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.

Establishment and characterization of Lesch-Nyhan syndrome rabbit model.

Lesch-Nyhan syndrome (LNS) is a congenital defect disease that results in defective purine metabolism. It is caused by pathogenic variants of the HPRT gene. Its clinical symptoms mainly include high uric acid levels, gout, and kidney stones and damage. The mechanism of LNS has not been fully elucidated, and no cure exists. Animal models have always played an important role in exploring causative mechanisms and new therapies. This study combined CRISPR/Cas9 and microinjection to knock out the HPRT gene to create an LNS rabbit model. A sgRNA targeting exon 3 of HPRT gene was designed. Subsequently, Cas9 mRNA and sgRNA were injected into rabbit zygotes, and injected embryos were transferred to the uterus. The genotype and phenotype of rabbits were analyzed after birth. Four infant rabbits (named R1, R2, R3 and R4), which showed varying levels of gene modification, were born. The gene-editing efficiency was 100%. No wild-type sequences at the target HPRT gene were detected in R4 rabbit. Next, 6-thioguanine drug testing confirmed that HPRT enzymatic activity was deficient in R4 infant rabbit. HE staining revealed kidney abnormalities in all infant rabbits. Overall, an sgRNA capable of knocking out the HPRT gene in rabbits was successfully designed, and HPRT gene-modified rabbits were successfully constructed by using CRISPR/Cas9 technology and microinjection. This study provides a new nonrodent animal model for studying LNS syndrome.

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.

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.

Lesch-Nyhan disease with no HPRT1 gene mutation? / ¿Síndrome de Lesch-Nyhan sin mutación genética HPRT1?

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De la mutación al fenotipo; variabilidad clínica en la enfermedad de Lesch-Nyhan. El papel de la epigenética / From genotype to phenotype; clinical variability in Lesch-Nyhan disease. The role of epigenetics

La enfermedad de Lesch-Nyhan es una enfermedad genética considerada dentro de las enfermedades raras, caracterizada por un déficit en la función de la enzima hipoxantina-guanina fosforribosil transferasa (HPRT). Los pacientes afectados presentan hiperuricemia, trastornos motores, retraso mental y, en los casos más graves, automutilaciones. Las manifestaciones clínicas dependen de la actividad enzimática de la HPRT, que clásicamente se vincula al tipo de alteración en el gen HPRT, habiéndose encontrado más de 400 mutaciones. En la actualidad uno de los aspectos controvertidos de la enfermedad es la relación existente entre el genotipo y el fenotipo, habiéndose descrito casos en ausencia de mutación (como el paciente que presentamos), así como familias que, a pesar de compartir un mismo defecto genético, muestran afectaciones con distinta gravedad. Los procesos epigenéticos, que modifican la expresión genética sin alterar la secuencia del ácido desoxirribonucleico (ADN), podrían explicar la variabilidad clínica observada en esta enfermedad (AU)

Lesch-Nyhan disease is a rare genetic disease characterized by a deficiency in the function of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Patients affected by this disease experience hyperuricemia, motor disorders, mental retardation and, in the most severe cases, self-mutilation. Its clinical manifestations depend on the enzymatic activity of HGPRT, which is classically linked to the type of alteration in the HGPRT gene. More than 400 mutations of this gene have been found. At present, one of the controversial aspects of the disease is the relationship between the genotype and phenotype; cases have been described lacking a mutation, such as the patient presented in this article, as well as families who despite sharing the same genetic defect show disorders with differing severity. Epigenetic processes, which modify the genetic expression without changing the sequence of the deoxyribonucleic acid (DNA), could explain the clinical variability observed in this disease (AU)

Fenotipo variante del síndrome de Lesch-Nyhan / Variant phenotype of Lesch-Nyhan syndrome

Fundamento y objetivo: El síndrome de Lesch-Nyhan (SLN) y sus «variantes» se deben a mutaciones del gen HPRT1, que ocasionan una deficiencia de la actividad enzimática hipoxantina-guanina-fosforribosiltransferasa (HPRT). El fenotipo variante se ha asociado a mutaciones puntuales y a deficiencia parcial. Presentamos un paciente con fenotipo variante pero con una mutación que afectaba al procesamiento del ácido ribonucleico (ARN). Nos preguntamos si el fenotipo podía deberse a que se procesara normalmente una pequeña cantidad de ARN mensajero (ARNm) con cierta actividad residual HPRT. Paciente y método: Analizamos la presencia y la cantidad de ARNm normal de la enzima HPRT en este paciente y en 2 enfermos con las características del SLN, y mutaciones que afectaban al procesamiento. Resultados: Los 3 enfermos tenían una pequeña cantidad de ARNm de HPRT normalmente procesado. Conclusiones: Este enfermo constituye la primera descripción de un defecto genético importante con expresión fenotípica variante de la deficiencia de HPRT. Nuestros resultados ponen en duda que un ARNm normalmente procesado sea la causa de un fenotipo parcial de la deficiencia de HPRT (AU)

Background and objective: Lesch-Nyhan syndrome (LNS) and LNS variants are due to mutations in the HPRT1 gene causing HPRT enzymatic activity deficiency. We report a patient presenting a variant phenotype and a major genetic defect. The mutation has been previously reported as always associated with complete Lesch-Nyhan phenotype. Patient and methods: We analyzed the presence of complete HPRT mRNA in this patient, in two patients with the complete Lesch Nyhan syndrome phenotype, and in control subjects. Results: We found a minor amount of normal HPRT mRNA in the present patient but also in the two patients with splice mutation and the complete Lesch Nyhan syndrome phenotype. Conclusions: To our knowledge, this patient is the first report of a major genetic defect, with no detectable enzymatic activity, and a partial HPRT deficiency phenotype. Our results question the hypothesis of a normally spliced HPRT cDNA as the sole cause of the patient partial phenotype (AU)

Uma nova estratégia para o diagnóstico de mulheres portadoras de mutações que causam a síndrome de LESCH-NYHAN

A síndrome de Lesch-Nyhan é uma entidade clínica que apresenta um padrão de herança recessivo ligado ao cromossomo X. Pertence ao grupo dos erros inatos do metabolismo e é originada por uma deficiência absoluta da enzima hipoxantina-guanina fosforibosiltransferase (HPRT1; EC 2.4.2.8). Mulheres heterozigotas para mutações HPRT1 que causam a síndrome de Lesch-Nyhan podem ser detectadas mediante análises moleculares de híbridos celulares somáticos derivados da fusão de Iinfócitos de sangue periférico e células Hprt-negativas provenientes de roedor com a subseqüente seleção em meio de cultura contendo hipoxantina, aminopterina e timidina (HAT). A origem parental do cromossomo X contendo o alelo HPRT1 normal em linhagens celulares híbridas HPRT1 + pode ser determinada por haplótipos moleculares demonstrados por marcadores altamente polimórficos ligados ao cromossomo X. Esta metodologia foi utilizada para estudar uma possível heterozigota cujo cromossomo X paterno ativo esta sempre presente nas linhagens celulares derivadas dela. Contrariamente, o cromossomo X materno esteve sistematicamente ausente na maioria dos híbridos, e quando presente, estava inativo coexistindo com um X paterno ativo. Sub-clones sem o X paterno ativo e contendo o X materno inativo, não apresentaram atividade HPRT1 quando tratados com 5-aza-citidina. A ausência de transcrito do HPRT1 após o tratamento foi comprovada mediante RT-PCR. Estes resultados claramente demonstraram que a probanda é portadora de uma mutação responsável pela deficiência de HPRT1

Lesch-Nyhan syndrome is a recessive X-Iinked metabolic disorder resulting from absolute deficiency of the enzyme hypoxanthine/guanine phosphoribosy/transferase (HPRT1; EC 2.4.2.8). Heterozygous carriers of HPRT1 mutations responsible for Lesch-Nyhan syndrome can be detected by analysis of somatic cell hybrids derived from peripheral blood Iymphocytes and Hprt-negative cells of rodent origin followed by selection in culture medium containing hypoxanthine, aminopterine, and thymidine (HAT). The parental origin of the X chromosome containing the normal HPRT1 allele in HPRT1 + hybrid cell lines can be determined by molecular haplotyping attributable to highly polymorphic X-Iinked markers. We used this procedure to study a presumed carrier whose paternal active X chromosome always segregated in the cell hybrids derived from her. Conversely, her maternal X chromosome was systematically absent in most of cell hybrids, or when present, it was inactive and coexisted with an active, paternal X chromosome. Sub-clones lacking the paternal, active X chromosome and containing the X inactive maternal chromosome were unable to reactivate the HPRT1 locus following treatment with 5-aza-eytidine. HPRT1 transcripts were found to be absent with RT -PCR. These results c1early demonstrated that the proband was a heterozygous carrier of a mutation responsible for HPRT1 deficiency

Identification of a new lesch syndrome mutation (HPRT BRASIL) and analysis of pontentially heterozygous females

The mutation in the hypoxanthine-guanine phosphoribosyltransfere (HPRT) gene has been determined in two brothers affected with Lesch-Nyhan syndrome. Female members of the family who are at risk for being heterozygous carriers of the HPRT mutation were also studied to determine whether they carry the mutation. DNA sequencing revealed that the boys' mother heterozygous for the mutation in her somatic cells, but that three maternal aunts are not heterozygous. Such carrier information is important for the future pregnancy plans of at-risk females. The mutation, an A_T transversion at cDNA base 590 (590 A_T), results in an amino acid change of glutamic acid to valine at codon 197, and has not been reported previously in a Lesch-Nyhan syndrome male. This mutation is designated HPRT.

Síndrome de Lesch-Nyhan incompleto : comunicación del primer caso argentino / Incomplete Lesch-Nyhan syndrome : communication of first case argentinian

El síndrome de Lesch-Nyhan es una infrecuente gota hereditaria vinculada al déficit virtualmente completo de la enzima hipoxantina guanina fosforribosil transferasa (HGFT) existiendo ocasionalmente déficit parciales, que se diferencian de las formas completas por presentar manifestaciones neuropsiquiátricas menores. Es nuestro objetivo presentar al que en nuestro conocimiento es el primer paciente argentino portador del síndrome de Lesh-Nyhan incompleto o de Kelley-Seegmiller. Paciente varón de 38 años con historia familiar de gota e insuficiencia renal que desarrolla una severa artritis gotosa tofácea de rápida evolución y déficit intelectual. Los análisis de laboratorio de rutina mostraron una marcada hiperuricemia y excesiva excreción de ácido úrico con una función renal conservada. La determinación de la actividad enzimática de la HGFT se halló francamente disminuida. (AU)

Síndrome de Lesch-Nyhan incompleto : comunicación del primer caso argentino / Incomplete Lesch-Nyhan syndrome : communication of first case argentinian

El síndrome de Lesch-Nyhan es una infrecuente gota hereditaria vinculada al déficit virtualmente completo de la enzima hipoxantina guanina fosforribosil transferasa (HGFT) existiendo ocasionalmente déficit parciales, que se diferencian de las formas completas por presentar manifestaciones neuropsiquiátricas menores. Es nuestro objetivo presentar al que en nuestro conocimiento es el primer paciente argentino portador del síndrome de Lesh-Nyhan incompleto o de Kelley-Seegmiller. Paciente varón de 38 años con historia familiar de gota e insuficiencia renal que desarrolla una severa artritis gotosa tofácea de rápida evolución y déficit intelectual. Los análisis de laboratorio de rutina mostraron una marcada hiperuricemia y excesiva excreción de ácido úrico con una función renal conservada. La determinación de la actividad enzimática de la HGFT se halló francamente disminuida.

Aspectos clínicos y bioquímicos del síndrome de Lesch-Nyhan

El síndrome de Lesch-Nyhan es una enfermedad genética ligada al cromosoma X, originada por un defecto en el gen que codifica la hipoxantia guanina fosforribosiltransferasa (HGPRT). Esta enzima participa en la recuperación de la guanina e hipoxantina. La deficiencia enzemática conlleva una acumulación exagerada del ácido úrico. La deficiencia total o casi total de la enzima, produce el síndrome de Lesch-Nyhan, el cual se caracteriza por hiperruricemia, hiperaciduria, coreoatetosis, hiperreflexia, retardo mental y autoagresividad. La deficiencia parcial de la enzima ocasiona artritis gotosa y nefrolitiasis sin daño neurológico.

Enfermedad de Lesch-Nyhan / Lesch-Nyhan disease

Se presenta un caso de Enfermedad de Lesch-Nyhan típico, cuyo diagnóstico fue confirmado a través de técnicas de crecimiento celular diferencial. Esta técnica permitió también establecer la condición de portadora en la madre y una de las tías en la que se realizó el diagnóstico prenatal de un nuevo caso. Se revisan algunos aspectos clínicos, metabólicos y terapéuticos de interés, enfatizando la importancia de los estudios diagnósticos específicos para el consejo genético adecuado de familias con esta rara pero grave enfermedad