Metabolic and neurobehavioral disturbances induced by purine recycling deficiency in Drosophila.

Adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) are two structurally related enzymes involved in purine recycling in humans. Inherited mutations that suppress HGPRT activity are associated with Lesch-Nyhan disease (LND), a rare X-linked metabolic and neurological disorder in children, characterized by hyperuricemia, dystonia, and compulsive self-injury. To date, no treatment is available for these neurological defects and no animal model recapitulates all symptoms of LND patients. Here, we studied LND-related mechanisms in the fruit fly. By combining enzymatic assays and phylogenetic analysis, we confirm that no HGPRT activity is expressed in Drosophila melanogaster, making the APRT homolog (Aprt) the only purine-recycling enzyme in this organism. Whereas APRT deficiency does not trigger neurological defects in humans, we observed that Drosophila Aprt mutants show both metabolic and neurobehavioral disturbances, including increased uric acid levels, locomotor impairments, sleep alterations, seizure-like behavior, reduced lifespan, and reduction of adenosine signaling and content. Locomotor defects could be rescued by Aprt re-expression in neurons and reproduced by knocking down Aprt selectively in the protocerebral anterior medial (PAM) dopaminergic neurons, the mushroom bodies, or glia subsets. Ingestion of allopurinol rescued uric acid levels in Aprt-deficient mutants but not neurological defects, as is the case in LND patients, while feeding adenosine or N6-methyladenosine (m6A) during development fully rescued the epileptic behavior. Intriguingly, pan-neuronal expression of an LND-associated mutant form of human HGPRT (I42T), but not the wild-type enzyme, resulted in early locomotor defects and seizure in flies, similar to Aprt deficiency. Overall, our results suggest that Drosophila could be used in different ways to better understand LND and seek a cure for this dramatic disease.

Chromosome Transplantation: Correction of the Chronic Granulomatous Disease Defect in Mouse Induced Pluripotent Stem Cells.

In spite of the progress in gene editing achieved in recent years, a subset of genetic diseases involving structural chromosome abnormalities, including aneuploidies, large deletions and complex rearrangements, cannot be treated with conventional gene therapy approaches. We have previously devised a strategy, dubbed chromosome transplantation (CT), to replace an endogenous mutated chromosome with an exogenous normal one. To establish a proof of principle for our approach, we chose as disease model the chronic granulomatous disease (CGD), an X-linked severe immunodeficiency due to abnormalities in CYBB (GP91) gene, including large genomic deletions. We corrected the gene defect by CT in induced pluripotent stem cells (iPSCs) from a CGD male mouse model. The Hprt gene of the endogenous X chromosome was inactivated by CRISPR/Cas9 technology thus allowing the exploitation of the hypoxanthine-aminopterin-thymidine selection system to introduce a normal donor X chromosome by microcell-mediated chromosome transfer. X-transplanted clones were obtained, and diploid XY clones which spontaneously lost the endogenous X chromosome were isolated. These cells were differentiated toward the myeloid lineage, and functional granulocytes producing GP91 protein were obtained. We propose the CT approach to correct iPSCs from patients affected by other X-linked diseases with large deletions, whose treatment is still unsatisfactory. Stem Cells 2019;37:876-887.

Induction of somatic mutations by low-dose X-rays: the challenge in recognizing radiation-induced events.

It is difficult to distinguish radiation-induced events from spontaneous events during induction of stochastic effects, especially in the case of low-dose or low-dose-rate exposures. By using a hypersensitive system for detecting somatic mutations at the HPRT1 locus, we investigated the frequency and spectrum of mutations induced by low-dose X-rays. The mutant frequencies induced by doses of >0.15 Gy were statistically significant when compared with the spontaneous frequency, and a clear dose dependency was also observed for mutant frequencies at doses of >0.15 Gy. In contrast, mutant frequencies at doses of <0.1 Gy occurred at non-significant levels. The mutation spectrum in HPRT-deficient mutants revealed that the type of mutations induced by low-dose exposures was similar to that seen in spontaneous mutants. An apparent change in mutation type was observed for mutants induced by doses of >0.2 Gy. Our observations suggest that there could be a critical dose for mutation induction at between 0.1 Gy and 0.2 Gy, where mutagenic events are induced by multiple DNA double-strand breaks (DSBs). These observations also suggest that low-dose radiation delivered at doses of <0.1 Gy may not result in DSB-induced mutations but may enhance spontaneous mutagenesis events.

Genetic background of uric acid metabolism in a patient with severe chronic tophaceous gout.

Hyperuricemia depends on the balance of endogenous production and renal excretion of uric acid. Transporters for urate are located in the proximal tubule where uric acid is secreted and extensively reabsorbed: secretion is principally ensured by the highly variable ABCG2 gene. Enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) plays a central role in purine metabolism and its deficiency is an X-linked inherited metabolic disorder associated with clinical manifestations of purine overproduction. Here we report the case of a middle-aged man with severe chronic tophaceous gout with a poor response to allopurinol and requiring repeated surgical intervention. We identified the causal mutations in the HPRT1 gene, variant c.481G>T (p.A161S), and in the crucial urate transporter ABCG2, a heterozygous variant c.421C>A (p.Q141K). This case shows the value of an analysis of the genetic background of serum uric acid.

Hypoxanthine deregulates genes involved in early neuronal development. Implications in Lesch-Nyhan disease pathogenesis.

Neurological manifestations in Lesch-Nyhan disease (LND) are attributed to the effect of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency on the nervous system development. HPRT deficiency causes the excretion of increased amounts of hypoxanthine into the extracellular medium and we hypothesized that HPRT deficiency related to hypoxanthine excess may then lead, directly or indirectly, to transcriptional aberrations in a variety of genes essential for the function and development of striatal progenitor cells. We have examined the effect of hypoxanthine excess on the differentiation of neurons in the well-established human NTERA-2 cl.D1 (NT2/D1) embryonic carcinoma neurogenesis model. NT2/D1 cells differentiate along neuroectodermal lineages after exposure to retinoic acid (RA). Hypoxanthine effects on RA-differentiation were examined by the changes on the expression of various transcription factor genes essential to neuronal differentiation and by the changes in tyrosine hydroxylase (TH), dopamine, adenosine and serotonin receptors (DRD, ADORA, HTR). We report that hypoxanthine excess deregulate WNT4, from Wnt/ß-catenin pathway, and engrailed homeobox 1 gene and increased TH and dopamine DRD1, adenosine ADORA2A and serotonin HTR7 receptors, whose over expression characterize early neuro-developmental processes.

Loss of dopamine phenotype among midbrain neurons in Lesch-Nyhan disease.

OBJECTIVE: Lesch-Nyhan disease (LND) is caused by congenital deficiency of the purine recycling enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt). Affected patients have a peculiar neurobehavioral syndrome linked with reductions of dopamine in the basal ganglia. The purpose of the current studies was to determine the anatomical basis for the reduced dopamine in human brain specimens collected at autopsy. METHODS: Histopathological studies were conducted using autopsy tissue from 5 LND cases and 6 controls. Specific findings were replicated in brain tissue from an HGprt-deficient knockout mouse using immunoblots, and in a cell model of HGprt deficiency by flow-activated cell sorting (FACS). RESULTS: Extensive histological studies of the LND brains revealed no signs suggestive of a degenerative process or other consistent abnormalities in any brain region. However, neurons of the substantia nigra from the LND cases showed reduced melanization and reduced immunoreactivity for tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. In the HGprt-deficient mouse model, immunohistochemical stains for TH revealed no obvious loss of midbrain dopamine neurons, but quantitative immunoblots revealed reduced TH expression in the striatum. Finally, 10 independent HGprt-deficient mouse MN9D neuroblastoma lines showed no signs of impaired viability, but FACS revealed significantly reduced TH immunoreactivity compared to the control parent line. INTERPRETATION: These results reveal an unusual phenomenon in which the neurochemical phenotype of dopaminergic neurons is not linked with a degenerative process. They suggest an important relationship between purine recycling pathways and the neurochemical integrity of the dopaminergic phenotype.

Late diagnosis of Lesch-Nyhan disease variant.

A 30-year-old man was referred for investigation and management of hyperuricaemia. History included recurrent nephrolithiasis and chronic gout with poor response to medical management. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme activity was investigated and found to be deficient confirming the diagnosis of Lesch-Nyhan disease. Hyperuricaemia was treated with allopurinol. To prevent nephrolithiasis, the patient was instructed to avoid dehydration and aim for a minimum urine output of 2 L/day. Urinary alkalinisation with potassium citrate was started. The patient was referred for genetic counselling. This case discusses the genetics, pathophysiology, clinical manifestations, diagnosis and management of HGPRT deficiency.

Quantitative evaluation of the clinical effects of S-adenosylmethionine on mood and behavior in Lesch-Nyhan patients.

UNLABELLED: BACKGROUND, RATIONALE, AND METHODS: Lesch-Nyhan disease is a rare, X-linked disorder due to hypoxanthine phosphoribosyltransferase deficiency. To evaluate reported benefit on mood and behavior obtained by the administration of S-adenosyl-L-methionine in this condition, we developed 2 quantitative evaluation tools, and used them to assess the effects of the drug in our population: the weekly questionnaire and the resistance to self-injurious behavior test. We performed an open-label, dose-escalation trial of the drug on 14 patients. RESULTS: Four patients tolerated the drug and reported beneficial effects. The majority experienced worsened behavior. The 2 assessment tools demonstrated effectiveness in quantitatively evaluating the self-injurious behavior.

HPRT-deficiency dysregulates cAMP-PKA signaling and phosphodiesterase 10A expression: mechanistic insight and potential target for Lesch-Nyhan Disease?

Lesch-Nyhan Disease (LND) is the result of mutations in the X-linked gene encoding the purine metabolic enzyme, hypoxanthine guanine phosphoribosyl transferase (HPRT). LND gives rise to severe neurological anomalies including mental retardation, dystonia, chorea, pyramidal signs and a compulsive and aggressive behavior to self injure. The neurological phenotype in LND has been shown to reflect aberrant dopaminergic signaling in the basal ganglia, however there are little data correlating the defect in purine metabolism to the neural-related abnormalities. In the present studies, we find that HPRT-deficient neuronal cell lines have reduced CREB (cAMP response element-binding protein) expression and intracellular cyclic AMP (cAMP), which correlates with attenuated CREB-dependent transcriptional activity and a reduced phosphorylation of protein kinase A (PKA) substrates such as synapsin (p-syn I). Of interest, we found increased expression of phosphodiesterase 10A (PDE10A) in HPRT-deficient cell lines and that the PDE10 inhibitor papaverine and PDE10A siRNA restored cAMP/PKA signaling. Furthermore, reconstitution of HPRT expression in mutant cells partly increased cAMP signaling synapsin phosphorylation. In conclusion, our data show that HPRT-deficiency alters cAMP/PKA signaling pathway, which is in part due to the increased of PDE10A expression and activity. These findings suggest a mechanistic insight into the possible causes of LND and highlight PDE10A as a possible therapeutic target for this intractable neurological disease.

Knockdown of HPRT for selection of genetically modified human hematopoietic progenitor cells.

The inability to obtain sufficient numbers of transduced cells remains a limitation in gene therapy. One strategy to address this limitation is in vivo pharmacologic selection of transduced cells. We have previously shown that knockdown of HPRT using lentiviral delivered shRNA facilitates efficient selection of transduced murine hematopoietic progenitor cells (HPC) using 6-thioguanine (6TG). Herein, we now extend these studies to human HPC. We tested multiple shRNA constructs in human derived cell lines and identified the optimal shRNA sequence for knockdown of HPRT and 6TG resistance. We then tested this vector in human umbilical cord blood derived HPC in vitro and in NOD/SCID recipients. Knockdown of HPRT effectively provided resistance to 6TG in vitro. 6TG treatment of mice resulted in increased percentages of transduced human CD45(+) cells in the peripheral blood and in the spleen in particular, in both myeloid and lymphoid compartments. 6TG treatment of secondary recipients resulted in higher percentages of transduced human cells in the bone marrow, confirming selection from the progeny of long-term repopulating HPCs. However, the extent of selection of cells in the bone marrow at the doses of 6TG tested and the toxicity of higher doses, suggest that this strategy may be limited to selection of more committed progenitor cells. Together, these data suggest that human HPC can be programmed to be resistant to purine analogs, but that HPRT knockdown/6TG-based selection may not be robust enough for in vivo selection.

Impairment of adenylyl cyclase 2 function and expression in hypoxanthine phosphoribosyltransferase-deficient rat B103 neuroblastoma cells as model for Lesch-Nyhan disease: BODIPY-forskolin as pharmacological tool.

Hypoxanthine phosphoribosyl transferase (HPRT) deficiency results in Lesch-Nyhan disease (LND). The link between the HPRT defect and the self-injurious behavior in LND is still unknown. HPRT-deficient rat B103 neuroblastoma cells serve as a model system for LND. In B103 cell membranes, HPRT deficiency is associated with a decrease of basal and guanosine triphosphate-stimulated adenylyl cyclase (AC) activity (Pinto and Seifert, J Neurochem 96:454-459, 2006). Since recombinant AC2 possesses a high basal activity, we tested the hypothesis that AC2 function and expression is impaired in HPRT deficiency. We examined AC regulation in B103 cell membranes, cAMP accumulation in intact B103 cells, AC isoform expression, and performed morphological studies. As most important pharmacological tool, we used 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene forskolin (BODIPY-FS) that inhibits recombinant AC2 but activates ACs 1 and 5 (Erdorf et al., Biochem Pharmacol 82:1673-1681, 2011). In B103 control membranes, BODIPY-FS reduced catalysis, but in HPRT(-) membranes, BODIPY-FS was rather stimulatory. 2'(3')-O-(N-methylanthraniloyl) (MANT)-nucleoside 5'-[γ-thio]triphosphates inhibit recombinant ACs 1 and 5 more potently than AC2. In B103 control membranes, MANT-guanosine 5'-[γ-thio]triphosphate inhibited catalysis in control membranes less potently than in HPRT(-) membranes. Quantitative real-time PCR revealed that in HPRT deficiency, AC2 was virtually absent. In contrast, AC5 was up-regulated. Forskolin (FS) and BODIPY-FS induced cell clustering and rounding and neurite extension in B103 cells. The effects of FS and BODIPY-FS were much more prominent in control than in HPRT(-) cells, indicative for a differentiation defect in HPRT deficiency. Neither FS nor BODIPY-FS significantly changed cAMP concentrations in intact B103 cells. Collectively, our data show that HPRT deficiency in B103 cells is associated with impaired AC2 function and expression and reduced sensitivity to differentiation induced by FS and BODIPY-FS. We discuss the pathophysiological implications of our data for LND.

Combined preconditioning and in vivo chemoselection with 6-thioguanine alone achieves highly efficient reconstitution of normal hematopoiesis with HPRT-deficient bone marrow.

Purine analogs such as 6-thioguanine (6TG) cause myelotoxicity upon conversion into nucleotides by hypoxanthine-guanine phosphoribosyltransferase (HPRT). Here we have developed a novel and highly efficient strategy employing 6TG as a single agent for both conditioning and in vivo chemoselection of HPRT-deficient hematopoietic stem cells. The dose-response and time course of 6TG myelotoxicity were first compared in HPRT wild-type mice and HPRT-deficient transgenic mice. Dosage and schedule parameters were optimized to employ 6TG for myelosuppressive conditioning, immediately followed by in vivo chemoselection of HPRT-deficient transgenic donor bone marrow (BM) transplanted into syngeneic HPRT wild-type recipients. At appropriate doses, 6TG induced selective myelotoxicity without any adverse effects on extrahematopoietic tissues in HPRT wild-type mice, while hematopoietic stem cells deficient in HPRT activity were highly resistant to its cytotoxic effects. Combined 6TG conditioning and post-transplantation chemoselection consistently achieved ∼95% engraftment of HPRT-deficient donor BM, with low overall toxicity. Long-term reconstitution of immunophenotypically normal BM was achieved in both primary and secondary recipients. Our results provide proof-of-concept that single-agent 6TG can be used for both myelosuppressive conditioning without requiring irradiation and for in vivo chemoselection of HPRT-deficient donor cells. Our results show that by applying the myelosuppressive effects of 6TG both before (as conditioning) and after transplantation (as chemoselection), highly efficient engraftment of HPRT-deficient hematopoietic stem cells can be achieved.

MicroRNA-mediated dysregulation of neural developmental genes in HPRT deficiency: clues for Lesch-Nyhan disease?

Mutations in the gene encoding the purine biosynthetic enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause the intractable neurodevelopmental Lesch-Nyhan disease (LND) associated with aberrant development of brain dopamine pathways. In the current study, we have identified an increased expression of the microRNA miR181a in HPRT-deficient human dopaminergic SH-SY5Y neuroblastoma cells. Among the genes potentially regulated by miR181a are several known to be required for neural development, including Engrailed1 (En1), Engrailed2 (En2), Lmx1a and Brn2. We demonstrate that these genes are down-regulated in HPRT-deficient SH-SY5Y cells and that over-expression of miR181a significantly reduces endogenous expression of these genes and inhibits translation of luciferase plasmids bearing the En1/2 or Lmx1a 3'UTR miRNA-binding elements. Conversely, inhibition of miR181a increases the expression of these genes and enhances translation of luciferase constructs bearing the En1/2 and Lmx1a 3'UTR miRNA-binding sequences. We also demonstrate that key neurodevelopmental genes (e.g. Nurr1, Pitx3, Wnt1 and Mash1) known to be functional partners of Lmx1a and Brn2 are also markedly down-regulated in SH-SY5Y cells over-expressing miR181a and in HPRT-deficient cells. Our findings in SH-SY5Y cells demonstrate that HPRT deficiency is accompanied by dysregulation of some of the important pathways that regulate the development of dopaminergic neurons and dopamine pathways and that this defect is associated with and possibly due at least partly to aberrant expression of miR181a. Because aberrant expression of miR181a is not as apparent in HPRT-deficient LND fibroblasts, the relevance of the SH-SY5Y neuroblastoma cells to human disease remains to be proven. Nevertheless, we propose that these pleiotropic neurodevelopment effects of miR181a may play a role in the pathogenesis of LND.

Impaired P2X and P2Y receptor-mediated signaling in HPRT-deficient B103 neuroblastoma cells.

Defect of hypoxanthine phosphoribosyl transferase (HPRT) causes Lesch-Nyhan disease (LND), but the link between HPRT deficiency and the self-injurious behavior of LND is unknown. In a previous study (Pinto et al., J. Neurochem. 72 (2005) 1579-1586) we reported on a decrease in nucleotidase activity in membranes of several HPRT(-) cell lines and fibroblasts from LND patients. Since nucleotidases are involved in ATP-induced signal transduction, in the present study, we tested the hypothesis that P2X and P2Y receptor-mediated signal transduction is impaired in HPRT deficiency. As model we studied rat B103 neuroblastoma cells. Compared to control cells, in HPRT(-) cells, NTP and NDP-induced Ca(2+) influx across the membrane and Ca(2+) mobilization from intracellular stores were impaired. Both P2X and P2Y receptors were involved in the responses. Quantitative real-time PCR revealed reduced expression of receptors P2X(3), P2X(5), P2Y(2), P2Y(4), P2Y(12), P2Y(13) and P2Y(14) in HPRT deficiency. Collectively, HPRT deficiency is associated with abnormal purinergic signaling, encompassing P2X and P2Y receptors and nucleotidases.

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.

HPRT deficiency coordinately dysregulates canonical Wnt and presenilin-1 signaling: a neuro-developmental regulatory role for a housekeeping gene?

We have used microarray-based methods of global gene expression together with quantitative PCR and Western blot analysis to identify dysregulation of genes and aberrant cellular processes in human fibroblasts and in SH-SY5Y neuroblastoma cells made HPRT-deficient by transduction with a retrovirus stably expressing an shRNA targeted against HPRT. Analysis of the microarray expression data by Gene ontology (GO) and Gene Set Enrichment Analysis (GSEA) as well as significant pathway analysis by GeneSpring GX10 and Panther Classification System reveal that HPRT deficiency is accompanied by aberrations in a variety of pathways known to regulate neurogenesis or to be implicated in neurodegenerative disease, including the canonical Wnt/ß-catenin and the Alzheimer's disease/presenilin signaling pathways. Dysregulation of the Wnt/ß-catenin pathway is confirmed by Western blot demonstration of cytosolic sequestration of ß-catenin during in vitro differentiation of the SH-SY5Y cells toward the neuronal phenotype. We also demonstrate that two key transcription factor genes known to be regulated by Wnt signaling and to be vital for the generation and function of dopaminergic neurons; i.e., Lmx1a and Engrailed 1, are down-regulated in the HPRT knockdown SH-SY5Y cells. In addition to the Wnt signaling aberration, we found that expression of presenilin-1 shows severely aberrant expression in HPRT-deficient SH-SY5Y cells, reflected by marked deficiency of the 23 kDa C-terminal fragment of presenilin-1 in knockdown cells. Western blot analysis of primary fibroblast cultures from two LND patients also shows dysregulated presenilin-1 expression, including aberrant proteolytic processing of presenilin-1. These demonstrations of dysregulated Wnt signaling and presenilin-1 expression together with impaired expression of dopaminergic transcription factors reveal broad pleitropic neuro-regulatory defects played by HPRT expression and suggest new directions for investigating mechanisms of aberrant neurogenesis and neuropathology in LND and potential new targets for restoration of effective signaling in this neuro-developmental defect.

In vivo 6-thioguanine-resistant T cells from melanoma patients have public TCR and share TCR beta amino acid sequences with melanoma-reactive T cells.

In vivo hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient T cells (MT) from melanoma patients are enriched for T cells with in vivo clonal amplifications that traffic between blood and tumor tissues. Melanoma is thus a model cancer to test the hypothesis that in vivo MT from cancer patients can be used as immunological probes for immunogenic tumor antigens. MT were obtained by 6-thioguanine (TG) selection of lymphocytes from peripheral blood and tumor tissues, and wild-type T cells (WT) were obtained analogously without TG selection. cDNA sequences of the T cell receptor beta chains (TRB) were used as unambiguous biomarkers of in vivo clonality and as indicators of T cell specificity. Public TRB were identified in MT from the blood and tumor of different melanoma patients. Such public TRB were not found in normal control MT or WT. As an indicator of T cell specificity for melanoma, the >2600 MT and WT TRB, including the public TRB from melanoma patients, were compared to a literature-derived empirical database of >1270 TRB from melanoma-reactive T cells. Various degrees of similarity, ranging from 100% conservation to 3-amino acid motifs (3-mer), were found between both melanoma patient MT and WT TRBs and the empirical database. The frequency of 3-mer and 4-mer TRB matching to the empirical database was significantly higher in MT compared with WT in the tumor (p=0.0285 and p=0.006, respectively). In summary, in vivo MT from melanoma patients contain public TRB as well as T cells with specificity for characterized melanoma antigens. We conclude that in vivo MT merit study as novel probes for uncharacterized immunogenic antigens in melanoma and other malignancies.

Deficiency of the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) dysregulates neurogenesis.

Neuronal transcription factors play vital roles in the specification and development of neurons, including dopaminergic (DA) neurons. Mutations in the gene encoding the purine biosynthetic enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause the resulting intractable and largely untreatable neurological impairment of Lesch-Nyhan disease (LND). The disorder is associated with a defect in basal ganglia DA pathways. The mechanisms connecting the purine metabolic defect and the central nervous system (CNS) phenotype are poorly understood but have been presumed to reflect a developmental defect of DA neurons. We have examined the effect of HPRT deficiency on the differentiation of neurons in the well-established human (NT2) embryonic carcinoma neurogenesis model. We have used a retrovirus expressing a small hairpin RNA (shRNA) to knock down HPRT gene expression and have examined the expression of a number of transcription factors essential for neuronal differentiation and marker genes involved in DA biosynthetic pathway. HPRT-deficient NT2 cells demonstrate aberrant expression of several transcription factors and DA markers. Although differentiated HPRT-deficient neurons also demonstrate a striking deficit in neurite outgrowth during differentiation, resulting neurons demonstrate wild-type electrophysiological properties. These results represent direct experimental evidence for aberrant neurogenesis in HPRT deficiency and suggest developmental roles for other housekeeping genes in neurodevelopmental disease.