Mild Choline Deficiency and MTHFD1 Synthetase Deficiency Interact to Increase Incidence of Developmental Delays and Defects in Mice

Folate and choline are interconnected metabolically. The MTHFD1 R653Q SNP is a risk factor for birth defects and there are concerns that choline deficiency may interact with this SNP and exacerbate health risks. 80–90% of women do not meet the Adequate Intake (AI) for choline. The objective of this study was to assess the effects of choline deficiency on maternal one-carbon metabolism and reproductive outcomes in the MTHFD1-synthetase deficient mouse (Mthfd1S), a model for MTHFD1 R653Q. Mthfd1S+/+ and Mthfd1S+/− females were fed control (CD) or choline-deficient diets (ChDD; 1/3 the amount of choline) before mating and during pregnancy. Embryos were evaluated for delays and defects at 10.5 days gestation. Choline metabolites were measured in the maternal liver, and total folate measured in maternal plasma and liver. ChDD significantly decreased choline, betaine, phosphocholine, and dimethylglycine in maternal liver (p < 0.05, ANOVA), and altered phosphatidylcholine metabolism. Maternal and embryonic genotype, and diet-genotype interactions had significant effects on defect incidence. Mild choline deficiency and Mthfd1S+/− genotype alter maternal one-carbon metabolism and increase incidence of developmental defects. Further study is required to determine if low choline intakes contribute to developmental defects in humans, particularly in 653QQ women.

A Neuro-metabolic Syndrome that Needs to Be Discovered: A Child with Late Onset Asparagine Synthetase Deficiency

Asparagine synthetase (ASNS) deficiency is a rare inborn error of metabolism caused by a defect in ASNS—a gene encoding asparagine synthetase. It has mainly been described as a neurological phenotype manifesting as severe developmental delay, congenital microcephaly, spasticity, and refractory seizures; it is not associated with any specific dysmorphisms. ASNS deficiency leads to the inability to synthesize a nonessential amino acid in the brain, this explains why the symptoms are primarily neurological. The accumulation of aspartate/glutamate causes increased neuronal apoptosis leading to brain atrophy and increased neuronal excitability leading to seizures. Asparagine levels in plasma and cerebrospinal fluid are not reliable biomarkers for this disorder, therefore diagnosis is mainly obtained by molecular genetics. This disorder is associated with a poor prognosis and there is no treatment except supportive therapy. Prenatal diagnosis is possible. We report a case of a later onset form, c.146G > A (p.Arg49Gln) variant in the ASNS gene detected by molecular analysis using next-generation sequencing; the patient's clinical presentation included microcephaly, regression of developmental milestones, epilepsy, and hyperthermia.

Leucyl-tRNA synthetase deficiency systemically induces excessive autophagy in zebrafish

Leucyl-tRNA synthetase (LARS) is an enzyme that catalyses the ligation of leucine with leucine tRNA. LARS is also essential to sensitize the intracellular leucine concentration to the mammalian target of rapamycin complex 1 (mTORC1) activation. Biallelic mutation in the LARS gene causes infantile liver failure syndrome type 1 (ILFS1), which is characterized by acute liver failure, anaemia, and neurological disorders, including microcephaly and seizures. However, the molecular mechanism underlying ILFS1 under LARS deficiency has been elusive. Here, we generated Lars deficient (larsb−/−) zebrafish that showed progressive liver failure and anaemia, resulting in early lethality within 12 days post fertilization. The atg5-morpholino knockdown and bafilomycin treatment partially improved the size of the liver and survival rate in larsb−/− zebrafish. These findings indicate the involvement of autophagy in the pathogenesis of larsb−/− zebrafish. Indeed, excessive autophagy activation was observed in larsb−/− zebrafish. Therefore, our data clarify a mechanistic link between LARS and autophagy in vivo. Furthermore, autophagy regulation by LARS could lead to development of new therapeutics for IFLS1.

Effects of A6E Mutation on Protein Expression and Supramolecular Assembly of Yeast Asparagine Synthetase

Asparagine synthetase deficiency (ASD) has been found to be caused by certain mutations in the gene encoding human asparagine synthetase (ASNS). Among reported mutations, A6E mutation showed the greatest reduction in ASNS abundance. However, the effect of A6E mutation has not yet been tested with yeast asparagine synthetase (Asn1/2p). Here, we constructed a yeast strain by deleting ASN2 from its genome, introducing the A6E mutation codon to ASN1, along with GFP downstream of ASN1. Our mutant yeast construct showed a noticeable decrease of Asn1p(A6E)-GFP levels as compared to the control yeast expressing Asn1p(WT)-GFP. At the stationary phase, the A6E mutation also markedly lowered the assembly frequency of the enzyme. In contrast to Asn1p(WT)-GFP, Asn1p(A6E)-GFP was insensitive to changes in the intracellular energy levels upon treatment with sodium azide during the log phase or fresh glucose at the stationary phase. Our study has confirmed that the effect of A6E mutation on protein expression levels of asparagine synthetase is common in both unicellular and multicellular eukaryotes, suggesting that yeast could be a model of ASD. Furthermore, A6E mutation could be introduced to the ASNS gene of acute lymphoblastic leukemia patients to inhibit the upregulation of ASNS by cancer cells, reducing the risk of developing resistance to the asparaginase treatment.

Leucyl-tRNA Synthetase Deficiency Systemically Induces Excessive Autophagy in Zebrafish

Leucyl-tRNA synthetase (LARS) is an enzyme that catalyses the ligation of leucine with leucine tRNA. LARS is also essential to sensitize the intracellular leucine concentration to the mammalian target of rapamycin complex 1 (mTORC1) activation. Biallelic mutation in the LARS gene causes infantile liver failure syndrome type 1 (ILFS1), which is characterized by acute liver failure, anaemia, and neurological disorders, including microcephaly and seizures. However, the molecular mechanism underlying ILFS1 under LARS deficiency has been elusive. Here, we generated Lars deficient (larsb-/-) zebrafish that showed progressive liver failure and anaemia, resulting in early lethality within 12 days post fertilization. The atg5-morpholino knockdown and bafilomycin treatment partially improved the size of the liver and survival rate in larsb-/- zebrafish. These findings indicate the involvement of autophagy in the pathogenesis of larsb-/- zebrafish. Indeed, excessive autophagy activation was observed in larsb-/- zebrafish. Therefore, our data clarify a mechanistic link between LARS and autophagy in vivo. Furthermore, autophagy regulation by LARS could lead to development of new therapeutics for IFLS1.

Asparagine Synthetase Deficiency with Intracranial Hemorrhage Can Mimic Molybdenum Cofactor Deficiency

Here we report a consanguineous Egyptian family with two siblings presented with congenital microcephaly, early-onset epileptic encephalopathy, feeding difficulties, and early lethality. The condition was initially diagnosed as molybdenum cofactor deficiency as the brain imaging for one of them showed brain edema and intracranial hemorrhage in addition to the hypoplastic corpus callosum, vermis hypoplasia, and small-sized pons. Subsequently, whole exome sequencing identified a novel homozygous missense variant in exon 4 of ASNS gene c.397_398GT > CA (p.Val133Gln) confirming the diagnosis of asparagine synthetase deficiency syndrome. No discernible alternative cause for the intracranial hemorrhage was found. Our patient is the second to show asparagine synthetase deficiency and intracranial hemorrhage, thus confirming the involvement of ASNS gene. As such, it is important to consider asparagine synthetase deficiency syndrome in patients with microcephaly, brain edema, and neonatal intracranial hemorrhage.