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Contains fulltext : 231549.pdf (Publisher’s version ) (Closed access) PURPOSE: The 2015 American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines for the interpretation of sequence variants provide a framework to standardize terminology in the classification of variants uncovered through genetic testing. We aimed to assess the validity of utilizing clinical response to therapies specifically targeted to a suspected disease in clarifying variant pathogenicity. METHODS: Five families with disparate clinical presentations and different genetic diseases evaluated and treated in multiple diagnostic settings are summarized. RESULTS: Extended evaluations indicated possible genetic diagnoses and assigned candidate causal variants, but the cumulative clinical, biochemical, and molecular information in each instance was not completely consistent with the identified disease. Initiation of treatment specific to the suspected diagnoses in the affected individuals led to clinical improvement in all five families. CONCLUSION: We propose that the effect of therapies that are specific and targeted to treatable genetic diseases embodies an in vivo physiological response and could be considered as additional criteria within the 2015 ACMG/AMP guidelines in determining genomic variant pathogenicity.

Untargeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomics strategies are being increasingly applied in metabolite screening for a wide variety of medical conditions. The long-standing "grand challenge" in the utilization of this approach is metabolite identification-confidently determining the chemical structures of m/z-detected unknowns. Here, we use a novel workflow based on the detection of molecular features of interest by high-throughput untargeted LC-MS analysis of patient body fluids combined with targeted molecular identification of those features using infrared ion spectroscopy (IRIS), effectively providing diagnostic IR fingerprints for mass-isolated targets. A significant advantage of this approach is that in silico-predicted IR spectra of candidate chemical structures can be used to suggest the molecular structure of unknown features, thus mitigating the need for the synthesis of a broad range of physical reference standards. Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine metabolism, resulting from a mutation in the ALDH7A1 gene that leads to an accumulation of toxic levels of alpha-aminoadipic semialdehyde (alpha-AASA), piperideine-6-carboxylate (P6C), and pipecolic acid in body fluids. While alpha-AASA and P6C are known biomarkers for PDE in urine, their instability makes them poor candidates for diagnostic analysis from blood, which would be required for application in newborn screening protocols. Here, we use combined untargeted metabolomics-IRIS to identify several new biomarkers for PDE-ALDH7A1 that can be used for diagnostic analysis in urine, plasma, and cerebrospinal fluids and that are compatible with analysis in dried blood spots for newborn screening. The identification of these novel metabolites has directly provided novel insights into the pathophysiology of PDE-ALDH7A1.

Item does not contain fulltext BACKGROUND: Cerebrotendinous xanthomatosis (CTX) is an autosomal recessively inherited inborn error of metabolism. Neurological symptoms are considered to be a clinical hallmark of untreated adult patients. We describe a 'milder CTX phenotype', without neurological involvement. METHODS: We performed a retrospective patient file study in 79 genetically confirmed Dutch patients with CTX (55 patients aged ≥ 21 years) to study the clinical heterogeneity of CTX. We studied the frequency of adult patients with CTX without neurological involvement at diagnosis, in our Dutch cohort, and included a family from South Africa and patients from Italy, USA, Chile and Asia from the literature. RESULTS: In total, we describe 19 adult patients with CTX from 16 independent families, without neurological symptoms at diagnosis. A relatively small percentage (21%, n = 4) had a history of cataract. The majority, 84% (n = 16), presented with tendon xanthomas as the sole or predominant feature. The majority of patients showed increased plasma cholesterol levels. No correlation was found between this 'milder phenotype', the cholestanol levels and the CYP27A1 genotype. In addition, we describe three novel mutations in the CYP27A1 gene. CONCLUSIONS: This study shows the clinical heterogeneity of CTX, highlighting the existence of a 'milder phenotype', that is without neurological involvement at diagnosis. Adult patients with CTX may present with tendon xanthomas as the sole or predominant feature, mimicking familial hypercholesterolemia. It is important to realize that the absence of neurological symptoms does not rule out the development of future neurological symptoms. As CTX is a treatable disorder, early diagnosis and initiation of treatment when additional clinical signs occur is therefore essential.

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Contains fulltext : 225031.pdf (Publisher’s version ) (Open Access) 3-Hydroxy-3-methylglutaryl-coenzyme A lyase deficiency (HMGCLD) is a rare autosomal recessively inherited metabolic disorder. Patients suffer from avoidable neurologically devastating metabolic decompensations and thus would benefit from newborn screening (NBS). The diagnosis is currently made by measuring dry blood spot acylcarnitines (C5OH and C6DC) followed by urinary organic acid profiling for the differential diagnosis from several other disorders. Using untargeted metabolomics (reversed-phase UHPLC coupled to an Orbitrap Elite hybrid mass spectrometer) of plasma samples from 5 HMGCLD patients and 19 age-matched controls, we found 3-methylglutaconic acid and 3-hydroxy-3-methylglutaric acid, together with 3-hydroxyisovalerylcarnitine as the most discriminating metabolites between the groups. In order to evaluate the NBS potential of these metabolites we quantified the most discriminating metabolites from untargeted metabolomics in 23 blood spots from 4 HMGCLD patients and 55 controls by UHPLC tandem mass spectrometry. The results provide a tool for expanded NBS of HMGCLD using tandem mass spectrometry. Selected reaction monitoring transition 262/85 could be used in a first-tier NBS analysis to screen for elevated 3-hydroxyisovalerylcarnitine. In a positive case, a second-tier analysis of 3-hydroxy-3-methylglutaric acid and 3-methylglutaconic acid in a dry blood spot using UHPLC tandem mass spectrometry instruments confirms the diagnosis. In conclusion, we describe the identification of new diagnostic biomarkers for HMGCLD and their application in NBS in dry blood spots. By using second-tier testing, all patients with HMGCLD were unequivocally and correctly diagnosed.

Liquid chromatography-mass spectrometry (LC-MS) is, due to its high sensitivity and selectivity, currently the method of choice in (bio)analytical studies involving the (comprehensive) profiling of metabolites in body fluids. However, as closely related isomers are often hard to distinguish on the basis of LC-MS(MS) and identification is often dependent on the availability of reference standards, the identification of the chemical structures of detected mass spectral features remains the primary limitation. Infrared ion spectroscopy (IRIS) aids identification of MS-detected ions by providing an infrared (IR) spectrum containing structural information for a detected MS-feature. Moreover, IR spectra can be routinely and reliably predicted for many types of molecular structures using quantum-chemical calculations, potentially avoiding the need for reference standards. In this work, we demonstrate a workflow for reference-free metabolite identification that combines experiments based on high-pressure liquid chromatography (HPLC), MS and IRIS with quantum-chemical calculations that efficiently generate IR spectra and give the potential to enable reference-standard free metabolite identification. Additionally, a scoring procedure is employed which shows the potential for automated structure assignment of unknowns. Via a simple, illustrative example where we identify lysine in the plasma of a hyperlysinemia patient, we show that this approach allows the efficient assignment of a database-derived molecular structure to an unknown.

Untargeted LC-MS based metabolomics strategies are being increasingly applied in metabolite screening for a wide variety of medical conditions. The long-standing “grand challenge” in the utilization of this approach is metabolite identification – confidently determining the chemical structures of m/z-detected unknowns. Here, we use a novel workflow based on the detection of molecular features of interest by high-throughput untargeted LC-MS analysis of patient body fluids combined with targeted molecular identification of those features using infrared ion spectroscopy (IRIS), effectively providing diagnostic IR fingerprints for mass-isolated targets. A significant advantage of this approach is that in silico predicted IR spectra of candidate chemical structures can be used to suggest the molecular structure of unknown features, thus mitigating the need for the synthesis of a broad range of physical reference standards. Pyridoxine dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine metabolism, resulting from a mutation in the ALDH7A1 gene that leads to an accumulation of toxic levels of α-aminoadipic semialdehyde (α-AASA), piperideine-6-carboxylate (P6C), and pipecolic acid in body fluids. While α-AASA and P6C are known biomarkers for PDE in urine, their instability makes them poor candidates for diagnostic analysis from blood, which would be required for application in newborn screening protocols. Here, we use combined untargeted metabolomics-IRIS to identify several new biomarkers for PDE-ALDH7A1 that can be used for diagnostic analysis in urine, plasma, and cerebrospinal fluids, and are compatible with analysis in dried blood spots for newborn screening. The identification of these novel metabolites has directly rendered novel insights in the pathophysiology of PDE-ALDH7A1.

Contains fulltext : 235312.pdf (Publisher’s version ) (Open Access) BACKGROUND: Reliable measurement of phenylalanine (Phe) is a prerequisite for adequate follow-up of phenylketonuria (PKU) patients. However, previous studies have raised concerns on the intercomparability of plasma and dried blood spot (DBS) Phe results. In this study, we made an inventory of differences in (pre-)analytical methodology used for Phe determination across Dutch laboratories, and compared DBS and plasma results. METHODS: Through an online questionnaire, we assessed (pre-)analytical Phe measurement procedures of seven Dutch metabolic laboratories. To investigate the difference between plasma and DBS Phe, participating laboratories received simultaneously collected plasma-DBS sets from 23 PKU patients. In parallel, 40 sample sets of DBS spotted from either venous blood or capillary fingerprick were analyzed. RESULTS: Our data show that there is no consistency on standard operating procedures for Phe measurement. The association of DBS to plasma Phe concentration exhibits substantial inter-laboratory variation, ranging from a mean difference of -15.5% to +30.6% between plasma and DBS Phe concentrations. In addition, we found a mean difference of +5.8% in Phe concentration between capillary DBS and DBS prepared from venous blood. CONCLUSIONS: The results of our study point to substantial (pre-)analytical variation in Phe measurements, implicating that bloodspot Phe results should be interpreted with caution, especially when no correction factor is applied. To minimize variation, we advocate pre-analytical standardization and analytical harmonization of Phe measurements, including consensus on application of a correction factor to adjust DBS Phe to plasma concentrations.

Pyridoxine-dependent epilepsy (PDE-ALDH7A1), also known as antiquitin deficiency, is an inborn error of lysine metabolism that presents with refractory epilepsy in newborns. Bi-allelic ALDH7A1 variants lead to deficiency of α-aminoadipic semialdehyde dehydrogenase, resulting in accumulation of piperideine-6-carboxylate (P6C), and secondary deficiency of the important co-factor pyridoxal-5’-phosphate (PLP, active vitamin B6) through its complexation with P6C. Vitamin B6 supplementation resolves epilepsy in patients, but despite this treatment, intellectual disability may occur. Early diagnosis and treatment, preferably based on newborn screening, potentially optimize long-term clinical outcome. However, the currently known diagnostic PDE-ALDH7A1 biomarkers are incompatible with newborn screening procedures. Using a combination of the innovative analytical methods untargeted metabolomics and infrared ion spectroscopy, we have been able to discover novel biomarkers for PDE-ALDH7A1: 2S,6S-and 2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP) and 6-oxopiperidine-2-carboxylic acid (6-oxoPIP). We demonstrate the applicability of 2-OPP as a PDE-ALDH7A1 biomarker in newborn screening. Additionally, we show that 2-OPP accumulates in brain tissue of patients and Aldh7a1 knock-out mice, and induces epilepsy-like behavior in a zebrafish model system. We speculate that 2-OPP may contribute to ongoing neurotoxicity, also in treated PDE-ALDH7A1 patients. As 2-OPP formation appears to increase upon ketosis, we emphasize the importance of avoiding catabolism in PDE-ALDH7A1 patients.

Contains fulltext : 237460.pdf (Publisher’s version ) (Open Access) Early detection of congenital disorders by newborn screening (NBS) programs is essential to prevent or limit disease manifestation in affected neonates. These programs balance between the detection of the highest number of true cases and the lowest number of false-positives. In this case report, we describe four unrelated cases with a false-positive NBS result for very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD). Three neonates presented with decreased but not deficient VLCAD enzyme activity and two of them carried a single heterozygous ACADVL c.1844G>A mutation. Initial biochemical investigations after positive NBS referral in these infants revealed acylcarnitine and organic acid profiles resembling those seen in multiple acyl-CoA dehydrogenase deficiency (MADD). Genetic analysis did not reveal any pathogenic mutations in the genes encoding the electron transfer flavoprotein (ETF alpha and beta subunits) nor in ETF dehydrogenase. Subsequent further diagnostics revealed decreased levels of riboflavin in the newborns and oral riboflavin administration normalized the MADD-like biochemical profiles. During pregnancy, the mothers followed a vegan, vegetarian or lactose-free diet which probably caused alimentary riboflavin deficiency in the neonates. This report demonstrates that a secondary (alimentary) maternal riboflavin deficiency in combination with reduced VLCAD activity in the newborns can result in an abnormal VLCADD/MADD acylcarnitine profile and can cause false-positive NBS. We hypothesize that maternal riboflavin deficiency contributed to the false-positive VLCADD neonatal screening results.

Purpose Pheochromocytomas and Paragangliomas (PPGL) result in chronic catecholamine excess and serious health complications. A recent study obtained a metabolic signature in plasma from PPGL patients; however, its targeted nature may have generated an incomplete picture and a broader approach could provide additional insights. We aimed to characterize the plasma metabolome of PPGL patients before and after surgery, using an untargeted approach, and to broaden the scope of the investigated metabolic impact of these tumors. Design A cohort of 36 PPGL patients was investigated. Blood plasma samples were collected before and after surgical tumor removal, in association with clinical and tumor characteristics. Methods Plasma samples were analyzed using untargeted nuclear magnetic resonance (NMR) spectroscopy metabolomics. The data were evaluated using a combination of uni- and multi-variate statistical methods. Results Before surgery, patients with a nonadrenergic tumor could be distinguished from those with an adrenergic tumor based on their metabolic profiles. Tyrosine levels were significantly higher in patients with high compared to those with low BMI. Comparing subgroups of pre-operative samples with their post-operative counterparts, we found a metabolic signature that included ketone bodies, glucose, organic acids, methanol, dimethyl sulfone and amino acids. Three signals with unclear identities were found to be affected. Conclusions Our study suggests that the pathways of glucose and ketone body homeostasis are affected in PPGL patients. BMI-related metabolite levels were also found to be altered, potentially linking muscle atrophy to PPGL. At baseline, patient metabolomes could be discriminated based on their catecholamine phenotype.

The identification of disease biomarkers plays a crucial role in developing diagnostic strategies for inborn errors of metabolism and understanding their pathophysiology. A primary metabolite that accumulates in the inborn error phenylketonuria is phenylalanine, however its levels do not always directly correlate with clinical outcomes. Here we combine infrared ion spectroscopy and NMR spectroscopy to identify the Phe-glucose Amadori rearrangement product as a biomarker for phenylketonuria. Additionally, we find analogous amino acid-glucose metabolites formed in the body fluids of patients accumulating methionine, lysine, proline and citrulline. Amadori rearrangement products are well-known intermediates in the formation of advanced glycation end-products and have been associated with the pathophysiology of diabetes mellitus and ageing, but are now shown to also form under conditions of aminoacidemia. They represent a general class of metabolites for inborn errors of amino acid metabolism that show potential as biomarkers and may provide further insight in disease pathophysiology., Rianne van Outersterp et al. combine mass spectrometry, NMR, and infrared ion spectroscopy to identify amino acid-hexose conjugates in the blood plasma from patients with metabolic disorders such as phenylketonuria (PKU). These conjugates, or Amadori rearrangement products, are generally not detectable in blood samples from unaffected individuals, and may therefore represent disease biomarkers.

Many inborn errors of metabolism (IEMs) are amenable to treatment, therefore early diagnosis is imperative. Whole-exome sequencing (WES) variant prioritization coupled with phenotype-guided clinical and bioinformatics expertise is typically used to identify disease-causing variants; however, it can be challenging to identify the causal candidate gene when a large number of rare and potentially pathogenic variants are detected. Here, we present a network-based approach, metPropagate, that uses untargeted metabolomics (UM) data from a single patient and a group of controls to prioritize candidate genes in patients with suspected IEMs. We validate metPropagate on 107 patients with IEMs diagnosed in Miller et al. (2015) and 11 patients with both CNS and metabolic abnormalities. The metPropagate method ranks candidate genes by label propagation, a graph-smoothing algorithm that considers each gene’s metabolic perturbation in addition to the network of interactions between neighbors. metPropagate was able to prioritize at least one causative gene in the top 20th percentile of candidate genes for 92% of patients with known IEMs. Applied to patients with suspected neurometabolic disease, metPropagate placed at least one causative gene in the top 20th percentile in 9/11 patients, and ranked the causative gene more highly than Exomiser’s phenotype-based ranking in 6/11 patients. Interestingly, ranking by a weighted combination of metPropagate and Exomiser scores resulted in improved prioritization. The results of this study indicate that network-based analysis of UM data can provide an additional mode of evidence to prioritize causal genes in patients with suspected IEMs.

Following publication of the original article, the authors would like to correct a sentence in the paragraph “1H-NMR spectra were recorded at 298 K…” under the heading “NMR experiments”. The sentence currently reads: “The LED pulse sequence had the form -RD-901-G1- 1801-G1-901-G2-T-901-G1-1801-G1-901-G2-t-901-acquire FID, where RD is a relaxation delay, 901 is a 901 RF pulse, G1 is the pulsed-field gradient that is applied to allow editing, 1801 is a 1801 RF pulse, G2 is a spoil gradient applied to remove unwanted magnetization components. The diffusion delay Δ is the time during which the molecules are allowed to diffuse—this is the period (901-G1-1801-G1- 901-G2-T-); and t is a delay to allow the longitudinal eddy currents caused within the sample to decay (Beckonert et al. 2007).” The sentence should read: “The LED pulse sequence had the form -RD-90°-G1- 180°-G1-90°-G2-T-90°-G1-180°-G1-90°-G2-t-90°-acquire FID, where RD is a relaxation delay, 90° is a 90° RF pulse, G1 is the pulsed-field gradient that is applied to allow editing, 180° is a 180° RF pulse, G2 is a spoil gradient applied to remove unwanted magnetization components. The diffusion delay ? is the time during which the molecules are allowed to diffuse—this is the period (90°-G1-180°-G1-90°-G2 T-); and t is a delay to allow the longitudinal eddy currents caused within the sample to decay (Beckonert et al. 2007).” This has been corrected with this erratum.

Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate diagnosis to guide proper management. Whole-exome sequencing was used to investigate the disease etiology in four children from independent families with intellectual disability and epilepsy, revealing bi-allelic GOT2 mutations. In-depth metabolic studies in individual 1 showed low plasma serine, hypercitrullinemia, hyperlactatemia, and hyperammonemia. The epilepsy was serine and pyridoxine responsive. Functional consequences of observed mutations were tested by measuring enzyme activity and by cell and animal models. Zebrafish and mouse models were used to validate brain developmental and functional defects and to test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate transaminase. GOT2 enzyme activity was deficient in fibroblasts with bi-allelic mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role in the intracellular NAD(H) redox balance. De novo serine biosynthesis was impaired in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the highly oxidized cytosolic NAD-redox state by pyruvate supplementation restored serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted in a brain developmental defect associated with seizure-like electroencephalography spikes, which could be rescued by supplying pyridoxine in embryo water. Both pyridoxine and serine synergistically rescued embryonic developmental defects in zebrafish got2a morphants. The two treated individuals reacted favorably to their treatment. Our data provide a mechanistic basis for the biochemical abnormalities in GOT2 deficiency that may also hold for other MAS defects.

The identification of molecular biomarkers is critical for diagnosing and treating patients and for establishing a fundamental understanding of the pathophysiology and underlying biochemistry of inborn errors of metabolism. Currently, liquid chromatography/high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy are the principle methods used for biomarker research and for structural elucidation of small molecules in patient body fluids. While both are powerful techniques, several limitations exist that often make the identification of unknown compounds challenging. Here, we describe how infrared ion spectroscopy has the potential to be a valuable orthogonal technique that provides highly-specific molecular structure information while maintaining ultra-high sensitivity. Here, we characterize and distinguish two well-known biomarkers of inborn errors of metabolism, glutaric acid for glutaric aciduria and ethylmalonic acid for short-chain acyl-CoA dehydrogenase deficiency, using infrared ion spectroscopy. In contrast to tandem mass spectra, in which ion fragments can hardly be predicted, we show that the prediction of an IR spectrum allows reference-free identification in the case that standard compounds are either commercially or synthetically unavailable. Finally, we illustrate how functional group information can be obtained from an IR spectrum for an unknown and how this is valuable information to, for example, narrow down a list of candidate structures resulting from a database query. Early diagnosis in inborn errors of metabolism is crucial for enabling treatment and depends on the identification of biomarkers specific for the disorder. Infrared ion spectroscopy has the potential to play a pivotal role in the identification of challenging biomarkers.

BACKGROUND: Cerebrotendinous xanthomatosis (CTX) is an autosomal recessively inherited inborn error of metabolism (IEM) due to mutations in the CYP27A1 gene. The clinical picture ranges from being nearly asymptomatic in early childhood, up to severe disability at adult age. Infantile-onset diarrhea and juvenile-onset cataract are the earliest symptoms in childhood. In the current study, we evaluated the presence of autism spectrum disorder (ASD) in a large cohort of CTX patients. METHODS: We performed a retrospective patient file study in 77 genetically confirmed Dutch CTX patients to determine the frequency of ASD. In addition, we compared plasma cholestanol levels in CTX patients with and without a diagnosis of ASD and tried to establish a relation between CYP27A1 genotype and ASD. RESULTS: In our CTX cohort, 10 patients (13%; nine pediatric and one adult) with ASD were identified. At the time of diagnosis of ASD, most patients only exhibited symptoms of diarrhea and/or intellectual disability without signs of cataract or neurological symptoms. No correlation was found between the presence of ASD and the level of cholestanol or CYP27A1 genotype. The behavioral problems stabilized or improved after treatment initiation with chenodeoxycholic acid (CDCA) in all pediatric patients. CONCLUSIONS: We conclude that ASD is an early and probably underestimated frequent feature in CTX. Metabolic screening for CTX should be performed in patients with ASD when accompanied by diarrhea, intellectual disability, juvenile cataract, and/or neurological involvement. Early recognition allows for earlier initiation of specific treatment and will improve clinical outcome. Our results add CTX to the list of treatable IEMs associated with ASD.

Specific diagnostic markers are the key to effective diagnosis and treatment of inborn errors of metabolism (IEM). Untargeted metabolomics allows for the identification of potential novel diagnostic biomarkers. Current separation techniques coupled to high-resolution mass spectrometry provide a powerful tool for structural elucidation of unknown compounds in complex biological matrices. This is a proof-of-concept study testing this methodology to determine the molecular structure of as yet uncharacterized m/z signals that were significantly increased in plasma samples from patients with phenylketonuria and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. A hybrid linear ion trap-orbitrap high resolution mass spectrometer, capable of multistage fragmentation, was used to acquire accurate masses and product ion spectra of the uncharacterized m/z signals. In order to determine the molecular structures, spectral databases were searched and fragmentation prediction software was used. This approach enabled structural elucidation of novel compounds potentially useful as biomarkers in diagnostics and follow-up of IEM patients. Two new conjugates, glutamyl-glutamyl-phenylalanine and phenylalanine-hexose, were identified in plasma of phenylketonuria patients. These novel markers showed high inter-patient variation and did not correlate to phenylalanine levels, illustrating their potential added value for follow-up. As novel biomarkers for 3-hydroxy-3-methylglutaryl-CoA lyase deficiency, three positional isomers of 3-methylglutaconyl carnitine could be detected in patient plasma. Our results highlight the applicability of current accurate mass multistage fragmentation techniques for structural elucidation of unknown metabolites in human biofluids, offering an unprecedented opportunity to gain further biochemical insights in known inborn errors of metabolism by enabling high confidence identification of novel biomarkers.

Many inborn errors of metabolism (IEMs) are amenable to treatment, therefore early diagnosis before irreversible damage occurs is imperative. Despite recent advances, the genetic basis of many metabolic phenotypes remains unknown. For discovery purposes, Whole Exome Sequencing (WES) variant prioritization coupled with phenotype-guided clinical and bioinformatics expertise is currently the primary method used to identify novel disease-causing variants; however, it can be challenging to identify the causal candidate gene given the large number of plausible variants. Using untargeted metabolomics (UM) to prioritize metabolically relevant candidate genes is a promising approach to diagnosing known or novel IEMs in a single patient. Here, we present a network-based bioinformatics approach, metPropagate, that uses UM data from a single patient and a group of controls to prioritize candidate genes. We validate metProp on 107 patients with diagnosed IEMs and 11 patients with novel IEMs diagnosed through the TIDE gene discovery project at BC Children’s Hospital. The metPropagate method ranks candidate genes by considering the network of interactions between them. This is done by using a graph smoothing algorithm called label propagation to quantify the metabolic disruption in genes’ local neighbourhood. metPropagate was able to prioritize the causative gene in the top 20th percentile of candidate genes for 91% of patients with known IEM disorders. For novel IEMs, metPropagate placed the causative gene in the top 20th percentile in 9/11 patients. Using metPropagate, the causative gene was ranked higher than Exomiser’s phenotype-based ranking in 6/11 patients. The results of this study indicate that for diagnostic and gene discovery purposes, network-based analysis of metabolomics data can lend support to WES gene-discovery methods by providing an additional mode of evidence to help identify causal genes.

Patellofemoral instability is a motion related disease, featured as the patella dislocating from the trochlear groove. Four dimensional computed tomography (4DCT) enables full assessment of the patellofemoral movement. Nevertheless, the quantitative measurements of patellofemoral instability are still under research and currently of limited practical use. The aim of this study is to develop a robust and semi-automatic workflow to quantitatively describe the patellofemoral movement in a patient group of eight suffering from patellofemoral instability. The initial results show agreement with manual observations of the tibial tubercle - trochlear groove (TT-TG) distance in routine practice, and the possibility to evaluate both TT-TG distance and patellar centre - trochlear groove (PC-TG) distance dynamically during active flexion-extension-flexion movement of the knee.

Introduction When analyzing the human plasma metabolome with Nuclear Magnetic Resonance (NMR) spectroscopy, the Carr–Purcell–Meiboom–Gill (CPMG) experiment is commonly employed for large studies. However, this process can lead to compromised statistical analyses due to residual macromolecule signals. In addition, the utilization of Trimethylsilylpropanoic acid (TSP) as an internal standard often leads to quantification issues, and binning, as a spectral summarization step, can result in features not clearly assignable to metabolites. Objectives Our aim was to establish a new complete protocol for large plasma cohorts collected with the purpose of describing the comparative metabolic profile of groups of samples. Methods We compared the conventional CPMG approach to a novel procedure that involves diffusion NMR, using the Longitudinal Eddy-Current Delay (LED) experiment, maleic acid (MA) as the quantification reference and peak picking for spectral reduction. This comparison was carried out using the ultrafiltration method as a gold standard in a simple sample classification experiment, with Partial Least Squares–Discriminant Analysis (PLS-DA) and the resulting metabolic signatures for multivariate data analysis. In addition, the quantification capabilities of the method were evaluated. Results We found that the LED method applied was able to detect more metabolites than CPMG and suppress macromolecule signals more efficiently. The complete protocol was able to yield PLS-DA models with enhanced classification accuracy as well as a more reliable set of important features than the conventional CPMG approach. Assessment of the quantitative capabilities of the method resulted in good linearity, recovery and agreement with an established amino acid assay for the majority of the metabolites tested. Regarding repeatability, ~ 85% of all peaks had an adequately low coefficient of variation ( Conclusion Overall, our comparison yielded a high-throughput untargeted plasma NMR protocol for optimized data acquisition and processing that is expected to be a valuable contribution in the field of metabolic biomarker discovery.

Contains fulltext : 194257.pdf (Publisher’s version ) (Open Access) Purpose: To present a complication of compressive optic neuropathy caused by a swollen hydrogel explant and posteriorly displaced silicone encircling band. Observations: A 72-year-old female patient presented with progressive visual loss and a tilted optic disc. Her medical history included a retinal detachment in 1993 that was treated with a hydrogel explant under a solid silicone encircling band. Visual acuity had decreased from 6/10 to 6/20 and perimetry showed a scotoma in the temporal superior quadrant. On Magnetic Resonance Imaging (MRI), compression of the optic nerve by a displaced silicone encircling band inferior nasally in combination with a swollen episcleral hydrogel explant was observed. Surgical removal of the hydrogel explant and silicone encircling band was uneventful and resulted in improvement of visual acuity and visual field loss. Conclusions and importance: This is the first report on compressive optic neuropathy caused by swelling of a hydrogel explant resulting in a dislocated silicone encircling band. The loss of visual function resolved upon removal of the explant and encircling band.

Combining the individual analytical strengths of mass spectrometry and infrared spectroscopy, infrared ion spectroscopy is increasingly recognized as a powerful tool for small-molecule identification in a wide range of analytical applications. Mass spectrometry is itself a leading analytical technique for small-molecule identification on the merit of its outstanding sensitivity, selectivity and versatility. The foremost shortcoming of the technique, however, is its limited ability to directly probe molecular structure, especially when contrasted against spectroscopic techniques. In infrared ion spectroscopy, infrared vibrational spectra are recorded for mass-isolated ions and provide a signature that can be matched to reference spectra, either measured from standards or predicted using quantum-chemical calculations. Here we present an overview of the potential for this technique to develop into a versatile analytical method for identifying molecular structures in mass spectrometry-based analytical workflows. In this tutorial perspective, we introduce the reader to the technique of infrared ion spectroscopy and highlight a selection of recent experimental advances and applications in current analytical challenges, in particular in the field of untargeted metabolomics. We report on the coupling of infrared ion spectroscopy with liquid chromatography and present experiments that serve as proof-of-principle ex- amples of strategies to address outstanding challenges.

ObjectiveTo evaluate the effect of chenodeoxycholic acid treatment on disease progression in cerebrotendinous xanthomatosis (CTX).MethodsIn this retrospective cohort study, we report the clinical long-term follow-up characteristics of 56 Dutch patients with CTX. Age at diagnosis was correlated with clinical characteristics and with the course of modified Rankin Scale (mRS) and Expanded Disability Status Scale (EDSS) scores at follow-up.ResultsMedian follow-up time was 8 years (6 months–31.5 years). Patients diagnosed and treated before the age of 24 years had a significantly better outcome at follow-up. When considering only patients with a good treatment adherence (n = 43), neurologic symptoms, if present, disappeared in all patients who were diagnosed before the age of 24 and treated since. Furthermore, treatment prevented the development of new neurologic symptoms during follow-up. In contrast, 61% of the patients diagnosed and treated after the age of 24 showed deterioration of the neurologic symptoms, with parkinsonism as a treatment-resistant feature. There was an improvement or stabilization in favor of patients diagnosed and treated before the age of 24 compared to those treated after the age of 24: 100% vs 58% for mRS scores and 100% vs 50% for EDSS scores, respectively.ConclusionsTreatment start at an early age can reverse and even prevent the development of neurologic symptoms in CTX. This study emphasizes the importance of early diagnosis in CTX and provides a rationale to include CTX in newborn screening programs.

The implementation of whole-exome sequencing in clinical diagnostics has generated a need for functional evaluation of genetic variants. In the field of inborn errors of metabolism (IEM), a diverse spectrum of targeted biochemical assays is employed to analyze a limited amount of metabolites. We now present a single-platform, high-resolution liquid chromatography quadrupole time of flight (LC-QTOF) method that can be applied for holistic metabolic profiling in plasma of individual IEM-suspected patients. This method, which we termed “next-generation metabolic screening” (NGMS), can detect >10,000 features in each sample. In the NGMS workflow, features identified in patient and control samples are aligned using the “various forms of chromatography mass spectrometry (XCMS)” software package. Subsequently, all features are annotated using the Human Metabolome Database, and statistical testing is performed to identify significantly perturbed metabolite concentrations in a patient sample compared with controls. We propose three main modalities to analyze complex, untargeted metabolomics data. First, a targeted evaluation can be done based on identified genetic variants of uncertain significance in metabolic pathways. Second, we developed a panel of IEM-related metabolites to filter untargeted metabolomics data. Based on this IEM-panel approach, we provided the correct diagnosis for 42 of 46 IEMs. As a last modality, metabolomics data can be analyzed in an untargeted setting, which we term “open the metabolome” analysis. This approach identifies potential novel biomarkers in known IEMs and leads to identification of biomarkers for as yet unknown IEMs. We are convinced that NGMS is the way forward in laboratory diagnostics of IEMs. Electronic supplementary material The online version of this article (10.1007/s10545-017-0131-6) contains supplementary material, which is available to authorized users.

Contains fulltext : 154390.pdf (Publisher’s version ) (Closed access) BACKGROUND: The urinary concentrations of monosaccharides and polyols are used for diagnosing inborn errors of metabolism and renal tubular disorders. Reference values are age-related and depend on the method of detection. However, the influence of the renal function is often still neglected. In this study we examined the urinary excretion of monosaccharides and polyols in children with various degrees of chronic kidney disease (CKD), but with no known metabolic or renal tubular disorders. CASE DIAGNOSIS/TREATMENT: In 25 patients with CKD stage 1-5, urinary concentrations of 18 monosaccharides and polyols were measured by gas chromatography-mass spectrometry (GC-MS) in random urinary samples and were compared with age-related reference values. Serum creatinine was measured at the time of the urine sample, and the height-independent estimated glomerular filtration rate (eGFR-Pottel) was calculated. Urinary excretions of monosaccharides and polyols were above the reference values in 8-88 % of all patients. A significant difference between CKD stage 1-2 compared with CKD stage 3-5 was found for allose, arabitol and sorbitol (p < 0.05) and for arabinose, fucose, myoinositol, ribitol, xylitol, and xylose (p < 0.01). CONCLUSIONS: We show that the excretion of polyols and sugars depends on eGFR, which warrants a cautious interpretation of the results in patients with CKD.

Corrigendum: NANS-mediated synthesis of sialic acid is required for brain and skeletal development

A reduced response of cystathionine beta-synthase (CBS) to its allosteric activator S-adenosylmethionine (SAM) has been reported to be a cause of CBS dysfunction in homocystinuria patients. In this work we performed a retrospective analysis of fibroblast data from 62 homocystinuria patients and found that 13 of them presented a disturbed SAM activation. Their genotypic background was identified and the corresponding CBS mutant proteins were produced in E. coli. Nine distinct mutations were detected in 22 independent alleles: the novel mutations p.K269del, p.P427L, p.S500L and p.L540Q; and the previously described mutations p.P49L, p.C165Rfs2, p.I278T, p.R336H and p.D444N. Expression levels and residual enzyme activities, determined in the soluble fraction of E. coli lysates, strongly correlated with the localization of the affected amino acid residue. C-terminal mutations lead to activities in the range of the wild-type CBS and to oligomeric forms migrating faster than tetramers, suggesting an abnormal conformation that might be responsible for the lack of SAM activation. Mutations in the catalytic core were associated with low protein expression levels, decreased enzyme activities and a higher content of high molecular mass forms. Furthermore, the absence of SAM activation found in the patients' fibroblasts was confirmed for all but one of the characterized recombinant proteins (p.P49L). Our study experimentally supports a deficient regulation of CBS by SAM as a frequently found mechanism in CBS deficiency, which should be considered not only as a valuable diagnostic tool but also as a potential target for the development of new therapeutic approaches in classical homocystinuria. © 2013 SSIEM and Springer Science+Business Media Dordrecht.

Contains fulltext : 174066.pdf (Publisher’s version ) (Open Access) BACKGROUND: Elevated urinary excretion of orotic acid is associated with treatable disorders of the urea cycle and pyrimidine metabolism. Establishing the correct and timely diagnosis in a patient with orotic aciduria is key to effective treatment. Uridine monophosphate synthase is involved in de novo pyrimidine synthesis. Uridine monophosphate synthase deficiency (or hereditary orotic aciduria), due to biallelic mutations in UMPS, is a rare condition presenting with megaloblastic anemia in the first months of life. If not treated with the pyrimidine precursor uridine, neutropenia, failure to thrive, growth retardation, developmental delay, and intellectual disability may ensue. METHODS AND RESULTS: We identified mild and isolated orotic aciduria in 11 unrelated individuals with diverse clinical signs and symptoms, the most common denominator being intellectual disability/developmental delay. Of note, none had blood count abnormalities, relevant hyperammonemia or altered plasma amino acid profile. All individuals were found to have heterozygous alterations in UMPS. Four of these variants were predicted to be null alleles with complete loss of function. The remaining variants were missense changes and predicted to be damaging to the normal encoded protein. Interestingly, family screening revealed heterozygous UMPS variants in combination with mild orotic aciduria in 19 clinically asymptomatic family members. CONCLUSIONS: We therefore conclude that heterozygous UMPS-mutations can lead to mild and isolated orotic aciduria without clinical consequence. Partial UMPS-deficiency should be included in the differential diagnosis of mild orotic aciduria. The discovery of heterozygotes manifesting clinical symptoms such as hypotonia and developmental delay are likely due to ascertainment bias.

BACKGROUND: Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level.METHODS: To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patient's clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes.RESULTS: We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%).CONCLUSIONS: Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Children's Hospital Foundation and others.).

Item does not contain fulltext Background: In order to correctly assess the efficacy of therapy or diet in intervention studies on the activity of cystathionine beta-synthase (CBS) a sensitive analytical method is necessary. Methods: An electrospray LC-MS/MS method preceded by a solid phase extraction step was developed for the measurement of CBS activity in cell extracts. Nonafluoropentanoic acid was used as an ionpair to provide the underivatized cystathionine the desired retention on a C18 column. Results: A detection limit of 50pmol cystathionine/h/mg protein was achieved. In fibroblasts, intra- and inter-assay CVs for the CBS activity were 5.2% and 14.7%, respectively. A K(m) value of 8mumol/L for homocysteine, and 2.5mumol/L for serine was calculated. In fibroblasts wildtype, heterozygous, and homozygous CBS activity ranges measured were 8.5-27.0, 4.2-13.4, 0.0-0.7nmol/hxmg protein, respectively. The method was applied to a study where rats were fed 2 diets. Increase of dietary methionine (7.7 versus 3.8mg/kg methionine) significantly increased the CBS activity in rat liver lysates from a median of 58.0 to a median of 71.5 (P=0.037)nmol/hxmg protein. In a lymphoblasts cell culture experiment, the addition of Hcy to the culture media increased the activity of CBS 3 fold. Conclusion: This LC-MS/MS is able to diagnose CBS deficiency at the enzyme level, and can accurately measure the effect diets or therapy might have on the CBS activity in a variety of cell types.

Contains fulltext : 95780.pdf (Publisher’s version ) (Closed access) Recent evidence implies that impaired metabolism of glutathione has a role in the pathogenesis of nephropathic cystinosis. This recessive inherited disorder is characterized by lysosomal cystine accumulation and results in renal Fanconi syndrome progressing to end stage renal disease in the majority of patients. The most common treatment involves intracellular cystine depletion by cysteamine, delaying the development of end stage renal disease by a yet elusive mechanism. However, cystine depletion does not arrest the disease nor cures Fanconi syndrome in patients, indicating involvement of other yet unknown pathologic pathways. Using a newly developed proximal tubular epithelial cell model from cystinotic patients, we investigate the effect of cystine accumulation and cysteamine on both glutathione and ATP metabolism. In addition to the expected increase in cystine and defective sodium-dependent phosphate reabsorption, we observed less negative glutathione redox status and decreased intracellular ATP levels. No differences between control and cystinosis cell lines were observed with respect to protein turnover, albumin uptake, cytosolic and mitochondrial ATP production, total glutathione levels, protein oxidation and lipid peroxidation. Cysteamine treatment increased total glutathione in both control and cystinotic cells and normalized cystine levels and glutathione redox status in cystinotic cells. However, cysteamine did not improve decreased sodium-dependent phosphate uptake. Our data implicate that cysteamine increases total glutathione and restores glutathione redox status in cystinosis, which is a positive side-effect of this agent next to cystine depletion. This beneficial effect points to a potential role of cysteamine as anti-oxidant for other renal disorders associated with enhanced oxidative stress.

Contains fulltext : 95893.pdf (Publisher’s version ) (Closed access) BACKGROUND: Muscle biopsy is the gold standard for diagnosis of mitochondrial disorders because of the lack of sensitive biomarkers in serum. Fibroblast growth factor 21 (FGF-21) is a growth factor with regulatory roles in lipid metabolism and the starvation response, and concentrations are raised in skeletal muscle and serum in mice with mitochondrial respiratory chain deficiencies. We investigated in a retrospective diagnostic study whether FGF-21 could be a biomarker for human mitochondrial disorders. METHODS: We assessed samples from adults and children with mitochondrial disorders or non-mitochondrial neurological disorders (disease controls) from seven study centres in Europe and the USA, and recruited healthy volunteers (healthy controls), matched for age where possible, from the same centres. We used ELISA to measure FGF-21 concentrations in serum or plasma samples (abnormal values were defined as >200 pg/mL). We compared these concentrations with values for lactate, pyruvate, lactate-to-pyruvate ratio, and creatine kinase in serum or plasma and calculated sensitivity, specificity, and positive and negative predictive values for all biomarkers. FINDINGS: We analysed serum or plasma from 67 patients (41 adults and 26 children) with mitochondrial disorders, 34 disease controls (22 adults and 12 children), and 74 healthy controls. Mean FGF-21 concentrations in serum were 820 (SD 1151) pg/mL in adult and 1983 (1550) pg/mL in child patients with respiratory chain deficiencies and 76 (58) pg/mL in healthy controls. FGF-21 concentrations were high in patients with mitochondrial disorders affecting skeletal muscle but not in disease controls, including those with dystrophies. In patients with abnormal FGF-21 concentrations in serum, the odds ratio of having a muscle-manifesting mitochondrial disease was 132.0 (95% CI 38.7-450.3). For the identification of muscle-manifesting mitochondrial disease, the sensitivity was 92.3% (95% CI 81.5-97.9%) and specificity was 91.7% (84.8-96.1%). The positive and negative predictive values for FGF-21 were 84.2% (95% CI 72.1-92.5%) and 96.1 (90.4-98.9%). The accuracy of FGF-21 to correctly identify muscle-manifesting respiratory chain disorders was better than that for all conventional biomarkers. The area under the receiver-operating-characteristic curve for FGF-21 was 0.95; by comparison, the values for other biomarkers were 0.83 lactate (p=0.037, 0.83 for pyruvate (p=0.015), 0.72 for the lactate-to-pyruvate ratio (p=0.0002), and 0.77 for creatine kinase (p=0.013). INTERPRETATION: Measurement of FGF-21 concentrations in serum identified primary muscle-manifesting respiratory chain deficiencies in adults and children and might be feasible as a first-line diagnostic test for these disorders to reduce the need for muscle biopsy. FUNDING: Sigrid Juselius Foundation, Jane and Aatos Erkko Foundation, Molecular Medicine Institute of Finland, University of Helsinki, Helsinki University Central Hospital, Academy of Finland, Novo Nordisk, Arvo and Lea Ylppo Foundation. 01 september 2011

Cystathionine β-synthase (CBS) deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. We investigated whether CBS is present in human plasma and whether determination of its activity in plasma could be used for diagnostic purposes. We developed an assay to measure CBS activity in 20 μL of plasma using a stable isotope substrate - 2,3,3-2H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-2H-cystathionine, using LC-MS/MS. The median enzyme activity in control plasma samples was 404 nmol/h/L (range 66–1,066; n = 57). In pyridoxine nonresponsive CBS deficient patients, the median plasma activity was 0 nmol/ho/L (range 0–9; n = 26), while in pyridoxine responsive patients the median activity was 16 nmol/hour/L (range 0–358; n = 28); this overlapped with the enzyme activity from control subject. The presence of CBS in human plasma was confirmed by an in silico search of the proteome database, and was further evidenced by the activation of CBS by S-adenosyl-L-methionine and pyridoxal 5′-phosphate, and by configuration of the detected reaction product, 3,3-2H-cystathionine, which was in agreement with the previously observed CBS reaction mechanism. We hypothesize that the CBS enzyme in plasma originates from liver cells, as the plasma CBS activities in patients with elevated liver aminotransferase activities were more than 30-fold increased. In this study, we have demonstrated that CBS is present in human plasma and that its catalytic activity is detectable by LC-MS/MS. CBS assay in human plasma brings new possibilities in the diagnosis of pyridoxine nonresponsive CBS deficiency. Electronic supplementary material The online version of this article (doi:10.1007/s10545-010-9178-3) contains supplementary material, which is available to authorized users.

In patients with guanidinoacetate methyltransferase (GAMT) deficiency several parameters may point towards the diagnosis of GAMT deficiency. These include the low levels of creatine and creatinine in urine, the high concentration of guanidinoacetic acid (GAA) in urine and the low levels of creatine and creatinine in the cerebrospinal fluid (CSF). In this study, body fluids from 10 GAMT deficient patients were analysed using (1)H NMR spectroscopy. The urine 1D (1)H NMR spectra of all the patients showed a doublet resonance at 3.98 ppm (pH 2.50) derived from GAA present in high concentration. For this compound, a good recovery and good correlation was found between an LC-MS/MS method and (1)H NMR spectroscopy. In CSF NMR spectra of these patients, the singlet resonances of creatine and creatinine (3.05 and 3.13 ppm, respectively) were absent (normally always present in (1)H NMR spectra of CSF). Due to overlap by other resonances, the doublet of GAA could not be observed. Our data demonstrate that (1)H NMR spectroscopy of urine and CSF can be used to diagnose patients with GAMT deficiency. ispartof: NMR in Biomedicine vol:22 issue:5 pages:538-544 ispartof: location:England status: published

Contains fulltext : 69271.pdf (Publisher’s version ) (Closed access) BACKGROUND: Plasma methylmalonic acid (MMA) is a specific marker for functional cobalamin deficiency. This deficiency can give rise to non-specific but serious symptoms in childhood such as developmental delay, convulsions and failure to thrive and may even lead to irreversible neurological damage. AIM OF THE STUDY: To analyse plasma MMA concentrations in Dutch children and to evaluate possible factors influencing its concentration. METHODS: A number of 186 Dutch children aged 0-19 years were analysed cross-sectionally. Blood was collected to measure MMA, total homocysteine (tHcy), cobalamin (Cbl) and serum creatinine concentrations. In addition, information about medical history, age and sex was recorded. RESULTS: The geometric mean (GM) plasma MMA concentration was 0.17 micromol/l (95% CI 0.07-0.42) and the GM tHcy was 6.6 micromol/l (95% CI 3.1-13.9). There is a slight positive correlation between plasma MMA and age in children >1 year (r = 0.211, P < 0.05). Plasma MMA concentrations were significantly higher in children with low Cbl concentrations. No significant difference in MMA, Cbl, tHcy or creatinine concentrations between sexes could be observed. Regression analysis showed that Cbl was the strongest determinant of plasma MMA (regression coefficient -0.414, P < 0.05). The association between MMA and Cbl is stronger at increasing age (P for trend 0.045). CONCLUSIONS: Plasma Cbl is the main determinant of MMA in this group of Dutch children. The strength of the association increased with increasing age.