Inborn Errors of Purine and Pyrimidine Metabolism: How Much We Owe to H. Anne Simmonds

Purines and pyrimidines, regarded for a long time merely as building blocks for nucleic acid synthesis and intermediates in the transfer of metabolic energy, have attracted increasing attention after genetically determined aberrations in their metabolism were linked to a range of symptoms from hyperuricemia and immunodeficiency to neurological disorders. The pathogenesis of such disorders involves cell or mitochondrial damage, but the molecular mechanisms underlying symptoms is often unclear. H. Anne Simmonds made major contributions to the metabolic, clinical, and molecular aspects of these disorders and the Purine Research Laboratory, which she established in London, became the world center for clinical and experimental studies in the field. We owe her gratitude not only for this direct contribution but also for her enthusiasm for purine and pyrimidine research that she transmitted to generations of scientists. Our research in this field stemmed from expertise in pyridine metabolism and its connection with purines, and from clinical involvement with biochemical diagnosis of enzyme deficiencies. We joined H. Anne Simmonds in studying the biochemical basis of altered NAD content in erythrocytes of PNP- and HPRT-deficient patients, discovering some alterations in NAD synthesis and breakdown.     

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.     

Metabolic fate of extracellular NAD in human skin fibroblasts

Extracellular NAD is degraded to pyridine and purine metabolites by different types of surface-located enzymes which are expressed differently on the plasmamembrane of various human cells and tissues. In a previous report, we demonstrated that NAD-glycohydrolase, nucleotide pyrophosphatase and 5'-nucleotidase are located on the outer surface of human skin fibroblasts. Nucleotide pyrophosphatase cleaves NAD to nicotinamide mononucleotide and AMP, and 5'-nucleotidase hydrolyses AMP to adenosine. Cells incubated with NAD, produce nicotinamide, nicotinamide mononucleotide, hypoxanthine and adenine. The absence of ADPribose and adenosine in the extracellular compartment could be due to further catabolism and/or uptake of these products. To clarify the fate of the purine moiety of exogenous NAD, we investigated uptake of the products of NAD hydrolysis using U-[(14)C]-adenine-NAD. ATP was found to be the main labeled intracellular product of exogenous NAD catabolism; ADP, AMP, inosine and adenosine were also detected but in small quantities. Addition of ADPribose or adenosine to the incubation medium decreased uptake of radioactive purine, which, on the contrary, was unaffected by addition of inosine. ADPribose strongly inhibited the activity of ecto-NAD-hydrolyzing enzymes, whereas adenosine did not. Radioactive uptake by purine drastically dropped in fibroblasts incubated with (14)C-NAD and dipyridamole, an inhibitor of adenosine transport. Partial inhibition of [(14)C]-NAD uptake observed in fibroblasts depleted of ATP showed that the transport system requires ATP to some extent. All these findings suggest that adenosine is the purine form taken up by cells, and this hypothesis was confirmed incubating cultured fibroblasts with (14)C-adenosine and analyzing nucleoside uptake and intracellular metabolism under different experimental conditions. Fibroblasts incubated with [(14)C]-adenosine yield the same radioactive products as with [(14)C]-NAD; the absence of inhibition of [(14)C]-adenosine uptake by ADPribose in the presence of alpha-beta methyleneADP, an inhibitor of 5' nucleotidase, demonstrates that ADPribose coming from NAD via NAD-glycohydrolase is finally catabolised to adenosine. These results confirm that adenosine is the NAD hydrolysis product incorporated by cells and further metabolized to ATP, and that adenosine transport is partially ATP dependent.     

Role of hyperhomocysteinemia in aortic disease.

A growing body of evidence has shown a strong association between elevated plasma homocysteine (Hcy) levels with vascular disease and thrombotic complications. Data available in literature also suggest a role of hyperhomocysteinemia in abdominal and thoracic aortic diseases. In particular, Hcy was investigated in patients with Marfan syndrome and it was demonstrated that Hcy levels were associated with the risk of severe cardiovascular manifestations or dissection. Hcy was significantly higher also in patients with abdominal aortic aneurysms and was associated with the size of aneurysms. It remains to be elucidated if this association is causal or simply an effect of the disease. A number of mechanisms may be evoked to explain these findings. Studies in animal models demonstrated that hyperhomocysteinemia could induce marked remodelling of the extracellular matrix of the arterial wall by inducing elastolysis through the activation of metalloproteinases. In addition, Hcy may directly affect fibrillin-1 or collagen by interfering with intra- and/or inter-molecular disulfide bonds through disulfide exchange, or binding to free sulphydryl groups. Further studies are needed to confirm the role of Hcy in aortic disease and the usefulness of including Hcy determination in the clinical evaluation of these patients.     

Pharmacokinetic analysis of plasma curves obtained after i.v. injection of the PET radioligand [11C] raclopride provides a likely explanation for rapid radioligand metabolism

Positron emission tomography (PET) is an imaging technique that provides direct measurements of receptor binding in neurons. The present study was performed to find reasons for the common observation of rapid metabolism of receptor radioligands during time of a brain PET scan. To this aim, the 1-h phase during which imaging-data are acquired was evaluated by using a pharmacokinetic approach. The values of half-lives, volumes of distribution, and dilution calculated for a set of metabolite corrected plasma curves of D2-receptor radioligand [(11)C]raclopride (PETc) during 50 min after radioligand injection in tracer dose were compared with the reference values obtained from a set of plasma curves (REFc) during 30 h after i.v. infusion of unlabelled raclopride in pharmacological doses. We found that the half-life of PETc correspond to the distribution half-life of REFc. Accordingly, the distribution volume during the terminal phase of PETc (13.6 ± 10.8 L) was significantly lower than that during the terminal phase (82.2 ± 30.5 L) and at steady state (59.4 ± 20 L) for REFc, and the dilution of raclopride in body for PETc at 50 min was 38 L, whereas it was 1015 L for REFc at 30 h. The [(11)C]raclopride in plasma at 50 min was higher (10% of dose) than the value for unlabelled raclopride at 30 h (4%). We concluded that the kinetic behavior of the radiolabelled drug [(11)C]raclopride during the 1 h time of a PET corresponds to the distribution phase. The high percentage of [(11)C]raclopride in plasma during this phase is a likely reason for the observed rapid radioligand metabolism.     

Neurofibromatosis type 2 attributable to gonosomal mosaicism in a clinically normal mother, and identification of seven novel mutations in the NF2 gene

Neurofibromatosis type 2 (NF2) is an autosomal dominant cancer syndrome that predisposes to the development of bilateral vestibular schwannomas sometimes associated with schwannomas at other locations, meningiomas, ependymomas and juvenile posterior subcapsular lenticular opacities. This disease is caused by inactivating mutations in the NF2 tumour-suppressor gene, located in 22q12. Recently, somatic mosaicism has been demonstrated in some "de novo" NF2 patients. We here report the genetic study of 33 NF2 patients from 33 unrelated Italian families. Twelve mutations were characterised, including seven newly identified mutations and five recurrent ones. Furthermore, we describe one patient with an inactivating mutation that lies in exon 13 but that is present in only a portion of the lymphocytes and, more importantly, a clinically normal individual carrying a somatic/germinal mosaicism for a nonsense mutation in exon 10 of the NF2 gene. Our results confirm the relatively high percentage of mosaicism for mutations in the NF2 gene and establish the importance of evaluating genomic DNA from several tissues, in addition to lymphocytes, so as to identify mosaicism in "de novo" NF2 patients and their relatives. In addition, the demonstration of somatic and/or gonadal mosaicism is an important tool for accurate genetic counselling in families with sporadic cases of NF2.