Expanding the clinical and genetic spectrum of CAD deficiency: an epileptic encephalopathy treatable with uridine supplementation.

PURPOSE: Biallelic CAD variants underlie CAD deficiency (or early infantile epileptic encephalopathy-50, [EIEE-50]), an error of pyrimidine de novo biosynthesis amenable to treatment via the uridine salvage pathway. We further define the genotype and phenotype with a focus on treatment. METHODS: Retrospective case series of 20 patients. RESULTS: Our study confirms CAD deficiency as a progressive EIEE with recurrent status epilepticus, loss of skills, and dyserythropoietic anemia. We further refine the phenotype by reporting a movement disorder as a frequent feature, and add that milder courses with isolated developmental delay/intellectual disability can occur as well as onset with neonatal seizures. With no biomarker available, the diagnosis relies on genetic testing and functional validation in patient-derived fibroblasts. Underlying pathogenic variants are often rated as variants of unknown significance, which could lead to underrecognition of this treatable disorder. Supplementation with uridine, uridine monophosphate, or uridine triacetate in ten patients was safe and led to significant clinical improvement in most patients. CONCLUSION: We advise a trial with uridine (monophosphate) in all patients with developmental delay/intellectual disability, epilepsy, and anemia; all patients with status epilepticus; and all patients with neonatal seizures until (genetically) proven otherwise or proven unsuccessful after 6 months. CAD deficiency might represent a condition for genetic newborn screening.

Nucleotide sugar profiles throughout development in wildtype and galt knockout zebrafish.

Nucleotide sugars (NS) are fundamental molecules in life and play a key role in glycosylation reactions and signal conduction. Several pathways are involved in the synthesis of NS. The Leloir pathway, the main pathway for galactose metabolism, is crucial for production of uridine diphosphate (UDP)-glucose and UDP-galactose. The most common metabolic disease affecting this pathway is galactose-1-phosphate uridylyltransferase (GALT) deficiency, that despite a lifelong galactose-restricted diet, often results in chronically debilitating complications. Alterations in the levels of UDP-sugars leading to galactosylation abnormalities have been hypothesized as a key pathogenic factor. However, UDP-sugar levels measured in patient cell lines have shown contradictory results. Other NS that might be affected, differences throughout development, as well as tissue specific profiles have not been investigated. Using recently established UHPLC-MS/MS technology, we studied the complete NS profiles in wildtype and galt knockout zebrafish (Danio rerio). Analyses of UDP-hexoses, UDP-hexosamines, CMP-sialic acids, GDP-fucose, UDP-glucuronic acid, UDP-xylose, CDP-ribitol, and ADP-ribose profiles at four developmental stages and in tissues (brain and gonads) in wildtype zebrafish revealed variation in NS levels throughout development and differences between examined tissues. More specifically, we found higher levels of CMP-N-acetylneuraminic acid, GDP-fucose, UDP-glucuronic acid, and UDP-xylose in brain and of CMP-N-glycolylneuraminic acid in gonads. Analysis of the same NS profiles in galt knockout zebrafish revealed no significant differences from wildtype. Our findings in galt knockout zebrafish, even when challenged with galactose, do not support a role for abnormalities in UDP-glucose or UDP-galactose as a key pathogenic factor in GALT deficiency, under the tested conditions.

Evolution of Dihydropyrimidine Dehydrogenase Diagnostic Testing in a Single Center during an 8-Year Period of Time.

OBJECTIVE: Fluoropyrimidine treatment can be optimized based on dihydropyrimidine dehydrogenase (DPD) activity. DPD dysfunction leads to increased exposure to active metabolites, which can result in severe or even fatal toxicity. METHODS: We provide an overview of 8 years of DPD diagnostic testing (n = 1194). RESULTS: Within the study period, our diagnostic test evolved from a single-enzyme measurement using first a radiochemical and then a nonradiochemical assay by ultra HPLC-MS in peripheral blood mononuclear cells with uracil, to a combined enzymatic and genetic test (ie, polymerase chain reaction) followed by Sanger sequence analysis of 4 variants of the DPYD gene (ie, DPYD*2A, DPYD*13, c.2846A>T, and 1129-5923C>G; allele frequencies 0.58%, 0.03%, 0.29%, and 1.35%, respectively). Patients who have 1 of the 4 variants tested (n = 814) have lower enzyme activity than the overall patient group. The majority of patients with the DPYD*2A variant (83%) consistently showed decreased enzyme activity. Only 24 (25.3%) of 95 patients (tested for 4 variants) with low enzyme activity carried a variant. Complete DPYD sequencing in a subgroup with low enzyme activity and without DPYD*2A variant (n = 47) revealed 10 genetic variants, of which 4 have not been described previously. We did not observe a strong link between DPYD genotype and enzyme activity. CONCLUSIONS: Previous studies have shown that DPD status should be determined before treatment with fluoropyrimidine agents to prevent unnecessary side effects with possible fatal consequences. Our study in combination with literature shows that there is a discrepancy between the DPD enzyme activity and the presence of clinically relevant single nucleotide polymorphisms. At this moment, a combination of a genetic and enzyme test is preferable for diagnostic testing. (Curr Ther Res Clin Exp. 2018; 79:XXX-XXX).

Classic Galactosemia: Study on the Late Prenatal Development of GALT Specific Activity in a Sheep Model.

Classic galactosemia results from deficient activity of galactose-1-phosphate uridylyltransferase (GALT), a key enzyme of galactose metabolism. Despite early diagnosis and early postnatal therapeutic intervention, patients still develop neurologic and fertility impairments. Prenatal developmental toxicity has been hypothesized as a determinant factor of disease. In order to shed light on the importance of prenatal GALT activity, several studies have examined GALT activity throughout development. GALT was shown to increase with gestational age in 7-28 weeks human fetuses; later stages were not investigated. Prenatal studies in animals focused exclusively on brain and hepatic GALT activity. In this study, we aim to examine GALT specific activity in late prenatal and adult stages, using a sheep model. Galactosemia acute target-organs-liver, small intestine and kidney-had the highest late prenatal activity, whereas the chronic target-organs-brain and ovary-did not exhibit a noticeable pre- or postnatal different activity compared with nontarget organs. This is the first study on GALT specific activity in the late prenatal stage for a wide variety of organs. Our findings suggest that GALT activity cannot be the sole pathogenic factor accounting for galactosemia long-term complications, and that some organs/cells might have a greater susceptibility to galactose toxicity. Anat Rec, 300:1570-1575, 2017. © 2017 Wiley Periodicals, Inc.

Effects of plant stanol esters supplied in low-fat yoghurt on serum lipids and lipoproteins, non-cholesterol sterols and fat soluble antioxidant concentrations.

Oil-based products enriched with plant stanol esters can lower low-density lipoprotein (LDL) cholesterol concentrations by 10-14%. Effectiveness of low-fat products, however, has never been evaluated, although such products fit into a healthy diet. We therefore examined the effects of plant stanol esters emulsified into low-fat yoghurt (0.7% fat) on fasting concentrations of plasma lipids and lipid-soluble antioxidants, which may also change by plant stanol consumption. Sixty non-hypercholesterolemic subjects first consumed daily three cups (3 x 150 ml) of placebo yoghurt for 3 weeks. For the next 4 weeks, 30 subjects continued with the placebo yoghurt, while the other 30 subjects received three cups of experimental yoghurt. Each cup provided 1 g of plant stanols (0.71 g sitostanol plus 0.29 g campestanol) as its fatty acid ester. LDL cholesterol (mean+/-S.D.) increased by 0.06+/-0.21 mmol/l in the placebo group, but decreased by -0.34+/-0.30 mmol/l in the experimental group. The difference in changes between the two groups of 0.40 mmol or 13.7% was highly significant (P<0.001; 95% confidence interval for the difference, (-)0.26 -(-)0.53 mmol/l). Effects were already maximal after 1 week. HDL cholesterol and triacylglycerol concentrations did not change. Total tocopherol levels increased by 1.43 micromol/mmol LDL cholesterol (14.0%, P=0.015). beta-carotene levels, however, decreased by -0.02 micromol/mmol LDL cholesterol (-14.4%, P=0.038). Decreases in absolute beta-carotene concentrations were found in all apoB-containing lipoproteins. LDL-cholesterol standardised phytofluene levels decreased by 21.4+/-25.7% (P<0.001), while other plasma carotenoid (lutein/zeaxanthin, beta-cryptoxanthin, lycopene and alpha-carotene) levels did not change significantly. We conclude that low-fat yoghurt enriched with plant stanol esters lowers within 1 week LDL cholesterol to the same extent as oil-based products. LDL-cholesterol standardised concentrations of tocopherol increased. The observed decrease in beta-carotene levels, as found in many other studies, appears not to be limited to the LDL fraction.

Erythrocyte inosine triphosphatase activity is decreased in HIV-seropositive individuals.

BACKGROUND: Inosine triphosphatase (ITPase) is encoded by the polymorphic gene ITPA and maintains low intracellular levels of the inosine nucleotides ITP and dITP. The most frequently reported polymorphisms are ITPA c.94C>A (rs 1127354) and ITPA c. 124+21 A>C (rs7270101). Some nucleoside-analogues used in the treatment of HIV-seropositive (HIV+) patients are potential substrates for ITPase. Therefore, the frequency of ITPA SNPs and ITPase activity were studied in a population of HIV+-patients. METHODS: The study population consisted of 222 patients, predominantly Caucasian males, >95% using HAART. Erythrocyte ITPase activity was determined by measuring the formation of IMP from ITP. ITPA genotype was determined by sequencing genomic DNA. Distribution of ITPase activity, genotype-phenotype correlation and allele frequencies were compared to 198 control subjects. The effect of nucleoside analogues on ITPase activity was studied using lymphoblastic T-cell cultures and human recombinant ITPase. Enzyme kinetic experiments were performed on erythrocyte ITPase from HIV+ patients and controls. RESULTS: No difference was observed in the allele frequencies between the HIV+-cohort (± HAART) and the control population. HIV+ carriers of the wild type and ITPA c.94C>A had significantly lower ITPase activities than control subjects with the same genotype (p<0.005). This was not observed in ITPA c. 124+21 A>C carriers. Nucleoside analogues did not affect ITPase activity in cell culture and human recombinant ITPase. CONCLUSION: ITPA population genetics were identical in HIV+ and control populations. However, the majority of HIV+-patients had decreased erythrocyte ITPase activity compared to controls, probably due to decreased amounts of ITPase protein. It seems unlikely that ITPase activity is decreased due to nucleoside analogues (HAART). Long-term effects of HIV-infection altering ITPase protein expression or stability may explain the phenomenon observed.

The effect of ITPA polymorphisms on the enzyme kinetic properties of human erythrocyte inosine triphosphatase toward its substrates ITP and 6-Thio-ITP.

The role of inosine triphosphatase (ITPase) in adverse drug reactions associated with thiopurine therapy is still under heavy debate. Surprisingly, little is known about the way thiopurines are handled by ITPase. We studied the effect of ITPA polymorphisms on the handling of inosine triphosphate (ITP) and thioinosine triphosphate (TITP) to gain more insight into this phenomenon. Human erythrocyte ITPase activity was measured by incubation with ITP using established protocols, and the generated inosine monophosphate (IMP) was measured using ion-pair RP-HPLC. Molecular analysis of the ITPA gene was performed to establish the genotype. Kinetic parameters were established for the two common polymorphisms for both ITP and TITP as substrates using the above mentioned protocol. Both ITP and TITP are substrates for ITPase and their enzyme activities are comparable. Substrate binding is not altered in the different ITPA polymorphisms. It is shown that the velocity of pyrophosphohydrolysis is compromised when the c.94C > A polymorphism is present, both in the heterozygous and in the homozygous state. TITP is handled by ITPase in a similar way as for ITP, which implies that TITP will accumulate in the erythrocytes of patients with an ITPase deficiency, resulting in adverse drug reactions (ADRs) on thiopurine therapy. In carriers of ITPA polymorphisms, the matter is more complex and the development of ADR may depend on additional epigenetic factors rather than on the accumulation of thiopurinenucleotides.

Direct non-radioactive assay of galactose-1-phosphate:uridyltransferase activity using high performance liquid chromatography.

BACKGROUND: Galactose-1-phosphate:uridyltransferase (GALT) catalyses the conversion of galactose-1-phosphate (Gal-1-P) and UDP-glucose (UDP-Glc) into glucose-6-phosphate and UDP-galactose (UDP-Gal). Complete, or near complete, deficiency of GALT causes classic galactosaemia. The diagnosis is confirmed by measuring GALT activity in erythrocytes. The most commonly used assays require radio labelled substrates or indirect coupled assays. METHODS: GALT activity was measured in erythrocyte lysates using optimal concentrations of the substrates galactose-1-phosphate and UDP-Glc. UDP-Gal and UDP-Glc were separated using reversed-phase high performance liquid chromatography with UV detection. Clinical validity was assessed using blood samples from galactosaemic patients. RESULTS: UDP-Gal and UDP-Glc were separated with HPLC. The assay was linear with incubation times up 80 min and between 0 and 42.5 nmol haemoglobin. Within-day and between-day imprecision at 50, 75 and 100% enzyme activity was <1.4% and <2.4%, respectively. Mean GALT activity in 33 individuals was 601+/-79 nmol UDP-Gal/(micromol Hb.h) (range 492-697). Patients with classical galactosaemia were easily detected by their extremely low activity. CONCLUSIONS: We have developed a reliable and convenient direct method to measure GALT activity in human erythrocytes using HPLC with UV detection.

Pharmacogenetic significance of inosine triphosphatase.

Inosine triphosphatase (ITPase) is the enzyme that catalyzes the conversion of inosine triphosphate (ITP) and deoxy-inosine triphosphate (dITP) to inosine monophosphate and deoxy-inosine monophosphate, respectively, thereby maintaining low intracellular concentrations of ITP and dITP. Individuals deficient in ITPase activity were first recognized over 30 years ago. For decades, no clinical significance could be attributed to this inborn error of metabolism whatsoever. In recent years, evidence has started to accumulate that polymorphisms in the gene encoding ITPase are associated with potentially severe adverse drug reactions towards the thiopurine drugs azathioprine and 6-mercaptopurine. The pharmacogenetic significance is presently being debated in the literature. However, the present state of knowledge is still insufficient to definitively determine the pharmacogenetic significance of ITPase. This article aims to review the current knowledge on the role of ITPase in thiopurine metabolism.

Cardiac fatty acid metabolism is preserved in the compensated hypertrophic rat heart.

Cardiac hypertrophy and failure are associated with alterations in cardiac substrate metabolism. It remains to be established, however, whether genomically driven changes in cardiac glucose and fatty acid (FA) metabolism represent a key event of the hypertrophic remodeling process. Accordingly, we investigated metabolic gene expression and substrate metabolism during compensatory hypertrophy, in relation to other cardiac remodeling processes. Thereto, cardiac hypertrophy was induced in rats by supra-renal aortic constriction to various degrees, resulting in increased heart/body weight ratios of 22% (Aob-1), 24% (Aob-2) and 32% (Aob-3) (p < 0.005) after 4 weeks. The unaltered ejection fraction in all groups indicated that the hypertrophy was still compensatory in nature. beta-Myosin Heavy Chain protein and ANF mRNA levels were increased in all groups. Only in Aob-3 rats were SERCA2a mRNA levels markedly reduced. In this group, glycolytic capacity was modestly elevated (+ 25%; p < 0.01). Notwithstanding these phenotypical changes, the expression of genes involved in FA metabolism and FA oxidation rate in cardiac homogenates was completely preserved, irrespective of the degree of hypertrophy. These findings indicate that cardiac FA oxidative capacity is preserved during compensatory hypertrophy, and that a decline in metabolic gene expression does not represent a hallmark of the development of hypertrophy.

Peroxisome proliferator-activated receptors: lipid binding proteins controling gene expression.

The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. Since their discovery in the beginning of the nineties the three isoforms (PPARalpha, beta/delta and gamma, encoded by different genes) have been implicated in the regulation of almost every single aspect of lipid metabolism and, consequently, in diseases that involve disturbances in lipid metabolism (obesity, diabetes, atherosclerosis, cardiac failure). Although their prominent role in these processes has hardly been disputed, the way in which the activity of these transcription factors is regulated under physiological and pathological conditions awaits further clarification. An unresolved issue has been the nature of the natural ligand of these receptors. Biochemical studies have shown that the PPAR isoforms are rather promiscuous with respect to ligand binding, with a large variety of naturally occurring lipid-like substances acting as low-affinity ligands. More recently this concept has been confirmed by crystallographic studies on the ligand-binding pocket. In addition to ligand availability, the trans-activating capacity likely depends on phosphorylation status of the PPARs and on the recruitment of auxiliary proteins (co-activators and corepressors). Accordingly, the biological activity of these key-regulators of metabolism is controlled at multiple levels, which enables each tissue to fine tune its metabolic machinery to the demands of the body in a specific fashion.