Next-Generation Sequencing and Quantitative Proteomics of Hutchinson-Gilford progeria syndrome-derived cells point to a role of nucleotide metabolism in premature aging.

Hutchinson-Gilford progeria syndrome (HGPS) is a very rare fatal disease characterized for accelerated aging. Although the causal agent, a point mutation in LMNA gene, was identified more than a decade ago, the molecular mechanisms underlying HGPS are still not fully understood and, currently, there is no cure for the patients, which die at a mean age of thirteen. With the aim of unraveling non-previously altered molecular pathways in the premature aging process, human cell lines from HGPS patients and from healthy parental controls were studied in parallel using Next-Generation Sequencing (RNAseq) and High-Resolution Quantitative Proteomics (iTRAQ) techniques. After selection of significant proteins and transcripts and crosschecking of the results a small set of protein/transcript pairs were chosen for validation. One of those proteins, ribose-phosphate pyrophosphokinase 1 (PRPS1), is essential for nucleotide synthesis. PRPS1 loss-of-function mutants present lower levels of purine. PRPS1 protein and transcript levels are detected as significantly decreased in HGPS cell lines vs. healthy parental controls. This modulation was orthogonally confirmed by targeted techniques in cell lines and also in an animal model of Progeria, the ZMPSTE24 knock-out mouse. In addition, functional experiments through supplementation with S-adenosyl-methionine (SAMe), a metabolite that is an alternative source of purine, were done. Results indicate that SAMe has a positive effect in the proliferative capacity and reduces senescence-associated Beta-galactosidase staining of the HPGS cell lines. Altogether, our data suggests that nucleotide and, specifically, purine-metabolism, are altered in premature aging, opening a new window for the therapeutic treatment of the disease.

A profound computational study to prioritize the disease-causing mutations in PRPS1 gene.

Charcot-Marie-Tooth disease (CMT) is one of the most commonly inherited congenital neurological disorders, affecting approximately 1 in 2500 in the US. About 80 genes were found to be in association with CMT. The phosphoribosyl pyrophosphate synthetase 1 (PRPS1) is an essential enzyme in the primary stage of de novo and salvage nucleotide synthesis. The mutations in the PRPS1 gene leads to X-linked Charcot-Marie-Tooth neuropathy type 5 (CMTX5), PRS super activity, Arts syndrome, X-linked deafness-1, breast cancer, and colorectal cancer. In the present study, we obtained 20 missense mutations from UniProt and dbSNP databases and applied series of comprehensive in silico prediction methods to assess the degree of pathogenicity and stability. In silico tools predicted four missense mutations (D52H, M115 T, L152P, and D203H) to be potential disease causing mutations. We further subjected the four mutations along with native protein to 50 ns molecular dynamics simulation (MDS) using Gromacs package. The resulting trajectory files were analyzed to understand the stability differences caused by the mutations. We used the Root Mean Square Deviation (RMSD), Radius of Gyration (Rg), solvent accessibility surface area (SASA), Covariance matrix, Principal Component Analysis (PCA), Free Energy Landscape (FEL), and secondary structure analysis to assess the structural changes in the protein upon mutation. Our study suggests that the four mutations might affect the PRPS1 protein function and stability of the structure. The proposed study may serve as a platform for drug repositioning and personalized medicine for diseases that are caused by the PRPS1 deficiency.

Expanding the phenotype of PRPS1 syndromes in females: neuropathy, hearing loss and retinopathy.

BACKGROUND: Phosphoribosyl pyrophosphate synthetase (PRS) I deficiency is a rare medical condition caused by missense mutations in PRPS1 that lead to three different phenotypes: Arts Syndrome (MIM 301835), X-linked Charcot-Marie-Tooth (CMTX5, MIM 311070) or X-linked non-syndromic sensorineural deafness (DFN2, MIM 304500). All three are X-linked recessively inherited and males affected display variable degree of central and peripheral neuropathy. We applied whole exome sequencing to a three-generation family with optic atrophy followed by retinitis pigmentosa (RP) in all three cases, and ataxia, progressive peripheral neuropathy and hearing loss with variable presentation. METHODS: Whole exome sequencing was performed in two affecteds and one unaffected member of the family. Sanger sequencing was used to validate and segregate the 12 candidate mutations in the family and to confirm the absence of the novel variant in PRPS1 in 191 controls. The pathogenic role of the novel mutation in PRPS1 was assessed in silico and confirmed by enzymatic determination of PRS activity, mRNA expression and sequencing, and X-chromosome inactivation. RESULTS: A novel missense mutation was identified in PRPS1 in the affected females. Age of onset, presentation and severity of the phenotype are highly variable in the family: both the proband and her mother have neurological and ophthalmological symptoms, whereas the phenotype of the affected sister is milder and currently confined to the eye. Moreover, only the proband displayed a complete lack of expression of the wild type allele in leukocytes that seems to correlate with the degree of PRS deficiency and the severity of the phenotype. Interestingly, optic atrophy and RP are the only common manifestations to all three females and the only phenotype correlating with the degree of enzyme deficiency. CONCLUSIONS: These results are in line with recent evidence of the existence of intermediate phenotypes in PRS-I deficiency syndromes and demonstrate that females can exhibit a disease phenotype as severe and complex as their male counterparts.

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.

Inherited hyperuricemic disorders.

Inherited hyperuricemic disorders fall into two major classes, metabolic overproduction of purines and renal tubular undersecretion. The aim was to explore both. Methodology was a combination of personal experience and review of relevant literature. The overproduction of hyperuricemias result from deficiency of hypoxanthine-guanine phosphoribosyl transferase, overactivity of phosphoribosylpyrophosphate synthetase and deficiency of glucose-6-phosphatase. The undersecretion disorders are autosomal dominantly inherited and are heterogeneous. A major number of these patients result from mutations in the gene that codes for uromodulin. Treatment is with allopurinol.

A simplified method for the determination of phosphoribosylpyrophosphate synthetase activity in hemolysates.

Methods currently employed for measurement of phosphoribosylpyrophosphate synthetase (ribophosphate pyrophosphokinase; PRPPs) activity are cumbersome and expensive, requiring an auxiliary enzyme reaction, a radioactive nucleobase and multiple incubations, or do not allow a complete kinetic study. The aim of the present study is to describe a simplified, single step, non-isotopic method for determination of PRPPs in hemolysate, appropriate for a complete screening of PRPPs activity disorders. Briefly, the charcoal-treated hemolysate is incubated with saturating amounts of substrates and P1 P5 di(adenosine 5') pentaphosphate (Ap5A), an inhibitor of human adenylate kinase, to prevent conversion of AMP to ADP. AMP generated in this reaction is then measured by HPLC. Adenylate kinase activity was fully inhibited by Ap5A, allowing the accurate determination of AMP. The method was sensitive and reproducible and mean and variance values compared closely with those reported using other, more complicated, assay procedures. The hyperbolic curve relating Pi concentration to initial reaction velocity was shifted to sigmoidal by addition of 0.02 mmol/l GDP which inhibited PRPPs activities only at inorganic phosphate concentrations < 2 mmol/l. This suggests that this method should provide sensitive and accurate screening for regulatory, as well as catalytic, defects underlying PRPPs superactivity.

Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase.

This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purin auxotroph by a procedure designed to select guanosine-utilizing mutants. Strain GP122 had roughly 15% of the PRPP synthetase activity and 25% of the PRPP pool of its parent strain. The mutant exhibited many of the predicted consequences of a decreased PRPP pool and a defective PRPP synthetase enzyme, including: poor growth on purine bases; decreased accumulation of 5-aminoimidazole ribonucleotide (the substrate of the blocked purE reaction) under conditions of purine starvation; excretion of anthranilic acid when grown in medium lacking tryptophan; increased resistance to inhibition by 5-fluorouracil; derepressed levels of aspartate transcarbamylase and orotate phosphoribosyltransferase, enzymes involved in the pyrimidine de novo biosynthetic pathway; growth stimulation by PRPP-sparing compounds (e.g. guanosine, histidine); poor growth in low phosphate medium; and increased heat lability of the defective enzyme. This mutant strain also had increased levels of guanosine 5'-monophosphate reductase. This genetic lesion, designated prs, was mapped by conjugation and phage P22-mediated transduction at 35 units on the Salmonella linkage map.

Disorders associated with purine and pyrimidine metabolism.

There has been an explosion of knowledge in disorders of purine and pyrimidine metabolism during the last 20 years. During this time, more than 10 diseases have been discovered and their metabolic bases studied. Hyperuricemia and gout remain the most common clinical disorder. Rarely these disorders are explainable by an inherited enzyme abnormally, such as hypoxanthine-guanine phosphoribosyltransferase deficiency, phosphoribosyl-pyrophosphate synthetase deficiency, or glucose-6-phosphatase deficiency. The description of immunodeficiency syndromes in association with purine enzyme deficiency has led to a novel area of investigation encompassing the biochemical basis for immune function. Although less information is available concerning the other diseases associated with renal calculi, myopathy, anemia, and central nervous system dysfunction, further research will elucidate important metabolic relationships. These will no doubt expand our understanding of the pathogenesis of these disorders and provide innovative therapeutic approaches.

Additional data from two kindreds with genetically induced deficiencies of erythrocyte pyrimidine nucleotidase.

Two subjects, not previously reported in detail, had severe inherited deficiencies of erythrocyte pyrimidine nucleotidase. This was manifested hematologically by moderate hemolytic anemia with splenomegaly, morphologically by punctate basophilic stippling of Wright's stained erythrocytes, and biochemically by intraerythrocytic accumulation of pyrimidine nucleotides, elevated concentrations of reduced glutathione, and partial deficiencies of ribosephosphate pyrophosphokinase. All 5 of their children were asymptomatic and phenotypically normal except for intermediate reductions in activities of pyrimidine nucleotidase consistent with heterozygosity for an autosomal recessive defect.

Hereditary hemolytic anemia with erythrocyte pyrimidine 5'-nucleotidase deficiency in Spain. Clinical, biological and familial studies.

We report a hereditary hemolytic anemia associated with a severe erythrocyte pyrimidine 5'-nucleotidase deficiency in a Spanish family of five members in which the parents are first cousins. Both parents exhibited decreased nucleotidase activity without clinical or hematologic abnormalities. Two children (a male and a female) showed severe pyrimidine 5'-nucleotidase deficiency with hemolytic anemia. The remaining child (a male) showed no signs of the disease. The findings strongly suggest an autosomal recessive mode of inheritance in this enzymopathy. This seems to be the first report of pyrimidine 5'-nucleotidase deficiency in Spain.

Electroencephalographic study of an infant with phosphoribosylpyrophosphate synthetase deficiency.

In a hypouricemic and mentally retarded infant due to a defect of 5-phosphoribosylpyrophosphate synthetase, electroencephalograms were recorded at the age of 4, 7, 10 and 11 months. Hypsarrhythmia was first observed at the age of 10 months, and markedly improved after ACTH therapy with concomitant increase in the enzyme activity of erythrocytes.