl buffer, pH 7.4. The eluate was added to substrate solutions and incubated at 37 degrees C. Reaction products were analysed by HPLC. RESULTS AND CONCLUSIONS: The enzyme activities tested in spot eluates were similar to those in erythrocyte lysates from the same subjects. None of the enzymatic activities tested were significantly affected by different storage temperatures. The main advantages of the proposed method are small blood volume required, easy sample collection and transfer, and accurate results. The method is therefore suitable for screening inborn errors of purine metabolism even in newborns.

Neurological disorders of purine and pyrimidine metabolism.

Purines and pyrimidines, regarded for a long time only as building blocks for nucleic acid synthesis and intermediates in the transfer of metabolic energy, gained increasing attention since genetically determined aberrations in their metabolism were associated clinically with various degrees of mental retardation and/or unexpected and often devastating neurological dysfunction. In most instances the molecular mechanisms underlying neurological symptoms remain undefined. This suggests that nucleotides and nucleosides play fundamental but still unknown roles in the development and function of several organs, in particular central nervous system. Alterations of purine and pyrimidine metabolism affecting brain function are spread along both synthesis (PRPS, ADSL, ATIC, HPRT, UMPS, dGK, TK), and breakdown pathways (5NT, ADA, PNP, GCH, DPD, DHPA, TP, UP), sometimes also involving pyridine metabolism. Explanations for the pathogenesis of disorders may include both cellular and mitochondrial damage: e.g. deficiency of the purine salvage enzymes hypoxanthine-guanine phosphoribosyltransferase and deoxyguanosine kinase are associated to the most severe pathologies, the former due to an unexplained adverse effect exerted on the development and/or differentiation of dopaminergic neurons, the latter due to impairment of mitochondrial functions. This review gathers the presently known inborn errors of purine and pyrimidine metabolism that manifest neurological syndromes, reporting and commenting on the available hypothesis on the possible link between specific enzymatic alterations and brain damage. Such connection is often not obvious, and though investigated for many years, the molecular basis of most dysfunctions of central nervous system associated to purine and pyrimidine metabolism disorders are still unexplained.

Poly(ADP-ribose) polymerase activity in systemic lupus erythematosus and systemic sclerosis.

The aim of this study is to investigate the role of poly(ADP-ribose) polymerase (PARP), involved in DNA repair and in autoimmune pathologic conditions such as systemic lupus erythematosus (SLE) and both limited systemic sclerosis (lSSc) and diffuse systemic sclerosis (dSSc), to assess its possible implication in the pathogenetic processes. The relationship between PARP activity and the intracellular concentration of its substrate nicotinamide adenine dinucleotide (NAD) is also investigated. Peripheral mononuclear cells (PMC) from controls and patients with SLE, lSSc, and dSSc were irradiated with ultraviolet light (UV) and PARP activity was assayed by a radiochemical method. Pyridine nucleotide concentrations were assayed by a high-performance liquid chromatography-linked method. PARP activity was detectable in nonirradiated cells and showed similar values in all groups. The activity significantly increased after UV irradiation in control, SLE, and lSSc cells, but not in dSSc cells. Irradiated PMC from both SLE and dSSc showed lower enzyme activity with respect to irradiated controls. Higher intracellular NAD content was found in all of the pathologic conditions in comparison to values in the control; this difference was statistically significant in dSSc. Our data demonstrate a lower PARP activity in response to UV damage in PMC from patients affected by the above pathologic conditions compared with controls. An inverse relationship between PARP activity and NAD content was also observed.