Lesch-Nyhan syndrome (LNS) is inherited as an X-linked recessive genetic disorder caused by mutations in hypoxanthine-guanine phosphoribosyl transferase 1 (HPRT1). Patients with LNS show various clinical phenotypes, including hyperuricemia, gout, devastating behavioral abnormality, intellectual disability, and self-harm. Although uric acid overproduction can be modulated with the xanthine oxidase inhibitor allopurinol, there exists no treatment for behavioral and neurological manifestations of LNS. In the current study, CRISPR-mediated base editors (BEs) and prime editors (PEs) were utilized to generate LNS-associated disease models and correct the disease models for therapeutic approach. Cytosine BEs (CBEs) were used to induce c.430C>T and c.508C>T mutations in HAP1 cells, and then adenine BEs (ABEs) were used to correct these mutations without DNA cleavage. PEs induced a c.333_334ins(A) mutation, identified in a Korean patient with LNS, in HAP1 cells, which was corrected in turn by PEs. Furthermore, improved PEs corrected the same mutation in LNS patient-derived fibroblasts by up to 14% without any unwanted mutations. These results suggest that CRISPR-mediated BEs and PEs would be suggested as a potential therapeutic strategy of this extremely rare, devastating genetic disease.

Self-injurious behavior is the most outstanding feature of Lesch-Nyhan syndrome and has recently been ascribed to an obsessive-compulsive behavior. Lesch-Nyhan syndrome results from the complete enzyme deficiency of hypoxanthine-guanine phosphoribosyl transferase (HPRT) but the link between abnormal purine metabolism and its neurological and behavioral manifestations remains largely unknown. Previous studies led us to hypothesize that adenosine and dopamine receptor expression could be altered in HPRT-deficient cells. To test this hypothesis, we examined mRNA expressions of adenosine (ADORA2A and ADORA2B) and dopamine receptors (DRD1 and DRD2 like), and dopamine transporter (DAT1) in peripheral blood lymphocytes (PBLs) from Lesch-Nyhan patients. We also examined the influence of hypoxanthine in these expressions. As compared to normal PBLs, both ADORA2A and DRD5 expression were abnormal in PBLs from Lesch-Nyhan patients. In contrast, DAT1 expression was similar to control values in HPRT deficient PBLs. These results indicate an abnormal adenosine and dopamine receptor expression in HPRT-deficient cells and suggest disrupted adenosine and dopamine neurotransmission may have a significant role in the pathogenesis of the neurological manifestations of Lesch-Nyhan syndrome.

Current therapies for the Lesch-Nyhan Syndrome (OMIM: 300322) are off-label and experimental, often leading to inconsistent outcomes. We here report the effects of an intrathecal baclofen therapy, carried out at the Scientific Institute Eugenio Medea (Lecco, Italy), on three patients who no longer received benefit from previous therapies. This treatment, as expected, ameliorated the motor symptoms and, unexpectedly, it also improved behavioural components. This result may involve a functional interaction between baclofen and dopamine, complemented by an anxiolytic effect. Our observations provide the rationale for the use of intrathecal baclofen administration in the therapy of the Lesch-Nyhan Syndrome.

Lesch-Nyhan syndrome (LNS, MIM300322) is a rare inherited disorder caused by mutations in HPRT1 gene. Here we describe the generation of induced pluripotent stem cells (iPSCs) from an infected child carrying the HPRT1 mutation c.508C > T(p.R170X) by reprogramming peripheral blood mononuclear cells (PBMCs) with episomal vectors. The obtained hiPSCs exhibited normal karyotype, expressed pluripotency markers, and possessed trilineage differentiation capacity.

The effects of footshock sensitization (priming), apomorphine (APO) priming and their combination on behavior and neostriatal and cortical catecholamines were examined in adult rats which had neonatally received bilateral intracerebroventricular injections with 6-hydroxydopamine (6-OHDA; a model of Lesch-Nyhan syndrome (LNS)) or vehicle (unlesioned rats). Lesioned (6-OHDA-treated) rats displayed self-biting (SB; 7/20 rats) and self-injurious behavior (SIB; 1/20 rats) during APO priming, but not during footshock priming. During subsequent acute cumulative APO dosing, 20-30% of lesioned rats primed with APO alone or footshock alone displayed SB and SIB. However, SB and SIB incidence in APO+footshock-primed lesioned rats was nearly tripled. Dopamine (DA) synthesis, metabolism and extracellular concentrations (disposition) in unlesioned rats and in cortices of lesioned animals were unaffected by priming. In lesioned rats primed with APO alone or footshock alone, only neostriatal 3-methoxytyramine (3-MT) was significantly increased. However, neostriatal DA and metabolite concentrations (and norepinephrine (NE)) were all significantly elevated in lesioned rats primed with both APO and footshock. These results confirm that neonatal 6-OHDA-induced neostriatal catecholamine depletion can be antagonized by experiential change, suggest that behavioral and neurochemical cross-sensitization between APO and footshock in such rats is unidirectional and support the view that stress can exacerbate the incidence of SIB in LNS.

Lesch-Nyhan Syndrome (LNS) is a neurodevelopmental disorder caused by mutations in the gene encoding the purine metabolic enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). This syndrome is characterized by an array of severe neurological impairments that in part originate from striatal dysfunctions. However, the molecular and cellular mechanisms underlying these dysfunctions remain largely unidentified. In this report, we demonstrate that HPRT-deficiency causes dysregulated expression of key genes essential for striatal patterning, most notably the striatally-enriched transcription factor B-cell leukemia 11b (Bcl11b). The data also reveal that the down-regulated expression of Bcl11b in HPRT-deficient immortalized mouse striatal (STHdh) neural stem cells is accompanied by aberrant expression of some of its transcriptional partners and other striatally-enriched genes, including the gene encoding dopamine- and cAMP-regulated phosphoprotein 32, (DARPP-32). Furthermore, we demonstrate that components of the BDNF/TrkB signaling, a known activator of DARPP-32 striatal expression and effector of Bcl11b transcriptional activation are markedly increased in HPRT-deficient cells and in the striatum of HPRT knockout mouse. Consequently, the HPRT-deficient cells display superior protection against reactive oxygen species (ROS)-mediated cell death upon exposure to hydrogen peroxide. These findings suggest that the purine metabolic defect caused by HPRT-deficiency, while it may provide neuroprotection to striatal neurons, affects key genes and signaling pathways that may underlie the neuropathogenesis of LNS.

We have shown functional complementation of a genetic deficiency in human cultured cells, using artificial chromosomes derived from cloned human genomic fragments. A 404-kb human-artificial-chromosome (HAC) vector, consisting of 220 kb of alphoid DNA from the centromere of chromosome 17, human telomeres, and the hypoxanthine guanine phosphoribosyltransferase (HPRT) genomic locus, was transferred to HPRT-deficient HT1080 fibrosarcoma cells. We generated several cell lines with low-copy-number, megabase-sized HACs containing a functional centromere and one or possibly several copies of the HPRT1 gene complementing the metabolic deficiency. The HACs consisted of alternating alphoid and nonalphoid DNA segments derived only from the input DNA (within the sensitivity limits of FISH detection), and the largest continuous alphoid segment was 158-250 kb. The study of both the structure and mitotic stability of these HACs offers insights into the mechanisms of centromere formation in synthetic chromosomes and will further the development of this human-gene-transfer technology.

Patients with gout have numerous comorbidities. We aimed to estimate the prevalence and incidence rates of renal and cardiovascular morbidities in trial-aligned patients with established gout in Germany (DE), the United Kingdom (UK), the United States (US), and France (FR).

Patients with congenital insensitivity to pain and anhydrosis syndrome are at risk for renal amyloidosis and inflammatory bowel disease. Physicians caring for such patients should be aware of these complications.

Lesch-Nyhan syndrome encompasses a host of neurological symptoms, caused by a deficiency of the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGPRT). How the absence of this enzymes activity affects development of the nervous system is unknown. In this study, we examined the ability of N2aTG, a HGPRT-deficient neuroblastoma and its HGPRT-positive counterpart to proliferate and differentiate at various densities. In summary, N2aTG cells proliferated less and differentiated more than N2a cells, with the former cells exhibiting enhanced sensitivity to the effects of low-density culture. Given the homogeneity of this neuroblastoma cell line and its use in studies of neuronal development, the present study indicates that N2aTG cells may prove a suitable in vitro model for the study of non-dopaminergic neuronal development in Lesch-Nyhan syndrome.

The extent to which non-coding mutations contribute to Mendelian disease is a major unknown in human genetics. Relatedly, the vast majority of candidate regulatory elements have yet to be functionally validated. Here, we describe a CRISPR-based system that uses pairs of guide RNAs (gRNAs) to program thousands of kilobase-scale deletions that deeply scan across a targeted region in a tiling fashion ("ScanDel"). We applied ScanDel to HPRT1, the housekeeping gene underlying Lesch-Nyhan syndrome, an X-linked recessive disorder. Altogether, we programmed 4,342 overlapping 1 and 2 kb deletions that tiled 206 kb centered on HPRT1 (including 87 kb upstream and 79 kb downstream) with median 27-fold redundancy per base. We functionally assayed programmed deletions in parallel by selecting for loss of HPRT function with 6-thioguanine. As expected, sequencing gRNA pairs before and after selection confirmed that all HPRT1 exons are needed. However, HPRT1 function was robust to deletion of any intergenic or deeply intronic non-coding region, indicating that proximal regulatory sequences are sufficient for HPRT1 expression. Although our screen did identify the disruption of exon-proximal non-coding sequences (e.g., the promoter) as functionally consequential, long-read sequencing revealed that this signal was driven by rare, imprecise deletions that extended into exons. Our results suggest that no singular distal regulatory element is required for HPRT1 expression and that distal mutations are unlikely to contribute substantially to Lesch-Nyhan syndrome burden. Further application of ScanDel could shed light on the role of regulatory mutations in disease at other loci while also facilitating a deeper understanding of endogenous gene regulation.

In this paper we report the application of an extensive database of symptoms, signs, laboratory findings and illnesses, to the diagnosis of an historical figure. The medical diagnosis of Augustus d'Este (1794-1848) - widely held to be the first documented case of multiple sclerosis - is reviewed, using the detailed symptom diary, which he kept over many years, as clinical data. Some of the reported features prompted the competing claim that d'Este suffered from acute porphyria, which in turn was used in support of the hypothesis that his grandfather, King George III, also suffered from the disease. We find that multiple sclerosis is statistically the most likely diagnosis, with neuromyelitis optica a strong alternative possibility. The database did not support a diagnosis of any of the acute porphyrias.

Learning disorders are often associated with persistent hyperactivity and aggression and are part of a spectrum of neurodevelopmental disorders. A potential clue to understanding these linked phenomena is that physical exercise and passive forms of stimulation are calming, enhance cognitive functions and learning, and are recommended as complementary treatments for these problems. The theory is proposed that hyperactivity and aggression are intense stimulation-seeking behaviors (SSBs) driven by increased brain retinergic activity, and the stimulation thus obtained activates opposing nitrergic systems which inhibit retinergic activity, induce a state of calm, and enhance cognition and learning. In persons with cognitive deficits and associated behavioral disorders, the retinergic system may be chronically overactivated and the nitrergic system chronically underactivated due to environmental exposures occurring pre- and/or postnatally that affect retinoid metabolism or expression. For such individuals, the intensity of stimulation generated by SSB may be insufficient to activate the inhibitory nitrergic system. A multidisciplinary research program is needed to test the model and, in particular, to determine the extent to which applied physical treatments can activate the nitrergic system directly, providing the necessary level of intensity of sensory stimulation to substitute for that obtained in maladaptive and harmful ways by SSB, thereby reducing SSB and enhancing cognitive skills and performance.

Proteins play important roles in biology, biotechnology and pharmacology, and missense variants are a common cause of disease. Discovering functionally important sites in proteins is a central but difficult problem because of the lack of large, systematic data sets. Sequence conservation can highlight residues that are functionally important but is often convoluted with a signal for preserving structural stability. We here present a machine learning method to predict functional sites by combining statistical models for protein sequences with biophysical models of stability. We train the model using multiplexed experimental data on variant effects and validate it broadly. We show how the model can be used to discover active sites, as well as regulatory and binding sites. We illustrate the utility of the model by prospective prediction and subsequent experimental validation on the functional consequences of missense variants in HPRT1 which may cause Lesch-Nyhan syndrome, and pinpoint the molecular mechanisms by which they cause disease.

Protein translation is a highly regulated process involving the interaction of numerous genes on every component of the protein translation machinery. Upregulated protein translation is a hallmark of cancer and is implicated in autism spectrum disorder, but the risks of developing each disease do not appear to be correlated with one another. In this study we identified two siblings from the NIH Undiagnosed Diseases Program with loss of function variants in PUS7, a gene previously implicated in the regulation of total protein translation. These patients exhibited a neurodevelopmental phenotype including autism spectrum disorder in the proband. Both patients also had features of Lesch-Nyhan syndrome, including hyperuricemia and self-injurious behavior, but without pathogenic variants in HPRT1. Patient fibroblasts demonstrated upregulation of protein synthesis, including elevated MYC protein, but did not exhibit increased rates of cell proliferation. Interestingly, the dysregulation of protein translation also resulted in mildly decreased levels of HPRT1 protein suggesting an association between dysregulated protein translation and the LNS-like phenotypic findings. These findings strengthen the correlation between neurodevelopmental disease, particularly autism spectrum disorders, and the rate of protein translation.

Lesch-Nyhan disease (LND) is a severe and incurable X-linked genetic syndrome caused by the deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT), resulting in severe alterations of central nervous system, hyperuricemia and subsequent impaired renal functions. Therapeutic options consist in supportive care and treatments of complications, but the disease remains largely untreatable. Enzyme replacement of the malfunctioning cytosolic protein might represent a possible therapeutic approach for the LND treatment. Protein transduction domains, such as the TAT peptide derived from HIV TAT protein, have been used to transduce macromolecules into cells in vitro and in vivo. The present study was aimed to the generation of TAT peptide fused to human HPRT for cell transduction in enzyme deficient cells. Here we document the construction, expression and delivery of a functional HPRT enzyme into deficient cells by TAT transduction domain and by liposome mediated protein transfer. With this approach we demonstrate the correction of the enzymatic defect in HPRT deficient cells. Our data show for the first time the feasibility of the enzyme replacement therapy for the treatment of LND.

Genetic changes in Hypoxanthine guanine phosphoribosyltransferace (HPRT1) gene can alter the expression of the dopamine neurotransmitter leads to abnormal neuron function, a disease called Lesch-Nyhan syndrome (LNS). Although different studies were conducted on LNS, information on codon usage bias (CUB) of HPRT1 gene is limited. The present study examines the genetic determinants of CUB in HPRT1 gene using twelve mammalian species. In the coding sequence of HPRT1 genes, A/T ending codons was most frequently used. A higher ENC value was observed indicating lower HPRT1 gene expression in the selected mammalian species. Correlation analysis indicates that compositional constraints under mutation pressure can involve in CUB of HPRT1 genes among the selected mammalian species. Relative synonymous codon usage (RSCU) value revealed that the codons such as ACT, AGG, ATT and AGC were over-represented in each of the mammalian species. Result from the analysis of the RSCU indicates that compositional constraint is a key driver for the variation in codon usage. Ratio of nonsynonymous (dN) and synonymous (dS) substitution further suggested that purifying selection occurs among the HPRT1 gene of studied mammals to maintain its protein function under the process of evolution. Our findings report an insight into the codon usage patterns of HPRT1 gene and will be useful for LNS management.

Severe 6-hydroxydopamine (6-OHDA)-induced neostriatal dopamine (DA) depletion is generally held to be irreversible. Adult rats administered 6-OHDA soon after weaning, or neonatally, respectively model Parkinson's disease (PD) and Lesch-Nyhan syndrome (LNS). Prior studies in our laboratory indicate that prolonged training on incrementally more difficult fixed-ratio (FR) discriminations can reverse 'irreversible' 6-OHDA-induced neostriatal DA depletion in adult LNS rats. The present study evaluated the effects of such training on neostriatal DA depletion and its functional consequences in adult PD and control (vehicle-injected) rats. After recovery from 6-OHDA-induced hypophagia, rats were sacrificed to assess neostriatal DA depletion magnitude, or were food-deprived and either subjected to food-maintained operant FR discrimination training or allowed to remain in their home cages. 6-OHDA treatment antagonized amphetamine (AMP)-induced increases in brief rearing behavior and locomotor activity in 3-month-old PD rats prior to training, and reduced operant response rates throughout training without affecting learning rates. One week after training, AMP-increased locomotor and brief-rearing frequencies were augmented in all groups except trained controls, and the prior inhibitory effect of 6-OHDA treatment on AMP-increased behavioral frequencies was essentially eliminated. Cumulative apomorphine (APO) dose-effect curve (0.1-3.2 mg/kg) construction 3 weeks post-training revealed that 6-OHDA treatment abolished APO-induced intense licking behavior. However, training eliminated the hyperresponsiveness of 6-OHDA-treated rats to the locomotor- and brief-rearing stimulant effects of APO but did not affect the depletion of neostriatal DA. Nevertheless, 6-OHDA-induced increases in neostriatal DOPAC/DA and HVA/DA ratios were normalized by age/food-deprivation while that of 3MT/DA was not. These findings suggest that training reduces the functional responsiveness of at least some central DA receptors, FR discrimination training could be a useful adjunct to PD replacement therapy and that the neostriatal DA-repleting action of training in 6-OHDA-treated rats depend on the age at which 6-OHDA is administered.

A single founder mutation resulting in a Ser163Arg substitution in the C1QTNF5 gene product causes autosomal dominant late-onset retinal macular degeneration (L-ORMD) in humans, which has clinical and pathological features resembling age-related macular degeneration. We generated and characterised a mouse "knock-in" model carrying the Ser163Arg mutation in the orthologous murine C1qtnf5 gene by site-directed mutagenesis and homologous recombination into mouse embryonic stem cells. Biochemical, immunological, electron microscopic, fundus autofluorescence, electroretinography and laser photocoagulation analyses were used to characterise the mouse model. Heterozygous and homozygous knock-in mice showed no significant abnormality in any of the above measures at time points up to 2 years. This result contrasts with another C1qtnf5 Ser163Arg knock-in mouse which showed most of the features of L-ORMD but differed in genetic background and targeting construct.

Understanding the genetic causes of kidney disease is essential for accurate diagnosis and could lead to improved therapeutic strategies and prognosis. To accurately and promptly identify the genetic background of kidney diseases, we applied a targeted next-generation sequencing gene panel including 203 genes associated with kidney disease, as well as diseases originating in other organs with mimicking symptoms of kidney disease, to analyze 51 patients with nonspecific nephrogenic symptoms, followed by validation of its efficacy as a diagnostic tool. We simultaneously screened for copy number variants (CNVs) in each patient to obtain a higher diagnostic yield (molecular diagnostic rate: 39.2%). Notably, one patient suspected of having Bartter syndrome presented with chloride-secreting diarrhea attributable to homozygous SLC26A3 variants. Additionally, in eight patients, NGS confirmed the genetic causes of undefined kidney diseases (8/20, 40%), and initial clinical impression and molecular diagnosis were matched in 11 patients (11/20, 55%). Moreover, we found seven novel pathogenic/likely pathogenic variants in PKD1, PKHD1, COL4A3, and SLC12A1 genes, with a possible pathogenic variant in COL4A3 (c.1229G>A) identified in two unrelated patients. These results suggest that targeted NGS-panel testing performed with CNV analysis might be advantageous for noninvasive and comprehensive diagnosis of suspected genetic kidney diseases.