Long term follow-up in GAMT deficiency - Correlation of therapy regimen, biochemical and in vivo brain proton MR spectroscopy data.

GAMT deficiency is a rare autosomal recessive disease within the group of cerebral creatine deficiency syndromes. Cerebral creatine depletion and accumulation of guanidinoacetate (GAA) lead to clinical presentation with intellectual disability, seizures, speech disturbances and movement disorders. Treatment consists of daily creatine supplementation to increase cerebral creatine, reduction of arginine intake and supplementation of ornithine for reduction of toxic GAA levels. This study represents the first long-term follow-up over a period of 14 years, with detailed clinical data, biochemical and multimodal neuroimaging findings. Developmental milestones, brain MRI, quantitative single voxel 1H magnetic resonance spectroscopy (MRS) and biochemical analyses were assessed. The results reveal insights into the dose dependent effects of creatine/ornithine supplementation and expand the phenotypic spectrum of GAMT deficiency. Of note, the creatine concentrations, which were regularly monitored over a long follow-up period, increased significantly over time, but did not reach age matched control ranges. Our patient is the second reported to show normal neurocognitive outcome after an initial delay, stressing the importance of early diagnosis and treatment initiation.

Semaphorin 3C exacerbates liver fibrosis.

BACKGROUND AND AIMS: Chronic liver disease is a growing epidemic, leading to fibrosis and cirrhosis. TGF-ß is the pivotal profibrogenic cytokine that activates HSC, yet other molecules can modulate TGF-ß signaling during liver fibrosis. Expression of the axon guidance molecules semaphorins (SEMAs), which signal through plexins and neuropilins (NRPs), have been associated with liver fibrosis in HBV-induced chronic hepatitis. This study aims at determining their function in the regulation of HSCs. APPROACH AND RESULTS: We analyzed publicly available patient databases and liver biopsies. We used transgenic mice, in which genes are deleted only in activated HSCs to perform ex vivo analysis and animal models. SEMA3C is the most enriched member of the semaphorin family in liver samples from patients with cirrhosis. Higher expression of SEMA3C in patients with NASH, alcoholic hepatitis, or HBV-induced hepatitis discriminates those with a more profibrotic transcriptomic profile. SEMA3C expression is also elevated in different mouse models of liver fibrosis and in isolated HSCs on activation. In keeping with this, deletion of SEMA3C in activated HSCs reduces myofibroblast marker expression. Conversely, SEMA3C overexpression exacerbates TGF-ß-mediated myofibroblast activation, as shown by increased SMAD2 phosphorylation and target gene expression. Among SEMA3C receptors, only NRP2 expression is maintained on activation of isolated HSCs. Interestingly, lack of NRP2 in those cells reduces myofibroblast marker expression. Finally, deletion of either SEMA3C or NRP2, specifically in activated HSCs, reduces liver fibrosis in mice. CONCLUSION: SEMA3C is a novel marker for activated HSCs that plays a fundamental role in the acquisition of the myofibroblastic phenotype and liver fibrosis.

Metabolomic Profiling in Patients with Different Hemodynamic Subtypes of Severe Aortic Valve Stenosis.

Severe aortic stenosis (AS) is a common pathological condition in an ageing population imposing significant morbidity and mortality. Based on distinct hemodynamic features, i.e., ejection fraction (EF), transvalvular gradient and stroke volume, four different AS subtypes can be distinguished: (i) normal EF and high gradient, (ii) reduced EF and high gradient, (iii) reduced EF and low gradient, and (iv) normal EF and low gradient. These subtypes differ with respect to pathophysiological mechanisms, cardiac remodeling, and prognosis. However, little is known about metabolic changes in these different hemodynamic conditions of AS. Thus, we carried out metabolomic analyses in serum samples of 40 AS patients (n = 10 per subtype) and 10 healthy blood donors (controls) using ultrahigh-performance liquid chromatography-tandem mass spectroscopy. A total of 1293 biochemicals could be identified. Principal component analysis revealed different metabolic profiles in all of the subgroups of AS (All-AS) vs. controls. Out of the determined biochemicals, 48% (n = 620) were altered in All-AS vs. controls (p < 0.05). In this regard, levels of various acylcarnitines (e.g., myristoylcarnitine, fold-change 1.85, p < 0.05), ketone bodies (e.g., 3-hydroxybutyrate, fold-change 11.14, p < 0.05) as well as sugar metabolites (e.g., glucose, fold-change 1.22, p < 0.05) were predominantly increased, whereas amino acids (e.g., leucine, fold-change 0.8, p < 0.05) were mainly reduced in All-AS. Interestingly, these changes appeared to be consistent amongst all AS subtypes. Distinct differences between AS subtypes were found for metabolites belonging to hemoglobin metabolism, diacylglycerols, and dihydrosphingomyelins. These findings indicate that relevant changes in substrate utilization appear to be consistent for different hemodynamic subtypes of AS and may therefore reflect common mechanisms during AS-induced heart failure. Additionally, distinct metabolites could be identified to significantly differ between certain AS subtypes. Future studies need to define their pathophysiological implications.

Cln5 represents a new type of cysteine-based S-depalmitoylase linked to neurodegeneration.

Genetic CLN5 variants are associated with childhood neurodegeneration and Alzheimer's disease; however, the molecular function of ceroid lipofuscinosis neuronal protein 5 (Cln5) is unknown. We solved the Cln5 crystal structure and identified a region homologous to the catalytic domain of members of the N1pC/P60 superfamily of papain-like enzymes. However, we observed no protease activity for Cln5; and instead, we discovered that Cln5 and structurally related PPPDE1 and PPPDE2 have efficient cysteine palmitoyl thioesterase (S-depalmitoylation) activity using fluorescent substrates. Mutational analysis revealed that the predicted catalytic residues histidine-166 and cysteine-280 are critical for Cln5 thioesterase activity, uncovering a new cysteine-based catalytic mechanism for S-depalmitoylation enzymes. Last, we found that Cln5-deficient neuronal progenitor cells showed reduced thioesterase activity, confirming live cell function of Cln5 in setting S-depalmitoylation levels. Our results provide new insight into the function of Cln5, emphasize the importance of S-depalmitoylation in neuronal homeostasis, and disclose a new, unexpected enzymatic function for the N1pC/P60 superfamily of proteins.

LC-MS Based Platform Simplifies Access to Metabolomics for Peroxisomal Disorders.

Peroxisomes are central hubs for cell metabolism and their dysfunction is linked to devastating human disorders, such as peroxisomal biogenesis disorders and single peroxisomal enzyme/protein deficiencies. For decades, biochemical diagnostics have been carried out using classical markers such as very long-chain fatty acids (VLCFA), which can be inconspicuous in milder and atypical cases. Holistic metabolomics studies revealed several potentially new biomarkers for peroxisomal disorders for advanced laboratory diagnostics including atypical cases. However, establishing these new markers is a major challenge in routine diagnostic laboratories. We therefore investigated whether the commercially available AbsoluteIDQ p180 kit (Biocrates Lifesciences), which utilizes flow injection and liquid chromatography mass spectrometry, may be used to reproduce some key results from previous global metabolomics studies. We applied it to serum samples from patients with mutations in peroxisomal target genes PEX1, ABCD1, and the HSD17B4 gene. Here we found various changes in sphingomyelins and lysophosphatidylcholines. In conclusion, this kit can be used to carry out extended diagnostics for peroxisomal disorders in routine laboratories, even without access to a metabolomics unit.

Targeted metabolomics revealed changes in phospholipids during the development of neuroinflammation in Abcd1tm1Kds mice and X-linked adrenoleukodystrophy patients.

X-linked adrenoleukodystrophy (X-ALD) is the most common leukodystrophy. Despite intensive research in recent years, it remains unclear, what drives the different clinical disease courses. Due to this missing pathophysiological link, therapy for the childhood cerebral disease course of X-ALD (CCALD) remains symptomatic; the allogenic hematopoietic stem cell transplantation or hematopoietic stem-cell gene therapy is an option for early disease stages. The inclusion of dried blood spot (DBS) C26:0-lysophosphatidylcholine to newborn screening in an increasing number of countries is leading to an increasing number of X-ALD patients diagnosed at risk for CCALD. Current follow-up in asymptomatic boys with X-ALD requires repetitive cerebral MRIs under sedation. A reliable and easily accessible biomarker that predicts CCALD would therefore be of great value. Here we report the application of targeted metabolomics by AbsoluteIDQ p180-Kit from Biocrates to search for suitable biomarkers in X-ALD. LysoPC a C20:3 and lysoPC a C20:4 were identified as metabolites that indicate neuroinflammation after induction of experimental autoimmune encephalitis in the serum of Abcd1tm1Kds mice. Analysis of serum from X-ALD patients also revealed different concentrations of these lipids at different disease stages. Further studies in a larger cohort of X-ALD patient sera are needed to prove the diagnostic value of these lipids for use as early biomarkers for neuroinflammation in CCALD patients.

A new CUL4B variant associated with a mild phenotype and an exceptional pattern of leukoencephalopathy.

Cabezas type of X-linked syndromic intellectual disability (MRXSC; MIM300354) is a rare X-linked recessive intellectual disability characterized primarily by intellectual disability, short stature, hypogonadism, and gait abnormalities. It is caused by a wide spectrum of hemizygous variants in CUL4B. In a 10-year-old boy with an exceptional leukoencephalopathy pattern, we identified a new missense variant p.Leu329Gln in CUL4B using "Mendeliome" sequencing. However, his phenotype does not include the severe characteristics currently known for MRXSC. We discuss the divergent phenotype and propose a potential connection between the different CUL4B variants and corresponding phenotypes in the context of the current literature as well as 3D homology modeling.

Global genetic analyses reveal strong inter-ethnic variability in the loss of activity of the organic cation transporter OCT1.

BACKGROUND: The organic cation transporter OCT1 (SLC22A1) mediates the uptake of vitamin B1, cationic drugs, and xenobiotics into hepatocytes. Nine percent of Caucasians lack or have very low OCT1 activity due to loss-of-function polymorphisms in OCT1 gene. Here we analyzed the global genetic variability in OCT1 to estimate the therapeutic relevance of OCT1 polymorphisms in populations beyond Caucasians and to identify evolutionary patterns of the common loss of OCT1 activity in humans. METHODS: We applied massively parallel sequencing to screen for coding polymorphisms in 1,079 unrelated individuals from 53 populations worldwide. The obtained data was combined with the existing 1000 Genomes data comprising an additional 1,092 individuals from 14 populations. The identified OCT1 variants were characterized in vitro regarding their cellular localization and their ability to transport 10 known OCT1 substrates. Both the population genetics data and transport data were used in tandem to generate a world map of loss of OCT1 activity. RESULTS: We identified 16 amino acid substitutions potentially causing loss of OCT1 function and analyzed them together with five amino acid substitutions that were not expected to affect OCT1 function. The variants constituted 16 major alleles and 14 sub-alleles. Six major alleles showed improper subcellular localization leading to substrate-wide loss in activity. Five major alleles showed correct subcellular localization, but substrate-specific loss of activity. Striking differences were observed in the frequency of loss of OCT1 activity worldwide. While most East Asian and Oceanian individuals had completely functional OCT1, 80 % of native South American Indians lacked functional OCT1 alleles. In East Asia and Oceania the average nucleotide diversity of the loss-of-function variants was much lower than that of the variants that do not affect OCT1 function (ratio of 0.03) and was significantly lower than the theoretically expected heterozygosity (Tajima's D = -1.64, P < 0.01). CONCLUSIONS: Comprehensive genetic analyses showed strong global variations in the frequency of loss of OCT1 activity with selection pressure for maintaining OCT1 activity in East Asia and Oceania. These results not only enable pharmacogenetically-based optimization of drug treatment worldwide, but may help elucidate the functional role of human OCT1.

Structure of sulfamidase provides insight into the molecular pathology of mucopolysaccharidosis IIIA.

Mucopolysaccharidosis type IIIA (Sanfilippo A syndrome), a fatal childhood-onset neurodegenerative disease with mild facial, visceral and skeletal abnormalities, is caused by an inherited deficiency of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH; sulfamidase). More than 100 mutations in the SGSH gene have been found to reduce or eliminate its enzymatic activity. However, the molecular understanding of the effect of these mutations has been confined by a lack of structural data for this enzyme. Here, the crystal structure of glycosylated SGSH is presented at 2 Å resolution. Despite the low sequence identity between this unique N-sulfatase and the group of O-sulfatases, they share a similar overall fold and active-site architecture, including a catalytic formylglycine, a divalent metal-binding site and a sulfate-binding site. However, a highly conserved lysine in O-sulfatases is replaced in SGSH by an arginine (Arg282) that is positioned to bind the N-linked sulfate substrate. The structure also provides insight into the diverse effects of pathogenic mutations on SGSH function in mucopolysaccharidosis type IIIA and convincing evidence for the molecular consequences of many missense mutations. Further, the molecular characterization of SGSH mutations will lay the groundwork for the development of structure-based drug design for this devastating neurodegenerative disorder.

Structure and activity of the only human RNase T2.

Mutations in the gene of human RNase T2 are associated with white matter disease of the human brain. Although brain abnormalities (bilateral temporal lobe cysts and multifocal white matter lesions) and clinical symptoms (psychomotor impairments, spasticity and epilepsy) are well characterized, the pathomechanism of RNase T2 deficiency remains unclear. RNase T2 is the only member of the Rh/T2/S family of acidic hydrolases in humans. In recent years, new functions such as tumor suppressing properties of RNase T2 have been reported that are independent of its catalytic activity. We determined the X-ray structure of human RNase T2 at 1.6 Å resolution. The α+ß core fold shows high similarity to those of known T2 RNase structures from plants, while, in contrast, the external loop regions show distinct structural differences. The catalytic features of RNase T2 in presence of bivalent cations were analyzed and the structural consequences of known clinical mutations were investigated. Our data provide further insight into the function of human RNase T2 and may prove useful in understanding its mode of action independent of its enzymatic activity.

Different expression of the catalytic alpha subunits of the AMP activated protein kinase--an immunohistochemical study in human tissue.

AMPK is an ubiquitously distributed multienzyme complex. It is an important energy sensor and regulator of cellular metabolic activity. In this study we analyzed for the first time the cellular distribution of the catalytically active subunits AMPKα1 and α2 in different human tissues by immunohistochemistry. We found different expression patterns for both isoforms. AMPKα2 expression clearly dominates in skeletal myocytes and cardiomyocytes, whereas AMPKα1 dominates in a number of secreting cells, like mammary glands, islets of langerhans and cells of the colon crypts.

Lymphoblastoid cell lines for diagnosis of peroxisome biogenesis disorders.

Peroxisome biogenesis disorders (PBDs) are a group of autosomal-recessive developmental and progressive metabolic diseases leading to the Zellweger spectrum (ZS) phenotype in most instances. Diagnosis of clinically suspected cases can be difficult because of extensive genetic heterogeneity and large spectrum of disease severity. Furthermore, a second group of peroxisomal diseases caused by deficiencies of single peroxisomal enzymes can show an indistinguishable clinical phenotype. The diagnosis of these peroxisomal disorders relies on the clinical presentation, the biochemical parameters in plasma and erythrocyte membranes, and genetic testing as the final step. Analysis of patients' cells is frequently required during the diagnostic process, e.g., for complementation analysis to identify the affected gene before sequencing. In the cases with unclear clinical or biochemical presentation, patients' cells are analyzed to prove PBD or to demonstrate biochemical abnormalities that might be elusive in plasma. Cell lines from skin fibroblast that are usually generated for diagnostic workup are not available in all instances, mainly because the required skin biopsy is invasive and sometimes denied by parents. An alternative cellular system has not been analyzed sufficiently. In this study, we evaluated the alternative use of lymphoblastoid cell lines (LCLs), derived from a peripheral blood sample, in the diagnostic process for PBD. LCLs were suitable for immunofluorescence visualization of peroxisomal enzymes, complementation analysis, and the biochemical analysis to differentiate between control and PBD LCL. LCLs are therefore an easily obtainable alternative cellular system for a detailed PBD diagnostic workup with a reliability of diagnostic results equal to those of skin fibroblasts.

Typical cMRI pattern as diagnostic clue for D-bifunctional protein deficiency without apparent biochemical abnormalities in plasma.

D-bifunctional protein deficiency (DBPD) is an autosomal recessive disease caused by a defect in peroxisomal ß-oxidation. The majority of patients suffer from a severe neurological disease with neonatal hypotonia and seizures and die within the first 2 years of life. Few patients show milder clinical phenotypes with prolonged survival. The diagnosis relies on the clinical presentation, measurement of peroxisomal markers, including very long chain fatty acids (VLCFA) in plasma, followed by enzymatic studies in fibroblasts and genetic testing. Diagnosis can be difficult to establish in milder cases, especially if VLCFA concentration in plasma is not or only mildly elevated. We report on siblings in which initial measurement of plasma VLCFA did not indicate a peroxisomal disease. Nevertheless, cMRI showed a pattern typical for an inborn peroxisomal disease with cerebral and cerebellar leukencephalopathy, perisylvic polymicrogyria, and frontoparietal pachygyria. Repeated measurements of peroxisomal metabolites in plasma prompted by the cMRI findings showed values in the upper normal or mildly elevated range and led to further diagnostic steps. The diagnosis of a type III DBPD with a missense mutation (T15A) in the HSD17B4 gene, coding for D-bifunctional protein (DBP), could be established. We conclude that a typical "peroxisomal pattern" in cMRI including cerebral and cerebellar leukencephalopathy, perisylvic polymicrogyria and pachygyria is a valuable clue to the diagnosis of DBPD, especially in cases with no or only very mild abnormalities in plasma.

RNASET2-deficient cystic leukoencephalopathy resembles congenital cytomegalovirus brain infection.

Congenital cytomegalovirus brain infection without symptoms at birth can cause a static encephalopathy with characteristic patterns of brain abnormalities. Here we show that loss-of-function mutations in the gene encoding the RNASET2 glycoprotein lead to cystic leukoencephalopathy, an autosomal recessive disorder with an indistinguishable clinical and neuroradiological phenotype. Congenital cytomegalovirus infection and RNASET2 deficiency may both interfere with brain development and myelination through angiogenesis or RNA metabolism.

The homeodomain of PAX6 is essential for PAX6-dependent activation of the rat glucagon gene promoter: evidence for a PH0-like binding that induces an active conformation.

The transcription factor PAX6 plays an important role in transcriptional regulation of the peptide hormone glucagon from pancreatic alpha-cells. PAX6 contains two DNA binding domains, the paired domain (PD) and the homeodomain (HD). While the interaction of the PD with the PAX6 responsive elements G1 and G3 in the rat glucagon gene promoter is well understood, the role of the PAX6 HD for PAX6 binding and function on G1 and G3 remains unclear. In EMSA studies the PAX6 HD was found to be mandatory for PAX6 binding to G1 but not to G3. Transient transfections with luciferase reporter gene constructs revealed the HD to be critical for proper function of PAX6 on both, G1 and G3. Transfection data with variant promoter constructs and limited proteolysis assays demonstrated that the DNA sequence located 5' to the PD binding site plays an important role for PAX6 function and its conformation on the elements G1 and G3. Taken together, our data indicate a PH0-like binding of PAX6 to the glucagon promoter elements G1 and G3 where the HD binding site is abutted directly to the PD binding motif. The data suggest that the PH0-like binding induces a transcriptionally active conformation of PAX6.

A peroxisome proliferator-activated receptor gamma-retinoid X receptor heterodimer physically interacts with the transcriptional activator PAX6 to inhibit glucagon gene transcription.

The peptide hormone glucagon stimulates hepatic glucose output, and its levels in the blood are elevated in type 2 diabetes mellitus. The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) has essential roles in glucose homeostasis, and thiazolidinedione PPARgamma agonists are clinically important antidiabetic drugs. As part of their antidiabetic effect, thiazolidinediones such as rosiglitazone have been shown to inhibit glucagon gene transcription through binding to PPARgamma and inhibition of the transcriptional activity of PAX6 that is required for cell-specific activation of the glucagon gene. However, how thiazolidinediones and PPARgamma inhibit PAX6 activity at the glucagon promoter remained unknown. After transient transfection of a glucagon promoter-reporter fusion gene into a glucagon-producing pancreatic islet alpha-cell line, ligand-bound PPARgamma was found in the present study to inhibit glucagon gene transcription also after deletion of its DNA-binding domain. Like PPARgamma ligands, also retinoid X receptor (RXR) agonists inhibited glucagon gene transcription in a PPARgamma-dependent manner. In glutathione transferase pull-down assays, the ligand-bound PPARgamma-RXR heterodimer bound to the transactivation domain of PAX6. This interaction depended on the presence of the ligand and RXR, but it was independent of the PPARgamma DNA-binding domain. Chromatin immunoprecipitation experiments showed that PPARgamma is recruited to the PAX6-binding proximal glucagon promoter. Taken together, the results of the present study support a model in which a ligand-bound PPARgamma-RXR heterodimer physically interacts with promoter-bound PAX6 to inhibit glucagon gene transcription. These data define PAX6 as a novel physical target of PPARgamma-RXR.

Laminin-alpha4 and integrin-linked kinase mutations cause human cardiomyopathy via simultaneous defects in cardiomyocytes and endothelial cells.

BACKGROUND: Extracellular matrix proteins, such as laminins, and endothelial cells are known to influence cardiomyocyte performance; however, the underlying molecular mechanisms remain poorly understood. METHODS AND RESULTS: We used a forward genetic screen in zebrafish to identify novel genes required for myocardial function and were able to identify the lost-contact (loc) mutant, which encodes a nonsense mutation in the integrin-linked kinase (ilk) gene. This loc/ilk mutant is associated with a severe defect in cardiomyocytes and endothelial cells that leads to severe myocardial dysfunction. Additional experiments revealed the epistatic regulation between laminin-alpha4 (Lama4), integrin, and Ilk, which led us to screen for mutations in the human ILK and LAMA4 genes in patients with severe dilated cardiomyopathy. We identified 2 novel amino acid residue-altering mutations (2828C>T [Pro943Leu] and 3217C>T [Arg1073X]) in the integrin-interacting domain of the LAMA4 gene and 1 mutation (785C>T [Ala262Val]) in the ILK gene. Biacore quantitative protein/protein interaction data, which have been used to determine the equilibrium dissociation constants, point to the loss of integrin-binding capacity in case of the Pro943Leu (Kd=5+/-3 micromol/L) and Arg1073X LAMA4 (Kd=1+/-0.2 micromol/L) mutants compared with the wild-type LAMA4 protein (Kd=440+/-20 nmol/L). Additional functional data point to the loss of endothelial cells in affected patients as a direct consequence of the mutant genes, which ultimately leads to heart failure. CONCLUSIONS: This is the first report on mutations in the laminin, integrin, and ILK system in human cardiomyopathy, which has consequences for endothelial cells as well as for cardiomyocytes, thus providing a new genetic basis for dilated cardiomyopathy in humans.

Structure of Ecballium elaterium trypsin inhibitor II (EETI-II): a rigid molecular scaffold.

The Ecballium elaterium trypsin inhibitor II (EETI-II) belongs to the family of squash inhibitors and is one of the strongest inhibitors known for trypsin. The eight independent molecules of EETI-II in the crystal structure reported here provide a good opportunity to test the hypothesis that this small cystine-knot protein (knottin) is sufficiently rigid to be used as a molecular scaffold for protein-engineering purposes. To extend this test, the structures of two complexes of EETI-II with trypsin have also been determined, one carrying a four-amino-acid mutation of EETI-II. The remarkable similarity of these structures confirms the rigidity of the molecular framework and hence its suitability as a molecular scaffold.

Structure of viscotoxin A3: disulfide location from weak SAD data.

The crystal structure of viscotoxin A3 (VT A3) extracted from European mistletoe (Viscum album L.) has been solved using the anomalous diffraction of the native S atoms measured in-house with Cu Kalpha radiation to a resolution of 2.2 A and truncated to 2.5 A. A 1.75 A resolution synchrotron data set was used for phase expansion and refinement. An innovation in the dual-space substructure-solution program SHELXD enabled the individual S atoms of the disulfide bonds to be located using the Cu Kalpha data; this resulted in a marked improvement in the phasing compared with the use of super-S atoms. The VT A3 monomer consists of 46 amino acids with three disulfide bridges and has an overall fold resembling the canonical architecture of the alpha- and beta-thionins, a capital letter L. The asymmetric unit consists of two monomers related by a local twofold axis and held together by hydrophobic interactions between the monomer units. One phosphate anion (confirmed by 31P-NMR and MS) is associated with each monomer.