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The article discusses lethal capecitabine toxicity in patients with complete dihydropyrimidine dehydrogenase deficiency due to ultra-rare DPYD variants. It emphasizes the importance of preemptive genetic analysis of DPYD to reduce fluoropyrimidine-related adverse events and highlights the need for comprehensive genotyping and phenotyping strategies for identifying patients at risk of severe toxicity. The study presents two cases of patients with complete DPYD deficiency due to ultra-rare DPYD variants and lethal capecitabine toxicity, underscoring the challenges in predicting severe toxicity in patients with rare DPYD variants. The research advocates for a combined genotyping/phenotyping approach to enhance the safety of fluoropyrimidine-based therapy, even in patients with complete DPYD deficiency. [Extracted from the article]

A DPD deficiency should be considered in case of severe toxicity even in the absence of common risk variants in DPYD. [ABSTRACT FROM AUTHOR]

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU). Genetic variations in DPD have emerged as predictive risk factors for severe fluoropyrimidine toxicity. Here, we report novel and rare genetic variants underlying DPD deficiency in 9 cancer patients presenting with severe fluoropyrimidine-associated toxicity. All patients possessed a strongly reduced DPD activity, ranging from 9 to 53% of controls. Analysis of the DPD gene ( DPYD ) showed the presence of 21 variable sites including 4 novel and 4 very rare aberrations: 3 missense mutations, 2 splice-site mutations, 1 intronic mutation, a deletion of 21 nucleotides and a genomic amplification of exons 9–12. Two novel/rare variants (c.2843T > C, c.321 + 1G > A) were present in multiple, unrelated patients. Functional analysis of recombinantly-expressed DPD mutants carrying the p.I948T and p.G284V mutation showed residual DPD activities of 30% and 0.5%, respectively. Analysis of a DPD homology model indicated that the p.I948T and p.G284V mutations may affect electron transfer and the binding of FAD, respectively. cDNA analysis showed that the c.321 + 1G > A mutation in DPYD leads to skipping of exon 4 immediately upstream of the mutated splice-donor site in the process of DPD pre-mRNA splicing. A lethal toxicity in two DPD patients suggests that fluoropyrimidines combined with other therapies such as radiotherapy might be particularly toxic for DPD deficient patients. Our study advocates a more comprehensive genotyping approach combined with phenotyping strategies for upfront screening for DPD deficiency to ensure the safe administration of fluoropyrimidines. [ABSTRACT FROM AUTHOR]

Introduction Neuroblastoma (NBL) accounts for 10% of the paediatric malignancies and is responsible for 15% of the paediatric cancer-related deaths. Vanillylmandelic acid (VMA) and homovanillic acid (HVA) are most commonly analysed in urine of NBL patients. However, their diagnostic sensitivity is suboptimal (82%). Therefore, we performed in-depth analysis of the diagnostic sensitivity of a panel of urinary catecholamine metabolites. Patients and methods Retrospective study of a panel of 8 urinary catecholamine metabolites (VMA, HVA, 3-methoxytyramine [3MT], dopamine, epinephrine, metanephrine, norepinephrine and normetanephrine [NMN]) from 301 NBL patients at diagnosis. Special attention was given to subgroups, metaiodobenzylguanidine (MIBG) non-avid tumours and VMA/HVA negative patients. Results Elevated catecholamine metabolites, especially 3MT, correlated with nine out of 12 NBL characteristics such as stage, age, MYCN amplification, loss of heterozygosity for 1p and bone-marrow invasion. The combination of the classical markers VMA and HVA had a diagnostic sensitivity of 84%. NMN was the most sensitive single diagnostic metabolite with overall sensitivity of 89%. When all 8 metabolites were combined, a diagnostic sensitivity of 95% was achieved. Among the VMA and HVA negative patients, were also 29% with stage 4 disease, which usually had elevation of other catecholamine metabolites (93%). Diagnostic sensitivity for patients with MIBG non-avid tumour was improved from 33% (VMA and/or HVA) to 89% by measuring the panel. Conclusions Our study demonstrates that analysis of a urinary catecholamine metabolite panel, comprising 8 metabolites, ensures the highest sensitivity to diagnose NBL patients. [ABSTRACT FROM AUTHOR]

Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine and cytidine as well as the pharmacological activation of several cytotoxic pyrimidine ribonucleoside analogues. In this study, we investigated the functional role of two isoforms of UCK in neuroblastoma cell lines. Analysis of mRNA coding for UCK1 and UCK2 showed that UCK2 is the most abundantly expressed UCK in a panel of neuroblastoma cell lines. Transient and stable overexpression of UCK2 in neuroblastoma cells increased the metabolism of uridine and cytidine as well as the cytotoxicity of 3-deazauridine. Knockdown of endogenous UCK2 as well as overexpression of UCK1 resulted in decreased metabolism of uridine and cytidine and protected the neuroblastoma cells from 3-deazauridine-induced toxicity. Subcellular localization studies showed that UCK1-GFP and UCK2-GFP were localized in the cell nucleus and cytosol, respectively. However, co-expression of UCK1 with UCK2 resulted in a nuclear localization of UCK2 instead of its normal cytosolic localization, thereby impairing its normal function. The physical association of UCK1 and UCK2 was further demonstrated through pull-down analysis using his-tagged UCK. The discovery that UCK2 is highly expressed in neuroblastoma opens the possibility for selectively targeting neuroblastoma cells using UCK2-dependent pyrimidine analogues, while sparing normal tissues. [ABSTRACT FROM AUTHOR]

Abstract: ß-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and catalyzes the conversion of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid to ß-alanine and ß-aminoisobutyric acid, ammonia and CO2. To date, only five genetically confirmed patients with a complete ß-ureidopropionase deficiency have been reported. Here, we report on the clinical, biochemical and molecular findings of 11 newly identified ß-ureidopropionase deficient patients as well as the analysis of the mutations in a three-dimensional framework. Patients presented mainly with neurological abnormalities (intellectual disabilities, seizures, abnormal tonus regulation, microcephaly, and malformations on neuro-imaging) and markedly elevated levels of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid in urine and plasma. Analysis of UPB1, encoding ß-ureidopropionase, showed 6 novel missense mutations and one novel splice-site mutation. Heterologous expression of the 6 mutant enzymes in Escherichia coli showed that all mutations yielded mutant ß-ureidopropionase proteins with significantly decreased activity. Analysis of a homology model of human ß-ureidopropionase generated using the crystal structure of the enzyme from Drosophila melanogaster indicated that the point mutations p.G235R, p.R236W and p.S264R lead to amino acid exchanges in the active site and therefore affect substrate binding and catalysis. The mutations L13S, R326Q and T359M resulted most likely in folding defects and oligomer assembly impairment. Two mutations were identified in several unrelated ß-ureidopropionase patients, indicating that ß-ureidopropionase deficiency may be more common than anticipated. [Copyright &y& Elsevier]

Abstract: To enhance the efficacy of fenretinide (4HPR)-induced reactive oxygen species (ROS) in neuroblastoma, 4HPR was combined with buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, in neuroblastoma cell lines and spheroids, the latter being a three-dimensional tumor model. 4HPR exposure (2.5–10μM, 24h) resulted in ROS induction (114–633%) and increased GSH levels (68–120%). A GSH depletion of 80% of basal levels was observed in the presence of BSO (25–100μM, 24h). The 4HPR–BSO combination resulted in slightly increased ROS levels (1.1- to 1.3-fold) accompanied by an increase in cytotoxicity (110–150%) compared to 4HPR treatment alone. A correlation was observed between the ROS-inducing capacity of each cell line and the increase in cytotoxicity induced by 4HPR–BSO compared to 4HPR. No significant correlation between baseline antioxidant levels and sensitivity to 4HPR or BSO was observed. In spheroids, 4HPR–BSO induced a strong synergistic growth retardation and induction of apoptosis. Our data show that BSO increased the cytotoxic effects of 4HPR in neuroblastoma monolayers and spheroids in ROS-producing cell lines. This indicates that the 4HPR–BSO combination might be a promising new strategy in the treatment of neuroblastoma. [Copyright &y& Elsevier]

Abstract: Dihydropyrimidinase (DHP) is the second enzyme of the pyrimidine degradation pathway and catalyses the ring opening of 5,6-dihydrouracil and 5,6-dihydrothymine. To date, only 11 individuals have been reported suffering from a complete DHP deficiency. Here, we report on the clinical, biochemical and molecular findings of 17 newly identified DHP deficient patients as well as the analysis of the mutations in a three-dimensional framework. Patients presented mainly with neurological and gastrointestinal abnormalities and markedly elevated levels of 5,6-dihydrouracil and 5,6-dihydrothymine in plasma, cerebrospinal fluid and urine. Analysis of DPYS, encoding DHP, showed nine missense mutations, two nonsense mutations, two deletions and one splice-site mutation. Seventy-one percent of the mutations were located at exons 5–8, representing 41% of the coding sequence. Heterologous expression of 11 mutant enzymes in Escherichia coli showed that all but two missense mutations yielded mutant DHP proteins without significant activity. Only DHP enzymes containing the mutations p.R302Q and p.T343A possessed a residual activity of 3.9% and 49%, respectively. The crystal structure of human DHP indicated that the point mutations p.R490C, p.R302Q and p.V364M affect the oligomerization of the enzyme. In contrast, p.M70T, p.D81G, p.L337P and p.T343A affect regions near the di-zinc centre and the substrate binding site. The p.S379R and p.L7V mutations were likely to cause structural destabilization and protein misfolding. Four mutations were identified in multiple unrelated DHP patients, indicating that DHP deficiency may be more common than anticipated. [Copyright &y& Elsevier]

Abstract: Dihydropyrimidinase (DHP) is the second enzyme of the pyrimidine degradation pathway and it catalyses the ring opening of 5,6-dihydrouracil and 5,6-dihydrothymine to N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyric acid, respectively. To date, only nine individuals have been reported suffering from a complete DHP deficiency. We report two siblings presenting with strongly elevated levels of 5,6-dihydrouracil and 5,6-dihydrothymine in plasma, cerebrospinal fluid and urine. One of the siblings had a severe delay in speech development and white matter abnormalities, whereas the other one was free of symptoms. Analysis of the DHP gene (DPYS) showed that both patients were compound heterozygous for the missense mutation 1078T>C (W360R) in exon 6 and a novel missense mutation 1235G>T (R412M) in exon 7. Heterologous expression of the mutant enzymes in Escherichia coli showed that both missense mutations resulted in a mutant DHP enzyme without residual activity. Analysis of the crystal structure of eukaryotic DHP from the yeast Saccharomyces kluyveri and the slime mold Dictyostelium discoideum suggests that the W360R and R412M mutations lead to structural instability of the enzyme which could potentially impair the assembly of the tetramer. [Copyright &y& Elsevier]

Abstract: Dihydropyrimidine dehydrogenase (DPD) plays a pivotal role in the metabolism of 5FU. The prognostic significance of DPD activity in peripheral blood mononuclear (PBM) cells and buccal mucosa cells with respect to toxicity was investigated in 44 patients treated with 5FU-leucovorin. Grade III/IV haematological and grade III/IV gastrointestinal toxicity were observed in 25% and 21% of the patients, respectively. No association was observed between the DPD activity in buccal mucosa cells and toxicity. In contrast, the mean DPD activity in PBM cells proved to be increased in patients experiencing grade I/II neutropenia when compared to patients without neutropenia and those suffering from grade III/IV neutropenia (P =0.002). Patients with a high-normal DPD activity proved to be at risk of developing mild toxicity upon treatment with 5FU-leucovorin, suggesting an important role of DPD in the aetiology of toxicity associated with catabolites of 5FU. [Copyright &y& Elsevier]

Abstract: Thymidine phosphorylase (TP) catalyses the conversion of thymidine into thymine. A non-radiochemical assay procedure for TP was developed in which thymine was detected at 265nm after separation with reversed-phase HPLC. A complete separation of thymidine and thymine was achieved in 6min and the minimum amount of thymine that could be detected was 0.8pmol. The assay was linear with reaction times, up to at least 4h, and protein concentrations up to at least 65μg/ml. Population analysis showed no differences in TP activity between man and women or with increasing age. [Copyright &y& Elsevier]

The identification of genetic factors associated with either responsiveness or resistance to 5-fluorouracil (5-FU) chemotherapy, as well as genetic factors predisposing patients to the development of severe 5-FU-associated toxicity, is increasingly being recognised as an important field of study. Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU). Although the role of tumoral levels as a prognostic factor for clinical responsiviness has not been firmly established, there is ample evidence that a deficiency of DPD is associated with severe toxicity after the administration of 5-FU. Patients with a partial DPD deficiency have an increased risk of developing grade IV neutropenia. In addition, the onset of toxicity occurred twice as fast compared with patients with a normal DPD activity. To date, 39 different mutations and polymorphisms have been identified in DPYD. The IVS14+1G>A mutation proved to be the most common one and was detected in 24–28% of all patients suffering from severe 5-FU toxicity. Thus, a deficiency of DPD appears to be an important pharmacogenetic syndrome. [Copyright &y& Elsevier]

The N-terminal amino acid sequences and the electrophoretic mobilities of the subunits VIIa, VIIb and VIIc of cytochrome c oxidase purified from human heart were investigated and compared with those from human skeletal muscle and from bovine heart. In purified human heart cytochrome c oxidase, both so-called 'heart-type' and 'liver-type' isoforms of subunit VIIa were found. The first 30 residues of the N-terminal amino acid sequences of these 'heart-type' and 'liver-type' subunits VIIa showed nine differences. The two isoforms of subunit VIIa in human heart were present in almost equal amounts, in contrast to the situation in skeletal muscle, where the 'heart-type' subunit VIIa was predominant. Therefore, our results imply that in human heart a cytochrome c oxidase isoform pattern is present that differs from that found in skeletal muscle. Subunits VIIb and Vile purified from human heart oxidase proved to be very similar to their bovine heart counterparts. Our direct demonstration of the presence of subunit VIIb, the sequence of which has only recently been identified in the bovine heart enzyme, suggests that human cytochrome c oxidase also contains 13 subunits. We found no evidence for the presence of different isoforms of subunit VIIc in cytochrome c oxidase from human heart and skeletal muscle. We observed clear differences in the electrophoretic mobility of the subunits VIIa,b,c between bovine and human cytochrome c oxidase. On Tricine/glycerol/SDS/polyacrylamide gels the 'hearttype' and 'liver-type' subunits VIIa present in human heart cytochrome c oxidase migrated with almost the same electrophoretic mobility. Subunit VIIb migrated only slightly faster than subunit VIIa, whereas Vile proved to have the highest electrophoretic mobility on Tricine/SDS/glycerol/ polyacrylamide gels. Our findings may have implications for the elucidation of certain tissue-specific cytochrome c oxidase deficiencies in man. [ABSTRACT FROM AUTHOR]

The treatment of cancer patients with 5-fluorouracil (5FU)-based chemotherapy can be accompanied by severe and sometimes lethal toxicity. Dihydropyrimidine dehydrogenase (DPD) plays a pivotal role in the metabolism of 5FU and as such, a deficiency of DPD has been recognized as an important risk factor, predisposing patients to the development of severe 5FU-associated toxicity. To date, screening of patients for the presence of a DPD deficiency prior to the treatment is not yet routinely performed. Taking into account the relatively small impact of adjuvant 5FU-based chemotherapy on survival, patients should be informed about the risks of the therapy and should be offered the possibility of testing for the presence of a DPD deficiency in advance of receiving such treatment. [ABSTRACT FROM AUTHOR]

Presents a correction to the article "Identification of Three Novel Mutations in the Dihydropyrimidine Dehydrogenase Gene Associated With Altered Pre-mRNA Splicing or Protein Function," by B. P. van Kuilenburg André, et al, published in the journal "Biological Chemistry."

Beta-ureidopropionase deficiency, caused by variants in UPB1 , has been reported in association with various neurodevelopmental phenotypes including intellectual disability, seizures and autism. We aimed to reassess the relationship between variants in UPB1 and a clinical phenotype. Literature review, calculation of carrier frequencies from population databases, long-term follow-up of a previously published case and reporting of additional cases. Fifty-three published cases were identified, and two additional cases are reported here. Of these, 14 were asymptomatic and four had transient neurological features; clinical features in the remainder were variable and included non-neurological presentations. Several of the variants previously reported as pathogenic are present in population databases at frequencies higher than expected for a rare condition. In particular, the variant most frequently reported as pathogenic, p.Arg326Gln, is very common among East Asians, with a carrier frequency of 1 in 19 and 1 in 907 being homozygous for the variant in gnomAD v2.1.1. Pending the availability of further evidence, UPB1 should be considered a 'gene of uncertain clinical significance'. Caution should be used in ascribing clinical significance to biochemical features of beta-ureidopropionase deficiency and/or UPB1 variants in patients with neurodevelopmental phenotypes. UPB1 is not currently suitable for inclusion in gene panels for reproductive genetic carrier screening. The relationship between beta-ureidopropionase deficiency due to UPB1 variants and clinical phenotypes is uncertain. • The relationship between damaging UPB1 variants and human phenotypes is uncertain. • Described beta-ureidopropionase deficiency cases do not have a consistent phenotype. • Loss of function UPB1 variants are common in the general population. • Care must be used in ascribing diagnostic value to variants in UPB1. • UPB1 should not be included on carrier screening panels. [ABSTRACT FROM AUTHOR]

β-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and catalyses the conversion of N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyric acid to β-alanine and β-aminoisobutyric acid, ammonia and CO 2. To date, only a limited number of genetically confirmed patients with a complete β-ureidopropionase deficiency have been reported. Here, we report on the clinical, biochemical and molecular findings of 10 newly identified β-ureidopropionase deficient individuals. Patients presented mainly with neurological abnormalities and markedly elevated levels of N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyric acid in urine. Analysis of UPB1, encoding β-ureidopropionase, showed 5 novel missense variants and two novel splice-site variants. Functional expression of the UPB1 variants in mammalian cells showed that recombinant ß-ureidopropionase carrying the p.Ala120Ser, p.Thr129Met, p.Ser300Leu and p.Asn345Ile variant yielded no or significantly decreased β-ureidopropionase activity. Analysis of the crystal structure of human ß-ureidopropionase indicated that the point mutations affect substrate binding or prevent the proper subunit association to larger oligomers and thus a fully functional β-ureidopropionase. A minigene approach showed that the intronic variants c.[364 + 6 T > G] and c.[916 + 1_916 + 2dup] led to skipping of exon 3 and 8, respectively, in the process of UPB1 pre-mRNA splicing. The c.[899C > T] (p.Ser300Leu) variant was identified in two unrelated Swedish β-ureidopropionase patients, indicating that β-ureidopropionase deficiency may be more common than anticipated. • β-Ureidopropionase patients presented mainly with neurological abnormalities. • Analysis of UPB1 showed 7 novel variants. • Missense mutations affected catalysis, global folding and structural integrity. • Intronic variants in UPB1 affected pre-mRNA splicing. [ABSTRACT FROM AUTHOR]

Summary: Background: Atopic dermatitis (AD) is characterized by immune dysregulations and an impaired skin barrier, including abnormalities in lipid organization. In the stratum corneum (SC), β‐glucocerebrosidase (GBA) mediates transformation of glucosylceramide (GlcCER) into ceramide (CER) and cholesterol into glucosylcholesterol (GlcChol). Alteration in GBA activity might contribute to skin barrier defects in AD. Objectives: To investigate GBA activity in the SC of children with AD before and after topical corticosteroid therapy and to compare it with healthy controls; to determine SC levels of GlcCER‐ and CER‐containing hydroxysphingosine base (GlcCER[H] and CER[H], respectively) and GlcChol; and to relate them to disease severity, skin barrier function and the local cytokine milieu. Methods: Lipid markers and cytokines of innate, T helper 1 and T helper 2 immunity were determined in SC collected from healthy children and from clinically unaffected skin of children with AD, before and after 6 weeks of therapy with topical corticosteroids. AD severity was assessed by Scoring Atopic Dermatitis and skin barrier function by transepidermal water loss (TEWL). Results: Baseline GBA activity and GlcChol levels were increased in children with AD but declined after therapy. CER[H] levels and the CER[H] to GlcCER[H] ratio were increased in AD. GBA activity and GlcChol correlated with TEWL and levels of multiple cytokines, especially interleukin‐1α and interleukin‐18. GlcChol was strongly associated with disease severity. Conclusions: We show increased GBA activity and levels of GlcChol in AD. Our data suggest an important role of inflammation in disturbed lipid processing. GBA activity or GlcChol might be useful biomarkers in the monitoring of therapeutic responses in AD. What is already known about this topic?Patients with atopic dermatitis (AD) have a reduced skin barrier, mainly caused by altered lipid organization.The mechanisms underlying these lipid anomalies are not fully understood but likely reflect both genetic abnormalities in AD skin and the local cutaneous inflammatory environment. What does this study add?We show increased activity of the ceramide‐generating enzyme β‐glucocerebrosidase in AD. Activity of this enzyme was correlated with the local cytokine milieu and declined after local corticosteroid therapy.We show that glucosylcholesterol levels in the stratum corneum are increased in AD.The function of glucosylcholesterol and the physiological consequences of increased levels are not clear yet; however, its levels were strongly correlated with skin barrier function: high transepidermal water loss strongly correlated with high levels of glucosylcholesterol. What is the translational message?Correction of cutaneous inflammation largely restores alterations in lipid metabolism in the stratum corneum of infants with AD. [ABSTRACT FROM AUTHOR]

Acid Sphingomyelinase Deficiency (ASMD), or Niemann-Pick type A/B disease, is a rare lipid storage disorder leading to accumulation of sphingomyelin and its precursors primarily in macrophages. The disease has a broad phenotypic spectrum ranging from a fatal infantile form with severe neurological involvement (the infantile neurovisceral type) to a primarily visceral form with different degrees of pulmonary, liver, spleen and skeletal involvement (the chronic visceral type). With the upcoming possibility of treatment with enzyme replacement therapy, the need for biomarkers that predict or reflect disease progression has increased. Biomarkers should be validated for their use as surrogate markers of clinically relevant endpoints. In this review, clinically important endpoints as well as biochemical and imaging markers of ASMD are discussed and potential new biomarkers are identified. We suggest as the most promising biomarkers that may function as surrogate endpoints in the future: diffusion capacity measured by spirometry, spleen volume, platelet count, low-density lipoprotein cholesterol, liver fibrosis measured with a fibroscan, lysosphingomyelin and walked distance in six minutes. Currently, no biomarkers have been validated. Several plasma markers of lipid-laden cells, fibrosis or inflammation are of high potential as biomarkers and deserve further study. Based upon current guidelines for biomarkers, recommendations for the validation process are provided. • ERT for ASMD is under development, necessitating biomarkers to monitor disease manifestations and response to therapy. • Currently most biochemical and imaging markers in ASMD have not been properly validated as biomarkers of the disease. • The best evaluated potential biomarkers for ASMD are diffusion capacity, spleen volume, platelet count, LDL cholesterol, liver fibrosis measured with fibroscan and LSM. • New markers that may be predictive of irreversible disease should be urgently explored to help decide when to initiate treatment. [ABSTRACT FROM AUTHOR]

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. • hCNT1 deficiency is a novel inborn error of pyrimidine metabolism • Variants affected the structure, glycosylation and half-life of hCNT1 • Patient with hCNT1 deficiency presented with uridine-cytidineuria • Combined deficiency of perforin and hCNT1 resulted in multi-organ failure [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Niemann-Pick disease type B is a hereditary rare condition caused by deficiency of the acid sphingomyelinase (ASM) that is needed for lysosomal hydrolysis of sphingomyelin to ceramide and phosphocholine. This deficiency leads to a massive accumulation of sphingomyelin in cells throughout the body, predominantly in the liver, spleen and lungs. Currently, there is no effective treatment available. Olipudase alfa (recombinant human acid sphingomyelinase; rhASM) is an investigational drug that has shown promising results. However, dose-dependent toxicity was observed in mice upon the intravenous administration of rhASM, potentially due to the systemic release of ceramide upon the extracellular degradation of sphingomyelin by rhASM. Using a nanocarrier to deliver the rhASM to cells could improve the therapeutic window by shielding the rhASM to prevent the off-target degradation of sphingomyelin. For this aim, we recombinantly expressed hASM in human cells and loaded it into different liposomal formulations at a drug-to-lipid ratio of 4% (w/w). Among four formulations, the liposomal rhASM formulation with the composition DPPC:DOPS:BMP:CHOL:DiD (59:20:10:10:1 mol%) was selected because of its superiority concerning the encapsulation efficiency of rhASM (21%) and cellular uptake by fibroblasts and macrophages. The selected liposomal rhASM formulation significantly reduced the accumulated lyso-sphingomyelin in NPD-B fibroblasts by 71%, part of this effect was stimulated by the used lipids, compared to 55% when using the free rhASM enzyme. More importantly, the undesired extracellular degradation of sphingomyelin was reduced when using the selected liposomal rhASM by 61% relative to the free rhASM. The presented in vitro data indicate that the liposomal rhASM is effective and may provide a safer intervention than free rhASM. [ABSTRACT FROM AUTHOR]

Obstructive cholestasis causes liver injury via accumulation of toxic bile acids (BAs). Therapeutic options for cholestatic liver disease are limited, partially because the available murine disease models lack translational value. Profiling of time-related changes following bile duct ligation (BDL) in Gold Syrian hamsters revealed a biochemical response similar to cholestatic patients in terms of BA pool composition, alterations in hepatocyte BA transport and signaling, suppression of BA production, and adapted BA metabolism. Hamsters tolerated cholestasis well for up to 28 days and progressed relatively slowly to fibrotic liver injury. Hepatocellular necrosis was absent, which coincided with preserved intrahepatic energy levels and only mild oxidative stress. The histological response to cholestasis in hamsters was similar to the changes seen in 17 patients with prolonged obstructive cholestasis caused by cholangiocarcinoma. Hamsters moreover upregulated hepatic fibroblast growth factor 15 ( Fgf15) expression in response to BDL, which is a cytoprotective adaptation to cholestasis that hitherto had only been documented in cholestatic human livers. Hamster models should therefore be added to the repertoire of animal models used to study the pathophysiology of cholestatic liver disease. [ABSTRACT FROM AUTHOR]

Dihydropyrimidinase (DHP) is the second enzyme of the pyrimidine degradation pathway and catalyzes the ring opening of 5,6-dihydrouracil and 5,6-dihydrothymine. To date, only 31 genetically confirmed patients with a DHP deficiency have been reported and the clinical, biochemical and genetic spectrum of DHP deficient patients is, therefore, still largely unknown. Here, we show that 4 newly identified DHP deficient patients presented with strongly elevated levels of 5,6-dihydrouracil and 5,6-dihydrothymine in urine and a highly variable clinical presentation, ranging from asymptomatic to infantile spasm and reduced white matter and brain atrophy. Analysis of the DHP gene (DPYS) showed the presence of 8 variants including 4 novel/rare missense variants and one novel deletion. Functional analysis of recombinantly expressed DHP mutants carrying the p.M250I, p.H295R, p.Q334R, p.T418I and the p.R490H variant showed residual DHP activities of 2.0%, 9.8%, 9.7%, 64% and 0.3%, respectively. The crystal structure of human DHP indicated that all point mutations were likely to cause rearrangements of loops shaping the active site, primarily affecting substrate binding and stability of the enzyme. The observation that the identified mutations were more prevalent in East Asians and the Japanese population indicates that DHP deficiency may be more common than anticipated in these ethnic groups. [ABSTRACT FROM AUTHOR]

A series of simplex cases have been reported under various diagnoses sharing early aging, especially evident in congenitally decreased subcutaneous fat tissue and sparse hair, bone dysplasia of the skull and fingers, a distinctive facial gestalt, and prenatal and postnatal growth retardation. For historical reasons, we suggest naming the entity Fontaine syndrome. Exome sequencing of four unrelated affected individuals showed that all carried the de novo missense variant c.649C>T (p.Arg217Cys) or c.650G>A (p.Arg217His) in SLC25A24 , a solute carrier 25 family member coding for calcium-binding mitochondrial carrier protein (SCaMC-1, also known as SLC25A24). SLC25A24 allows an electro-neutral and reversible exchange of ATP-Mg and phosphate between the cytosol and mitochondria, which is required for maintaining optimal adenine nucleotide levels in the mitochondrial matrix. Molecular dynamic simulation studies predict that p.Arg217Cys and p.Arg217His narrow the substrate cavity of the protein and disrupt transporter dynamics. SLC25A24 -mutant fibroblasts and cells expressing p.Arg217Cys or p.Arg217His variants showed altered mitochondrial morphology, a decreased proliferation rate, increased mitochondrial membrane potential, and decreased ATP-linked mitochondrial oxygen consumption. The results suggest that the SLC25A24 mutations lead to impaired mitochondrial ATP synthesis and cause hyperpolarization and increased proton leak in association with an impaired energy metabolism. Our findings identify SLC25A24 mutations affecting codon 217 as the underlying genetic cause of human progeroid Fontaine syndrome. [ABSTRACT FROM AUTHOR]

Background The level of plasma globotriaosylsphingosine (lysoGb3) is an indication of disease severity in Fabry disease (FD) and its decrease during enzyme replacement therapy could be a reflection of treatment efficacy. Early treatment of FD may improve clinical outcome, but data to support this hypothesis are scarce. In this study we compared lysoGb3 decrease after ERT initiation in men with classical FD who started ERT before the age of 25 (early-treatment) with those who started later in life (late-treatment). Methods Treatment naïve men with classical FD from three centers of excellence in Europe were included. Measurements of lysoGb3 levels by tandem mass spectroscopy and antibodies by an inhibitory assay were performed in a single laboratory. Results were adjusted for lysoGb3 at baseline, first ERT (i.e. agalsidase alfa or beta) and the average ERT dose. Results 85 patients were included, 21 in the early-treatment and 64 in the late-treatment group. LysoGb3 level at baseline was not different between the two groups (112 vs 114 nmol/L, p = 0.92). The adjusted odds ratio for reaching a lysoGb3 level < 20 nmol/L was 7.38 for the early-treatment versus late-treatment group (95% CI: 1.91–34.04, p = 0.006). The adjusted lysoGb3 levels one year after ERT initiation was 12.9 nmol/L lower in the early-treatment (95% CI: − 20.1–− 5.8, p < 0.001) compared to the late-treatment group. Conclusion The current retrospective cohort study shows that initiation of ERT at younger age in men with classical Fabry disease results in a better biochemical response. [ABSTRACT FROM AUTHOR]

I DPYD i screening showed that the patient was homozygous for I DPYD i *2A; measurement of DPD activity in PBMCs of this patient indicated absence of DPD activity. Pretreatment identification of the patient homozygous for I DPYD i *2A with complete DPD deficiency saved this patient from receipt of a full fluoropyrimidine dose, which most likely would have been fatal. For patients 2 and 3, samples were collected only on C1D1, on the same time points as for patient 1, except for the latest time point (the sample 10 hours after capecitabine intake was not collected for patient 2, and the last sample was collected at 12 hours instead of at 10 hours for patient 3). For patients 2 and 3, uracil levels were increased compared with reference levels (a value of 28.7 ng/mL for patient 2 and of 35.6 ng/mL for patient 3), and both values were within the top 1% of reference values. [Extracted from the article]

Abstract: The efficacy and mechanism of action of cisplatin and gemcitabine were investigated in a panel of neuroblastoma cell lines and multicellular tumor spheroids. In neuroblastoma spheroids, the combination of cisplatin and gemcitabine induced a complete cytostasis at clinical relevant concentrations. A synergistic effect was observed when cells were coincubated with both drugs or preincubated with gemcitabine first. These administration sequences resulted in NASS cells in decreased ERCC1 and XPA expression, two key proteins of the NER DNA repair system, and increased platinum adduct formation in DNA. Most of these phenomena were not observed in SJNB8 cells which might explain the lack of synergy between cisplatin and gemcitabine in SJNB8 cells. Our results showed favorable interactions between cisplatin and gemcitabine in 4 out of 5 cell lines. Therefore, we feel that inclusion of gemcitabine into cisplatin-containing regiments might be a promising new strategy for the treatment of neuroblastoma. [Copyright &y& Elsevier]

Abstract: Type 2 diabetes mellitus (T2DM) is by far the most common type of diabetes and is characterized by insulin resistance and altered insulin secretion. Some genes, such as the vitamin D receptor gene (VDR, NM_001017535; GI: 7421), involved in its metabolic pathway have been regarded as good candidates for T2DM. In this study, we investigated whether there was an association of VDR: g.59979G>T or c.1025-49G>T (ApaIG>T) and g.60058T>C or c.1056T>C (TaqIT>C) polymorphisms in the 3′ untranslated region of VDR with T2DM in a Turkish population. We collected blood samples from 241 individuals (72 patients with T2DM and 169 healthy individuals), and their DNA was isolated. Polymorphisms of the VDR were analyzed by DNA amplification with polymerase chain reaction and endonuclease digestion with ApaI and TaqI. Body mass index was higher in T2DM patients than in control individuals. However, the frequency of g.59979TT genotype in T2DM patients was not significantly increased compared to healthy subjects (37.5% vs. 36.1%, respectively). Although the VDR g.60058CC genotype in T2DM patients (19.4%) was higher than that in healthy individuals (11.2%), there was no significant difference. In the same way, there was no difference between the groups in allele frequencies. In conclusion, our study did not provide evidence for the association of two examined VDR polymorphisms with T2DM in a Turkish population. [Copyright &y& Elsevier]

Abstract: Additive to synergistic induction of apoptosis has been reported as a result of sequential incubation of cancer cells with a histone deacetylase inhibitor (HDACi) and gemcitabine (dFdC), a deoxycytidine analogue with proven anti-tumour activity. This study shows that sequential treatment of two neuroblastoma cell lines with BL1521, an HDACi, and dFdC resulted in strong antagonism despite a minor increase of dFdCTP incorporation into the DNA of one cell line. Furthermore, no difference in the deoxycytidine kinase activity was observed in response to BL1521. In conclusion, cancer cells that respond to HDACi with a cell cycle arrest and differentiation may no longer be sensitive to dFdC. [Copyright &y& Elsevier]

Abstract: Neuroblastoma is a childhood tumor with a poor survival in advanced stage disease despite intensive chemotherapeutic regimes. The new histone deacetylase (HDAC) inhibitor BL1521 has shown promising results in neuroblastoma. Inhibition of HDAC resulted in a decrease in proliferation and metabolic activity, induction of apoptosis and differentiation of neuroblastoma cells. In order to elucidate the mechanism mediating the effects of BL1521 on neuroblastoma cells, we investigated the gene expression profile of an MYCN single copy (SKNAS) and an MYCN amplified (IMR32) neuroblastoma cell line after treatment with BL1521 using the Affymetrix oligonucleotide array U133A. An altered expression of 255 genes was observed in both neuroblastoma cell lines. The majority of these genes were involved in gene expression, cellular metabolism, and cell signaling. We observed changes in the expression of vital genes belonging to the cell cycle (cyclin D1 and CDK4) and apoptosis (BNIP3, BID, and BCL2) pathway in response to BL1521. The expression of 37 genes was altered by both BL1521 and Trichostatin A, which could indicate a common gene set regulated by different HDAC inhibitors. BL1521 treatment changed the expression of a number of MYCN-associated genes. Several genes in the Wnt and the Delta/Notch pathways were changed in response to BL1521 treatment, suggesting that BL1521 is able to induce the differentiation of neuroblastoma cells into a more mature phenotype. [Copyright &y& Elsevier]

Abstract: Histone deacetylase inhibitors (HDACi) have been discovered as potential drugs for cancer treatment. The effect of BL1521, a novel HDACi, on the cell cycle distribution and the induction of apoptosis was investigated in a panel of MYCN single copy and MYCN amplified neuroblastoma cell lines. BL1521 arrested neuroblastoma cells in the G1 phase and induced up to 30% apoptosis. Downregulation of CDK4, upregulation of p21WAF1/CIP1 and an increase of hypophosphorylated retinoblastoma protein were observed, indicating a possible mechanism for the cell-cycle arrest. BL1521 also induced downregulation of p27, which may underlie the observed induction of apoptosis. [Copyright &y& Elsevier]

Neuroblastoma is a childhood cancer arising from the sympathetic nervous system. Disseminated neuroblastoma has a poor prognosis despite intensive multimodality treatment. Histone deacetylases (HDACs) were recently discovered as a potential target for pharmacological gene therapy in cancer. HDACs have an important function in regulating DNA packaging in chromatin, thereby affecting the transcription of genes.In this paper, we tested the efficacy of a newly developed histone deacetylase inhibitor, BL1521, on neuroblastoma in vitro by investigating the changes in: acetylation of histone H3, in situ HDAC activity, p21WAF1/CIP1 and MYCN expression, metabolic activity, proliferation, morphology and the amount of apoptosis present.BL1521 inhibited the in situ HDAC activity of a panel of neuroblastoma cell lines by at least 85%. Western analysis showed an increase of histone H3 acetylation in neuroblastoma cells after incubation with BL1521. Northern analysis showed an increase in the expression of p21WAF1/CIP1 and a decrease in the expression of MYCN in neuroblastoma cells after incubation with BL1521. Proliferation as well as the metabolic activity of neuroblastoma cells decreased significantly in response to treatment with BL1521, regardless of the MYCN status of the cells. BL1521 induced poly-(ADP-ribose) polymerase cleavage in a time- and dose-dependent manner, indicating the induction of apoptosis. Furthermore, when compared to the HDAC inhibitors Trichostatin A and 4-phenylbutyrate, BL1521 has an intermediate efficacy. Our results show that BL1521 is a potent inhibitor of HDAC and that HDACs are an attractive target for selective chemotherapy in neuroblastoma. [Copyright &y& Elsevier]

A rapid yet reliable chemical diagnosis for dihydropyrimidine dehydrogenase (DHPD) deficiency, and possibly dihydropyrimidinase (DHP) deficiency in cancer patients, prior to therapy with pyrimidine analogues such as 5-fluorouracil, is desired for prevention of severe side-effects by these drugs. We have reported the basic separation and quantitation technology for pyrimidine metabolites using gas chromatography–mass spectrometry. A proposal to use the number (n) of standard deviations (SD) above the normal mean, as the index of the excessive urinary excretion of the metabolites appears not to be commonly used. When used, the values were too small, such as two or three, even in genetic disorders. Here, we applied the method to 11 urine specimens from proven cases including two DHP carriers and proved how specific the method is, because “n”-values were markedly large for thymine (T), uracil (U) and/or dihydrothymine (DHT) and dihydrouracil (DHU). In three cases with DHPD deficiency, two were siblings, one with symptoms and the other without, n was 12 for T and 5.9 for U, and 5-hydroxymethyluracil was distinctly detected. These values indicate that the nature of genetic mutation relates closely to the degree of metabolite accumulation in pyrimidine disorders. In six patients with DHP deficiency, n was 8.4–12 for DHT and 7.2–11 for DHU. Many mutations are known for both genes and the assay of residual enzyme activity may be time-consuming or invasive especially for those with DHP deficiency. Thus, this noninvasive yet comprehensive urinalysis has great value for those without a family history, as the first trial, before DNA or the enzyme assay. Our findings again raise the question whether the metabolic block really causes the symptoms found in pyrimidine disorders. [Copyright &y& Elsevier]

In this paper, it is demonstrated that all-trans, 9-cis and 13-cis retinoic acid (RA) decreased the sensitivity of SK-N-BE(2)c neuroblastoma cells towards the chemotherapeutic agent cyclopentenyl cytosine (CPEC), a potent inhibitor of cytosine-5′-triphosphate synthetase. Retinoic acid attenuated CPEC-induced apoptosis as reflected by a decreased caspase-3 induction. Retinoic acid decreased the accumulation of CPEC, whereas the salvage of cytidine was strongly increased. Metabolic labeling studies using [3H]uridine showed a strongly decreased biosynthesis of CTP via CTP synthetase. Retinoic acid likely confers resistance of neuroblastoma cells to CPEC in part by slowing down proliferation, and in part by shifting the synthesis of CTP towards the salvage of cytidine, thereby bypassing CTP synthetase. [Copyright &y& Elsevier]

Abstract: Objectives: Evaluation of a non-synonymous mutation associated with dihydropyrimidine dehydrogenase (DPD) deficiency. Design and methods: DPD enzyme analysis, mutation analysis and molecular dynamics simulations based on the 3D-model of DPD. Results: The substitution Lys63Glu is likely to affect the FAD binding pocket within the DPD protein and contributes to a near-complete DPD deficiency in a patient with developmental retardation. Conclusions: Like other DPD variants attenuating FAD binding, Lys63Glu should be included in screening for DPD deficiency. [Copyright &y& Elsevier]

Fabry Disease (FD) is a rare, X-linked, lysosomal storage disease that mainly causes renal, cardiac and cerebral complications. Enzyme replacement therapy (ERT) with recombinant alpha-galactosidase A is available, but approximately 50% of male patients with classical FD develop inhibiting anti-drug antibodies (iADAs) that lead to reduced biochemical responses and an accelerated loss of renal function. Once immunization has occurred, iADAs tend to persist and tolerization is hard to achieve. Here we developed a pre-treatment prediction model for iADA development in FD using existing data from 120 classical male FD patients from three European centers, treated with ERT. We found that nonsense and frameshift mutations in the α-galactosidase A gene (p = 0.05), higher plasma lysoGb3 at baseline (p < 0.001) and agalsidase beta as first treatment (p = 0.006) were significantly associated with iADA development. Prediction performance of a Random Forest model, using multiple variables (AUC-ROC: 0.77) was compared to a logistic regression (LR) model using the three significantly associated variables (AUC-ROC: 0.77). The LR model can be used to determine iADA risk in individual FD patients prior to treatment initiation. This helps to determine in which patients adjusted treatment and/or immunomodulatory regimes may be considered to minimize iADA development risk. [ABSTRACT FROM AUTHOR]