Kidney failure secondary to hereditary xanthinuria due to a homozygous deletion of the XDH gene, in the absence of overt kidney stone disease.

Hereditary xanthinuria (HXAN) is a rare metabolic disorder that results from mutations in either the xanthine dehydrogenase (XDH) or the molybdenum cofactor sulfurase genes (MOCOS), respectively defining HXAN type I and type II. Hypouricemia, hypouricosuria, and abnormally high plasma and urine levels of xanthine, causing susceptibility to xanthine nephrolithiasis and deposition of xanthine crystals in tissues, are the metabolic hallmarks of HXAN. Several pathogenic variants in the XDH gene have so far been identified in patients with HXAN type I, but the clinical phenotype associated with the whole deletion of the human XDH gene is unknown. Herein, we report the case of a woman diagnosed with HXAN, whose molecular genetic testing revealed a homozygous microdeletion involving the XDH gene. Distinctive features of her medical history were the diagnosis of arterial hypertension and microalbuminuria at 22 years of age; a single pregnancy, at the age of 25, complicated by proteinuria and transient kidney function deterioration in the third trimester; unexplained severe hypouricaemia incidentally discovered during pregnancy; inability to breastfeed her newborn daughter due to primary agalactia; chronic kidney disease (CKD) stage 3 diagnosed at age 35; and progression to end-stage kidney disease over the next 12 years. Protocol non-invasive laboratory and imaging investigation was not informative as to the cause of CKD. This is the first description of the clinical phenotype associated with a natural knockout of the human XDH gene. Despite the lack of kidney histopathology data, the striking similarities with the phenotypes exhibited by comparable murine models validates the latter as useful sources of mechanistic insights for the pathogenesis of the human disease, supporting the hypothesis that the absence of xanthine dehydrogenase activity might represent a susceptibility factor for chronic tubulointerstitial nephritis, even in patients without kidney stones.

Anticipatory banking of samples enables diagnosis of adenylosuccinase deficiency following molecular autopsy in an infant with vacuolating leukoencephalopathy.

Adenylosuccinase deficiency is a rare inborn error of metabolism. We present a newborn who died at 52 days of age with clinical features suggestive of severe epileptic encephalopathy and leukodystrophy of unknown cause. Post-mortem examination showed an unusual vacuolar appearance of the brain. A molecular autopsy performed via singleton clinical exome analysis revealed a known pathogenic and a variant of uncertain significance in ADSL that encodes adenylosuccinase. Tests on previously stored plasma samples showed elevated succinyladenosine and succinylaminoimidazole carboxamide riboside levels. Adenylosuccinase activity in stored fibroblasts was only ~5% of control confirming the diagnosis of adenylosuccinase deficiency in the child. The parents opted for a chorionic villus biopsy in a subsequent pregnancy and had a child unaffected by adenylosuccinase deficiency. This report adds vacuolating leukodystrophy as a novel feature of adenylosuccinase deficiency and shows the power of biochemical investigations directed by genomic studies to achieve accurate diagnosis. Importantly, this case demonstrates the importance of anticipatory banking of biological samples for reverse biochemical phenotyping in individuals with undiagnosed disorders who may not survive.

Treating gout - can hypouricemia produced by moderate doses of combined oral treatment give early treatment success? Preliminary observations.

Six male patients with gout were treated with combined oral medication (febuxostat, 120 mg/d, and benzbromarone, 50 or 100 mg/d), aiming at a more rapid success of uric acid lowering treatment (ULT) compared to guideline suggestions. By combined oral medication in moderate dosage, the sUA was reduced to <2 mg/dl in all cases. We conclude that, by the treatment schedule outlined, the majority of patients with gout can be cured within 1 - 2 years, with uricase treatment being necessary very rarely only.

Metabolite and thymocyte development defects in ADA-SCID mice receiving enzyme replacement therapy.

Deficiency of adenosine deaminase (ADA, EC3.5.4.4), a housekeeping enzyme intrinsic to the purine salvage pathway, leads to severe combined immunodeficiency (SCID) both in humans and mice. Lack of ADA results in the intracellular accumulation of toxic metabolites which have effects on T cell development and function. While untreated ADA-SCID is a fatal disorder, there are different therapeutic options available to restore ADA activity and reconstitute a functioning immune system, including enzyme replacement therapy (ERT). Administration of ERT in the form of pegylated bovine ADA (PEG-ADA) has proved a life-saving though non-curative treatment for ADA-SCID patients. However, in many patients treated with PEG-ADA, there is suboptimal immune recovery with low T and B cell numbers. Here, we show reduced thymus cellularity in ADA-SCID mice despite weekly PEG-ADA treatment. This was associated with lack of effective adenosine (Ado) detoxification in the thymus. We also show that thymocyte development in ADA-deficient thymi is arrested at the DN3-to-DN4 stage transition with thymocytes undergoing dATP-induced apoptosis rather than defective TCRß rearrangement or ß-selection. Our studies demonstrate at a detailed level that exogenous once-a-week enzyme replacement does not fully correct intra-thymic metabolic or immunological abnormalities associated with ADA deficiency.

Co-therapy with S-adenosylmethionine and nicotinamide riboside improves t-cell survival and function in Arts Syndrome (PRPS1 deficiency).

Arts syndrome or phosphoribosyl-pyrophosphate-synthetase-1 (PRPS1) deficiency is caused by loss-of-function mutations in the PRPS1 gene (Xq22.3). PRPS1 is an initial and essential step for the synthesis of the nucleotides of purines, pyrimidines, and nicotinamide. Classically, affected males present with sensorineural hearing loss, optic atrophy, muscular hypotonia, developmental impairment, and recurrent severe respiratory infections early in life. Treatment of a 3-year old boy with S-adenosylmethionine (SAM) replenished erythrocyte purine nucleotides of adenosine and guanosine, while SAM and nicotinamide riboside co-therapy further improved his clinical phenotype as well as T-cell survival and function.

Lesch-Nyhan Disease and Its Variants: Phenotypic and Mutation Spectrum of Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency in Argentine Patients

Abstract Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency is a disorder of purine metabolism responsible for Lesch-Nyhan Disease (LND) and its variants, HPRT-related hyperuricemia with neurologic dysfunction (HND) and HPRT-related hyperuricemia (HRH). The objective of this study was to characterize a cohort of Argentine patients with HPRT deficiency diagnosed in a single center. Results: Twenty nine patients were studied, including 12 LND, 15 HND and 2 HRH. The average onset age was 0.64 years for LND with motor delay as the main manifestation, 8.84 years for HND and 2.5 years for HRH; nephrological manifestations predominated as presenting features in these variants. The average diagnosis age was 3.58 years for LND, 17.21 years for HND and 2.5 years for HRH. Clinical heterogeneity was more evident in HND, even in members of the same family. All patients presented hyperuricemia and no detectable HPRT activity in erythrocyte lysate. The molecular study allowed to identify 9 different mutations in HPRT1 gene from 24 patients (11 independent pedigrees) and to establish genotype-phenotype correlation. In conclusion, this study describes the genotypic/phenotypic spectrum of HPRT deficiency in Argentine patients and highlights the need to increase awareness about the suspicion of these diseases, especially the LND variants with high clinical heterogeneity.

Refractory gout - does it exist?

Refractory gout (RG) has been increasingly recognized to be a major problem in clinical care. Patients diagnosed with RG have been assumed to be non-adherent, or under-dosed, to the greater part. In a minority, pathophysiological mechanisms have been discussed. During the last two decades, however, none of the studies differentiated non-adherence from impaired response to drug treatment. A definition of adherence has been proposed in the case of allopurinol treatment (oxipurinol in serum, >20 µmol/l), which would seem to confirm a dose of about 50 mg/d being taken by the patients. Guidelines for treating gout published by national or international rheumatology societies do provide very little, if any, information on how to evaluate patients with RG. Coinciding with the development of the xanthine oxidase inhibitor, febuxostat, a moderate increase in the number of publications on RG was observed, with a sharp rise following after its approval for clinical use. It was demonstrated recently that intensive training and supervision of patients with gout resulted in very low numbers of patients not reaching treatment targets. It should be remembered that allopurinol, is an ideal instrument for differentiating non-adherence from true impaired response. We conclude that, apart from very rare patients, needing confirmation of such a diagnosis by metabolic ward studies, RG does not exist, and with close to hundred percent, treatment failure is due to patient and physician behavior.

Quantification of Plasma and Urine Thymidine and 2'-Deoxyuridine by LC-MS/MS for the Pharmacodynamic Evaluation of Erythrocyte Encapsulated Thymidine Phosphorylase in Patients with Mitochondrial Neurogastrointestinal Encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare disorder caused by mutations in TYMP, leading to a deficiency in thymidine phosphorylase and a subsequent systemic accumulation of thymidine and 2'-deoxyuridine. Erythrocyte-encapsulated thymidine phosphorylase (EE-TP) is under clinical development as an enzyme replacement therapy for MNGIE. Bioanalytical methods were developed according to regulatory guidelines for the quantification of thymidine and 2'-deoxyuridine in plasma and urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for supporting the pharmacodynamic evaluation of EE-TP. Samples were deproteinized with 5% perchloric acid (v/v) and the supernatants analyzed using a Hypercarb column (30 × 2.1 mm, 3 µm), with mobile phases of 0.1% formic acid in methanol and 0.1% formic acid in deionized water. Detection was conducted using an ion-spray interface running in positive mode. Isotopically labelled thymidine and 2'-deoxyuridine were used as internal standards. Calibration curves for both metabolites showed linearity (r > 0.99) in the concentration ranges of 10-10,000 ng/mL for plasma, and 1-50 µg/mL for urine, with method analytical performances within the acceptable criteria for quality control samples. The plasma method was successfully applied to the diagnosis of two patients with MNGIE and the quantification of plasma metabolites in three patients treated with EE-TP.

Erythrocyte Encapsulated Thymidine Phosphorylase for the Treatment of Patients with Mitochondrial Neurogastrointestinal Encephalomyopathy: Study Protocol for a Multi-Centre, Multiple Dose, Open Label Trial.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder which primarily affects the gastrointestinal and nervous systems. This disease is caused by mutations in the nuclear TYMP gene, which encodes for thymidine phosphorylase, an enzyme required for the normal metabolism of deoxynucleosides, thymidine, and deoxyuridine. The subsequent elevated systemic concentrations of deoxynucleosides lead to increased intracellular concentrations of their corresponding triphosphates, and ultimately mitochondrial failure due to progressive accumulation of mitochondrial DNA (mtDNA) defects and mtDNA depletion. Currently, there are no treatments for MNGIE where effectiveness has been evidenced in clinical trials. This Phase 2, multi-centre, multiple dose, open label trial without a control will investigate the application of erythrocyte-encapsulated thymidine phosphorylase (EE-TP) as an enzyme replacement therapy for MNGIE. Three EE-TP dose levels are planned with patients receiving the dose level that achieves metabolic correction. The study duration is 31 months, comprising 28 days of screening, 90 days of run-in, 24 months of treatment and 90 days of post-dose follow-up. The primary objectives are to determine the safety, tolerability, pharmacodynamics, and efficacy of multiple doses of EE-TP. The secondary objectives are to assess EE-TP immunogenicity after multiple dose administrations and changes in clinical assessments, and the pharmacodynamics effect of EE-TP on clinical assessments.

Safety and Efficacy of Erythrocyte Encapsulated Thymidine Phosphorylase in Mitochondrial Neurogastrointestinal Encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare autosomal recessive disorder of nucleoside metabolism that is caused by mutations in the nuclear thymidine phosphorylase gene (TYMP) gene, encoding for the enzyme thymidine phosphorylase. There are currently no approved treatments for MNGIE. The aim of this study was to investigate the safety, tolerability, and efficacy of an enzyme replacement therapy for the treatment of MNGIE. In this single centre study, three adult patients with MNGIE received intravenous escalating doses of erythrocyte encapsulated thymidine phosphorylase (EE-TP; dose range: 4 to 108 U/kg/4 weeks). EE-TP was well tolerated and reductions in the disease-associated plasma metabolites, thymidine, and deoxyuridine were observed in all three patients. Clinical improvements, including weight gain and improved disease scores, were observed in two patients, suggesting that EE-TP is able to reverse some aspects of the disease pathology. Transient, non-serious adverse events were observed in two of the three patients; these did not lead to therapy discontinuation and they were managed with pre-medication prior to infusion of EE-TP. To conclude, enzyme replacement therapy with EE-TP demonstrated biochemical and clinical therapeutic efficacy with an acceptable clinical safety profile.

Uric acid, an important screening tool to detect inborn errors of metabolism: a case series.

BACKGROUND: Uric acid is the metabolic end product of purine metabolism in humans. Altered serum and urine uric acid level (both above and below the reference ranges) is an indispensable marker in detecting rare inborn errors of metabolism. We describe different case scenarios of 4 Sri Lankan patients related to abnormal uric acid levels in blood and urine. CASE 1: A one-and-half-year-old boy was investigated for haematuria and a calculus in the bladder. Xanthine crystals were seen in microscopic examination of urine sediment. Low uric acid concentrations in serum and low urinary fractional excretion of uric acid associated with high urinary excretion of xanthine and hypoxanthine were compatible with xanthine oxidase deficiency. CASE 2: An 8-month-old boy presented with intractable seizures, feeding difficulties, screaming episodes, microcephaly, facial dysmorphism and severe neuro developmental delay. Low uric acid level in serum, low fractional excretion of uric acid and radiological findings were consistent with possible molybdenum cofactor deficiency. Diagnosis was confirmed by elevated levels of xanthine, hypoxanthine and sulfocysteine levels in urine. CASE 3: A 3-year-10-month-old boy presented with global developmental delay, failure to thrive, dystonia and self-destructive behaviour. High uric acid levels in serum, increased fractional excretion of uric acid and absent hypoxanthine-guanine phosphoribosyltransferase enzyme level confirmed the diagnosis of Lesch-Nyhan syndrome. CASE 4: A 9-year-old boy was investigated for lower abdominal pain, gross haematuria and right renal calculus. Low uric acid level in serum and increased fractional excretion of uric acid pointed towards hereditary renal hypouricaemia which was confirmed by genetic studies. CONCLUSION: Abnormal uric acid level in blood and urine is a valuable tool in screening for clinical conditions related to derangement of the nucleic acid metabolic pathway.

Adenine phosphoribosyltransferase deficiency in the United Kingdom: two novel mutations and a cross-sectional survey.

BACKGROUND: Adenine phosphoribosyltransferase deficiency is an inborn error of metabolism that can cause kidney disease from crystalline nephropathy or kidney stones. METHODS: We present three cases from a single centre with varied presentations to illustrate how increasing awareness led to better patient identification. We then undertook a cross-sectional survey of all the patients identified from the Purine Research Laboratory in the UK since 1974. RESULTS: Our index case presented with recurrent nephrolithiasis and was diagnosed on stone analysis, the second case presented with acute kidney injury and the third case was identified from a biopsy undertaken for acute on chronic kidney injury. Genetic studies identified two novel mutations. Twenty patients were retrospectively identified. The mean age at diagnosis was 25 years (range 2-70); eight were <20 years, seven were 20-40 years and five were >40 years. Five of the 20 patients were deceased, 3 after end-stage renal disease (ESRD). Twelve have normal renal function, one had CKD stage 3, one had severe kidney disease and one was on dialysis. CONCLUSIONS: Adenine phosphoribosyltransferase deficiency presents in a wide spectrum in all age groups. Patients can be completely asymptomatic and kidney disease may be incorrectly attributed to other conditions. Outcome is poor in late diagnosis and there is a high prevalence of ESRD. Patients with unexplained renal stone disease or deterioration in kidney function should be considered for screening. Identification and surveillance of patients in the UK can improve. There is now a rare disease registry with meetings organized that include patients, families and health care providers to improve awareness.

IL-12 and IL-4 activate a CD39-dependent intrinsic peripheral tolerance mechanism in CD8(+) T cells.

Immune responses to protein antigens involve CD4(+) and CD8(+) T cells, which follow distinct programs of differentiation. Naïve CD8 T cells rapidly develop cytotoxic T-cell (CTL) activity after T-cell receptor stimulation, and we have previously shown that this is accompanied by suppressive activity in the presence of specific cytokines, i.e. IL-12 and IL-4. Cytokine-induced CD8(+) regulatory T (Treg) cells are one of several Treg-cell phenotypes and are Foxp3(-) IL-10(+) with contact-dependent suppressive capacity. Here, we show they also express high level CD39, an ecto-nucleotidase that degrades extracellular ATP, and this contributes to their suppressive activity. CD39 expression was found to be upregulated on CD8(+) T cells during peripheral tolerance induction in vivo, accompanied by release of IL-12 and IL-10. CD39 was also upregulated during respiratory tolerance induction to inhaled allergen and on tumor-infiltrating CD8(+) T cells. Production of IL-10 and expression of CD39 by CD8(+) T cells was independently regulated, being respectively blocked by extracellular ATP and enhanced by an A2A adenosine receptor agonist. Our results suggest that any CTL can develop suppressive activity when exposed to specific cytokines in the absence of alarmins. Thus negative feedback controls CTL expansion under regulation from both nucleotide and cytokine environment within tissues.

Spectrum of mutations in a cohort of UK patients with ADA deficient SCID: Segregation of genotypes with specific ethnicities.

Severe combined immunodeficiency (SCID) arises from a number of different genetic defects, one of the most common being mutations in the gene encoding adenosine deaminase (ADA). In the UK, ADA deficient SCID compromises approximately 20% of all known cases of SCID. We carried out a retrospective analysis of the ADA gene in 46 known ADA deficient SCID patients on whom DNA had been stored. Here, we report a high frequency of two previously reported mutations and provide a link between the mutations and patient ethnicity within our patient cohort. We also report on 9 novel mutations that have been previously unreported.

Pleural fluid adenosine deaminase (pfADA) in the diagnosis of tuberculous effusions in a low incidence population.

INTRODUCTION: Previous studies have assessed the diagnostic ability of pleural fluid adenosine deaminase (pfADA) in detecting tuberculous pleural effusions, with good specificity and sensitivity reported. However, in North Western Europe pfADA is not routinely used in the investigation of a patient with an undiagnosed pleural effusion, mainly due to a lack of evidence as to its utility in populations with low mycobacterium tuberculosis (mTB) incidence. METHODS: Patients presenting with an undiagnosed pleural effusion to a tertiary pleural centre in South-West England over a 3 year period, were prospectively recruited to a pleural biomarker study. Pleural fluid from consecutive patients with robust 12-month follow up data and confirmed diagnosis were sent for pfADA analysis. RESULTS: Of 338 patients enrolled, 7 had confirmed tuberculous pleural effusion (2%). All mTB effusions were lymphocyte predominant with a median pfADA of 72.0 IU/L (range- 26.7 to 91.5) compared to a population median of 12.0 IU/L (range- 0.3 to 568.4). The optimal pfADA cut off was 35 IU/L, which had a negative predictive value (NPV) of 99.7% (95% CI; 98.2-99.9%) for the exclusion of mTB, and sensitivity of 85.7% (95% CI; 42.2-97.6%) with an area under the curve of 0.88 (95% CI; 0.732-1.000). DISCUSSION: This is the first study examining the diagnostic utility of pfADA in a low mTB incidence area. The chance of an effusion with a pfADA under 35 IU/L being of tuberculous aetiology was negligible. A pfADA of over 35 IU/L in lymphocyte-predominant pleural fluid gives a strong suspicion of mTB.