Deep postnatal phenotyping of a new mouse model of nonketotic hyperglycinemia.

Nonketotic hyperglycinemia due to deficient glycine cleavage enzyme activity causes a severe neonatal epileptic encephalopathy. Current therapies based on mitigating glycine excess have only limited impact. An animal model with postnatal phenotyping is needed to explore new therapeutic approaches. We developed a Gldc p.Ala394Val mutant model and bred it to congenic status in 2 colonies on C57Bl/6J (B6) and J129X1/SvJ (J129) backgrounds. Mutant mice had reduced P-protein and enzyme activity indicating a hypomorphic mutant. Glycine levels were increased in blood and brain regions, exacerbated by dietary glycine, with higher levels in female than male J129 mice. Birth defects were more prevalent in mutant B6 than J129 mice, and hydrocephalus was more frequent in B6 (40%) compared to J129 (none). The hydrocephalus rate was increased by postnatal glycine challenge in B6 mice, more so when delivered from the first neonatal week than from the fourth. Mutant mice had reduced weight gain following weaning until the eighth postnatal week, which was exacerbated by glycine loading. The electrographic spike rate was increased in mutant mice following glycine loading, but no seizures were observed. The alpha/delta band intensity ratio was decreased in the left cortex in female J129 mice, which were less active in an open field test and explored less in a Y-maze, suggesting an encephalopathic effect. Mutant mice showed no evidence of memory dysfunction. This partial recapitulation of human symptoms and biochemistry will facilitate the evaluation of new therapeutic approaches with an early postnatal time window likely most effective. Take home message: A mouse model of nonketotic hyperglycinemia is described that shows postnatal abnormalities in glycine levels, neural tube defects, body weight, electroencephalographic recordings, and in activity in young mice making it amenable for the evaluation of novel treatment interventions. Author contributions: Study concept and design: JVH, MHM, NB, KNMAnimal study data: MAS, HJ, NB, MHM, JC, CBBiochemical and genetic studies: MAS, RAVH, MWFStatistical analysis: NB, JVHFirst draft writing: JVH, NB, MHMCritical rewriting: MAS, NB, MHM, TAB, JC, MWF, KNM, JVHFinal responsibility, guarantor, and communicating author: JVH. Competing interest statement: The University of Colorado (JVH, MS, KNM, HJ) has the intention to file Intellectual property protection for certain biochemical treatments of NKH. Otherwise, the authors have stated that they had no interests that might be perceived as posing a conflict or bias to this subject matter. Funding support: Financial support is acknowledged form the NKH Crusaders, Brodyn's Friends, Nora Jane Almany Foundation, the Dickens Family Foundation, the Lucas John Foundation, Les Petits Bourdons, Joseph's Fund, the Barnett Family, Maud & Vic Foundation, Lucy's BEElievers fund, Hope for NKH, Madi's Mission NKH fund, and from Dr. and Ms. Shaw, and the University of Colorado Foundation NKH research fund. The study was supported by a grant (CNS-X-19-103) from the University of Colorado School of Medicine and the Colorado Clinical Translational Science Institute, which is supported by NIH/NCATS Colorado CTSA Grant Number UL1 TR002535. Contents are the authors' sole responsibility and do not necessarily represent official NIH views. All funding sources had no role in the design or execution of the study, the interpretation of data, or the writing of the study. Ethics approval on Laboratory Animal Studies: Mouse studies were carried out with approval from the Institutional Animal Care and Use Committee of the University of Colorado Anschutz Medical Campus (IACUC# 00413). Data sharing statement: The data that support the findings of this study are available from the corresponding author upon reasonable request.

Protein biomarkers GDF15 and FGF21 to differentiate mitochondrial hepatopathies from other pediatric liver diseases.

BACKGROUND: Mitochondrial hepatopathies (MHs) are primary mitochondrial genetic disorders that can present as childhood liver disease. No recognized biomarkers discriminate MH from other childhood liver diseases. The protein biomarkers growth differentiation factor 15 (GDF15) and fibroblast growth factor 21 (FGF21) differentiate mitochondrial myopathies from other myopathies. We evaluated these biomarkers to determine if they discriminate MH from other liver diseases in children. METHODS: Serum biomarkers were measured in 36 children with MH (17 had a genetic diagnosis); 38 each with biliary atresia, α1-antitrypsin deficiency, and Alagille syndrome; 20 with NASH; and 186 controls. RESULTS: GDF15 levels compared to controls were mildly elevated in patients with α1-antitrypsin deficiency, Alagille syndrome, and biliary atresia-young subgroup, but markedly elevated in MH (p<0.001). FGF21 levels were mildly elevated in NASH and markedly elevated in MH (p<0.001). Both biomarkers were higher in patients with MH with a known genetic cause but were similar in acute and chronic presentations. Both markers had a strong performance to identify MH with a molecular diagnosis with the AUC for GDF15 0.93±0.04 and for FGF21 0.90±0.06. Simultaneous elevation of both markers >98th percentile of controls identified genetically confirmed MH with a sensitivity of 88% and specificity of 96%. In MH, independent predictors of survival without requiring liver transplantation were international normalized ratio and either GDF15 or FGF21 levels, with levels <2000 ng/L predicting survival without liver transplantation (p<0.01). CONCLUSIONS: GDF15 and FGF21 are significantly higher in children with MH compared to other childhood liver diseases and controls and, when combined, were predictive of MH and had prognostic implications.

Pathogenic variants in NUBPL result in failure to assemble the matrix arm of complex I and cause a complex leukoencephalopathy with thalamic involvement.

Disorders of the white matter are genetically very heterogeneous including several genes involved in mitochondrial bioenergetics. Diagnosis of the underlying cause is aided by pattern recognition on neuroimaging and by next-generation sequencing. Recently, genetic changes in the complex I assembly factor NUBPL have been characterized by a consistent recognizable pattern of leukoencephalopathy affecting deep white matter including the corpus callosum and cerebellum. Here, we report twin boys with biallelic variants in NUBPL, an unreported c.351 G > A; p.(Met117Ile) and a previously reported pathological variant c. 693 + 1 G > A. Brain magnetic resonance imaging showed abnormal T2 hyperintense signal involving the periventricular white matter, external capsule, corpus callosum, and, prominently, the bilateral thalami. The neuroimaging pattern evolved over 18 months with marked diffuse white matter signal abnormality, volume loss, and new areas of signal abnormality in the cerebellar folia and vermis. Magnetic resonance spectroscopy showed elevated lactate. Functional studies in cultured fibroblasts confirmed pathogenicity of the genetic variants. Complex I activity of the respiratory chain was deficient spectrophotometrically and on blue native gel with in-gel activity staining. There was absent assembly and loss of proteins of the matrix arm of complex I when traced with an antibody to NDUFS2, and incomplete assembly of the membrane arm when traced with an NDUFB6 antibody. There was decreased NUBPL protein on Western blot in patient fibroblasts compared to controls. Compromised NUBPL activity impairs assembly of the matrix arm of complex I and produces a severe, rapidly-progressive leukoencephalopathy with thalamic involvement on MRI, further expanding the neuroimaging phenotype.

The complete mitochondrial genomes of five lichenized fungi in the genus Usnea (Ascomycota: Parmeliaceae).

Known colloquially as 'Old Man's Beard', Usnea is a genus of lichenized Ascomycete fungi characterized by having a fruticose growth form and cartilaginous central axis. The complete mitochondrial genomes of Usnea halei, U. mutabilis, U. subfusca, U. subgracilis, and U. subscabrosa were sequenced using Illumina data and then assembled de novo. These mitogenomes ranged in size from 52,486 bp (U. subfusca) to 94,464 bp (U. subgracilis). All were characterized by having high levels of intronic and intergenic variation, such as ORFs that encode proteins with homology to two homing endonuclease types, LAGLIDADG and GIY-YIG. Genes annotated within these mitogenomes include 14 protein-coding genes, the large and small ribosomal subunits (LSU and SSU), and 23-26 tRNAs. Notably, the atp9 gene was absent from each genome. Genomic synteny was highly conserved across the five species. Five conserved mitochondrial genes (nad2, nad4, cox1, cox2, and cox3) were used to infer a best estimate maximum likelihood phylogeny among these five Usnea and other relatives, which yielded relationships consistent with prior published phylogenies.