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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.

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.

Contains fulltext : 225118.pdf (Publisher’s version ) (Open Access) Maple syrup urine disease (MSUD) leads to severe neurological deterioration unless diagnosed early and treated immediately. We have evaluated the effectiveness of 11 years of MSUD newborn screening (NBS) in the Netherlands (screening >72 hours, referral if both total leucine (Xle) and valine ≥400 μmol/L blood) and have explored possibilities for improvement by combining our data with a systematic literature review and data from Collaborative Laboratory Integrated Reports (CLIR). Dutch MSUD NBS characteristics and accuracy were determined. The hypothetical referral numbers in the Dutch population of additional screening markers suggested by CLIR were calculated. In a systematic review, articles reporting NBS leucine concentrations of confirmed patients were included. Our data showed that NBS of 1 963 465 newborns identified 4 MSUD patients and led to 118 false-positive referrals (PPV 3.28%; incidence 1:491 000 newborns). In literature, leucine is the preferred NBS parameter. Total leucine (Xle) concentrations (mass-spectrometry) of 53 detected and 8 false-negative patients (sampling age within 25 hours in 3 patients) reported in literature ranged from 288 to 3376 (median 900) and 42 to 325 (median 209) μmol/L blood respectively. CLIR showed increasing Xle concentrations with sampling age and early NBS sampling and milder variant MSUD phenotypes with (nearly) normal biochemical profiles are causes of false-negative NBS results. We evaluated the effect of additional screening markers and established the Xle/phenylalanine ratio as a promising additional marker ratio for increasing the PPV, while maintaining high sensitivity in the Dutch MSUD NBS.

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.

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.

Background: NANS-CDG is a recently described congenital disorder of glycosylation caused by biallelic genetic variants in NANS, encoding an essential enzyme in de novo sialic acid synthesis. Sialic acid at the end of glycoconjugates plays a key role in biological processes such as brain and skeletal development. Here, we present an observational cohort study to delineate the genetic, biochemical, and clinical phenotype and assess possible correlations.Methods: Medical and laboratory records were reviewed with retrospective extraction and analysis of genetic, biochemical, and clinical data (2016–2020).Results: Nine NANS-CDG patients (nine families, six countries) referred to the Radboudumc CDG Center of Expertise were included. Phenotyping confirmed the hallmark features including intellectual developmental disorder (IDD) (n = 9/9; 100%), facial dysmorphisms (n = 9/9; 100%), neurologic impairment (n = 9/9; 100%), short stature (n = 8/9; 89%), skeletal dysplasia (n = 8/9; 89%), and short limbs (n = 8/9; 89%). Newly identified features include ophthalmological abnormalities (n = 6/9; 67%), an abnormal septum pellucidum (n = 6/9; 67%), (progressive) cerebral atrophy and ventricular dilatation (n = 5/9; 56%), gastrointestinal dysfunction (n = 5/9; 56%), thrombocytopenia (n = 5/9; 56%), and hypo–low-density lipoprotein cholesterol (n = 4/9; 44%). Biochemically, elevated urinary excretion of N-acetylmannosamine (ManNAc) is pathognomonic, the concentrations of which show a significant correlation with clinical severity. Genotypically, eight novel NANS variants were identified. Three severely affected patients harbored identical compound heterozygous pathogenic variants, one of whom was initiated on experimental prenatal and postnatal treatment with oral sialic acid. This patient showed markedly better psychomotor development than the other two genotypically identical males.Conclusions: ManNAc screening should be considered in all patients with IDD, short stature with short limbs, facial dysmorphisms, neurologic impairment, and an abnormal septum pellucidum +/– congenital and neurodegenerative lesions on brain imaging, to establish a precise diagnosis and contribute to prognostication. Personalized management includes accurate genetic counseling and access to proper supports and tailored care for gastrointestinal symptoms, thrombocytopenia, and epilepsy, as well as rehabilitation services for cognitive and physical impairments. Motivated by the short-term positive effects of experimental treatment with oral sialic, we have initiated this intervention with protocolized follow-up of neurologic, systemic, and growth outcomes in four patients. Research is ongoing to unravel pathophysiology and identify novel therapeutic targets.

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.

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.

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

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.

The multidrug resistance gene 1 product, P-glycoprotein (P-gp), is expressed in several excretory organs, including the apical membrane of proximal tubules. After inducing acute renal failure, P-gp expression is upregulated and this might be a protective function by pumping out toxicants and harmful products of oxidative stress. We characterized renal function of P-gp knockout mice and studied its consequences in renal ischemic damage. Compared with wild-type mice, knockout mice have a lower glomerular filtration rate and renal plasma flow. An augmented urinary excretion of sodium, numerous amino acids, calcium, glucose, and low molecular weight proteins was observed along with an increased diuresis. A higher lithium plasma clearance in the knockout mice suggested proximal tubular dysfunction. Electron microscopy showed mitochondrial abnormalities in proximal tubular cells that could account for decreased adenosine triphosphate levels in the cortex. After inducing ischemia, wild-type mice showed a decrease in creatinine clearance and severe proximal tubular necrosis. In contrast, knockout mice had no signs of tubular damage. Our data indicate that P-gp knockout mice have impaired renal function but are protected against ischemic renal injury. © 2007 International Society of Nephrology. Chemicals/CAS: adenosine triphosphate, 15237-44-2, 56-65-5, 987-65-5; amino acid, 65072-01-7; calcium, 7440-70-2; creatinine, 19230-81-0, 60-27-5; glucose, 50-99-7, 84778-64-3; lithium, 7439-93-2; protein, 67254-75-5; sodium, 7440-23-5; Amino Acids; Calcium, 7440-70-2; P-Glycoprotein; Sodium, 7440-23-5

Contains fulltext : 167788.pdf (Publisher’s version ) (Closed access) Aminoacylase 1 (ACY1) deficiency is an organic aciduria due to mutations in the ACY1 gene. It is considered much underdiagnosed. Most individuals known to be affected by ACY1 deficiency have presented with neurologic symptoms. We report here a cognitively normal 63-year-old woman who around the age of 12 years had developed dystonic symptoms that gradually evolved into generalized dystonia. Extensive investigations, including metabolic diagnostics and diagnostic exome sequencing, were performed to elucidate the cause of dystonia. Findings were only compatible with a diagnosis of ACY1 deficiency: the urinary metabolite pattern with N-acetylated amino acids was characteristic, there was decreased ACY1 activity in immortalized lymphocytes, and two compound heterozygous ACY1 mutations were detected, one well-characterized c.1057C>T (p.Arg353Cys) and the other novel c.325A>G (p.Arg109Gly). Expression analysis in HEK293 cells revealed high residual activity of the enzyme with the latter mutation. However, following co-transfection of cells with stable expression of the c.1057C>T variant with either wild-type ACY1 or the c.325A>G mutant, only the wild-type enhanced ACY1 activity and ACY1 presence in the Western blot, suggesting an inhibiting interference between the two variants. Our report extends the clinical spectrum of ACY1 deficiency to include dystonia and indicates that screening for organic acidurias deserves consideration in patients with unexplained generalized dystonia.

Contains fulltext : 50339.pdf (Publisher’s version ) (Closed access)

Contains fulltext : 50036.pdf (Publisher’s version ) (Closed access) A diagnosis of 3-methylglutaconic aciduria type I (OMIM: 250950) based on elevated urinary excretion of 3-methylglutaconic acid (3MGA), 3-methylglutaric acid (3MG) and 3-hydroxyisovaleric acid (3HIVA) was made in a 61-year-old female patient presenting with leukoencephalopathy slowly progressing over more than 30 years. The diagnosis was confirmed at the enzymatic and molecular level. In vivo brain MR spectroscopic imaging (MRSI) was performed at 3.0 T, and one-dimensional and two-dimensional in vitro NMR spectroscopy of body fluids of the patient was performed at 11.7 T. Additionally, we measured 1D (1)H-NMR spectra of urine of seven patients with a total of four different inborn errors of leucine metabolism. Increased concentrations of 3HIVA, 3MGA (cis and trans) and 3MG were observed in the NMR spectra of the patient's urine. In the cerebrospinal fluid, the 3HIVA concentration was 10 times higher than in the plasma of the patient and only the cis isomer of 3MGA was observed. In vivo brain MRSI showed an abnormal resonance at 1.28 ppm that may be caused by 3HIVA. Comparison of (1)H-NMR spectra of urine samples from all eight patients studied, representing five different inborn errors of leucine metabolism, showed that each disease has typical NMR characteristics. Our leukoencephalopathy patient suffers from a late-onset form of 3-methylglutaconic aciduria type I. In the literature, only very few adult patients with this conditions have been described, and 3HIVA accumulation in white matter in the brain has not been presented before in these patients. Our data demonstrate that (1)H-NMR spectroscopy of urine can easily discriminate between the known inborn errors of leucine metabolism and provide the correct diagnosis.

Contains fulltext : 59288.pdf (Publisher’s version ) (Closed access) Thymidylate synthase (TYMS) and 5,10-methylenetetrahydrofolate reductase (MTHFR) may compete for their common cofactor 5,10-methylenetetrahyhdrofolate (5,10-meTHF). Limiting 5,10-meTHF results in elevated homocysteine, especially in individuals homozygous for the T allele of the MTHFR C677T polymorphism. The TYMS gene has a tandem repeat polymorphism (two repeats or three repeats, designated 2R or 3R, respectively), which may also affect homocysteine concentrations. The 3R allele is subject to increased translational efficiency in vitro and the 3R3R genotype is associated with both decreased serum folate and elevated plasma homocysteine (tHcy) in a population of Singapore Chinese. We assessed the relationship between TYMS genotype and key biochemical and genetic variables in a random sample of 392 healthy young Northwestern European men and women. The tHcy concentrations for 3R3R homozygotes (median 8.5 micromol/l) did not differ significantly from those for 2R2R homozygotes (median 8.7 micromol/l) or 2R3R heterozygotes (median 9.3 micromol/l) in the population as a whole (P=0.43), or in subsets of subjects with low serum folate (P=0.60) or the MTHFR 677TT genotype (P=0.90). Furthermore, there was no trend toward elevated tHcy in 3R3R homozygotes. Similarly, the TYMS tandem repeat polymorphism was not associated with serum folate concentrations. Our findings indicate that the TYMS 3R3R genotype is not a determinant of homocysteine in this sample of healthy young Caucasian adults from Northern Ireland.

Objective— Mild hyperhomocystenemia is an independent, graded risk factor for cardiovascular disease. Genetic determinants of hyperhomocystenemia include functional polymorphisms in several folate/homocysteine metabolic enzymes. Nitric oxide may also modulate plasma homocysteine (tHcy) concentrations, either by direct inhibition of methionine synthase or via an indirect effect on folate catabolism. Methods and Results— The hypothesis that the endothelial nitric oxide synthase ( NOS3 ) G894T polymorphism is a genetic determinant of tHcy concentrations was tested in 2 independent healthy adult populations. In both populations, NOS3 genotype was significantly associated with tHcy concentrations in nonsmokers with low folate ( P =0.03 for each). Models were constructed to adjust for known determinants of tHcy concentrations and test for interactions between NOS3 genotype and these determinants in nonsmokers from each population. NOS3 genotype remained a significant determinant of tHcy concentrations after adjustment. Interactions between NOS3 genotype and serum folate were significant in both populations, and the interaction between NOS3 genotype and MTHFR C677T genotype was significant in the larger population. Conclusions— These data indicate that the NOS3 894TT genotype is a risk factor for elevated tHcy in healthy nonsmoking adults with low serum folate and supports the hypothesis that nitric oxide modulates homocysteine through an effect on folate catabolism.

Item does not contain fulltext Periconceptional folic acid supplementation may reduce the risk of cleft lip with or without cleft palate (CL(P)). Polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene reduce availability of 5-methyltetrahydrofolate, the predominant circulating form of folate. To determine the effect of MTHFR C677T and MTHFR A1298C genotypes and haplotypes on CL(P) risk and the interaction with maternal periconceptional dietary folate and folic acid supplement intake, the authors conducted a case-control triad study in the Netherlands (1998-2000) among 179 CL(P) and 204 control families. Infant and parental MTHFR C677T and MTHFR A1298C genotypes and haplotypes were not associated with CL(P) risk in the case-control and transmission disequilibrium test analyses. Mothers carrying the MTHFR 677TT genotype and who either did not use folic acid supplements periconceptionally or had a low dietary folate intake, or both, had an increased risk of delivering a CL(P) child (odds ratio (OR) = 5.9, 95% confidence interval (CI): 1.1, 30.9; OR = 2.8, 95% CI: 0.7, 10.5; OR = 10.0, 95% CI: 1.3, 79.1, respectively). No supplement use, low dietary folate intake, and maternal MTHFR 1298CC genotype increased the risk of CL(P) offspring almost sevenfold (OR = 6.5, 95% CI: 1.4, 30.2). Thus, the detrimental effect of low periconceptional folate intake on the risk of giving birth to a CL(P) child was more pronounced in mothers with the MTHFR 677TT or MTHFR 1298CC genotype.

Item does not contain fulltext Hyperhomocysteinaemia is generally accepted as an independent and graded risk factor for both arterial occlusive disease and venous thrombosis. The only way of homocysteine degradation is conversion to cysteine in the transsulfuration pathway in which the regulating step is catalysed by cystathionine beta-synthase (CBS). Mild impairment of CBS function could therefore affect homocysteine concentration, in particular after methionine loading, and consequently cardiovascular disease (CVD) risk. We analysed two silent polymorphisms and one short tandem repeat in the CBS gene (ie 699C-->T, 1080C-->T and -5697 (GT) STR) as genetic markers potentially in linkage disequilibrium with a functional polymorphism. We assessed their association with fasting and post-methionine load homocysteine in 190 patients with arterial occlusive disease, and in 381 controls. No differences in CBS genotype frequencies between cases and controls were found, nor was a particular CBS genotype associated with an elevated risk of arterial occlusive disease. Although we did find a high rate of linkage disequilibrium between the two single nucleotide polymorphisms and the GT STR, none of the genotypes defined by the three CBS variants studied showed an association with elevated fasting, post-load or increase upon methionine loading homocysteine concentrations. In conclusion, we did not find any indication that genetic variation in the CBS gene is associated with increased homocysteine concentrations.

Item does not contain fulltext In hyperhomocysteinemia (HHcy), an independent risk factor for cardiovascular diseases, endothelial dysfunction due to reduced bioavailability of nitric oxide is a consistent finding. However, the underlying mechanisms remain unknown. Increased levels of the nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) have been associated with HHcy, and may contribute, at least in part, for the homocysteine-induced endothelial dysfunction, but whether cystathionine beta-synthase (CBS) deficiency is associated with increased ADMA has hardly been investigated. To address this question, we measured total homocysteine (tHcy), ADMA and symmetric dimethylarginine (SDMA) in plasma of 22 adult CBS deficient patients, using established HPLC techniques. Results showed that in CBS deficient patients with elevated levels of tHcy (median (total range): 33 (14-237) mumol/L), both ADMA and SDMA levels were normal. Moreover, tHcy and ADMA concentrations were not correlated (r(s)=0.017, p=0.94). Our results favor the hypothesis that the negative vascular effects of HHcy have an ADMA-independent etiology. 01 juni 2012

Background: Hyperhomocysteinemia is an independent risk factor for cardiovascular disease (CVD). Intracellular vitamin B12 deficiency may lead to increased plasma total homocysteine (tHcy) concentrations and because transcobalamin (TC) is the plasma transporter that delivers vitamin B12 to cells, genetic variation in the TC gene may affect intracellular vitamin B12 availability and, consequently, tHcy concentrations.Methods: We examined five sequence variants, i.e., I23V, G94S, P259R, S348F, and R399Q, in the TC gene as possible determinants of tHcy and, concordantly, as possible risk factors for CVD in 190 vascular disease patients and 601 controls. We also studied potential effect-modification of vitamin B12 by genotype.Results: In individuals with high vitamin B12, 259PP individuals had lower tHcy concentrations than 259PR and 259RR individuals. Homozygous 23VV individuals had lower fasting tHcy concentrations than their 23IV and 23II peers. None of the genotypes defined by the three other sequence variants showed an association with tHcy concentrations, nor was any TC genotype associated with an increased CVD risk.Conclusions: In individuals in the highest quartile of the vitamin B12 distribution (>299 pmol/L), tHcy concentrations are lower in 259PP homozygotes than in 259PR and 259RR individuals. Therefore, 259PP individuals, who represent >25% of the general population, may be more susceptible to reduction of plasma tHcy concentrations by increasing the vitamin B12 status.

Item does not contain fulltext Cardiovascular diseases (CVD), especially coronary heart disease (CHD), are the most important causes of death in industrialized countries. Increased concentrations of total plasma homocysteine (tHcy) have been associated with an increased risk of CHD. Assuming that this relation is causal, a lower tHcy concentration will reduce the occurrence and recurrence of CHD. Therefore, it is important to know which factors determine the tHcy concentration. In the general population, the most important modifiable determinants of tHcy are folate intake and coffee consumption. Smoking and alcohol consumption are also associated with the tHcy concentration, but more research is necessary to elucidate whether these relations are not originating from residual confounding due to other lifestyle factors. The most important nonmodifiable determinant is the 677 C>T polymorphism in the gene that encodes methylenetetrahydrofolate reductase (MTHFR), a regulating enzyme in homocysteine metabolism. Especially subjects with the homozygous form of this polymorphism (i.e., 677TT genotype) and a low folate status have elevated tHcy concentrations. Specific clinical conditions like the use of antiepileptic drugs or methotrexate, renal failure, cancer, rheumatoid arthritis, and hypothyroidism may lead to elevated tHcy concentrations. The available epidemiological evidence indicates that an increased tHcy concentration is not an important risk factor for CHD in healthy subjects. However, prospective studies, which included subjects at high risk of CHD, and secondary prevention trials with intermediary endpoints consistently show that elevations in the tHcy concentration may be an important risk factor in these subjects for a (recurrent) CHD event. The induction of vascular endothelial dysfunction by homocysteine may underlie this increased risk. Ongoing intervention trials will indicate whether homocysteine-lowering through vitamin supplementation, prevents CHD in the treatment groups.

BACKGROUND: Cystinosis is an autosomal recessive disorder marked by intralysosomal cystine accumulation. Patients present with generalized proximal tubular dysfunction called renal Fanconi syndrome. Urinary carnitine loss results in plasma and muscle carnitine deficiency, but no clinical signs of carnitine deficiency have been described. Also, the optimal dose of carnitine supplementation is undefined. This study aimed to determine whether currently recommended carnitine doses result in adequate correction of plasma carnitine. METHODS: Five cystinosis patients with renal Fanconi syndrome, aged 2-18 years, were included. L-carnitine was prescribed 50 mg/kg/day since diagnosis: median 36 (range 18-207) months. Total and free plasma and urine carnitine and carnitine profiles were measured at study onset, after stopping L-carnitine for 3 months and 3 months after reintroducing L-carnitine 50 mg/kg/day. RESULTS: At study onset, plasma free carnitine was normal in all patients, total carnitine (1/5), acetylcarnitine (3/5), and several short- and medium-chain acylcarnitines ≤10 carbons (5/5) were increased indicating carnitine over-supplementation. Three months after cessation, carnitine profiles normalized and 3/5 patients showed plasma carnitine deficiency. Three months after reintroduction, plasma free carnitine normalized in all patients, however, carnitine profiles were disturbed in 4/5 patients. Urine free carnitine, acetylcarnitine, and acylcarnitines ≤10 carbons were increased in all patients independent of carnitine supplementation. CONCLUSION: Administration of recommended doses L-carnitine (50 mg/kg/day) resulted in over-supplementation. Although the drug is considered to be rather safe, long-term effects of over-supplementation remain unknown warranting cautious use of high doses. Plasma carnitine profile might be used as a monitor, to prevent overdosing.

Item does not contain fulltext Epidemiological evidence has revealed that an elevated plasma homocysteine level (hyperhomocysteinemia) confers an increased risk of cardiovascular disease and neural tube defects. Hyperhomocysteinemia is caused by both nutritional (e.g. folate, vitamins B(6) and B(12)) and genetic factors, including functional polymorphisms of key enzymes involved in homocysteine metabolism. One such enzyme, methionine synthase reductase (MTRR), maintains adequate levels of methylcob(III)alamin, the activated cofactor for methionine synthase, which catalyzes the remethylation of homocysteine to methionine. A common MTRR polymorphism, i.e. a 66 A-->G substitution specifying an isoleucine to methionine substitution (I22M), was recently identified. To assess the influence of this polymorphism on total plasma homocysteine (tHcy), we undertook a genotype/phenotype analysis in a study population of 601 Northern-Irish men, aged 30--49, for which biochemical and genetic data relevant to folate/homocysteine metabolism had already been acquired. The 66AA genotype has a frequency of 29% in this population. We established that there was a significant influence of MTRR genotype on tHcy ranking (P=0.004) and that the 66AA genotype contributes to a moderate increase in tHcy levels across the distribution [OR 1.59 (95% CI: 1.10--2.25) for the 66AA genotype to be in the upper half of the tHcy distribution, P=0.03]. The homocysteine-elevating effect of the 66AA genotype is independent of serum folate, vitamin B(12) and vitamin B(6) levels. Based on published estimates of the enhanced cardiovascular disease risk conferred by defined increments of plasma tHcy, we estimate that 66AA homozygotes have, on average, an approximately 4% increase in cardiovascular disease risk compared to 66GG homozygotes. This study provides the first evidence that the MTRR A66G polymorphism significantly influences the circulating tHcy concentration.

Contains fulltext : 51920.pdf (Publisher’s version ) (Closed access)

Severe hyperhomocysteinemia in its most frequent form, is caused by a homozygous enzymatic deficiency of cystathionine β-synthase (CBS). A major complication in CBS deficiency is deep venous thrombosis or pulmonary embolism. A recent report by Mandel et al (N Engl J Med 334:763, 1996) postulated factor V Leiden (FVL) to be an absolute prerequisite for the development of thromboembolism in patients with severe hyperhomocysteinemia. We studied 24 patients with homocystinuria caused by homozygous CBS deficiency from 18 unrelated kindreds for FVL and for the 677C→T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene and investigated their possible interaction in the risk of venous thrombosis. Thrombotic complications were diagnosed in six patients, of whom only one was a carrier of FVL. On the contrary, thermolabile MTHFR caused by the 677C→T mutation, was frequently observed among homocystinuria patients, especially among those with thromboembolic complications: three of six homocystinuria patients who had suffered from a thromboembolic event had thermolabile MTHFR. These data indicate that FVL is not an absolute prerequisite and probably not even a major determinant of venous thrombosis in homocystinuria, but, interestingly, thermolabile MTHFR may constitute a significant risk factor for thromboembolic complications in this inborn error of methionine metabolism.

SummaryMild hyperhomocysteinemia is an established risk factor for both arteriosclerosis and thrombosis, and may be caused by genetic and environmental factors. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the cofactor for the methylation of homocysteine to methionine. Individuals with the thermolabile variant of MTHFR have decreased MTHFR activities, resulting in elevated plasma homocysteine concentrations. A homozygous 677C→T transition in the MTHFR gene has recently been identified as the cause of reduced enzyme activity and thermolability of the protein. We studied the frequency of the homozygous mutant (+/+) genotype in 471 patients with deep-vein thrombosis and 474 healthy controls enrolled in The Leiden Thrombophilia Study (LETS), its interaction with factor V Leiden, and assessed the association between the MTHFR genotypes and plasma homocysteine concentration. Homozygosity for the 677C→T polymorphism was observed in 47 (10%) patients, and in 47 (9.9%) controls (OR 1.01 [95% CI: 0.7-1.5]). No modified risk of the (+/+) genotype was observed in carriers of factor V Leiden. Our data suggest that, although the homozygous mutant genotype is associated with elevated plasma homocysteine concentrations, this homozygous mutation itself is not a genetic risk factor for deep-vein thrombosis, irrespective of factor V Leiden genotype.

Contains fulltext : 136381.pdf (Publisher’s version ) (Open Access) Total parenteral nutrition (TPN) is associated with the development of parenteral nutrition-associated liver disease (PNALD) in infants. Fish oil-based lipid emulsions can reverse PNALD, yet it is unknown if they can prevent PNALD. We studied preterm pigs administered TPN for 14 days with either 100% soybean oil (IL), 100% fish oil (OV), or a mixture of soybean oil, medium chain triglycerides (MCTs), olive oil, and fish oil (SL); a group was fed formula enterally (ENT). In TPN-fed pigs, serum direct bilirubin, gamma glutamyl transferase (GGT), and plasma bile acids increased after the 14 day treatment but were highest in IL pigs. All TPN pigs had suppressed hepatic expression of farnesoid X receptor (FXR), cholesterol 7-hydroxylase (CYP7A1), and plasma 7alpha-hydroxy-4-cholesten-3-one (C4) concentrations, yet hepatic CYP7A1 protein abundance was increased only in the IL versus ENT group. Organic solute transporter alpha (OSTalpha) gene expression was the highest in the IL group and paralleled plasma bile acid levels. In cultured hepatocytes, bile acid-induced bile salt export pump (BSEP) expression was inhibited by phytosterol treatment. We show that TPN-fed pigs given soybean oil developed cholestasis and steatosis that was prevented with both OV and SL emulsions. Due to the presence of phytosterols in the SL emulsion, the differences in cholestasis and liver injury among lipid emulsion groups in vivo were weakly correlated with plasma and hepatic phytosterol content.

Currently, six inborn errors of metabolism with 3-methylglutaconic aciduria as discriminative feature are known. The “Primary 3-methylglutaconic aciduria,” 3-methylglutaconyl-CoA hydratase deficiency or AUH defect, is a disorder of leucine catabolism. For all other subtypes, also denoted “Secondary 3-methylglutaconic acidurias” (TAZ defect or Barth syndrome, SERAC1 defect or MEGDEL syndrome, OPA3 defect or Costeff syndrome, DNAJC19 defect or DCMA syndrome, TMEM70 defect, “not otherwise specified (NOS) 3-MGA-uria”), the origin of 3-methylglutaconic aciduria remains enigmatic but is hypothesized to be independent from leucine catabolism. Here we show the results of leucine loading test in 21 patients with different inborn errors of metabolism who present with 3-methylglutaconic aciduria. After leucine loading urinary 3-methylglutaconic acid levels increased only in the patients with an AUH defect. This strongly supports the hypothesis that 3-methylglutaconic aciduria is independent from leucine breakdown in other inborn errors of metabolism with 3-methylglutaconic aciduria and also provides a simple test to discriminate between primary and secondary 3-methylglutaconic aciduria in regular patient care.

Item does not contain fulltext Amyloid-beta (Abeta) deposition, one of the main hallmarks of Alzheimer's disease (AD), has been linked to glutamatergic dysfunction, i.e., increased stimulation of synaptic glutamate receptors that may ultimately result in neuronal loss. It was our aim to study the effect of Abeta on multiple components of the glutamatergic system, and therefore we assessed the expression of several glutamate-related proteins and amino acids in the TgSwDI mouse model for Abeta pathology. We determined that in TgSwDI mice, levels of several amino acids are altered, in particular that of glycine. Protein changes were only found in 9-month-old TgSwDI mice with extensive Abeta deposits, with the most prominent change an increased expression of vesicular glutamate transporter 1 (vGlut1). Autoradiography experiments demonstrated that, while the number of activated N-methyl-D-aspartic acid (NMDA) receptors was unchanged in TgSwDI mice, binding of the NMDA receptor radioligand [3H]MDL-105,519 to the glycine-binding site of these receptors was increased. Although there are some discrepancies between our results and those found in AD patients, our results suggest that several components of the glutamatergic system might serve as meaningful markers to monitor the progression of AD.

Background Hyperhomocysteinemia, an independent and graded risk factor for coronary artery disease (CAD), may result from both environmental and hereditary factors. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of methylenetetrahydrofolate to methyltetrahydrofolate, the methyl donor in the remethylation of homocysteine to methionine. A 677C→T mutation in the MTHFR gene has been associated with elevated homocysteine concentrations in homozygous (+/+) individuals. Methods and Results We assessed the frequency of this common mutation in 735 CAD patients from the Regression Growth Evaluation Statin Study (REGRESS), a lipid-lowering coronary-regression trial, and in 1250 population-based control subjects. Furthermore, the association between the mutation and serum homocysteine concentrations was studied. The frequency of the homozygous (+/+) mutation was 9.5% among patients versus 8.5% among control subjects, resulting in an odds ratio of 1.21 (95% confidence interval [CI], 0.87 to 1.68), relative to the (−/−) genotype. Homocysteine concentrations were significantly elevated in both (+/+) and (+/−) individuals compared with (−/−) individuals (median homocysteine levels, 15.4, 13.4, and 12.6 μmol/L, for (+/+), (+/−), and (−/−) individuals, respectively). For a summary estimation of the risk of the (+/+) genotype for CAD, we performed a meta-analysis on 8 different case-control studies on thermolabile MTHFR in CAD. In the meta-analysis, the homozygous (+/+) genotype was present in 299 of 2476 patients (12.1%) and in 257 (10.4%) of 2481 control subjects, resulting in a significant odds ratio of 1.22 (95% CI, 1.01 to 1.47) relative to the (−/−) genotype. Conclusions Both the homozygous (+/+) and heterozygous (+/−) genotype result in elevated homocysteine concentrations. From our meta-analysis, we conclude that the homozygous (+/+) genotype is a modest but significant risk factor for CAD.

Elevated homocysteine (Hcy) levels are observed in two apparently unrelated diseases: neural-tube defects (NTD) and premature vascular disease. Defective human methionine synthase (MS) could result in elevated Hcy levels. We sequenced the coding region of MS in 8 hyperhomocysteinaemic patients (4 NTD patients and 4 patients with pregnancies complicated by spiral arterial disease, SAD). We identified only one mutation resulting in an amino acid substitution: an A-->G transition at bp 2756, converting an aspartic acid (D919) into a glycine (G). We screened genomic DNA for the presence of this mutation in 56 NTD patients, 69 mothers of children with NTD, 108 SAD patients and 364 controls. There was no increased prevalence of the GG and AG genotypes in NTD patients, their mothers or SAD patients. The D919G mutation does not seem to be a risk factor for NTD or vascular disease. We then examined the mean Hcy levels for each MS genotype. There was no correlation between GG- or AG-genotype and Hcy levels. The D919G mutation is thus a fairly prevalent, and probably benign polymorphism. This study, though limited, provides no evidence for a major involvement of MS in the aetiology of homocysteine-related diseases such as NTD or vascular disease.

Contains fulltext : 135973.pdf (Publisher’s version ) (Closed access) Mutations in PCBD1 are causative for transient neonatal hyperphenylalaninemia and primapterinuria (HPABH4D). Until now, HPABH4D has been regarded as a transient and benign neonatal syndrome without complications in adulthood. In our study of three adult patients with homozygous mutations in the PCBD1 gene, two patients were diagnosed with hypomagnesemia and renal Mg(2+) loss, and two patients developed diabetes with characteristics of maturity onset diabetes of the young (MODY), regardless of serum Mg(2+) levels. Our results suggest that these clinical findings are related to the function of PCBD1 as a dimerization cofactor for the transcription factor HNF1B. Mutations in the HNF1B gene have been shown to cause renal malformations, hypomagnesemia, and MODY. Gene expression studies combined with immunohistochemical analysis in the kidney showed that Pcbd1 is expressed in the distal convoluted tubule (DCT), where Pcbd1 transcript levels are upregulated by a low Mg(2+)-containing diet. Overexpression in a human kidney cell line showed that wild-type PCBD1 binds HNF1B to costimulate the FXYD2 promoter, the activity of which is instrumental in Mg(2+) reabsorption in the DCT. Of seven PCBD1 mutations previously reported in HPABH4D patients, five mutations caused proteolytic instability, leading to reduced FXYD2 promoter activity. Furthermore, cytosolic localization of PCBD1 increased when coexpressed with HNF1B mutants. Overall, our findings establish PCBD1 as a coactivator of the HNF1B-mediated transcription necessary for fine tuning FXYD2 transcription in the DCT and suggest that patients with HPABH4D should be monitored for previously unrecognized late complications, such as hypomagnesemia and MODY diabetes.

Item does not contain fulltext BACKGROUND: Cystinosis is an autosomal recessive disease caused by intralysosomal cystine accumulation, treated with cysteamine. Recently, new adverse effects of cysteamine were reported. Skin biopsies showed microvascular proliferation (angioendotheliomatosis). To examine the mechanism of angioendotheliomatosis associated with cysteamine toxicity, we examined the effect of cysteamine on human dermal microvascular endothelial cells (HDMVEC). METHODS: After cysteamine exposure (range 0-3.0 mM) during 24 h, cell viability was measured using water soluble tetrazolium salt-1 (WST-1) in both control HDMVEC and fibroblasts. Cell proliferation and apoptosis rate were measured in HDMVEC by bromodeoxyuridine (BrdU) incorporation and caspase 3 and caspase 7 activity, respectively. Intracellular glutathione (GSH) was measured in HDMVEC after cysteamine exposure of 0, 0.1 or 1.0 mM. Medium and cysteamine were refreshed every 6 h to mimic the in vivo situation. Next, cell viability in HDMVEC was measured after 24 h of GSH exposure (range 0-10.0 mM). RESULTS: HDMVEC viability and proliferation increased after cysteamine exposure 0.03-3.0 mM (p < 0.01) and 0.03-1.0 mM (p = 0.01) respectively; cell viability in fibroblasts was not affected by incubation with cysteamine. Apoptosis remained unaffected by incubation with 0-1.0 mM cysteamine, 3.0 mM caused increased apoptosis. Intracellular GSH was significantly increased after incubation with cysteamine 0.1 mM (p = 0.02) and 1.0 mM (p < 0.01). HDMVEC viability increased after exposure to GSH 1.0-5.0 mM (p < 0.01). CONCLUSION: Cysteamine concentrations, similar to those described in plasma of cystinosis patients, stimulate HDMVEC viability and proliferation and increase intracellular GSH content. We postulate that this mechanism might underlie angioendotheliomatosis induced by cysteamine.

Contains fulltext : 166204.pdf (Publisher’s version ) (Closed access) Staphylococcus aureus (S. aureus) is a common bacterium infecting children with cystic fibrosis (CF). Since current detection methods are difficult to perform in children, there is need for an alternative. This proof of concept study investigates whether breath profiles can discriminate between S. aureus infected and non-infected CF patients based on volatile organic compounds (VOCs). We collected exhaled breath of CF patients with and without S. aureus airways infections in which VOCs were identified using gas chromatography-mass spectrometry. We classified these VOC profiles with sparse partial least squares discriminant analysis. Multivariate breath VOC profiles discriminated infected from non-infected CF patients with high sensitivity (100%) and specificity (80%). We identified the nine compounds most important for this discrimination. We successfully detected S. aureus infection in CF patients, using breath VOC profiles. Nine highlighted compounds can be used as a focus point in further biomarker identification research. The results show considerable potential for non-invasive diagnosis of airway infections.

Although the diagnosis of a primary carnitine deficiency is usually based on a very low level of free and total carnitine (free carnitine: 1–5 μM, normal 20–55 μM) (Longo et al. 2006), we detected a patient via newborn screening with a total carnitine level 67 % of the normal value. At the age of 1 year, after interruption of carnitine supplementation for a 4-week period the carnitine profile was assessed and the free carnitine level had dropped to 10.4 μmol/l (normal: 20–55 μM) and total carnitine level had dropped to 12.7 μmol/l (normal: 25–65 μM). Transient carnitine deficiency was not likely anymore and DNA mutation analysis of the OCTN2 (SLC22A5) gene showed a homozygous c.136C>T (p.P46S) mutation, confirming the diagnosis of primary carnitine deficiency. We would like to emphasize that neonates with a primary carnitine deficiency might present with relatively high levels of total carnitine due to placental carnitine transfer, and also draw the attention to the importance of regular follow-up and the significance of genetic diagnostics in patients with a nonclassical presentation.

Item does not contain fulltext Elevated urinary excretion of 3-methylglutaconic acid is considered rare in patients suspected of a metabolic disorder. In 3-methylglutaconyl-CoA hydratase deficiency (mutations in AUH), it derives from leucine degradation. In all other disorders with 3-methylglutaconic aciduria the origin is unknown, yet mitochondrial dysfunction is thought to be the common denominator. We investigate the biochemical, clinical and genetic data of 388 patients referred to our centre under suspicion of a metabolic disorder showing 3-methylglutaconic aciduria in routine metabolic screening. Furthermore, we investigate 591 patients with 50 different, genetically proven, mitochondrial disorders for the presence of 3-methylglutaconic aciduria. Three percent of all urine samples of the patients referred showed 3-methylglutaconic aciduria, often in correlation with disorders not reported earlier in association with 3-methylglutaconic aciduria (e.g. organic acidurias, urea cycle disorders, haematological and neuromuscular disorders). In the patient cohort with genetically proven mitochondrial disorders 11 % presented 3-methylglutaconic aciduria. It was more frequently seen in ATPase related disorders, with mitochondrial DNA depletion or deletion, but not in patients with single respiratory chain complex deficiencies. Besides, it was a consistent feature of patients with mutations in TAZ, SERAC1, OPA3, DNAJC19 and TMEM70 accounting for mitochondrial membrane related pathology. 3-methylglutaconic aciduria is found quite frequently in patients suspected of a metabolic disorder, and mitochondrial dysfunction is indeed a common denominator. It is only a discriminative feature of patients with mutations in AUH, TAZ, SERAC1, OPA3, DNAJC19 TMEM70. These conditions should therefore be referred to as inborn errors of metabolism with 3-methylglutaconic aciduria as discriminative feature.

Contains fulltext : 108785.pdf (Publisher’s version ) (Closed access) Using exome sequencing, we identify SERAC1 mutations as the cause of MEGDEL syndrome, a recessive disorder of dystonia and deafness with Leigh-like syndrome, impaired oxidative phosphorylation and 3-methylglutaconic aciduria. We localized SERAC1 at the interface between the mitochondria and the endoplasmic reticulum in the mitochondria-associated membrane fraction that is essential for phospholipid exchange. A phospholipid analysis in patient fibroblasts showed elevated concentrations of phosphatidylglycerol-34:1 (where the species nomenclature denotes the number of carbon atoms in the two acyl chains:number of double bonds in the two acyl groups) and decreased concentrations of phosphatidylglycerol-36:1 species, resulting in an altered cardiolipin subspecies composition. We also detected low concentrations of bis(monoacyl-glycerol)-phosphate, leading to the accumulation of free cholesterol, as shown by abnormal filipin staining. Complementation of patient fibroblasts with wild-type human SERAC1 by lentiviral infection led to a decrease and partial normalization of the mean ratio of phosphatidylglycerol-34:1 to phosphatidylglycerol-36:1. Our data identify SERAC1 as a key player in the phosphatidylglycerol remodeling that is essential for both mitochondrial function and intracellular cholesterol trafficking. 01 juli 2012

Contains fulltext : 80876.pdf (Publisher’s version ) (Closed access) Patients with SUCLA2 gene defects characteristically develop the trias of early hypotonia, progressive dystonia and sensori-neural deafness. We describe the clinical course and biochemical phenotype in 16 children from the Faroe Islands with a homozygous SUCLA2 splice site mutation. Elevated urinary 3-hydroxyisovaleric acid is a novel biochemical feature in patients. Progressive hearing loss, in combination with a characteristic metabolite profile (increased lactate, methylmalonic acid, C4-dicarboxylic carnitine, 3-hydroxyisovaleric acid) should lead the clinician to the correct diagnosis even in patients with only intermittent lactic acidemia. Direct SUCLA2 sequence analysis is suggested instead of an invasive muscle biopsy to obtain the diagnosis. Nutritional intervention may be considered in SUCLA2 patients.

Contains fulltext : 71010.pdf (Publisher’s version ) (Closed access) OBJECTIVE: The physicochemical approach, described by Stewart to investigate the acid-base balance, includes the strong ion gap (SIG), a quantitative measure of "unmeasured" anions, which strongly correlates to the corrected anion gap. The chemical nature of these anions is for the most part unknown. We hypothesized that amino acids, uric acid, and organic acids could contribute to the SIG. DESIGN: Prospective observational study. SETTING: Intensive care department of an academic hospital. PATIENTS: Consecutive intensive care unit patients (n = 31) with metabolic acidosis, defined as a pH of < 7.35 and a base excess of < or = -5 mmol/L. INTERVENTIONS: A single arterial blood sample was collected. MEASUREMENTS: The SIG was calculated and two groups were compared: patients with SIG of < or = 2 mEq/L and patients with SIG of > or = 5 mEq/L. "Unmeasured" anions were examined by ion-exchange column chromatography, reverse-phase high-performance liquid chromatography, and gas chromatography/mass spectrometry measuring amino acids, uric acid, and organic acids, respectively. MAIN RESULTS: Comparison of patient characteristics of both SIG groups showed that age, sex, Acute Physiology and Chronic Health Evaluation II, pH, base excess, and lactate were not significantly different. Renal insufficiency and sepsis were more prevalent in the SIG > or = 5 mEq/L group (n = 12; median SIG, 8.3 mEq/L), associated with higher mortality. Concentrations of the anionic compounds aspartic acid, uric acid, succinic acid, pyroglutamic acid, p-hydroxyphenyllactic acid, and the semiquantified organic acid homovanillic acid were all statistically significantly elevated in the SIG > or = 5 mEq/L group compared with the SIG < or = 2 mEq/L group (n = 8; median SIG, 0.6 mEq/L). Overall, the averaged difference between both SIG groups in total anionic amino acids, uric acid, and organic acids concentrations contributed to the SIG for, respectively, 0.07% (5 microEq/L, p = not significant), 2.2% (169 microEq/L, p = .021), and 5.6% (430 microEq/L, p = .025). CONCLUSIONS: Amino acids, uric acid, and organic acids together accounted for only 7.9% of the SIG in intensive care unit patients with metabolic acidosis.

Contains fulltext : 53236.pdf (Publisher’s version ) (Closed access) One pedigree with four patients has been recently described with mitochondrial DNA depletion and mutation in SUCLA2 gene leading to succinyl-CoA synthase deficiency. Patients had a Leigh-like encephalomyopathy and deafness but besides the presence of lactic acidosis, the profile of urine organic acid was not reported. We have studied 14 patients with mild 'unlabelled' methylmalonic aciduria (MMA) from 11 families. Eight of the families are from the Faroe Islands, having a common ancestor, and three are from southern Italy. Since the reaction catalysed by succinyl-CoA synthase in the tricarboxylic acid (TCA) cycle represents a distal step of the methylmalonic acid pathway, we investigated the SUCLA2 gene as a candidate gene in our patients. Genetic analysis of the gene in the 14 patients confirmed the defect in all patients and led to the identification of three novel mutations (p.Gly118Arg; p.Arg284Cys; c.534 + 1G --> A). The defect could be convincingly shown at the protein level and our data also confirm the previously described mitochondrial DNA depletion. Defects in SUCLA2 can be found at the metabolite level and are defined by mildly elevated methylmalonic acid and C4-dicarboxylic carnitine concentrations in body fluids in association with variable lactic acidosis. Clinically the diagnosis should be considered in patients with early/neonatal onset encephalomyopathy, dystonia, deafness and Leigh-like MRI abnormalities mainly affecting the putamen and the caudate nuclei. The frequency of the mutated allele in the Faroese population amounted to 2%, corresponding with an estimated homozygote frequency of 1 : 2500. Our data extend knowledge on the genetic defects causing MMA. Our patients present with an early infantile Leigh-like encephalomyopathy with deafness, and later on a progressive dystonia. Mild MMA, lactic acidosis and specific abnormalities in the carnitine ester profile are the biochemical hallmarks of the disease. In view of the frequency of the mutated allele on the Faroe Islands, measures become feasible to prevent the occurrence of the disease on the islands. We confirm and extend the findings on this inborn error of metabolism in the TCA cycle that must be carefully investigated by accurate metabolite analyses.

Contains fulltext : 50968.pdf (Publisher’s version ) (Closed access) OBJECTIVE: Familial combined hyperlipidemia (FCH) is characterized by elevated levels of total cholesterol (TC), triglycerides (TG) and apolipoprotein B (apo B) and is associated with premature cardiovascular disease (CVD). Other features of FCH are obesity and insulin resistance. Serum leptin levels have also been associated with obesity, insulin resistance and atherosclerosis. Leptin exerts its effect through the leptin receptor (LEPR). The aim of this study is to determine whether the Gln223Arg polymorphism in the LEPR gene contributes to FCH and its associated phenotypes. METHODS: The study population consists of 37 families, comprising 644 subjects, of whom 158 subjects were diagnosed as FCH. The FCH diagnosis was based on plasma TC and TG levels, adjusted for age and gender, and absolute apo B levels, according to our recently published nomogram. The Gln223Arg polymorphism was studied by restriction fragment length polymorphism-PCR. RESULTS: Carriers of one or two Arg alleles had an increased risk of FCH, compared to subjects homozygous for the Gln allele (OR=1.6 [95% CI 1.0-2.4]). A difference in high-density lipoprotein cholesterol (HDL-c) levels was present between carriers and non-carriers of an Arg allele, 1.21 vs 1.28 mmol/l, respectively (P=0.04), but no differences in obesity, insulin resistance and other lipid parameters were found. CONCLUSION: The Gln223Arg polymorphism in the LEPR gene is associated with FCH, which is supported by a significant association between HDL-c levels and the LEPR gene.

Contains fulltext : 50783.pdf (Publisher’s version ) (Closed access) Elevated total plasma homocysteine concentrations (tHcy), both fasting and post-methionine load, have been established as risk factors for vascular disease. Recently, we described the association of a 31 bp variable number of tandem repeats (VNTR) in the cystathionine beta-synthase (CBS) gene with both CBS enzyme activity and tHcy concentrations. In the present study, we determined the 31 bp VNTR genotypes in 2598 individuals of the Framingham Offspring Study and studied the association between this genotype and fasting, 2-h post-methionine load and delta (ie increase upon methionine loading) tHcy concentrations in 1416 subjects. We observed a positive association between the number of repeat units of the CBS 31 bp VNTR and both postload and delta tHcy concentrations. Adjustment for possible effect modifying factors like age, sex and vitamin (B6, B12 and folate) status did not change this observation. We hereby confirm the results of our earlier study, in which we found that this 31 bp VNTR is a genetic determinant of post-methionine load tHcy concentrations. Since also post-methionine load tHcy concentrations are found to be associated with an increased risk for cardiovascular disease (CVD), this 31 bp VNTR may be considered a risk factor for CVD.

Contains fulltext : 51287.pdf (Publisher’s version ) (Closed access) In this paper, we describe a distinct clinical subtype of 3-methylglutaconic aciduria. 3-Methylglutaconic aciduria is a group of different metabolic disorders biochemically characterized by increased urinary excretion of 3-methylglutaconic acid. We performed biochemical and genetic investigations, including urine organic acid analysis, NMR spectroscopy, measurement of 3-methylglutaconyl-CoA hydratase activity, cardiolipin levels, OPA3 gene analysis and measurement of the oxidative phosphorylation in four female patients with 3-methylglutaconic aciduria. 3-Methylglutaconic aciduria type I, Barth syndrome, and Costeff syndrome were excluded as the activity of 3-methylglutaconyl-CoA hydratase, the cardiolipin levels, and molecular analysis of the OPA3 gene, respectively, showed no abnormalities. The children presented with characteristic association of hearing loss and the neuro-radiological evidence of Leigh disease. They also had neonatal hypotonia, recurrent lactic acidemia, episodes with hypoglycemia and severe recurrent infections, feeding difficulties, failure to thrive, developmental delay, and progressive spasticity with extrapyramidal symptoms. Our patients were further biochemically characterized by a mitochondrial dysfunction and persistent urinary excretion of 3-methylglutaconic acid.

Contains fulltext : 51023.pdf (Publisher’s version ) (Closed access) The methionine synthase reductase (MTRR) enzyme restores methionine synthase (MTR) enzyme activity and therefore plays an essential role in homocysteine remethylation. In some studies, the 66A>G polymorphism in the MTRR gene was associated with increased neural tube defect (NTD) risk. Using a case-control design, we studied the association between the MTRR 66A>G polymorphism and spina bifida risk in 121 mothers, 109 spina bifida patients, 292 control women, and 234 pediatric controls. Possible interactions between the MTRR 66A>G variant and the MTR 2756A>G polymorphism, the MTHFR 677C>T variant, plasma vitamin B12, and plasma methylmalonic acid (MMA) levels were examined in the 121 mothers and 292 control women. Meta-analyses were conducted to set the results of the case-control study in the context of eligible literature on the relation between the MTRR 66A>G variant and NTD risk. Finally, a transmission disequilibrium test was performed for 82 complete mother-father-child triads to test for preferential transmission of the MTRR risk allele. In our case-control study, the MTRR 66A>G polymorphism had no influence on spina bifida risk in children [odds ratio (OR) 0.6, 95% confidence interval (CI) 0.4-1.1]. The MTRR 66GG genotype increased maternal spina bifida risk by 2.1-fold (OR 2.1, 95% CI 1.3-3.3). This risk became more pronounced in combination with the MTHFR 677TT genotype (OR 4.0, 95% CI 1.3-12.5). Moreover, we demonstrate a possible interaction between the MTRR 66GG genotype and high plasma MMA levels (OR 5.5, 95% CI 2.2-13.5). The meta-analyses demonstrated that the maternal MTRR 66GG genotype was associated with an overall 55% (95% CI 1.04-2.30) increase in NTD risk and that the MTRR 66GG genotype did not increase NTD risk in children (OR 0.96, 95% CI 0.46-2.01). These data show that the MTRR 66GG genotype is a maternal risk factor for spina bifida especially when intracellular vitamin B12 status is low.

Contains fulltext : 48177.pdf (Publisher’s version ) (Closed access) We present a patient, who experienced recurrent episodes of rhabdomyolysis. Her beneficial response to treatment with dantrolene sodium was previously reported. Adult onset Very Long Chain Acyl-CoA Dehydrogenase (VLCAD) deficiency has been diagnosed only recently. In adults, VLCAD deficiency results in recurrent fasting-, exercise-, or infection-induced muscle stiffness, muscle pain and myoglobinuria caused by rhabdomyolysis. This case illustrates for the first time the beneficial effect of dantrolene in VLCAD deficiency. We discuss the therapeutic mechanism of dantrolene sodium and its possible role as additional treatment modality for patients with VLCAD deficiency.

Evidence for an involvement of aberrant homocysteine metabolism in the aetiology of schizophrenia is limited and controversial. A case-control study was performed to quantify the risk of schizophrenia in the presence of elevated homocysteine concentrations or homozygosity for the 677C --> T polymorphism (677TT) in the methylenetetrahydrofolate reductase (MTHFR) gene in subjects of Dutch ancestry. We determined the 677C --> T MTHFR genotype distribution in 254 well-defined patients and 414 healthy controls. Plasma homocysteine concentrations were measured in 62 patients with schizophrenia and 432 control subjects. When homocysteine concentrations were stratified into quartiles of the control distribution, we calculated an increased risk for schizophrenia in the fourth and third quartile versus the lowest quartile [odds ratio (OR) = 3.3; 95% confidence interval (CI): 1.2-9.2, and OR = 3.1; 95% CI: 1.2-8.0, respectively]. A significant dose-response relation of increasing homocysteine levels and increasing risk for schizophrenia was observed (P = 0.036). The 677TT genotype was associated with an OR of 1.6 [95% CI: 0.96-2.8] of having schizophrenia. Heterozygosity for the T allele compared to 677CC subjects accounted for an OR of 1.3 [95% CI: 0.91-1.8]. Elevated homocysteine levels and the MTHFR 677TT genotype are associated with an increased risk for schizophrenia. These observations support a causal relation between disturbed homocysteine metabolism and schizophrenia.

Contains fulltext : 47727.pdf (Publisher’s version ) (Closed access) Elevated levels of homocysteine are a risk factor for vascular disease, thrombosis, neural tube defects and dementia. The 677C>T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene appears to be the most important single determinant of plasma homocysteine concentration. In the current study, we estimated heritability and fit a series of models of inheritance for both fasting and postmethionine-load homocysteine levels in the HOFAM-study (HOmocysteine in FAMilies study), which included 306 participants from 51 pedigrees, ascertained through a hyperhomocysteinemic proband. The crude heritability was 21.6% for fasting and 67.5% for postloading homocysteine. After adjustment for MTHFR 677C>T genotype, heritability dropped to 5.2 and 63.9%, respectively. Segregation analysis revealed that a nongenetic model with equal transmission was the best fitting and most parsimonious model for fasting homocysteine levels, while a two-distribution, Mendelian model with residual familial correlation was best for postmethionine-load homocysteine levels. This study shows that postload homocysteine levels have a stronger genetic determination than do fasting homocysteine levels. The heritability of postload homocysteine levels were not strongly affected by adjustment for MTHFR 677C>T genotype, in contrast to fasting homocysteine levels. Further studies are needed to identify the genes responsible for the inheritance of postload homocysteine levels.