Apolline Imbard

Neural Tube Defects, Folic Acid and Methylation

Neural tube defects (NTDs) are common complex congenital malformations resulting from failure of the neural tube closure during embryogenesis. It is established that folic acid supplementation decreases the prevalence of NTDs, which has led to national public health policies regarding folic acid. To date, animal studies have not provided sufficient information to establish the metabolic and/or genomic mechanism(s) underlying human folic acid responsiveness in NTDs. However, several lines of evidence suggest that not only folates but also choline, B12 and methylation metabolisms are involved in NTDs. Decreased B12 vitamin and increased total choline or homocysteine in maternal blood have been shown to be associated with increased NTDs risk. Several polymorphisms of genes involved in these pathways have also been implicated in risk of development of NTDs. This raises the question whether supplementation with B12 vitamin, betaine or other methylation donors in addition to folic acid periconceptional supplementation will further reduce NTD risk. The objective of this article is to review the role of methylation metabolism in the onset of neural tube defects.

Molecular characterization of 82 patients with pyruvate dehydrogenase complex deficiency. Structural implications of novel amino acid substitutions in E1 protein

BACKGROUNDnPyruvate dehydrogenase complex (PDHc) deficiencies are an important cause of primary lactic acidosis. Most cases result from mutations in the X-linked gene for the pyruvate dehydrogenase E1α subunit (PDHA1) while a few cases result from mutations in genes for E1β (PDHB), E2 (DLAT), E3 (DLD) and E3BP (PDHX) subunits or PDH-phosphatase (PDP1).nnnAIMnTo report molecular characterization of 82 PDHc-deficient patients and analyze structural effects of novel missense mutations in PDHA1.nnnMETHODSnPDHA1 variations were investigated first, by exon sequencing using a long range PCR product, gene dosage assay and cDNA analysis. Mutation scanning in PDHX, PDHB, DLAT and DLD cDNAs was further performed in unsolved cases. Novel missense mutations in PDHA1 were located on the tridimensional model of human E1 protein to predict their possible functional consequences.nnnRESULTSnPDHA1 mutations were found in 30 girls and 35 boys. Three large rearrangements, including two contiguous gene deletion syndrome were identified. Novel missense, frameshift and splicing mutations were also delineated and a nonsense mutation in a mosaic male. Mutations p.Glu75Ala, p.Arg88Ser, p.Arg119Trp, p.Gly144Asp, p.Pro217Arg, p.Arg235Gly, p.Tyr243Cys, p.Tyr243Ser, p.Arg245Gly, p.Pro250Leu, p.Gly278Arg, p.Met282Val, p.Gly298Glu in PDHA1 were predicted to impair active site channel conformation or subunit interactions. Six out of the seven patients with PDHB mutations displayed the recurrent p.Met101Val mutation; 9 patients harbored PDHX mutations and one patient DLD mutations.nnnCONCLUSIONnWe provide an efficient stepwise strategy for mutation screening in PDHc genes and expand the growing list of PDHA1 mutations analyzed at the structural level.

Biallelic Mutations in DNAJC12 Cause Hyperphenylalaninemia, Dystonia, and Intellectual Disability

Phenylketonuria (PKU, phenylalanine hydroxylase deficiency), an inborn error of metabolism, can be detected through newborn screening for hyperphenylalaninemia (HPA). Most individuals with HPA harbor mutations in the gene encoding phenylalanine hydroxylase (PAH), and a small proportion (2%) exhibit tetrahydrobiopterin (BH4) deficiency with additional neurotransmitter (dopamine and serotonin) deficiency. Here we report six individuals from four unrelated families with HPA who exhibited progressive neurodevelopmental delay, dystonia, and a unique profile of neurotransmitter deficiencies without mutations in PAH or BH4 metabolism disorder-related genes. In these six affected individuals, whole-exome sequencing (WES) identified biallelic mutations in DNAJC12, which encodes a heat shock co-chaperone family member that interacts with phenylalanine, tyrosine, and tryptophan hydroxylases catalyzing the BH4-activated conversion of phenylalanine into tyrosine, tyrosine into L-dopa (the precursor of dopamine), and tryptophan into 5-hydroxytryptophan (the precursor of serotonin), respectively. DNAJC12 was undetectable in fibroblasts from the individuals with null mutations. PAH enzyme activity was reduced in the presence of DNAJC12 mutations. Early treatment with BH4 and/or neurotransmitter precursors had dramatic beneficial effects and resulted in the prevention of neurodevelopmental delay in the one individual treated before symptom onset. Thus, DNAJC12 deficiency is a preventable and treatable cause of intellectual disability that should be considered in the early differential diagnosis when screening results are positive for HPA. Sequencing of DNAJC12 may resolve any uncertainty and should be considered in all children with unresolved HPA.

Riboflavin-Responsive and -Non-responsive Mutations in FAD Synthase Cause Multiple Acyl-CoA Dehydrogenase and Combined Respiratory-Chain Deficiency

Multiple acyl-CoA dehydrogenase deficiencies (MADDs) are a heterogeneous group of metabolic disorders with combined respiratory-chain deficiency and a neuromuscular phenotype. Despite recent advances in understanding the genetic basis of MADD, a number of cases remain unexplained. Here, we report clinically relevant variants in FLAD1, which encodes FAD synthase (FADS), as the cause of MADD and respiratory-chain dysfunction in nine individuals recruited from metabolic centers in six countries. In most individuals, we identified biallelic frameshift variants in the molybdopterin binding (MPTb) domain, located upstream of the FADS domain. Inasmuch as FADS is essential for cellular supply of FAD cofactors, the finding of biallelic frameshift variants was unexpected. Using RNA sequencing analysis combined with protein mass spectrometry, we discovered FLAD1 isoforms, which only encode the FADS domain. The existence of these isoforms might explain why affected individuals with biallelic FLAD1 frameshift variants still harbor substantial FADS activity. Another group of individuals with a milder phenotype responsive to riboflavin were shown to have single amino acid changes in the FADS domain. When produced in E. coli, these mutant FADS proteins resulted in impaired but detectable FADS activity; for one of the variant proteins, the addition of FAD significantly improved protein stability, arguing for a chaperone-like action similar to what has been reported in other riboflavin-responsive inborn errors of metabolism. In conclusion, our studies identify FLAD1 variants as a cause of potentially treatable inborn errors of metabolism manifesting with MADD and shed light on the mechanisms by which FADS ensures cellular FAD homeostasis.

Global protein and histone arginine methylation are affected in a tissue-specific manner in a rat model of diet-induced hyperhomocysteinemia

Accumulation of S-adenosylhomocysteine (AdoHcy), the homocysteine (Hcy) precursor and a potent methyltransferase inhibitor, may mediate the neurological and vascular complications associated with elevated Hcy. Protein arginine methylation is a crucial post-translational modification and generates monomethylarginine (MMA) and dimethylarginine (asymmetric, ADMA, and symmetric, SDMA) residues. We aimed at determining whether protein arginine methylation status is disturbed in an animal model of diet-induced hyperhomocysteinemia (HHcy). HHcy was achieved by dietary manipulation of Wistar rats: methionine-enrichment (HM), B vitamins deficiency (LV), or both (HMLV). Total Hcy, S-adenosylmethionine (AdoMet), AdoHcy, MMA, ADMA and SDMA concentrations in plasma or tissues (heart, brain and liver) were determined by adequate high-performance liquid chromatography or liquid chromatography-electrospray ionization-tandem mass spectrometry methods. Moreover, in tissues from the HMLV group, histone arginine asymmetric dimethylation was evaluated by Western blotting, and the histone methylation marks H3R17me2a, H3R8me2a and H4R3me2a were studied. HHcy was induced by all special diets, with elevation of AdoHcy concentrations in liver (LV and HMLV) and heart (HMLV) (all versus control). Plasma ADMA levels were lower in all hyperhomocysteinemic animals. Protein-incorporated ADMA levels were decreased in brain and in heart (both for the LV and HMLV groups). Moreover, in brain of animals exposed to the HMLV diet, the H3R8me2a mark was profoundly decreased. In conclusion, our results show that diet-induced Hcy elevation disturbs global protein arginine methylation in a tissue-specific manner and affects histone arginine methylation in brain. Future research is warranted to disclose the functional implications of the global protein and histone arginine hypomethylation triggered by Hcy elevation.

Measurement of free and total sialic acid by isotopic dilution liquid chromatography tandem mass spectrometry method.

The measurement of urine sialic acid (N Acetylneuraminic Acid: Neu5Ac) is useful for screening sialic acid storage disorders. We developed a new LC MS/MS method for the determination of a sialic acid. Urine samples were analyzed, after an HCl n-Butanol derivatization step, by a reverse phase based high-performance liquid chromatography method using 1,2,3-(13)C(3) N-Acetyl-D-neuraminic Acid ((13)C-Neu5Ac) as an internal standard. Selective detection was performed by tandem mass spectrometry using an electrospray source operating in positive ionization mode employing multiple reactions monitoring to monitor N-Acetylneuraminic Acid and the internal standard. The transitions m/z 366→330 and 369→333 for Neu5Ac and (13)C-Neu5Ac were respectively monitored. The limit of the method quantification was 1.40 μM of N-Acetylneuraminic Acid and the calibration curve showed a good linearity up to 1000 μM. The within assay precision and accuracy of the method ranged from 3.22 to 5.95% and 98.69 to 109.18%, respectively and the between assay precision and accuracy ranged, respectively, from 5.15 to 7.65% and 96.14 to 102.30%. The method can be applied for the determination of N-Acetylneuraminic Acid concentrations in urine and other biological fluids (e.g., amniotic and peritoneal fluids).

New spastic paraplegia phenotype associated to mutation of NFU1

Recently an early onset lethal encephalopathy has been described in relation to mutations of NFU1, one of the genes involved in iron-sulfur cluster metabolism. We report a new NFU1 mutated patient presenting with a milder phenotype characterized by a later onset, a slowly progressive spastic paraparesis with relapsing-remitting episodes, mild cognitive impairment and a long survival. The early white matter abnormalities observed on MRI was combined with a mixed sensory-motor neuropathy in the third decade. Our case clearly suggests the importance of considering NFU1 mutation in slowly evolving leukoencephalopathy with high glycine concentration.

Protein arginine hypomethylation in a mouse model of cystathionine β-synthase deficiency

Accumulation of the homocysteine (Hcy) precursor S‐adenosylhomocysteine (AdoHcy) may cause cellular hypomethylation in the setting of hyperhomocysteinemia because of cystathionine β‐synthase (CBS) deficiency, an inborn error of metabolism. To test this hypothesis, DNA and protein arginine methylation status were assessed in liver, brain, heart, and kidney obtained from a previously described mouse model of CBS deficiency. Metabolite levels in tissues and serum were determined by high‐performance liquid chromatography or liquid chromatography‐electrospray ionization‐tandem mass spectrometry. Global DNA and protein arginine methylation status were evaluated as the contents of 5‐methyldeoxycytidine in DNA and of methylarginines in proteins, respectively. In addition, histone arginine methylation was assessed by Western blotting. CBS‐deficient mice exhibited increased (>6‐fold) Hcy and AdoHcy levels in all tissues examined compared with control levels. In addition, global DNA methylation status was not affected, but global protein arginine methylation status was decreased (10–35%) in liver and brain. Moreover, asymmetric dimethylation of arginine 3 on histone H4 (H4R3me2a) content was markedly decreased in liver, and no differences were observed for the other histone arginine methylation marks examined. Our results show that CBS‐deficient mice present severe accumulation of tissue Hcy and AdoHcy, protein arginine hypomethylation in liver and brain, and decreased H4R3me2a content in liver. Therefore, protein arginine hypomethylation arises as a potential player in the pathophysiology of CBS deficiency.—Esse, R., Imbard, A., Florindo, C., Gupta, S., Quinlivan, E. P., Davids, M., Teerlink, T., Tavares de Almeida, I., Kruger, W. D., Blom, H. J., Castro, R. Protein arginine hypomethylation in a mouse model of cystathionine β‐synthase deficiency. FASEB J. 28, 2686–2695 (2014). www.fasebj.org

QIL1 mutation causes MICOS disassembly and early onset fatal mitochondrial encephalopathy with liver disease

Previously, we identified QIL1 as a subunit of mitochondrial contact site (MICOS) complex and demonstrated a role for QIL1 in MICOS assembly, mitochondrial respiration, and cristae formation critical for mitochondrial architecture (Guarani et al., 2015). Here, we identify QIL1 null alleles in two siblings displaying multiple clinical symptoms of early-onset fatal mitochondrial encephalopathy with liver disease, including defects in respiratory chain function in patient muscle. QIL1 absence in patients’ fibroblasts was associated with MICOS disassembly, abnormal cristae, mild cytochrome c oxidase defect, and sensitivity to glucose withdrawal. QIL1 expression rescued cristae defects, and promoted re-accumulation of MICOS subunits to facilitate MICOS assembly. MICOS assembly and cristae morphology were not efficiently rescued by over-expression of other MICOS subunits in patient fibroblasts. Taken together, these data provide the first evidence of altered MICOS assembly linked with a human mitochondrial disease and confirm a central role for QIL1 in stable MICOS complex formation. DOI: http://dx.doi.org/10.7554/eLife.17163.001

Maternal and fetal tyrosinemia type I

A 22xa0year-old woman with tyrosinemia type I (HT1) married her first cousin who is heterozygous for the same FAH mutation for which the patient is homozygous. During her pregnancy she was treated with diet (prescribed tyrosine intake 300xa0mg/day), and nitisinone (60xa0mg/day). Median plasma tyrosine levels were 560xa0μmol/L (range: 375–838, nu2009=u200921) and nitisinone 51xa0μmol/L (range: 41–57, nu2009=u20093) during pregnancy. She gave birth to a clinically healthy girl affected with tyrosinemia type 1. Birth was normal (birth weight 2615xa0g) and the baby had normal liver function, normal plasma alpha-fetoprotein concentrations, low urinary excretion of phenolic acids and no detectable succinylacetone. At birth, the baby had hypertyrosinemia (860xa0μmol/L in blood cord) and nitisinone levels of 14xa0μmol/L. Following molecular confirmation of the diagnosis of HT1 specific treatment began on day 15 by which time she had detectable urinary succinylacetone.