Biochemical Characterizations of Human TMPK Mutations Identified in Patients with Severe Microcephaly: Single Amino Acid Substitutions Impair Dimerization and Abolish Their Catalytic Activity

Deoxythymidylate kinase (TMPK) is a key enzyme in the synthesis of deoxythymidine triphosphate (dTTP). Four TMPK variants (P81L, A99T, D128N, and a frameshift) have been identified in human patients who suffered from severe neurodegenerative diseases. However, the impact of these mutations on TMPK function has not been clarified. Here we show that in fibroblasts derived from a patient, the P81L and D128N mutations led to a complete loss of TMPK activity in mitochondria and extremely low and unstable TMPK activity in cytosol. Despite the lack of TMPK activity, the patient-derived fibroblasts apparently grew normal. To investigate the impact of the mutations on the enzyme function, the mutant TMPKs were expressed, purified, and characterized. The wild-type TMPK mainly exists as a dimer with high substrate binding affinity, that is, low KM value and high catalytic efficiency, that is, kcat/KM. In contrast, all mutants were present as monomers with dramatically reduced substrate binding affinity and catalytic efficiencies. Based on the human TMPK structure, none of the mutated amino acids interacted directly with the substrates. By structural analysis, we could explain why the respective amino acid substitutions could drastically alter the enzyme structure and catalytic function. In conclusion, TMPK mutations identified in patients represent loss of function mutations but surprisingly the proliferation rate of the patient-derived fibroblasts was normal, suggesting the existence of an alternative and hitherto unknown compensatory TMPK-like enzyme for dTTP synthesis. Further studies of the TMPK enzymes will help to elucidate the role of TMPK in neuropathology

Serum bile acids associate with liver volume in polycystic liver disease and decrease upon treatment with lanreotide

Background: Polycystic liver disease (PLD) is a common extrarenal manifestation of autosomal dominant polycystic kidney disease (ADPKD). Bile acids may play a role in PLD pathogenesis. We performed a post-hoc exploratory analysis of bile acids in ADPKD patients, who had participated in a trial on the effect of a somatostatin analogue. Our hypothesis was that serum bile acid levels increase in PLD, and that lanreotide, which reduces liver growth, may also reduce bile acid levels. Furthermore, in PLD, urinary excretion of bile acids might contribute to renal disease. Methods:With liquid chromatography-mass spectrometry, 11 bile acids in serum and 6 in urine were quantified in 105 PLD ADPKD patients and 52 age-, sex-, mutation- and eGFR-matched non-PLD ADPKD patients. Sampling was done at baseline and after 120 weeks of either lanreotide or standard care. Results: Baseline serum levels of taurine- and glycine-conjugated bile acids were higher in patients with larger livers. In PLD patients, multiple bile acids decreased upon treatment with lanreotide but remained stable in untreated subjects. Changes over time did not correlate with changes in liver volume. Urine bile acid levels did not change and did not correlate with renal disease progression. Conclusion: In ADPKD patients with PLD, baseline serum bile acids were associated with liver volume. Lanreotide reduced bile acid levels and has previously been shown to reduce liver volume. However, in this study, the decrease in bile acids was not associated with the change in liver volume.</p

Erythrocyte Inosine triphosphatase activity: A potential biomarker for adverse events during combination antiretroviral therapy for HIV

The purine analogues tenofovir and abacavir are precursors of potential substrates for the enzyme Inosine 5’-triphosphate pyrophosphohydrolase (ITPase). Here, we investigated the association of ITPase activity and ITPA genotype with the occurrence of adverse events (AEs) during combination antiretroviral therapy (cART) for human immunodeficiency virus (HIV) infection. In 393 adult HIV-seropositive patients, AEs were defined as events that led to stop of cART regimen. ITPase activity 4 mmol IMP/mmol Hb/hour was considered as normal. ITPA genotype was determined by testing two ITPA polymorphisms: c.94C>A (p. Pro32Thr, rs1127354) and c.124+21A>C (rs7270101). Logistic regression analysis determined odds ratios for developing AEs. In tenofovir-containing regimens decreased ITPase activity was associated with less AEs (p = 0.01) and longer regimen duration (p = 0.001). In contrast, in abacavir-containing regimens decreased ITPase activity was associated with more AEs (crude p = 0.02) and increased switching of medication due to AEs (p = 0.03). ITPA genotype wt/wt was significantly associated with an increase in the occurrence of AEs in tenofovir-containing regimens. Decreased ITPase activity seems to be protective against occurrence of AEs in tenofovir-containing cART, while it is associated with an increase in AEs in abacavir-containing regimens

Kidney and vascular function in adult patients with hereditary fructose intolerance

Objective: Previous studies have shown that patients with hereditary fructose intolerance (HFI) are characterized by a greater intrahepatic triglyceride content, despite a fructose-restricted diet. The present study aimed to examine the long-term consequences of HFI on other aldolase-B-expressing organs, i.e. the kidney and vascular endothelium. Methods: Fifteen adult HFI patients were compared to healthy control individuals matched for age, sex and body mass index. Aortic stiffness was assessed by carotid-femoral pulse wave velocity (cf-PWV) and endothelial function by peripheral arterial tonometry, skin laser doppler flowmetry and the endothelial function biomarkers soluble E-selectin [sE-selectin] and von Willebrand factor. Serum creatinine and cystatin C were measured to estimate the glomerular filtration rate (eGFR). Urinary glucose and amino acid excretion and the ratio of tubular maximum reabsorption of phosphate to GFR (TmP/GFR) were determined as measures of proximal tubular function. Results: Median systolic blood pressure was significantly higher in HFI patients (127 versus 122 mmHg, p = .045). Pulse pressure and cf-PWV did not differ between the groups (p = .37 and p = .49, respectively). Of all endothelial function markers, only sE-selectin was significantly higher in HFI patients (p = .004). eGFR was significantly higher in HFI patients than healthy controls (119 versus 104 ml/min/1.73m2, p = .001, respectively). All measurements of proximal tubular function did not differ significantly between the groups. Conclusions: Adult HFI patients treated with a fructose-restricted diet are characterized by a higher sE-selectin level and slightly higher systolic blood pressure, which in time could contribute to a greater cardiovascular risk. The exact cause and, hence, clinical consequences of the higher eGFR in HFI patients, deserves further study.</p

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<lt;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

Impaired fertility and motor function in a zebrafish model for classic galactosemia

Classic galactosemia is a genetic disorder of galactose metabolism, caused by severe deficiency of galactose-1-phosphate uridylyltransferase (GALT) enzyme activity due to mutations of the GALT gene. Its pathogenesis is still not fully elucidated, and a therapy that prevents chronic impairments is lacking. In order to move research forward, there is a high need for a novel animal model, which allows organ studies throughout development and high-throughput screening of pharmacologic compounds. Here, we describe the generation of a galt knockout zebrafish model and present its phenotypical characterization. Using a TALEN approach, a galt knockout line was successfully created. Accordingly, biochemical assays confirm essentially undetectable galt enzyme activity in homozygotes. Analogous to humans, galt knockout fish accumulate galactose-1-phosphate upon exposure to exogenous galactose. Furthermore, without prior exposure to exogenous galactose, they exhibit reduced motor activity and impaired fertility (lower egg quantity per mating, higher number of unsuccessful crossings), resembling the human phenotype(s) of neurological sequelae and subfertility. In conclusion, our galt knockout zebrafish model for classic galactosemia mimics the human phenotype(s) at biochemical and clinical levels. Future studies in our model will contribute to improved understanding and management of this disorder. Electronic supplementary material The online version of this article (doi:10.1007/s10545-017-0071-1) contains supplementary material, which is available to authorized users

Persistent metabolic changes in HIV-infected patients during the first year of combination antiretroviral therapy

The HIV-human metabolic relationship is a complex interaction convoluted even more by antiretroviral therapy (cART) and comorbidities. The ability of cART to undo the HIV induced metabolic dysregulation is unclear and under-investigated. Using targeted metabolomics and multiplex immune biomarker analysis, we characterized plasma samples obtained from 18 untreated HIV-1-infected adult patients and compared these to a non-HIV infected (n = 23) control population. The biogenic amine perturbations during an untreated HIV infection implicated altered tryptophan- nitrogen- and muscle metabolism. Furthermore, the lipid profiles of untreated patients were also significantly altered compared to controls. In untreated HIV infection, the sphingomyelins and phospholipids correlated negatively to markers of infection IP-10 and sIL-2R whereas a strong association was found between triglycerides and MCP-1. In a second cohort, we characterized plasma samples obtained from 28 HIV-1-infected adult patients before and 12 months after the start of cART, to investigate the immune-metabolic changes associated with cART. The identified altered immune-metabolic pathways of an untreated HIV infection showed minimal change after 12 months of cART. In conclusion, 12 months of cART impacts only mildly on the metabolic dysregulation underlying an untreated HIV infection and provide insights into the comorbidities present in virally suppressed HIV patients

DTYMK is essential for genome integrity and neuronal survival

Nucleotide metabolism is a complex pathway regulating crucial cellular processes such as nucleic acid synthesis, DNA repair and proliferation. This study shows that impairment of the biosynthesis of one of the building blocks of DNA, dTTP, causes a severe, early-onset neurodegenerative disease. Here, we describe two unrelated children with bi-allelic variants in DTYMK, encoding dTMPK, which catalyzes the penultimate step in dTTP biosynthesis. The affected children show severe microcephaly and growth retardation with minimal neurodevelopment. Brain imaging revealed severe cerebral atrophy and disappearance of the basal ganglia. In cells of affected individuals, dTMPK enzyme activity was minimal, along with impaired DNA replication. In addition, we generated dtymk mutant zebrafish that replicate this phenotype of microcephaly, neuronal cell death and early lethality. An increase of ribonucleotide incorporation in the genome as well as impaired responses to DNA damage were observed in dtymk mutant zebrafish, providing novel pathophysiological insights. It is highly remarkable that this deficiency is viable as an essential component for DNA cannot be generated, since the metabolic pathway for dTTP synthesis is completely blocked. In summary, by combining genetic and biochemical approaches in multiple models we identified loss-of-function of DTYMK as the cause of a severe postnatal neurodegenerative disease and highlight the essential nature of dTTP synthesis in the maintenance of genome stability and neuronal survival