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.

A new physiological medium uncovers biochemical and cellular alterations in Lesch-Nyhan disease fibroblasts.

BACKGROUND: Lesch-Nyhan disease (LND) is a severe neurological disorder caused by the genetic deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGprt), an enzyme involved in the salvage synthesis of purines. To compensate this deficiency, there is an acceleration of the de novo purine biosynthetic pathway. Most studies have failed to find any consistent abnormalities of purine nucleotides in cultured cells obtained from the patients. Recently, it has been shown that 5-aminoimidazole-4-carboxamide riboside 5'-monophosphate (ZMP), an intermediate of the de novo pathway, accumulates in LND fibroblasts maintained with RPMI containing physiological levels (25 nM) of folic acid (FA), which strongly differs from FA levels of regular cell culture media (2200 nM). However, RPMI and other standard media contain non-physiological levels of many nutrients, having a great impact in cell metabolism that does not precisely recapitulate the in vivo behavior of cells. METHODS: We prepared a new culture medium containing physiological levels of all nutrients, including vitamins (Plasmax-PV), to study the potential alterations of LND fibroblasts that may have been masked by the usage of non-physiological media. We quantified ZMP accumulation under different culture conditions and evaluated the activity of two known ZMP-target proteins (AMPK and ADSL), the mRNA expression of the folate carrier SLC19A1, possible mitochondrial alterations and functional consequences in LND fibroblasts. RESULTS: LND fibroblasts maintained with Plasmax-PV show metabolic adaptations such a higher glycolytic capacity, increased expression of the folate carrier SCL19A1, and functional alterations such a decreased mitochondrial potential and reduced cell migration compared to controls. These alterations can be reverted with high levels of folic acid, suggesting that folic acid supplements might be a potential treatment for LND. CONCLUSIONS: A complete physiological cell culture medium reveals new alterations in Lesch-Nyhan disease. This work emphasizes the importance of using physiological cell culture conditions when studying a metabolic disorder.

Generation of an iPSC line (SDQLCHi030-A) derived from PBMCs of a patient with Lesch-Nyhan syndrome caused by HPRT1 mutation.

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.

CRISPR/Cas9-mediated generation of human embryonic stem cell sub-lines with HPRT1 gene knockout to model Lesch Nyhan disease.

Lesch-Nyhan disease (LND) is a X-linked genetic disease affecting boys characterized by complex neurological and neuropsychiatric symptoms. LND is caused by loss of function mutations in the HPRT1 gene leading to decrease activity of hypoxanthine-guanine phosphoribosyl transferase enzyme (HGPRT) and altered purine salvage pathway (Lesch and Nyhan, 1964). This study describes the generation of isogenic clones with deletions in HPRT1 produced from one male human embryonic stem cell line using CRISPR/Cas9 strategy. Differentiation of these cells into different neuronal subtypes will help elucidating the neurodevelopmental events leading to LND and develop therapeutic strategies for this devastating neurodevelopmental disorder.

[Genetic analysis of a Chinese pedigree with Lesch-Nyhan syndrome].

OBJECTIVE: To explore the genetic etiology for a Chinese pedigree affected with Lesch-Nyhan syndrome. METHODS: Members of the pedigree who had visited the Genetic Counseling Clinic of Linyi People's Hospital on February 10, 2022 were selected as the study subjects. Clinical data and family history of the proband were collected, and trio-whole exome sequencing (trio-WES) was carried out for the proband and his parents. Candidate variants were verified by Sanger sequencing. RESULTS: Trio-WES revealed that both the proband and his cousin brother had harbored a hemizygous c.385-1G>C variant in intron 4 of the HPRT1 gene, which was unreported previously. A heterozygous c.385-1G>C variant of the HPRT1 gene was also found in the proband's mother, grandmother, two aunts, and a female cousin, whilst all phenotypically normal males in his pedigree were found to have a wild type for the locus, which has conformed to an X-linked recessive inheritance. CONCLUSION: The heterozygous c.385-1G>C variant of the HPRT1 gene probably underlay the Lesch-Nyhan syndrome in this pedigree.

Genetic analysis of a Chinese pedigree with Lesch-Nyhan syndrome / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic etiology for a Chinese pedigree affected with Lesch-Nyhan syndrome.@*METHODS@#Members of the pedigree who had visited the Genetic Counseling Clinic of Linyi People's Hospital on February 10, 2022 were selected as the study subjects. Clinical data and family history of the proband were collected, and trio-whole exome sequencing (trio-WES) was carried out for the proband and his parents. Candidate variants were verified by Sanger sequencing.@*RESULTS@#Trio-WES revealed that both the proband and his cousin brother had harbored a hemizygous c.385-1G>C variant in intron 4 of the HPRT1 gene, which was unreported previously. A heterozygous c.385-1G>C variant of the HPRT1 gene was also found in the proband's mother, grandmother, two aunts, and a female cousin, whilst all phenotypically normal males in his pedigree were found to have a wild type for the locus, which has conformed to an X-linked recessive inheritance.@*CONCLUSION@#The heterozygous c.385-1G>C variant of the HPRT1 gene probably underlay the Lesch-Nyhan syndrome in this pedigree.

[Analysis of HPRT1 gene variant and prenatal diagnosis for a Chinese pedigree with Lesch-Nyhan syndrome but no specimen from affected probands].

OBJECTIVE: To carry out genetic testing and prenatal diagnosis for a Chinese pedigree with Lesch-Nyhan syndrome (LNS) but no specimen from the affected probands. METHODS: All affected individuals in this pedigrees were male and had deceased during childhood, with no biological specimen left. Based on their typical neurological dysfunction and tendency for self-mutilation, the diagnosis of LNS was suspected. Sanger sequencing was carried out to detect potential variant of the HPRT1 gene among female members from the pedigree. Following the identification of the pathogenic variant, prenatal diagnosis was provided for a high-risk fetus. RESULTS: The proband's mother and three other females were found to harbor heterozygous c.500_501delGGinsC (p.Arg167fs*23) variant of the HPRT1 gene, which was unreported previously. Prenatal diagnosis showed that the fetus was a male and had inherited the same pathogenic variant. CONCLUSION: The c.500_501delGGinsC variant of the HPRT1 gene probably underlay the LNS in this pedigree. Above finding has provided a basis for prenatal diagnosis and genetic counseling for this pedigree.

Abnormalities of neural stem cells in Lesch-Nyhan disease.

Lesch-Nyhan disease (LND) is a neurodevelopmental disorder caused by variants in the HPRT1 gene, which encodes the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGprt). HGprt deficiency provokes numerous metabolic changes which vary among different cell types, making it unclear which changes are most relevant for abnormal neural development. To begin to elucidate the consequences of HGprt deficiency for developing human neurons, neural stem cells (NSCs) were prepared from 6 induced pluripotent stem cell (iPSC) lines from individuals with LND and compared to 6 normal healthy controls. For all 12 lines, gene expression profiles were determined by RNA-seq and protein expression profiles were determined by shotgun proteomics. The LND lines revealed significant changes in expression of multiple genes and proteins. There was little overlap in findings between iPSCs and NSCs, confirming the impact of HGprt deficiency depends on cell type. For NSCs, gene expression studies pointed towards abnormalities in WNT signaling, which is known to play a role in neural development. Protein expression studies pointed to abnormalities in the mitochondrial F0F1 ATPase, which plays a role in maintaining cellular energy. These studies point to some mechanisms that may be responsible for abnormal neural development in LND.

Ethical implications of early genetic diagnosis in an infant with Lesch-Nyhan syndrome.

Pathogenic variants in HPRT1 lead to deficiency in hypoxanthine-guanine phosphoribosyltransferase and are responsible for a spectrum of disorders. The severe phenotype is termed Lesch-Nyhan syndrome (LNS) and is inherited in an X-linked recessive manner. Most individuals with LNS have profound intellectual and physical disabilities throughout life including self-mutilating behaviors. Here, we present the case of a male infant who was diagnosed with LNS at 3 weeks of age via rapid exome sequencing (ES), which revealed a hemizygous maternally inherited deletion of at least 1.3 Mb of Xq26.3, including exons 2 to 9 of HPRT1. We discuss the critical time points leading to this diagnosis while highlighting his parents' values that guided the decision-making. Genetic testing provided an early diagnosis for this infant that led to important considerations regarding goals of care in addition to raising new ethical concerns. This highlights the important role that early and rapid diagnostic genetic testing can play in helping families make difficult decisions. Additionally, this case highlights the complexity of discussing rare genetic diagnoses with families and facilitating critical discussions to empower the family toward making an informed decision.

HGprt deficiency disrupts dopaminergic circuit development in a genetic mouse model of Lesch-Nyhan disease.

In Lesch-Nyhan disease (LND), deficiency of the purine salvage enzyme hypoxanthine guanine phosphoribosyl transferase (HGprt) leads to a characteristic neurobehavioral phenotype dominated by dystonia, cognitive deficits and incapacitating self-injurious behavior. It has been known for decades that LND is associated with dysfunction of midbrain dopamine neurons, without overt structural brain abnormalities. Emerging post mortem and in vitro evidence supports the hypothesis that the dopaminergic dysfunction in LND is of developmental origin, but specific pathogenic mechanisms have not been revealed. In the current study, HGprt deficiency causes specific neurodevelopmental abnormalities in mice during embryogenesis, particularly affecting proliferation and migration of developing midbrain dopamine (mDA) neurons. In mutant embryos at E14.5, proliferation was increased, accompanied by a decrease in cell cycle exit and the distribution and orientation of dividing cells suggested a premature deviation from their migratory route. An abnormally structured radial glia-like scaffold supporting this mDA neuronal migration might lie at the basis of these abnormalities. Consequently, these abnormalities were associated with an increase in area occupied by TH+ cells and an abnormal mDA subpopulation organization at E18.5. Finally, dopaminergic innervation was disorganized in prefrontal and decreased in HGprt deficient primary motor and somatosensory cortices. These data provide direct in vivo evidence for a neurodevelopmental nature of the brain disorder in LND. Future studies should not only focus the specific molecular mechanisms underlying the reported neurodevelopmental abnormalities, but also on optimal timing of therapeutic interventions to rescue the DA neuron defects, which may also be relevant for other neurodevelopmental disorders.

Congenital anomalies-associated Riga-Fede disease as an early manifestation of Lesch-Nyhan syndrome: rare entities in the same pediatric patient-a case report.

BACKGROUND: Riga-Fede disease is a rare begnin disorder of the oral tissues, it can be associated with congenital anomalies and neurological disturbances. Lesch-Nyhan syndrome is a rare X-linked recessive disorder characterized by neurological and behavioral manifestations. A patient can rarely be diagnosed with both diseases in a lifetime. Therefore, reporting manifestations from such disorders is important to avoid misdiagnosis and help in timely intervention. CASE PRESENTATION: This case report presents an 8-months-old male infant with traumatic oral ulcers from deciduous teeth. A diagnosis of Riga-Fede disease was made. Teeth grinding was performed and the oral lesions were healed. At the age of 2.5 years, the patient presented with neurological manifestations as well as facial tissue and premature teeth loss from self mutilation. Genetic sequencing revealed a variant of uncertain significance in the Hypoxanthine Phosphoribosyltransferase 1 gene. He was diagnosed with Lesch-Nyhan syndrome. Cleft palate, ventricular septal defect, congenitally undescended testis and ectopic left iliac kidney were also reported. The patient was scheduled on psychiatric treatment and after about six months of follow-up, both the behavioral and neurological symptoms were improved. CONCLUSIONS: Riga-Fede disease can be an early manifestation of Lesch-Nyhan syndrome. To the best of our knowledge, this is the first reported case with the incidence of all the mentioned entities in one pediatric patient.

PUS7 deficiency in human patients causes profound neurodevelopmental phenotype by dysregulating protein translation.

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 syndrome due to a splice-site mutation in a 14-month-old boy presenting as acute renal failure.

Lesch-Nyhan disease (LND) is a rare X-linked recessive inherited disorder caused by mutations in HPRT1 gene resulting in deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). LND is characterized by hyperuricemia and a spectrum of neurological and behavioral manifestations. We describe a rare case of a 14-month-old boy presenting with acute renal failure and hyperuricemia. The patient exhibited all features of LNS apart from self-injurious behavior. The enzymatic analysis demonstrated total inactivity of the HPRT, and the molecular analysis revealed a splice-site mutation in intron 3 leading to exon 4 exclusion. This splice-site mutation has been previously reported only twice.

Induced pluripotent stem cells from subjects with Lesch-Nyhan disease.

Lesch-Nyhan disease (LND) is an inherited disorder caused by pathogenic variants in the HPRT1 gene, which encodes the purine recycling enzyme hypoxanthine-guanine phosphoribosyltransferase (HGprt). We generated 6 induced pluripotent stem cell (iPSC) lines from 3 individuals with LND, along with 6 control lines from 3 normal individuals. All 12 lines had the characteristics of pluripotent stem cells, as assessed by immunostaining for pluripotency markers, expression of pluripotency genes, and differentiation into the 3 primary germ cell layers. Gene expression profiling with RNAseq demonstrated significant heterogeneity among the lines. Despite this heterogeneity, several anticipated abnormalities were readily detectable across all LND lines, including reduced HPRT1 mRNA. Several unexpected abnormalities were also consistently detectable across the LND lines, including decreases in FAR2P1 and increases in RNF39. Shotgun proteomics also demonstrated several expected abnormalities in the LND lines, such as absence of HGprt protein. The proteomics study also revealed several unexpected abnormalities across the LND lines, including increases in GNAO1 decreases in NSE4A. There was a good but partial correlation between abnormalities revealed by the RNAseq and proteomics methods. Finally, functional studies demonstrated LND lines had no HGprt enzyme activity and resistance to the toxic pro-drug 6-thioguanine. Intracellular purines in the LND lines were normal, but they did not recycle hypoxanthine. These cells provide a novel resource to reveal insights into the relevance of heterogeneity among iPSC lines and applications for modeling LND.

Clinical and molecular findings in a Turkish family with an ultra-rare condition, ELP2-related neurodevelopmental disorder.

Elongator is a multi-subunit protein complex bearing six different protein subunits, Elp1 to -6, that are highly conserved among eukaryotes. Elp2 is the second major subunit of Elongator and, together with Elp1 and Elp3, form the catalytic core of this essential complex. Pathogenic variants that affect the structure and function of the Elongator complex may cause neurodevelopmental disorders. Here, we report on a new family with three children affected with a severe form of intellectual disability along with spastic tetraparesis, choreoathetosis, and self injury. Molecular genetic analyses reveal a homozygous missense variant in the ELP2 gene (NM_018255.4 (ELP2): c.1385G > A (p.Arg462Gln)), while in silico studies suggest a loss of electrostatic interactions that may contribute to the overall stability of the encoded protein. We also include a comparison of the patients with ELP2-related neurodevelopmental disorder to those previously reported in the literature. Apart from being affected with intellectual disability, we have extremely limited clinical knowledge about patients harboring ELP2 variants. Besides providing support to the causal role of p.Arg462Gln in ELP2-related neurodevelopmental disorder, we add self-injurious behavior to the clinical phenotypic repertoire of the disease.

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.

Delayed emergence from propofol anesthesia in a patient with Lesch-Nyhan syndrome: A case report.

RATIONALE: Lesch-Nyhan syndrome (LNS) is an X-linked recessive disorder presenting with uric acid overproduction, neurocognitive disability, and behavioral disturbances. Inhalational anesthesia has been frequently used in LNS patients undergoing surgery. Characteristic compulsive self-injurious behavior and high risk of emesis may hinder inhalational induction. Propofol may be beneficial for these patients because of its easy and rapid titration for anesthetic depth during induction, early recovery from anesthesia, and antiemetic effect as well as uricosuric effect. PATIENT CONCERNS: A 16-year-old male adolescent was scheduled for percutaneous nephrolithotomy. He exhibited poorly controlled muscle, self-injurious behaviors and intellectual disability. DIAGNOSIS: The patient presented with neurodevelopmental delay in the first year of life, and was diagnosed with LNS, with a substitution of phenylalanine to leucine in hypoxanthine-guanine phosphoribosyltransferase (HPRT) 1 gene on the X-chromosome at 3 years of age. INTERVENTIONS: Total intravenous anesthesia was used for induction and maintenance of anesthesia with propofol and remifentanil using target-controlled infusion. OUTCOMES: Time to recovery of consciousness was prolonged after uneventful surgery. Serum uric acid levels gradually increased during postoperative period. LESSONS: Propofol anesthesia using target-controlled infusion does not provide significant clinical advantages in rapid emergence from anesthesia and management of hyperuricemia in LNS patients undergoing urological surgery.

Lesch-Nyhan disease: I. Construction of expression vectors for hypoxanthine-guanine phosphoribosyltransferase (HGprt) enzyme and amyloid precursor protein (APP).

Lesch-Nyhan disease (LND) is a rare X-linked inherited neurogenetic disorder of purine metabolism in which the enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt) is defective. Despite having been characterized over 50 years ago, it remains unclear precisely how deficits in HGprt enzyme activity can lead to the neurological syndrome, especially the self-injury of LND. Several studies have proposed different hypotheses regarding the etiology of this disease, and several treatments have been tried in patients. However, up to now, there is no satisfactory explanation of the disease and for many LND patients, efficacious treatment for persistent self-injurious behavior remains unreachable. A role for epistasis between mutated hypoxanthine phosphoribosyltransferase 1 (HPRT1) and amyloid precursor protein (APP) genes has been recently suggested. This finding may provide new directions not only for investigating the role of APP in neuropathology associated with HGprt-deficiency in LND but also for the research in neurodevelopmental and neurodegenerative disorders in which the APP gene is involved in the pathogenesis of diseases and may pave the way for new strategies applicable to rational antisense drugs design. It is therefore necessary to study the HGprt enzyme and APP using expression vectors for exploring their impacts on LND as well as other human diseases, especially the ones related to APP such as Alzheimer's disease in which the physiologic function and the structure of the entire APP remain largely unclear until now. For such a purpose, we report here the construction of expression vectors as the first step (Part I) of our investigation.

Late diagnosis of Lesch-Nyhan disease complicated with end-stage renal disease and tophi burst: a case report.

Background: Lesch-Nyhan disease (LND) is a rare X-linked recessive inborn error of purine metabolism. Late diagnosis of LND may cause significant morbidity. LND cases have never been reported in Indonesia.Case report: A 15-year-old male who had been diagnosed with cerebral palsy was referred to our hospital due to renal failure requiring emergency dialysis. The patient presented with three classic manifestations of LND: increased uric acid levels, neurological disorders, and self-injurious behaviors. LND was suspected because of an abscess-like lump on the left ankle that was confirmed to be a tophus, which had burst and discharged thick masses containing blood, debris, and white crystal materials. The diagnosis of LND was confirmed by the presence of a deletion to exon 1 of the HPRT1 gene. The patient received oral allopurinol daily and treatment for end-stage renal disease (ESRD), which included regular dialysis and subcutaneous administration of erythropoietin. At a 2-month follow-up, he improved clinically with a 71% decrease in uric acid levels after regular dialysis and allopurinol treatment.Conclusion: In developed countries, LND can be diagnosed as early as 3 days after birth. However, diagnosis in the present case was delayed due to the rarity of the disease and the limited number of facilities in Indonesia that offer genetic counseling. Late diagnosis of LND leads to ESRD and irreversible abnormalities. This is the first case of LND presenting with a unique clinical presentation of tophus burst reported in Indonesia.