Disorders of purine biosynthesis metabolism.

Purines are essential molecules that are components of vital biomolecules, such as nucleic acids, coenzymes, signaling molecules, as well as energy transfer molecules. The de novo biosynthesis pathway starts from phosphoribosylpyrophosphate (PRPP) and eventually leads to the synthesis of inosine monophosphate (IMP) by means of 10 sequential steps catalyzed by six different enzymes, three of which are bi-or tri-functional in nature. IMP is then converted into guanosine monophosphate (GMP) or adenosine monophosphate (AMP), which are further phosphorylated into nucleoside di- or tri-phosphates, such as GDP, GTP, ADP and ATP. This review provides an overview of inborn errors of metabolism pertaining to purine synthesis in humans, including either phosphoribosylpyrophosphate synthetase (PRS) overactivity or deficiency, as well as adenylosuccinate lyase (ADSL), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), and adenylosuccinate synthetase (ADSS) deficiencies. ITPase deficiency is being described as well. The clinical spectrum of these disorders is broad, including neurological impairment, such as psychomotor retardation, epilepsy, hypotonia, or microcephaly; sensory involvement, such as deafness and visual disturbances; multiple malformations, as well as muscle presentations or consequences of hyperuricemia, such as gouty arthritis or kidney stones. Clinical signs are often nonspecific and, thus, overlooked. It is to be hoped that this is likely to be gradually overcome by using sensitive biochemical investigations and next-generation sequencing technologies.

Inborn errors of purine and pyrimidine metabolism: A guide to diagnosis.

Inborn errors of purine and pyrimidine (P/P) metabolism are under-reported and rarely mentioned in the general literature or in clinical practice, as well as in reviews dedicated to other inborn errors of metabolism (IEMs). However, their diagnosis is important because genetic counseling can be provided and, in some cases, specific treatment exists that may slow or even reverse clinical signs. The purpose of this review is to provide a practical guideline on the suspicion and investigation of inborn errors of P/P metabolism. Failure of a physician to recognize the presence of these disorders may be devastating for affected infants and children because of its permanent effects in the patient, and for their parents because of implications for future offspring. Diagnosis is crucial because genetic counseling can be provided and, in some cases, specific treatment can be offered that may slow or even reverse clinical symptoms. This review highlights the risk factors in the history, the important examination findings, and the appropriate biochemical investigation of the child. Herein we describe the approach to the diagnosis of P/P disorders and emphasize clinical situations in which physicians should consider these diseases as diagnostic possibilities.

Clinical, biochemical, mitochondrial, and metabolomic aspects of methylmalonate semialdehyde dehydrogenase deficiency: Report of a fifth case.

Methylmalonate semialdehyde dehydrogenase deficiency (MMSDD; MIM 614105) is a rare autosomal recessive defect of valine and pyrimidine catabolism. Four prior MMSDD cases are published. We present a fifth case, along with functional and metabolomic analysis. The patient, born to non-consanguineous parents of East African origin, was admitted at two weeks of age for failure to thrive. She was nondysmorphic, had a normal brain MRI, and showed mild hypotonia. Gastroesophageal reflux occurred with feeding. Urine organic acid assessment identified excess 3-hydroxyisobutyrate and 3-hydroxypropionate, while urine amino acid analysis identified elevated concentrations of ß-aminoisobutyrate and ß-alanine. Plasma amino acids showed an elevated concentration of ß-aminoisobutyrate with undetectable ß-alanine. ALDH6A1 gene sequencing identified a homozygous variant of uncertain significance, c.1261C > T (p.Pro421Ser). Management with valine restriction led to reduced concentration of abnormal analytes in blood and urine, improved growth, and reduced gastroesophageal reflux. Western blotting of patient fibroblast extracts demonstrated a large reduction of methylmalonate semialdehyde dehydrogenase (MMSD) protein. Patient cells displayed compromised mitochondrial function with increased superoxide production, reduced oxygen consumption, and reduced ATP production. Metabolomic profiles from patient fibroblasts demonstrated over-representation of fatty acids and fatty acylcarnitines, presumably due to methylmalonate semialdehyde shunting to ß-alanine and subsequently to malonyl-CoA with ensuing increase of fatty acid synthesis. Previously reported cases of MMSDD have shown variable clinical presentation. Our case continues the trend as clinical phenotypes diverge from prior cases. Recognition of mitochondrial dysfunction and novel metabolites in this patient provide the opportunity to assess future patients for secondary changes that may influence clinical outcome.

Novel PRPS1 gain-of-function mutation in a patient with congenital hyperuricemia and facial anomalies.

Phosphoribosylpyrophosphate synthetase (PRPPS) superactivity (OMIM 300661) is a rare inborn error of purine metabolism that is caused by gain-of-function mutations in the X-chromosomal gene PRPS1 (Xq22.3). Clinical characteristics include congenital hyperuricemia and hyperuricosuria, gouty arthritis, urolithiasis, developmental delay, hypotonia, recurrent infections, short stature, and hearing loss. Only eight families with PRPPS superactivity and PRPS1 gain-of-function mutations have been reported to date. We report on a 7-year-old boy with congenital hyperuricemia, urolithiasis, developmental delay, short stature, hypospadias, and facial dysmorphisms. His mother also suffered from hyperuricemia that was diagnosed at age 13 years. A novel PRPS1 missense mutation (c.573G>C, p.[Leu191Phe]) was detected in the proband and his mother. Enzyme activity analysis confirmed superactivity of PRPP synthetase. Analysis of the crystal structure of human PRPPS suggests that the Leu191Phe mutation affects the architecture of both allosteric sites, thereby preventing the allosteric inhibition of the enzyme. The family reported here broadens the clinical spectrum of PRPPS superactivity and indicates that this rare metabolic disorder might be associated with a recognizable facial gestalt.

Mild orotic aciduria in UMPS heterozygotes: a metabolic finding without clinical consequences.

BACKGROUND: Elevated urinary excretion of orotic acid is associated with treatable disorders of the urea cycle and pyrimidine metabolism. Establishing the correct and timely diagnosis in a patient with orotic aciduria is key to effective treatment. Uridine monophosphate synthase is involved in de novo pyrimidine synthesis. Uridine monophosphate synthase deficiency (or hereditary orotic aciduria), due to biallelic mutations in UMPS, is a rare condition presenting with megaloblastic anemia in the first months of life. If not treated with the pyrimidine precursor uridine, neutropenia, failure to thrive, growth retardation, developmental delay, and intellectual disability may ensue. METHODS AND RESULTS: We identified mild and isolated orotic aciduria in 11 unrelated individuals with diverse clinical signs and symptoms, the most common denominator being intellectual disability/developmental delay. Of note, none had blood count abnormalities, relevant hyperammonemia or altered plasma amino acid profile. All individuals were found to have heterozygous alterations in UMPS. Four of these variants were predicted to be null alleles with complete loss of function. The remaining variants were missense changes and predicted to be damaging to the normal encoded protein. Interestingly, family screening revealed heterozygous UMPS variants in combination with mild orotic aciduria in 19 clinically asymptomatic family members. CONCLUSIONS: We therefore conclude that heterozygous UMPS-mutations can lead to mild and isolated orotic aciduria without clinical consequence. Partial UMPS-deficiency should be included in the differential diagnosis of mild orotic aciduria. The discovery of heterozygotes manifesting clinical symptoms such as hypotonia and developmental delay are likely due to ascertainment bias.

Development and validation of a 2nd tier test for identification of purine nucleoside phosphorylase deficiency patients during expanded newborn screening by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Purine nucleoside phosphorylase (PNP) deficiency has been recently introduced in the newborn screening program in Tuscany. In order to improve the PNP screening efficiency, we developed a 2nd tier test to quantify PNP primary markers deoxyguanosine (dGuo) and deoxyinosine (dIno). METHODS: Dried blood spots (DBS) samples were extracted with 200 µL of methanol and 100 µL of water (by two steps). Internal standards were added at a final concentration of 10 µmol/L. After extraction, samples were analysed by LC-MS/MS. The chromatographic run was performed in gradient mode by using a Synergi Fusion column. RESULTS: The assay was linear over a concentration range of 0.05-50 µmol/L (R2>0.999) for dGuo and 0.5-50 µmol/L (R2>0.998) for dIno. Intra- and interassay imprecision (mean CVs) for dIno and dGuo ranged from 2.9% to 12%. Limit of quantitaion (LOQ) were found to be 0.05 µmol/L and 0.5 µmol/L for dGuo and dIno, respectively. The reference ranges, obtained by measuring dGuo and dIno concentrations on DBS, were close to zero for both biomarkers. Moreover, DBS samples from seven patients with confirmed PNP were retrospectively evaluated and correctly identified. CONCLUSIONS: The LC-MS/MS method can reliably measure dIno and dGuo in DBS for the diagnosis of PNP. Validation data confirm the present method is characterised by good reproducibility, accuracy and imprecision for the quantitation of dIno and dGuo. The assay also appears suitable for use in monitoring treatment of PNP patients.

Neurological presentations of inborn errors of purine and pyrimidine metabolism.

Purines and pyrimidines are essential components as they are the building blocks of vital molecules, such as nucleic acids, coenzymes, signalling molecules, as well as energy transfer molecules. Purine and pyrimidine metabolism defects are characterised by abnormal concentrations of purines, pyrimidines and/or their metabolites in cells or body fluids. This phenomenon is due to a decreased or an increased activity of enzymes involved in this metabolism and has been reported in humans for over 60 years. This review provides an overview of neurological presentations of inborn errors of purine and pyrimidine metabolism. These conditions can lead to psychomotor retardation, epilepsy, hypotonia, or microcephaly; sensory involvement, such as deafness and visual disturbances; multiple malformations, as well as muscular symptoms. Clinical signs are often nonspecific and thus overlooked, but some diseases are treatable and early diagnosis may improve the child's future. Although these metabolic hereditary diseases are rare, they are most probably under-diagnosed. When confronted with suggestive clinical or laboratory signs, clinicians should prescribe genetic testing in association with a biochemical screening including thorough purine and pyrimidine metabolites analysis and/or specific enzyme evaluation. This is most likely going to increase the number of confirmed patients.

Genetic and metabolomic analysis of AdeD and AdeI mutants of de novo purine biosynthesis: cellular models of de novo purine biosynthesis deficiency disorders.

Purines are molecules essential for many cell processes, including RNA and DNA synthesis, regulation of enzyme activity, protein synthesis and function, energy metabolism and transfer, essential coenzyme function, and cell signaling. Purines are produced via the de novo purine biosynthesis pathway. Mutations in purine biosynthetic genes, for example phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS, E.C. 6.3.2.6/E.C. 4.1.1.21), can lead to developmental anomalies in lower vertebrates. Alterations in PAICS expression in humans have been associated with various types of cancer. Mutations in adenylosuccinate lyase (ADSL, E.C. 4.3.2.2) or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC, E.C. 2.1.2.3/E.C. 3.5.4.10) lead to inborn errors of metabolism with a range of clinical symptoms, including developmental delay, severe neurological symptoms, and autistic features. The pathogenetic mechanism is unknown for these conditions, and no effective treatments exist. The study of cells carrying mutations in the various de novo purine biosynthesis pathway genes provides one approach to analysis of purine disorders. Here we report the characterization of AdeD Chinese hamster ovary (CHO) cells, which carry genetic mutations encoding p.E177K and p.W363* variants of PAICS. Both mutations impact PAICS structure and completely abolish its biosynthesis. Additionally, we describe a sensitive and rapid analytical method for detection of purine de novo biosynthesis intermediates based on high performance liquid chromatography with electrochemical detection. Using this technique we detected accumulation of AIR in AdeD cells. In AdeI cells, mutant for the ADSL gene, we detected accumulation of SAICAR and SAMP and, somewhat unexpectedly, accumulation of AIR. This method has great potential for metabolite profiling of de novo purine biosynthesis pathway mutants, identification of novel genetic defects of purine metabolism in humans, and elucidating the regulation of this critical metabolic pathway.

Urate nephropathy associated with impaired kinetic properties of hypoxanthine phosphoribosyl transferase in a 45-day-old infant.

We report a 45-day-old male infant who presented with anuric renal failure and fluid overload due to urate nephropathy consequent upon hyperuricemia with hyperuricosuria. His maternal uncle had undergone renal transplantation for chronic renal failure secondary to uric acid nephrolithiasis. The levels of hypoxanthine phosphoribosyl transferase (HPRT) and adenine phosphoribosyl transferase activity in the baby were found to be quantitatively normal. However, when the HPRT activity was measured at low substrate concentrations [phosphoribosyl pyrophosphate (PRPP) and hypoxanthine] and compared with usual assay conditions, the HPRT activity at lower PRPP was less in the propositus, suggesting altered enzyme kinetics. Apparent K (m(PRPP)) and V (max), but not K (m(hypoxanthine)), were then found to be higher in the propositus than the control range. This is the first case of urate nephropathy secondary to altered enzyme kinetics presenting as early as 45 days. Uric acid nephropathy should be considered in the differential diagnosis of unexplained acute kidney injury in infants. In such cases, quantitative tests for HPRT enzyme activity may not be sufficient and altered enzyme kinetics should also be investigated.

Mutations associated with functional disorder of xanthine oxidoreductase and hereditary xanthinuria in humans.

Xanthine oxidoreductase (XOR) catalyzes the conversion of hypoxanthine to xanthine and xanthine to uric acid with concomitant reduction of either NAD+ or O(2). The enzyme is a target of drugs to treat hyperuricemia, gout and reactive oxygen-related diseases. Human diseases associated with genetically determined dysfunction of XOR are termed xanthinuria, because of the excretion of xanthine in urine. Xanthinuria is classified into two subtypes, type I and type II. Type I xanthinuria involves XOR deficiency due to genetic defect of XOR, whereas type II xanthinuria involves dual deficiency of XOR and aldehyde oxidase (AO, a molybdoflavo enzyme similar to XOR) due to genetic defect in the molybdenum cofactor sulfurase. Molybdenum cofactor deficiency is associated with triple deficiency of XOR, AO and sulfite oxidase, due to defective synthesis of molybdopterin, which is a precursor of molybdenum cofactor for all three enzymes. The present review focuses on mutation or chemical modification studies of mammalian XOR, as well as on XOR mutations identified in humans, aimed at understanding the reaction mechanism of XOR and the relevance of mutated XORs as models to estimate the possible side effects of clinical application of XOR inhibitors.

[Asymptomatic classical hereditary xanthinuria type 1].

We report on a girl who was diagnosed with classical hereditary xanthinuria due to an incidental finding of extremely low Levels of uric acid in the blood. The girl is compLetely asymptomatic. Hereditary xanthinuria is a rare autosomal recessive disease that usually causes early urolithiasis but may cause rheumatoid arthritis-like disease and even be associated with defects in the formation of bone, hair and teeth. In Israel it has mostly been described in patients of Bedouin origin. Throughout the world, only about 150 cases have been described; about two thirds of these patients were asymptomatic. Since the clinical presentation and age of symptom appearance are diverse, the case raises questions as to the required follow-up of these patients and as to whether a low oxalate diet should be initiated.

New insights in research on purine and pyrimidine metabolism.

In June 2021, the Purine and Pyrimidine Society (PPS) organized the 19th biennial symposium on Purine and Pyrimidine metabolism (PP21). Due to the ongoing pandemic, the conference was organized as a webinar over 3 days with sessions dealing with enzymes, cancer, inborn errors, gout among others. The current issue of Nucleosides, Nucleotides & Nucleic Acids is a special issue covering proceedings from PP21-presentations and other PPS-related manuscripts, and in this editorial, we will give an overview of the scientific program of the meeting.

Pathway-specific effects of ADSL deficiency on neurodevelopment.

Adenylosuccinate lyase (ADSL) functions in de novo purine synthesis (DNPS) and the purine nucleotide cycle. ADSL deficiency (ADSLD) causes numerous neurodevelopmental pathologies, including microcephaly and autism spectrum disorder. ADSLD patients have normal serum purine nucleotide levels but exhibit accumulation of dephosphorylated ADSL substrates, S-Ado, and SAICAr, the latter being implicated in neurotoxic effects through unknown mechanisms. We examined the phenotypic effects of ADSL depletion in human cells and their relation to phenotypic outcomes. Using specific interventions to compensate for reduced purine levels or modulate SAICAr accumulation, we found that diminished AMP levels resulted in increased DNA damage signaling and cell cycle delays, while primary ciliogenesis was impaired specifically by loss of ADSL or administration of SAICAr. ADSL-deficient chicken and zebrafish embryos displayed impaired neurogenesis and microcephaly. Neuroprogenitor attrition in zebrafish embryos was rescued by pharmacological inhibition of DNPS, but not increased nucleotide concentration. Zebrafish also displayed phenotypes commonly linked to ciliopathies. Our results suggest that both reduced purine levels and impaired DNPS contribute to neurodevelopmental pathology in ADSLD and that defective ciliogenesis may influence the ADSLD phenotypic spectrum.

Unbalanced deoxynucleotide pools cause mitochondrial DNA instability in thymidine phosphorylase-deficient mice.

Replication and repair of DNA require equilibrated pools of deoxynucleoside triphosphate precursors. This concept has been proven by in vitro studies over many years, but in vivo models are required to demonstrate its relevance to multicellular organisms and to human diseases. Accordingly, we have generated thymidine phosphorylase (TP) and uridine phosphorylase (UP) double knockout (TP(-/-)UP(-/-)) mice, which show severe TP deficiency, increased thymidine and deoxyuridine in tissues and elevated mitochondrial deoxythymidine triphosphate. As consequences of the nucleotide pool imbalances, brains of mutant mice developed partial depletion of mtDNA, deficiencies of respiratory chain complexes and encephalopathy. These findings largely account for the pathogenesis of mitochondrial neurogastrointestinal encephalopathy (MNGIE), the first inherited human disorder of nucleoside metabolism associated with somatic DNA instability.

Nephrolithiasis related to inborn metabolic diseases.

Nephrolithiasis associated with inborn metabolic diseases is a very rare condition with some common characteristics: early onset of symptoms, family history, associated tubular impairment, bilateral, multiple and recurrent stones, and association with nephrocalcinosis. The prognosis of such diseases may lead to life threatening conditions, not only because of unabated kidney damage but also because of progressive extra-renal involvement, either in a systemic form (e.g. primary hyperoxaluria type 1, requiring combined liver and kidney transplantation), or in a neurological form (Lesch-Nyhan syndrome leading to auto-mutilation and disability, phosphoribosyl pyrophosphate synthetase superactivity, which is associated with mental retardation). Patients with other inborn metabolic diseases present only with recurrent stone formation, such as cystinuria, adenine phosphoribosyl-transferase deficiency, xanthine deficiency. Finally, nephrolithiasis may be secondarily part of some other metabolic diseases, such as glycogen storage disease type 1 or inborn errors of metabolism leading to Fanconi syndrome (nephropathic cystinosis, tyrosinaemia type 1, fructose intolerance, Wilson disease, respiratory chain disorders, etc.). The diagnosis is based on highly specific investigations, including crystal identification, biochemical analyses and DNA study. The treatment of nephrolithiasis requires hydration as well as specific measures. Compliance is a major issue regarding the progression of renal damage, but the overall outcome mainly depends on extra-renal involvement in relation to the metabolic defect.

Partial Purine Nucleoside Phosphorylase Deficiency Helps Determine Minimal Activity Required for Immune and Neurological Development.

Introduction: Complete or near complete absence of the purine nucleoside phosphorylase (PNP) enzyme causes a profound T cell immunodeficiency and neurological abnormalities that are often lethal in infancy and early childhood. We hypothesized that patients with partial PNP deficiency, characterized by a late and mild phenotype due to residual PNP enzyme, would provide important information about the minimal PNP activity needed for normal development. Methods: Three siblings with a homozygous PNP gene mutation (c.769C>G, p.His257Asp) resulting in partial PNP deficiency were investigated. PNP activity was semi-quantitively assayed by the conversion of [14C]inosine in hemolysates, mononuclear cells, and lymphoblastoid B cells. PNP protein expression was determined by Western Blotting in lymphoblastoid B cells. DNA repair was quantified by measuring viability of lymphoblastoid B cells following ionizing irradiation. Results: A 21-year-old female was referred for recurrent sino-pulmonary infections while her older male siblings, aged 25- and 28- years, did not suffer from significant infections. Two of the siblings had moderately reduced numbers of T, B, and NK cells, while the other had near normal lymphocyte subset numbers. T cell proliferations were normal in the two siblings tested. Hypogammaglobulinemia was noted in two siblings, including one that required immunoglobulin replacement. All siblings had typical (normal) neurological development. PNP activity in various cells from two patients were 8-11% of the normal level. All siblings had normal blood uric acid and increased PNP substrates in the urine. PNP protein expression in cells from the two patients examined was similar to that observed in cells from healthy controls. The survival of lymphoblastoid B cells from 2 partial PNP-deficient patients after irradiation was similar to that of PNP-proficient cells and markedly higher than the survival of cells from a patient with absent PNP activity or a patient with ataxia telangiectasia. Conclusions: Patients with partial PNP deficiency can present in the third decade of life with mild-moderate immune abnormalities and typical development. Near-normal immunity might be achieved with relatively low PNP activity.

Inborn errors of purine and pyrimidine metabolism.

Genetic disorders of purine and pyrimidine (PP) metabolism are under-reported and infrequently mentioned in the general literature, as well as in reviews dedicated to other inborn errors of metabolism. Owing to limited awareness, relatively recent recognition, as well as considerable phenotypic variation, these disorders may often be misdiagnosed or remain undiagnosed. Disorders that arise as a result of dysfunction in PP metabolism represent some of the most challenging diagnostic problems in medicine. In addition to their low prevalence rates, they also present with extremely variable signs and symptoms. They may affect any system in a variety of manners, and often mimic other, more recognizable disorders. The diagnostic problem is compounded by the fact that some biochemically affected patients are symptom-free. Rapidly evolving laboratory techniques such as high-performance liquid chromatography coupled to tandem mass spectrometry are now well established as the preferred method for detection for these defects, but currently the most important step in diagnosis consists of suspecting the disorder. Diagnosis is vital because genetic counselling can be provided and, in some cases, specific treatment can be offered that may slow or even reverse clinical symptoms. If undiagnosed, these disorders can be devastating to patients and their families, resulting in early death or institutionalization for the rest of patient's life. This article describes the current state of knowledge about inborn errors of purine and pyrimidine metabolism, focusing on the varying clinical presentations, the laboratory findings and discusses indications for selective screening for these disorders.

Orotic aciduria and uridine monophosphate synthase: a reappraisal.

Three subtypes of hereditary orotic aciduria are described in the literature, all related to deficiencies in uridine monophosphate synthase, the multifunctional enzyme that contains both orotate: pyrophosphoryl transferase and orotidine monophosphate decarboxylase activities. The type of enzyme defect present in the subtypes has been re-examined by steady-state modelling of the relative outputs of the three enzymic products, uridine monophosphate, urinary orotic acid and urinary orotidine. It is shown that the ratio of urinary outputs of orotidine to orotate provides a means of testing for particular forms of enzyme defect. It is confirmed that the type I defect is caused by loss of uridine monophosphate synthase activity. Cells and tissue of type I cases have a residual amount of activity that is qualitatively unchanged: the relative rates of the transferase and decarboxylase do not differ from those of wild-type enzyme. The single claimed case of type II, thought to be due to specific inactivation of orotidine monophosphate decarboxylase, is shown to have a product spectrum inconsistent with that claim. It is proposed that this type II form does not differ sufficiently to be accepted as separate from type I. The third subtype, hereditary orotic aciduria without megaloblastic anaemia, occurs in two cases. It has the product spectrum expected of a defect in orotidine monophosphate decarboxylase. This form is the only one that appears to have a qualitatively different uridine monophosphate synthase. The possibility that orotidine monophosphate may control flux through the pyrimidine biosynthesis pathway in hereditary orotic aciduria is discussed.

Deficiency of perforin and hCNT1, a novel inborn error of pyrimidine metabolism, associated with a rapidly developing lethal phenotype due to multi-organ failure.

Pyrimidine nucleotides are essential for a vast number of cellular processes and dysregulation of pyrimidine metabolism has been associated with a variety of clinical abnormalities. Inborn errors of pyrimidine metabolism affecting enzymes in the pyrimidine de novo and degradation pathway have been identified but no patients have been described with a deficiency in proteins affecting the cellular import of ribonucleosides. In this manuscript, we report the elucidation of the genetic basis of the observed uridine-cytidineuria in a patient presenting with fever, hepatosplenomegaly, persistent lactate acidosis, severely disturbed liver enzymes and ultimately multi-organ failure. Sequence analysis of genes encoding proteins directly involved in the metabolism of uridine and cytidine showed two variants c.1528C > T (p.R510C) and c.1682G > A (p.R561Q) in SLC28A1, encoding concentrative nucleotide transporter 1 (hCNT1). Functional analysis showed that these variants affected the three-dimensional structure of hCNT1, altered glycosylation and decreased the half-life of the mutant proteins which resulted in impaired transport activity. Co-transfection of both variants, mimicking the trans disposition of c.1528C > T (p.R510C) and c.1682G > A (p.R561Q) in the patient, significantly impaired hCNT1 biological function. Whole genome sequencing identified two pathogenic variants c.50delT; p.(Leu17Argfs*34) and c.853_855del; p.(Lys285del) in the PRF1 gene, indicating that our patient was also suffering from Familial Hemophagocytic Lymphohistiocytosis type 2. The identification of two co-existing monogenic defects might have resulted in a blended phenotype. Thus, the clinical presentation of isolated hCNT1 deficiency remains to be established.