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Item does not contain fulltext, BACKGROUND: Newborn screening (NBS) programmes identify a wide range of disease phenotypes, which raises the question whether early identification and treatment is beneficial for all. This study aims to answer this question for primary carnitine deficiency (PCD) taking into account that NBS for PCD identifies newborns with PCD and also until then undiagnosed mothers. METHODS: We investigated clinical, genetic (variants in SLC22A5 gene) and functional (carnitine transport activity in fibroblasts) characteristics of all referred individuals through NBS (newborns and mothers) and clinically diagnosed patients with PCD (not through NBS). Disease phenotype in newborns was predicted using data from PCD mothers and cases published in literature with identical SLC22A5 variants. RESULTS: PCD was confirmed in 19/131 referred newborns, 37/82 referred mothers and 5 clinically diagnosed patients. Severe symptoms were observed in all clinically diagnosed patients, 1 newborn and none of the mothers identified by NBS. PCD was classified as severe in all 5 clinically diagnosed patients, 3/19 newborns and 1/37 mothers; as benign in 8/19 newborns and 36/37 mothers and as unknown in 8/19 newborns. Carnitine transport activity completely separated severe phenotype from benign phenotype (median (range): 4.0% (3.5-5.0)] vs 26% (9.5-42.5), respectively). CONCLUSION: The majority of mothers and a significant proportion of newborns with PCD identified through NBS are likely to remain asymptomatic without early treatment. Conversely, a small proportion of newborns with predicted severe PCD could greatly benefit from early treatment. Genetic variants and carnitine transport activity can be used to distinguish between these groups.

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Item does not contain fulltext, Classical galactosemia (CG) is one of the more frequent inborn errors of metabolism affecting approximately 1:40.000 people. Despite a life-saving galactose-restricted diet, patients develop highly variable long-term complications including intellectual disability and movement disorders. The pathophysiology of these complications is still poorly understood and development of new therapies is hampered by a lack of valid prognostic biomarkers. Multi-omics approaches may discover new biomarkers and improve prediction of patient outcome. In the current study, (semi-)targeted mass-spectrometry based metabolomics and lipidomics were performed in erythrocytes of 40 patients with both classical and variant phenotypes and 39 controls. Lipidomics did not show any significant changes or deficiencies. The metabolomics analysis revealed that CG does not only compromise the Leloir pathway, but also involves other metabolic pathways including glycolysis, the pentose phosphate pathway, and nucleotide metabolism in the erythrocyte. Moreover, the energy status of the cell appears to be compromised, with significantly decreased levels of ATP and ADP. This possibly is the consequence of two different mechanisms: impaired formation of ATP from ADP possibly due to reduced flux though the glycolytic pathway and trapping of phosphate in galactose-1-phosphate (Gal-1P) which accumulates in CG. Our findings are in line with the current notion that the accumulation of Gal-1P plays a key role in the pathophysiology of CG not only by depletion of intracellular phosphate levels but also by decreasing metabolite abundance downstream in the glycolytic pathway and affecting other pathways. New therapeutic options for CG could be directed towards the restoration of intracellular phosphate homeostasis.

Mitochondrial diseases are a group of inherited diseases with highly varied and complex clinical presentations. Here, we report four individuals, including two siblings, affected by a progressive mitochondrial encephalopathy with biallelic variants in the cardiolipin biosynthesis gene CRLS1. Three affected individuals had a similar infantile presentation comprising progressive encephalopathy, bull’s eye maculopathy, auditory neuropathy, diabetes insipidus, autonomic instability, cardiac defects and early death. The fourth affected individual presented with chronic encephalopathy with neurodevelopmental regression, congenital nystagmus with decreased vision, sensorineural hearing loss, failure to thrive and acquired microcephaly. Using patient-derived fibroblasts, we characterized cardiolipin synthase 1 (CRLS1) dysfunction that impaired mitochondrial morphology and biogenesis, providing functional evidence that the CRLS1 variants cause mitochondrial disease. Lipid profiling in fibroblasts from two patients further confirmed the functional defect demonstrating reduced cardiolipin levels, altered acyl-chain composition and significantly increased levels of phosphatidylglycerol, the substrate of CRLS1. Proteomic profiling of patient cells and mouse Crls1 knockout cell lines identified both endoplasmic reticular and mitochondrial stress responses, and key features that distinguish between varying degrees of cardiolipin insufficiency. These findings support that deleterious variants in CRLS1 cause an autosomal recessive mitochondrial disease, presenting as a severe encephalopathy with multi-systemic involvement. Furthermore, we identify key signatures in cardiolipin and proteome profiles across various degrees of cardiolipin loss, facilitating the use of omics technologies to guide future diagnosis of mitochondrial diseases.

Contains fulltext : 245422.pdf (Publisher’s version ) (Open Access)

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.

Mutations in the short-chain enoyl-CoA hydratase (SCEH) gene, ECHS1, cause a rare autosomal recessive disorder of valine catabolism. Patients usually present with developmental delay, regression, dystonia, feeding difficulties, and abnormal MRI with bilateral basal ganglia involvement. We present clinical, biochemical, molecular, and functional data for four affected patients from two unrelated families of Samoan descent with identical novel compound heterozygous mutations. Family 1 has three affected boys while Family 2 has an affected daughter, all with clinical and MRI findings of Leigh syndrome and intermittent episodes of acidosis and ketosis. WES identified a single heterozygous variant in ECHS1 at position c.832G > A (p.Ala278Thr). However, western blot revealed significantly reduced ECHS1 protein for all affected family members. Decreased SCEH activity in fibroblasts and a mild increase in marker metabolites in urine further supported ECHS1 as the underlying gene defect. Additional investigations at the DNA (aCGH, WGS) and RNA (qPCR, RT-PCR, RNA-Seq, RNA-Array) level identified a silent, common variant at position c.489G > A (p.Pro163=) as the second mutation. This substitution, present at high frequency in the Samoan population, is associated with decreased levels of normally spliced mRNA. To our understanding, this is the first report of a novel, hypomorphic allele c.489G > A (p.Pro163=), associated with SCEH deficiency.

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.

Contains fulltext : 225114.pdf (publisher's version ) (Open Access), Early diagnosis and dietary treatment do not prevent long-term complications, which mostly affect the central nervous system in classical galactosemia patients. The clinical outcome of patients is highly variable, and there is an urgent need for prognostic biomarkers. The aim of this study was first to increase knowledge on the natural history of classical galactosemia by studying a cohort of patients with varying geno- and phenotypes and second to study the association between clinical outcomes and two possible prognostic biomarkers. In addition, the association between abnormalities on brain MRI and clinical outcomes was investigated. Classical galactosemia patients visiting the galactosemia expertise outpatient clinic of the Amsterdam University Medical Centre were evaluated according to the International Classical Galactosemia guideline with the addition of an examination by a neurologist, serum immunoglobulin G N-glycan profiling and a brain MRI. The biomarkers of interest were galactose-1-phosphate levels and N-glycan profiles, and the clinical outcomes studied were intellectual outcome and the presence or absence of movement disorders and/or primary ovarian insufficiency. Data of 56 classical galactosemia patients are reported. The intellectual outcome ranged from 45 to 103 (mean 77 ± 14) and was <85 in 62%. Movement disorders were found in 17 (47%) of the 36 tested patients. In females aged 12 years and older, primary ovarian insufficiency was diagnosed in 12 (71%) of the 17 patients. Significant differences in N-glycan peaks were found between controls and patients. However, no significant differences in either N-glycans or galactose-1-phosphate levels were found between patients with a poor (intellectual outcome < 85) and normal intellectual outcome (intellectual outcome ≥ 85), and with or without movement disorders or primary ovarian insufficiency. The variant patients detected by newborn screening, with previously unknown geno- and phenotypes and current

Contains fulltext : 220549.pdf (publisher's version ) (Open Access), Sjögren-Larsson syndrome (SLS) is a rare inborn error of lipid metabolism. The syndrome is caused by mutations in the ALDH3A2 gene, resulting in a deficiency of fatty aldehyde dehydrogenase. Most patients have a clearly recognizable severe phenotype, with congenital ichthyosis, intellectual disability, and spastic diplegia. In this study, we describe two patients with a remarkably mild phenotype. In both patients, males with actual ages of 45 and 61 years, the diagnosis was only established at an adult age. Their skin had been moderately affected from childhood onward, and both men remained ambulant with mild spasticity of their legs. Cognitive development, as reflected by school performance and professional career, had been unremarkable. Magnetic resonance spectroscopy of the first patient was lacking the characteristic lipid peak. We performed a literature search to identify additional SLS patients with a mild phenotype. We compared the clinical, radiologic, and molecular features of the mildly affected patients with the classical phenotype. We found 10 cases in the literature with a molecular proven diagnosis and a mild phenotype. Neither a genotype-phenotype correlation nor an alternative explanation for the strikingly mild phenotypes was found. New biochemical techniques to study the underlying metabolic defect in SLS, like lipidomics, may in the future help to unravel the reasons for the exceptionally mild phenotypes. In the meantime, it is important to recognize these mildly affected patients to provide them with appropriate care and genetic counseling, and to increase our insights in the true disease spectrum of SLS.

Contains fulltext : 229584.pdf (Publisher’s version ) (Open Access), Sjögren-Larsson syndrome (SLS) is a rare neurometabolic syndrome caused by deficient fatty aldehyde dehydrogenase. Patients exhibit intellectual disability, spastic paraplegia, and ichthyosis. The accumulation of fatty alcohols and fatty aldehydes has been demonstrated in plasma and skin but never in brain. Brain magnetic resonance imaging and spectroscopy studies, however, have shown an abundant lipid peak in the white matter of patients with SLS, suggesting lipid accumulation in the brain as well. Using histopathology, mass spectrometry imaging, and lipidomics, we studied the morphology and the lipidome of a postmortem brain of a 65-year-old female patient with genetically confirmed SLS and compared the results with a matched control brain. Histopathological analyses revealed structural white matter abnormalities with the presence of small lipid droplets, deficient myelin, and astrogliosis. Biochemically, severely disturbed lipid profiles were found in both white and gray matter of the SLS brain, with accumulation of fatty alcohols and ether lipids. Particularly, long-chain unsaturated ether lipid species accumulated, most prominently in white matter. Also, there was a striking accumulation of odd-chain fatty alcohols and odd-chain ether(phospho)lipids. Our results suggest that the central nervous system involvement in SLS is caused by the accumulation of fatty alcohols leading to a disbalance between ether lipid and glycero(phospho)lipid metabolism resulting in a profoundly disrupted brain lipidome. Our data show that SLS is not a pure leukoencephalopathy, but also a gray matter disease. Additionally, the histopathological abnormalities suggest that astrocytes and microglia might play a pivotal role in the underlying disease mechanism, possibly contributing to the impairment of myelin maintenance.

Background: The high variability in clinical outcome of patients with Classical Galactosemia (CG) is poorly understood and underlines the importance of prognostic biomarkers, which are currently lacking. The aim of this study was to investigate if residual galactose metabolism capacity is associated with clinical and biochemical outcomes in CG patients with varying geno- and phenotypes. Methods: Galactose Metabolite Profiling (GMP) was used to determine residual galactose metabolism in fibroblasts of CG patients. The association between the galactose index (GI) defined as the ratio of the measured metabolites [U13C]Gal-1-P/ [13C6]UDP-galactose, and both intellectual and neurological outcome and galactose-1-phosphate (Gal-1-P) levels was investigated. Results: GMP was performed in fibroblasts of 28 patients and 3 control subjects. The GI of the classical phenotype patients (n = 22) was significantly higher than the GI of four variant patients detected by newborn screening (NBS) (p = .002), two homozygous p.Ser135Leu patients (p = .022) and three controls (p = .006). In the classical phenotype patients, 13/18 (72%) had a poor intellectual outcome (IQ < 85) and 6/12 (50%) had a movement disorder. All the NBS detected variant patients (n = 4) had a normal intellectual outcome (IQ ≥ 85) and none of them has a movement disorder. In the classical phenotype patients, there was no significant difference in GI between patients with a poor and normal clinical outcome. The NBS detected variant patients had significantly lower GI levels and thus higher residual galactose metabolism than patients with classical phenotypes. There was a clear correlation between Gal-1-P levels in erythrocytes and the GI (p = .001). Conclusions: The GI was able to distinguish CG patients with varying geno- and phenotypes and correlated with Gal-1-P. The data of the NBS detected variant patients demonstrated that a higher residual galactose metabolism may result in a more favourable clinical outcome.

Classical galactosemia (CG) patients frequently develop long-term complications despite early dietary treatment. The highly variable clinical outcome is poorly understood and a lack of prognostic biomarkers hampers individual prognostication and treatment. The aim of this study was to investigate the association between residual galactose oxidation capacity and clinical and biochemical outcomes in CG patients with varying geno- and phenotypes. The noninvasive 1-13C galactose breath test was used to assess whole body galactose oxidation capacity. Participants received a 7 mg/kg oral dose of 1-13C labelled galactose. The galactose oxidation capacity was determined by calculating the cumulative percentage dose of the administered galactose (CUMPCD) recovered as 13CO2 in exhaled air. Forty-one CG patients (5–47 years) and four adult controls were included. The median galactose oxidation capacity after 120 minutes (CUMPCDT120) of 34 classical patients (0.29; 0.08–7.51) was significantly lower when compared to two homozygous p.Ser135Leu patients (9.44; 8.66–10.22), one heterozygous p. Ser135Leu patient 18.59, four NBS detected variant patients (13.79; 12.73–14.87) and four controls (9.29; 8.94–10.02). There was a clear correlation between Gal-1-P levels and CUMPCDT120 (P < .0005). In the classical patients, the differences in CUMPCDT120 were small and did not distinguish between patients with poor and normal clinical outcomes. The galactose breath test distinguished classical patients from homo- and heterozygous p.Ser135Leu and NBS detected variant patients, but was not able to predict clinical outcomes in classical patients. Future studies are warranted to enable individualised prognostication and treatment, especially in NBS variants with galactose oxidation capacities in the control range.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is an ultra-rare inborn error of mitochondrial fatty acid oxidation (FAO) and amino acid metabolism. Individual phenotypes and treatment response can vary markedly. We aimed to identify markers that predict MADD phenotypes. We performed a retrospective nationwide cohort study; then developed an MADD-disease severity scoring system (MADD-DS3) based on signs and symptoms with weighed expert opinions; and finally correlated phenotypes and MADD-DS3 scores to FAO flux (oleate and myristate oxidation rates) and acylcarnitine profiles after palmitate loading in fibroblasts. Eighteen patients, diagnosed between 1989 and 2014, were identified. The MADD-DS3 entails enumeration of eight domain scores, which are calculated by averaging the relevant symptom scores. Lifetime MADD-DS3 scores of patients in our cohort ranged from 0 to 29. FAO flux and [U-13C]C2-, C5-, and [U-13C]C16-acylcarnitines were identified as key variables that discriminated neonatal from later onset patients (all P <.05) and strongly correlated to MADD-DS3 scores (oleate: r = −.86; myristate: r = −.91; [U-13C]C2-acylcarnitine: r = −.96; C5-acylcarnitine: r =.97; [U-13C]C16-acylcarnitine: r =.98, all P <.01). Functional studies in fibroblasts were found to differentiate between neonatal and later onset MADD-patients and were correlated to MADD-DS3 scores. Our data may improve early prediction of disease severity in order to start (preventive) and follow-up treatment appropriately. This is especially relevant in view of the inclusion of MADD in population newborn screening programs.

Background: Patients with very long chain acyl-CoA dehydrogenase deficiency (VLCADD), a long chain fatty acid oxidation disorder, are traditionally treated with a long chain triglyceride (LCT) restricted and medium chain triglyceride (MCT) supplemented diet. Introduction of VLCADD in newborn screening (NBS) programs has led to the identification of asymptomatic newborns with VLCADD, who may have a more attenuated phenotype and may not need dietary adjustments. Objective: To define dietary strategies for individuals with VLCADD based on the predicted phenotype. Method: We evaluated long-term dietary histories of a cohort of individuals diagnosed with VLCADD identified before the introduction of VLCADD in NBS and their beta-oxidation (LC-FAO) flux score (rate of oleate oxidation) in cultured skin fibroblasts in relation to the clinical outcome. Based on these results a dietary strategy is proposed. Results: Sixteen individuals with VLCADD were included. One had an LC-FAO flux score >90%, was not on a restricted diet and is asymptomatic to date. Four patients had an LC-FAO flux score <10%, and significant VLCADD related symptoms despite the use of strict diets including LCT restriction, MCT supplementation and nocturnal gastric drip feeding. Patients with an LC-FAO flux score between 10 and 90% (n = 11) showed a more heterogeneous phenotype. Conclusions: This study shows that a strict diet cannot prevent poor clinical outcome in severely affected patients and that the LC-FAO flux is a good predictor of clinical outcome in individuals with VLCADD identified before its introduction in NBS. Hereby, we propose an individualized dietary strategy based on the LC-FAO flux score.

Short-chain enoyl-CoA hydratase (SCEH or ECHS1) deficiency is a rare inborn error of metabolism caused by biallelic mutations in the gene ECHS1 (OMIM 602292). Clinical presentation includes infantile-onset severe developmental delay, regression, seizures, elevated lactate, and brain MRI abnormalities consistent with Leigh syndrome (LS). Characteristic abnormal biochemical findings are secondary to dysfunction of valine metabolism. We describe four patients from two consanguineous families (one Pakistani and one Irish Traveler), who presented in infancy with LS. Urine organic acid analysis by GC/MS showed increased levels of erythro-2,3-dihydroxy-2-methylbutyrate and 3-methylglutaconate (3-MGC). Increased urine excretion of methacrylyl-CoA and acryloyl-CoA related metabolites analyzed by LC-MS/MS, were suggestive of SCEH deficiency; this was confirmed in patient fibroblasts. Both families were shown to harbor homozygous pathogenic variants in the ECHS1 gene; a c.476A > G (p.Gln159Arg) ECHS1variant in the Pakistani family and a c.538A > G, p.(Thr180Ala) ECHS1 variant in the Irish Traveler family. The c.538A > G, p.(Thr180Ala) ECHS1 variant was postulated to represent a Canadian founder mutation, but we present SNP genotyping data to support Irish ancestry of this variant with a haplotype common to the previously reported Canadian patients and our Irish Traveler family. The presence of detectable erythro-2,3-dihydroxy-2-methylbutyrate is a nonspecific marker on urine organic acid analysis but this finding, together with increased excretion of 3-MGC, elevated plasma lactate, and normal acylcarnitine profile in patients with a Leigh-like presentation should prompt consideration of a diagnosis of SCEH deficiency and genetic analysis of ECHS1. ECHS1 deficiency can be added to the list of conditions with 3-MGA.

Item does not contain fulltext, Dilated cardiomyopathy (DCM) is extremely heterogeneous with a large proportion due to dominantly inherited disease-causing variants in sarcomeric genes. Recessive metabolic diseases may cause DCM, usually with onset in childhood, and in the context of systemic disease. Whether metabolic defects can also cause adult-onset DCM is currently unknown. Therefore, we performed an extensive metabolic screening in 36 consecutive adult-onset DCM patients. Diagnoses were confirmed by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Measurement of propionyl-CoA carboxylase (PCC) activity was done in fibroblasts. Whole exome sequencing (WES) data of 157 additional DCM patients were analyzed for genetic defects. We found a metabolic profile characteristic for propionic acidemia in a patient with severe DCM from 55 years of age. Genetic analysis demonstrated compound heterozygous variants in PCCA. Enzymatic activity of PCC in fibroblasts was markedly reduced. A targeted analysis of the PCCA and PCCB genes using available WES data from 157 further DCM patients subsequently identified another patient with propionic acidemia. This patient had compound heterozygous variants in PCCB, and developed severe DCM from 42 years of age. Adult-onset DCM can be caused by propionic acidemia, an autosomal recessive inheritable metabolic disorder usually presenting as neonatal or childhood disease. Current guidelines advise a low-protein diet to ameliorate or prevent detrimental aspects of the disease. Long-term follow-up of a larger group of patients may show whether this diet would also ameliorate DCM. Our results suggest that diagnostic metabolic screening to identify propionic acidemia and related disorders in DCM patients is justified.

Contains fulltext : 169755.pdf (publisher's version ) (Closed access), EXTL3 regulates the biosynthesis of heparan sulfate (HS), important for both skeletal development and hematopoiesis, through the formation of HS proteoglycans (HSPGs). By whole-exome sequencing, we identified homozygous missense mutations c.1382C>T, c.1537C>T, c.1970A>G, and c.2008T>G in EXTL3 in nine affected individuals from five unrelated families. Notably, we found the identical homozygous missense mutation c.1382C>T (p.Pro461Leu) in four affected individuals from two unrelated families. Affected individuals presented with variable skeletal abnormalities and neurodevelopmental defects. Severe combined immunodeficiency (SCID) with a complete absence of T cells was observed in three families. EXTL3 was most abundant in hematopoietic stem cells and early progenitor T cells, which is in line with a SCID phenotype at the level of early T cell development in the thymus. To provide further support for the hypothesis that mutations in EXTL3 cause a neuro-immuno-skeletal dysplasia syndrome, and to gain insight into the pathogenesis of the disorder, we analyzed the localization of EXTL3 in fibroblasts derived from affected individuals and determined glycosaminoglycan concentrations in these cells as well as in urine and blood. We observed abnormal glycosaminoglycan concentrations and increased concentrations of the non-sulfated chondroitin disaccharide D0a0 and the disaccharide D0a4 in serum and urine of all analyzed affected individuals. In summary, we show that biallelic mutations in EXTL3 disturb glycosaminoglycan synthesis and thus lead to a recognizable syndrome characterized by variable expression of skeletal, neurological, and immunological abnormalities.