Improved diagnostics of purine and pyrimidine metabolism disorders using LC-MS/MS and its clinical application.

OBJECTIVES: To develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify 41 different purine and pyrimidine (PuPy) metabolites in human urine to allow detection of most known disorders in this metabolic pathway and to determine reference intervals. METHODS: Urine samples were diluted with an aqueous buffer to minimize ion suppression. For detection and quantification, liquid chromatography was combined with electrospray ionization, tandem mass spectrometry and multiple reaction monitoring. Transitions and instrument settings were established to quantify 41 analytes and nine stable-isotope-labeled internal standards (IS). RESULTS: The established method is precise (intra-day CV: 1.4-6.3%; inter-day CV: 1.3-15.2%), accurate (95.2% external quality control results within ±2 SD and 99.0% within ±3 SD; analyte recoveries: 61-121%), sensitive and has a broad dynamic range to quantify normal and pathological metabolite concentrations within one run. All analytes except aminoimidazole ribonucleoside (AIr) are stable before, during and after sample preparation. Moreover, analytes are not affected by five cycles of freeze-thawing (variation: -5.6 to 7.4%), are stable in thymol (variation: -8.4 to 12.9%) and the lithogenic metabolites also in HCl conserved urine. Age-dependent reference intervals from 3,368 urine samples were determined and used to diagnose 11 new patients within 7 years (total performed tests: 4,206). CONCLUSIONS: The presented method and reference intervals enable the quantification of 41 metabolites and the potential diagnosis of up to 25 disorders of PuPy metabolism.

Peripheral biopterin and neopterin in schizophrenia and depression.

Increasing evidence points to a causal involvement of inflammation in the pathogenesis of neuropsychiatric disorders, including major depressive disorder (MDD) and schizophrenia (SZ). Neopterin and biopterin may link peripheral immune system activation and central neurotransmitter alterations. However, it is not fully established whether these alterations are transdiagnostic or disorder-specific and whether they are associated with reward-related psychopathologies. We investigated group differences in neopterin and biopterin in the plasma of healthy comparison (HC) (n=19), SZ (n=45) and MDD (n=43) participants. We then correlated plasma proteins with CRP as a measure for inflammation. Lastly, plasma proteins were correlated with the reward-related psychopathological domain apathy. We found a trend-level difference in biopterin levels and no significant difference in neopterin levels between groups. Within both patient groups, but not HC, we show a significant positive correlation of CRP with neopterin but not with biopterin. Further, we observed no significant correlations of plasma proteins with reward-related psychopathology in HC, MDD or SZ. While our study shows trend-level alterations of biopterin with relevance for future research, it does not support the hypothesis that peripheral neopterin or biopterin are associated with reward-related psychopathology.

Distinct Proteomic, Transcriptomic, and Epigenetic Stress Responses in Dorsal and Ventral Hippocampus.

BACKGROUND: Acutely stressful experiences can trigger neuropsychiatric disorders and impair cognitive processes by altering hippocampal function. Although the intrinsic organization of the hippocampus is highly conserved throughout its long dorsal-ventral axis, the dorsal (anterior) hippocampus mediates spatial navigation and memory formation, whereas the ventral (posterior) hippocampus is involved in emotion regulation. To understand the molecular consequences of stress, detailed genome-wide screens are necessary and need to distinguish between dorsal and ventral hippocampal regions. While transcriptomic screens have become a mainstay in basic and clinical research, proteomic methods are rapidly evolving and hold even greater promise to reveal biologically and clinically relevant biomarkers. METHODS: Here, we provide the first combined transcriptomic (RNA sequencing) and proteomic (sequential window acquisition of all theoretical mass spectra [SWATH-MS]) profiling of dorsal and ventral hippocampus in mice. We used three different acute stressors (novelty, swim, and restraint) to assess the impact of stress on both regions. RESULTS: We demonstrated that both hippocampal regions display radically distinct molecular responses and that the ventral hippocampus is particularly sensitive to the effects of stress. Separately analyzing these structures greatly increased the sensitivity to detect stress-induced changes. For example, protein interaction cluster analyses revealed a stress-responsive epigenetic network around histone demethylase Kdm6b restricted to the ventral hippocampus, and acute stress reduced methylation of its enzymatic target H3K27me3. Selective Kdm6b knockdown in the ventral hippocampus led to behavioral hyperactivity/hyperresponsiveness. CONCLUSIONS: These findings underscore the importance of considering dorsal and ventral hippocampus separately when conducting high-throughput molecular analyses, which has important implications for fundamental research as well as clinical studies.

Tetrahydrobiopterin treatment reduces brain L-Phe but only partially improves serotonin in hyperphenylalaninemic ENU1/2 mice.

Hyperphenylalaninemia (HPA) caused by hepatic phenylalanine hydroxylase (PAH) deficiency has severe consequences on brain monoamine neurotransmitter metabolism. We have studied monoamine neurotransmitter status and the effect of tetrahydrobiopterin (BH4) treatment in Pahenu1/enu2 (ENU1/2) mice, a model of partial PAH deficiency. These mice exhibit elevated blood L-phenylalanine (L-Phe) concentrations similar to that of mild hyperphenylalaninemia (HPA), but brain levels of L-Phe are still ~5-fold elevated compared to wild-type. We found that brain L-tyrosine, L-tryptophan, BH4 cofactor and catecholamine concentrations, and brain tyrosine hydroxylase (TH) activity were normal in these mice but that brain serotonin, 5-hydroxyindolacetic acid (5HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) content, and brain TH protein, as well as tryptophan hydroxylase type 2 (TPH2) protein levels and activity were reduced in comparison to wild-type mice. Parenteral L-Phe loading conditions did not lead to significant changes in brain neurometabolite concentrations. Remarkably, enteral BH4 treatment, which normalized brain L-Phe levels in ENU1/2 mice, lead to only partial recovery of brain serotonin and 5HIAA concentrations. Furthermore, indirect evidence indicated that the GTP cyclohydrolase I (GTPCH) feedback regulatory protein (GFRP) complex may be a sensor for brain L-Phe elevation to ameliorate the toxic effects of HPA. We conclude that BH4 treatment of HPA toward systemic L-Phe lowering reverses elevated brain L-Phe content but the recovery of TPH2 protein and activity as well as serotonin levels is suboptimal, indicating that patients with mild HPA and mood problems (depression or anxiety) treated with the current diet may benefit from supplementation with BH4 and 5-OH-tryptophan.

Heterogeneous clinical spectrum of DNAJC12-deficient hyperphenylalaninemia: from attention deficit to severe dystonia and intellectual disability.

BACKGROUND: Autosomal recessive mutations in DNAJC12, encoding a cochaperone of HSP70 with hitherto unknown function, were recently described to lead to hyperphenylalaninemia, central monoamine neurotransmitter (dopamine and serotonin) deficiency, dystonia and intellectual disability in six subjects affected by homozygous variants. OBJECTIVE: Patients exhibiting hyperphenylalaninemia in whom deficiencies in hepatic phenylalanine hydroxylase and tetrahydrobiopterin cofactor metabolism had been excluded were subsequently analysed for DNAJC12 variants. METHODS: To analyse DNAJC12, genomic DNA from peripheral blood (Sanger sequencing), as well as quantitative messenger RNA (Real Time Quantitative Polymerase Chain Reaction (RT-qPCR)) and protein expression (Western blot) from primary skin fibroblasts were performed. RESULTS: We describe five additional patients from three unrelated families with homozygosity/compound heterozygosity in DNAJC12 with three novel variants: c.85delC/p.Gln29Lysfs*38, c.596G>T/p.*199Leuext*42 and c.214C>T/p.(Arg72*). In contrast to previously reported DNAJC12-deficient patients, all five cases showed a very mild neurological phenotype. In two subjects, cerebrospinal fluid and primary skin fibroblasts were analysed showing similarly low 5-hydroxyindolacetic acid and homovanillic acid concentrations but more reduced expressions of mRNA and DNAJC12 compared with previously described patients. All patients responded to tetrahydrobiopterin challenge by lowering blood phenylalanine levels. CONCLUSIONS: DNAJC12 deficiency appears to result in a more heterogeneous neurological phenotype than originally described. While early identification and institution of treatment with tetrahydrobiopterin and neurotransmitter precursors is crucial to ensure optimal neurological outcome in DNAJC12-deficient patients with a severe phenotype, optimal treatment for patients with a milder phenotype remains to be defined.

Mildly compromised tetrahydrobiopterin cofactor biosynthesis due to Pts variants leads to unusual body fat distribution and abdominal obesity in mice.

Tetrahydrobiopterin (BH4) is an essential cofactor for the aromatic amino acid hydroxylases, alkylglycerol monooxygenase, and nitric oxide synthases (NOS). Inborn errors of BH4 metabolism lead to severe insufficiency of brain monoamine neurotransmitters while augmentation of BH4 by supplementation or stimulation of its biosynthesis is thought to ameliorate endothelial NOS (eNOS) dysfunction, to protect from (cardio-) vascular disease and/or prevent obesity and development of the metabolic syndrome. We have previously reported that homozygous knock-out mice for the 6-pyruvolytetrahydropterin synthase (PTPS; Pts-ko/ko) mice with no BH4 biosynthesis die after birth. Here we generated a Pts-knock-in (Pts-ki) allele expressing the murine PTPS-p.Arg15Cys with low residual activity (15% of wild-type in vitro) and investigated homozygous (Pts-ki/ki) and compound heterozygous (Pts-ki/ko) mutants. All mice showed normal viability and depending on the severity of the Pts alleles exhibited up to 90% reduction of PTPS activity concomitant with neopterin elevation and mild reduction of total biopterin while blood L-phenylalanine and brain monoamine neurotransmitters were unaffected. Yet, adult mutant mice with compromised PTPS activity (i.e., Pts-ki/ko, Pts-ki/ki or Pts-ko/wt) had increased body weight and elevated intra-abdominal fat. Comprehensive phenotyping of Pts-ki/ki mice revealed alterations in energy metabolism with proportionally higher fat content but lower lean mass, and increased blood glucose and cholesterol. Transcriptome analysis indicated changes in glucose and lipid metabolism. Furthermore, differentially expressed genes associated with obesity, weight loss, hepatic steatosis, and insulin sensitivity were consistent with the observed phenotypic alterations. We conclude that reduced PTPS activity concomitant with mildly compromised BH4-biosynthesis leads to abnormal body fat distribution and abdominal obesity at least in mice. This study associates a novel single gene mutation with monogenic forms of obesity.

Brain catecholamine depletion and motor impairment in a Th knock-in mouse with type B tyrosine hydroxylase deficiency.

Tyrosine hydroxylase catalyses the hydroxylation of L-tyrosine to l-DOPA, the rate-limiting step in the synthesis of catecholamines. Mutations in the TH gene encoding tyrosine hydroxylase are associated with the autosomal recessive disorder tyrosine hydroxylase deficiency, which manifests phenotypes varying from infantile parkinsonism and DOPA-responsive dystonia, also termed type A, to complex encephalopathy with perinatal onset, termed type B. We generated homozygous Th knock-in mice with the mutation Th-p.R203H, equivalent to the most recurrent human mutation associated with type B tyrosine hydroxylase deficiency (TH-p.R233H), often unresponsive to l-DOPA treatment. The Th knock-in mice showed normal survival and food intake, but hypotension, hypokinesia, reduced motor coordination, wide-based gate and catalepsy. This phenotype was associated with a gradual loss of central catecholamines and the serious manifestations of motor impairment presented diurnal fluctuation but did not improve with standard l-DOPA treatment. The mutant tyrosine hydroxylase enzyme was unstable and exhibited deficient stabilization by catecholamines, leading to decline of brain tyrosine hydroxylase-immunoreactivity in the Th knock-in mice. In fact the substantia nigra presented an almost normal level of mutant tyrosine hydroxylase protein but distinct absence of the enzyme was observed in the striatum, indicating a mutation-associated mislocalization of tyrosine hydroxylase in the nigrostriatal pathway. This hypomorphic mouse model thus provides understanding on pathomechanisms in type B tyrosine hydroxylase deficiency and a platform for the evaluation of novel therapeutics for movement disorders with loss of dopaminergic input to the striatum.

Prolonged survival upon ultrasound-enhanced doxorubicin delivery in two syngenic glioblastoma mouse models.

Glioblastoma multiforme (GBM) is the most common and most aggressive malignant primary brain tumor in humans with a very poor prognosis. Chemotherapeutical treatment of GBMs is limited by the blood-brain barrier (BBB). This physical and metabolic barrier separates the blood from the brain parenchyma and prevents the entry of toxins but also of potentially useful chemotherapeutics from the blood into the brain. Microbubble-enhanced focused ultrasound (MB-FUS) has been proposed to disrupt locally and reversibly the BBB to facilitate diffusion of drugs from the micro vasculature into brain tissue. The present study investigates the feasibility and the safety of such an approach in two syngenic mouse models of GBM (GL261 and SMA-560). Local doxorubicin (DOX) concentration in MB-FUS sonicated normal brain tissue as well as in brain tumor tissue was increased as compared to the unsonicated control tissue in the contralateral hemisphere. Moreover, ultrasound mediated BBB disruption, in combination with DOX therapy, resulted in a significant increase of survival and in a slower disease progression in the two syngenic GBM mouse models. In conclusion, our results confirm that MB-ultrasound might ultimately be an effective technology to improve the therapy of GBM, and they provide for the first time evidence that combining MB-FUS with DOX treatment is effective in syngenic mouse models for GBM which can serve as preclinical models to study the impact of immune system on the therapeutic application of MB-FUS chemotherapy.

Pseudoexon exclusion by antisense therapy in 6-pyruvoyl-tetrahydropterin synthase deficiency.

Antisense oligonucleotide therapy to modulate splicing mutations in inherited diseases is emerging as a treatment option also for metabolic defects. In this article, we report the effect of cellular antisense therapy to suppress pseudoexon activation in primary dermal fibroblasts from patients with mutations in the PTS gene encoding 6-pyruvoyltetrahydropterin synthase (PTPS), which leads to tetrahydrobiopterin and monoamine neurotransmitter deficiency. Pathogenic inclusion of SINE or LINE-derived cryptic exons in different PTPS patients due to the intronic mutations c.84-322A>T, c.163 + 695_163 + 751del57, or c.164-712A>T was demonstrated by transcript analysis in fibroblasts and minigene ex vivo assays. Antisense morpholino oligonucleotides (AMOs) directed to the pseudoexons 3' or 5' splice sites were designed with the aim of preventing the pathological pseudoexon inclusion. At the time of AMO transfection, we investigated patients' cells for correct PTS-mRNA splicing and functional recovery of the PTPS protein. Transcriptional profiling after 24 hr posttransfection revealed a dose- and sequence-specific recovery of normal splicing. Furthermore, PTPS enzyme activity in all three patients' fibroblasts and the pterin profile were close to normal values after antisense treatment. Our results demonstrate proof-of-concept for pseudoexon exclusion therapy using AMO in inherited metabolic disease.

Diagnosis of tetrahydrobiopterin deficiency using filter paper blood spots: further development of the method and 5 years experience.

In every newborn with even mild hyperphenylalaninemia (HPA) tetrahydrobiopterin (BH(4)) deficiencies need to be excluded as soon as possible. Differential diagnosis is most commonly performed by analysis of urinary neopterin and biopterin. In 2005 a new method for the measurement of neopterin, biopterin and other pterins in dried blood spot (DBS) on filter paper was introduced. In order to evaluate the usefulness of this method as a standard tool for differential diagnosis of HPAs we analyzed neopterin, biopterin, pterin and dihydropteridine reductase activity in DBS from 362 patients with HPA over the period of five years. Age-dependent reference values were established for the HPA population. Sixty-four patients with BH(4) deficiency (27 patients with 6-pyruvoyl-tetrahydropterin synthase deficiency, seven with GTP cyclohydrolase I deficiency, and 30 with dihydropteridine reductase) were identified. Reference values for neopterin and biopterin in DBS were calculated for each of the variants. 6-pyruvoyl-tetrahydropterin synthase and GTP cyclohydrolase I deficiency can be diagnosed by neopterin and biopterin analysis alone, while for diagnosis of dihydropteridine reductase deficiency additional determination of enzyme activity from the same DBS is essential. Regarding test sensitivity, the interpretation of neopterin and biopterin concentration per hemoglobin is more valid than the interpretation of neopterin and biopterin per liter. Percentage of biopterin, of the sum of neopterin and biopterin should always be calculated. In addition, determination of hemoglobin concentration is essential as a measure for efficient extraction of neopterin and biopterin. Although the measurement of neopterin and biopterin in urine is more sensitive due to the higher concentrations present, our data prove the usefulness of their measurement from DBS for the routine diagnosis of BH(4) deficiencies.