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.

Hypoxanthine phosphoribosyltransferase (HPRT)-deficiency is associated with impaired fertility in the female rat.

The purine hypoxanthine plays important role in regulating oocyte maturation and early embryonic development. The enzyme hypoxanthine phosphoribosyltransferase (HPRT) recycles hypoxanthine to generate substrates for nucleotide synthesis and key metabolites, and here we show that HPRT deficiency in the rat disrupts early embryonic development and causes infertility in females.

Clinical, biochemical and genetic characteristics of a cohort of 101 French and Italian patients with HPRT deficiency.

BACKGROUND: HPRT deficiency is a rare disorder of purine metabolism whose natural history is not fully understood. No optimal management recommendations exist. The objective of the present study is to characterize a large cohort of patients with HPRT deficiency, comparing Lesch-Nyhan Disease (LND) and its attenuated variants, with the purpose of helping clinicians in disease management and prognostic definition. METHODS: Genetic and clinical features of French and Italian patients with a confirmed diagnosis of HPRT deficiency were collected. RESULTS: A hundred and one patients were studied, including 66 LND, 22 HND (HPRT-related Neurological Dysfunction) and 13 HRH (HPRT-Related Hyperuricemia) patients. The clinical manifestations at onset were not specific, but associated with an orange coloration of diapers in 22% of patients. The overall neurological involvement was more severe in LND than in HND patients. Behavioural disturbances were not limited to self-injuries and were not exclusive of LND. Median age of involuntary movements and self-injuries appearance in LND was 1.0 and 3 years, respectively. Renal manifestations (66.3% of patients) occurred at any age with a median onset age of 1.1 years, while gout (25.7% of patients) appeared later in disease course (median onset age 18 years) and was more frequent in attenuated variants than in LND. HPRT activity and genotype showed a significant correlation with the severity of the neurological disease. On the contrary, there were no significant differences in the development of nephropathy or gout. For the treatment of neurological aspects, botulinum toxin injections, oral or intrathecal baclofen and gabapentin were partially efficacious and well tolerated, while deep brain stimulation was associated to a worsening of patients' condition. CONCLUSIONS: The present study improves the knowledge of the natural history of HPRT deficiency and could represent a starting point for the development of future management guidelines.

Kelley-Seegmiller Syndrome: Urolithiasis, Renal Uric Acid Deposits, and Gout: What is the Role of the Urologist?

Kelley-Seegmiller syndrome (KSS) is a disorder that occurs when there is a partial deficiency of the enzyme hypoxanthine guanine phosphoribosyl transferase. It is involved in the metabolism of purines, clinically manifesting as hyperuricemia, hyperuricosuria, gout arthritis, and urolithiasis. The aim of this article is to present the case of a 33-year-old male with KSS, with left ureteral colic, and a 5-mm, 323-HU ureteral calculi, successfully managed with conservative management. It is critical to recognize that most urologists are not familiar with this inborn metabolic error and 75% of these patients will be affected by urolithiasis, thus making it a very critical and significant disease in our practice.

A Trivalent Enzymatic System for Uricolytic Therapy of HPRT Deficiency and Lesch-Nyhan Disease.

PURPOSE: Because of the evolutionary loss of the uricolytic pathway, humans accumulate poorly soluble urate as the final product of purine catabolism. Restoration of uricolysis through enzyme therapy is a promising treatment for severe hyperuricemia caused by deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). To this end, we studied the effect of PEG conjugation on the activity and stability of the enzymatic complement required for conversion of urate into the more soluble (S)-allantoin. METHODS: We produced in recombinant form three zebrafish enzymes required in the uricolytic pathway. We carried out a systematic study of the effect of PEGylation on the function and stability of the three enzymes by varying PEG length, chemistry and degree of conjugation. We assayed in vitro the uricolytic activity of the PEGylated enzymatic triad. RESULTS: We defined conditions that allow PEGylated enzymes to retain native-like enzymatic activity even after lyophilization or prolonged storage. A combination of the three enzymes in an appropriate ratio allowed efficient conversion of urate to (S)-allantoin with no accumulation of intermediate metabolites. CONCLUSIONS: Pharmaceutical restoration of the uricolytic pathway is a viable approach for the treatment of severe hyperuricemia.

Do clinical features of Lesch-Nyhan disease correlate more closely with hypoxanthine or guanine recycling?

Lesch-Nyhan disease (LND) is a rare, X-linked recessive neurodevelopmental disorder caused by deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGprt), an enzyme in the purine salvage pathway. HGprt has two functions; it recycles hypoxanthine and guanine. Which of these two functions is more relevant for pathogenesis is unclear because some evidence points to hypoxanthine recycling, but other evidence points to guanine recycling. In this study, we selectively assayed hypoxanthine (Hprt) and guanine (Gprt) recycling in skin fibroblasts from 17 persons with LND, 11 with an attenuated variant of the disease (LNV), and 19 age-, sex-, and race-matched healthy controls (HC). Activity levels of both enzymes differed across groups (p < 0.0001), but only Gprt distinguished patients with LND from those with LNV (p < 0.05). Gprt also showed slightly stronger correlations than Hprt with 13 of 14 measures of the clinical phenotype, including the severity of dystonia, cognitive impairment, and behavioral abnormalities. These findings suggest that loss of guanine recycling might be more closely linked to the LND/LNV phenotype than loss of hypoxanthine recycling.

Deficiency of the purine metabolic gene HPRT dysregulates microRNA-17 family cluster and guanine-based cellular functions: a role for EPAC in Lesch-Nyhan syndrome.

Lesch-Nyhan syndrome (LNS) is a neurodevelopmental disorder caused by mutations in the gene encoding the purine metabolic enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). A series of motor, cognitive and neurobehavioral anomalies characterize this disease phenotype, which is still poorly understood. The clinical manifestations of this syndrome are believed to be the consequences of deficiencies in neurodevelopmental pathways that lead to disordered brain function. We have used microRNA array and gene ontology analysis to evaluate the gene expression of differentiating HPRT-deficient human neuron-like cell lines. We set out to identify dysregulated genes implicated in purine-based cellular functions. Our approach was based on the premise that HPRT deficiency affects preeminently the expression and the function of purine-based molecular complexes, such as guanine nucleotide exchange factors (GEFs) and small GTPases. We found that several microRNAs from the miR-17 family cluster and genes encoding GEF are dysregulated in HPRT deficiency. Most notably, our data show that the expression of the exchange protein activated by cAMP (EPAC) is blunted in HPRT-deficient human neuron-like cell lines and fibroblast cells from LNS patients, and is altered in the cortex, striatum and midbrain of HPRT knockout mouse. We also show a marked impairment in the activation of small GTPase RAP1 in the HPRT-deficient cells, as well as differences in cytoskeleton dynamics that lead to increased motility for HPRT-deficient neuron-like cell lines relative to control. We propose that the alterations in EPAC/RAP1 signaling and cell migration in HPRT deficiency are crucial for neuro-developmental events that may contribute to the neurological dysfunctions in LNS.

Clinical severity in Lesch-Nyhan disease: the role of residual enzyme and compensatory pathways.

Mutations in the HPRT1 gene, which encodes the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGprt), cause Lesch-Nyhan disease (LND) and more mildly affected Lesch-Nyhan variants. Prior studies have suggested a strong correlation between residual hypoxanthine recycling activity and disease severity. However, the relevance of guanine recycling and compensatory changes in the de novo synthesis of purines has received little attention. In the current studies, fibroblast cultures were established for 21 healthy controls and 36 patients with a broad spectrum of disease severity related to HGprt deficiency. We assessed hypoxanthine recycling, guanine recycling, steady-state purine pools, and de novo purine synthesis. There was a strong correlation between disease severity and either hypoxanthine or guanine recycling. Intracellular purines were normal in the HGprt-deficient fibroblasts, but purine wasting was evident as increased purine metabolites excreted from the cells. The normal intracellular purines in the HGprt-deficient fibroblasts were likely due in part to a compensatory increase in purine synthesis, as demonstrated by a significant increase in purinosomes. However, the increase in purine synthesis did not appear to correlate with disease severity. These results refine our understanding of the potential sources of phenotypic heterogeneity in LND and its variants.

Consequences of impaired purine recycling on the proteome in a cellular model of Lesch-Nyhan disease.

The importance of specific pathways of purine metabolism for normal brain function is highlighted by several inherited disorders, such as Lesch-Nyhan disease (LND). In this disorder, deficiency of the purine recycling enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt), causes severe neurological and behavioral abnormalities. Despite many years of research, the mechanisms linking the defect in purine recycling to the neurobehavioral abnormalities remain unclear. In the current studies, an unbiased approach to the identification of potential mechanisms was undertaken by examining changes in protein expression in a model of HGprt deficiency based on the dopaminergic rat PC6-3 line, before and after differentiation with nerve growth factor (NGF). Protein expression profiles of 5 mutant sublines carrying different mutations affecting HGprt enzyme activity were compared to the HGprt-competent parent line using the method of stable isotopic labeling by amino acids in cell culture (SILAC) followed by denaturing gel electrophoresis with liquid chromatography and tandem mass spectrometry (LC-MS/MS) of tryptic digests, and subsequent identification of affected biochemical pathways using the Database for Annotation, Visualization and Integrated Discovery (DAVID) functional annotation chart analysis. The results demonstrate that HGprt deficiency causes broad changes in protein expression that depend on whether the cells are differentiated or not. Several of the pathways identified reflect predictable consequences of defective purine recycling. Other pathways were not anticipated, disclosing previously unknown connections with purine metabolism and novel insights into the pathogenesis of LND.

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.

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.

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.

Genotypic and phenotypic spectrum in attenuated variants of Lesch-Nyhan disease.

Lesch-Nyhan disease and its attenuated variants are caused by deficiency of the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt). All patients exhibit excessive production of uric acid, which increases the risk for nephrolithiasis, renal failure, gouty arthritis and tophi. The mildest phenotype includes only problems related to overproduction of uric acid. The most severe clinical phenotype includes prominent neurological abnormalities and the universal feature is self-injurious behavior. In between the mildest and most severe syndromes is a broad spectrum of phenotypes with varying degrees of neurological, neurocognitive and behavioral abnormalities. The effect of HPRT1 gene mutations on residual HGprt enzyme activity is the most relevant factor contributing to disease phenotype. Attenuated clinical phenotypes are associated with residual enzyme function, whereas the most severe phenotype is usually associated with null activity. In cases of gouty arthritis with urate overproduction, a careful evaluation for motor impairments or neurocognitive abnormalities may help to identify attenuated variants of Lesch-Nyhan disease for better management.

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.

HIV-1 TAT-mediated protein transduction of human HPRT into deficient cells.

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.

Phenotypic variation among seven members of one family with deficiency of hypoxanthine-guanine phosphoribosyltransferase.

We describe a family of seven boys affected by Lesch-Nyhan disease with various phenotypes. Further investigations revealed a mutation c.203T>C in the gene encoding HGprt of all members, with substitution of leucine to proline at residue 68 (p.Leu68Pro). Thus patients from this family display a wide variety of symptoms although sharing the same mutation. Mutant HGprt enzyme was prepared by site-directed mutagenesis and the kinetics of the enzyme revealed that the catalytic activity of the mutant was reduced, in association with marked reductions in the affinity towards phosphoribosylpyrophosphate (PRPP). Its Km for PRPP was increased 215-fold with hypoxanthine as substrate and 40-fold with guanine as substrate with associated reduced catalytic potential. Molecular modeling confirmed that the most prominent defect was the dramatically reduced affinity towards PRPP. Our studies suggest that the p.Leu68Pro mutation has a strong impact on PRPP binding and on stability of the active conformation. This suggests that factors other than HGprt activity per se may influence the phenotype of Lesch-Nyhan patients.

A site-directed chromosomal translocation induced in embryonic stem cells by Cre-loxP recombination.

We have developed a strategy for chromosome engineering in embryonic stem (ES) cells that relies on sequential gene targeting and Cre-loxP site-specific recombination. Gene targeting was first used to integrate loxP sites at the desired positions in the genome. Transient expression of Cre recombinase was then used to mediate the chromosomal rearrangement. A genetic selection relying on reconstruction of a selectable marker from sequences co-integrated with the loxP sites allowed detection of cells containing the Cre-mediated rearrangement. A programmed translocation between the c-myc and immunoglobulin heavy chain genes on chromosomes 15 and 12 was created by this method. This strategy will allow the design of a variety of chromosome rearrangements that can be selected and verified in ES cells or activated in ES cell-derived mice.

Production of a model for Lesch-Nyhan syndrome in hypoxanthine phosphoribosyltransferase-deficient mice.

The inherited disease Lesch-Nyhan syndrome, which is caused by a deficiency of the enzyme hypoxanthine phosphoribosyltransferase (HPRT), is characterized by behavioural alterations, including self-injurious behaviour and mental retardation. Although HPRT-deficient mice have been generated using the embryonic stem cell system, no spontaneous behavioural abnormalities had been reported. We examined whether mice were more tolerant of HPRT deficiency because they were more reliant on adenine phosphoribosyltransferase (APRT) than HPRT for their purine salvage. The administration of an APRT inhibitor to HPRT-deficient mice induced persistent self-injurious behaviour. This combined genetic and biochemical model will facilitate the study of Lesch-Nyhan syndrome and the evaluation of novel therapies.

Direct evidence of allele-specific binding of CTCF and MeCP2 to Tsix in a HPRT-deficient female F1 hybrid mouse cell line.

Mammalian dosage compensation requires silencing of one of the two X chromosomes in females and is controlled by the X inactivation center (Xic). Xic contains many of the regulatory elements for the mutual interplay of X-inactive specific transcript (Xist) and Tsix, the antisense counterpart of Xist. The regulatory elements control X chromosome inactivation (XCI) via the formation of DNA-DNA and DNA-protein complexes with cis- and trans-acting factors. However, the process-dependent regulation of Xist/Tsix by these elements in each XCI process remains largely unknown. In this study, a 6-thioguanine-resistant female F(1) hybrid mouse cell line (designated HOBMSKI2) was constructed from a cross between a female HPRT-deficient transgenic mouse (designated BM3) and a male wild type Mus spretus mouse (designated MS), which enabled the direct discrimination of both allele-specific expression of X-linked genes and allele-specific binding of proteins associated with XCI due to DNA polymorphisms between BM3 and MS. Using this cell line, we found that Tsix on the active X chromosome (Xa) was not expressed in somatic cells despite the fact that CTCF, which ensures Tsix expression in embryonic stem cells, was still bound to the 5' end of Tsix on Xa, implying that CTCF may function differently during each XCI process and its trans-activating activity for Tsix expression may be lost in the maintenance process. In addition, the monoallelic expression of Tsix on Xa was inhibited by epigenetic modification of the chromatin in the maintenance process, which was mediated by protein complexes recruited by MeCP2. The results indicate the value of HOBMSKI2 in directly detecting the allele-specific binding of CTCF and MeCP2 to the 5' end of Tsix. The HOBMSKI2 mouse line is a versatile and useful resource for studying the molecular mechanism of the XCI process.