Background/Objectives: We have previously demonstrated that fatty acid oxidation (FAO) enzymes physically and functionally interact with electron transfer chain supercomplexes (ETC-SC) at two contact points. The FAO trifunctional protein (TFP) and electron transfer flavoprotein dehydrogenase (ETFDH) interact with the NADH+-binding domain of ETC complex I (com I) and the core 2 subunit of complex III (com III), respectively. In addition, the FAO enzyme very-long-chain acyl-CoA dehydrogenase (VLCAD) interacts with TFP. These interactions define a functional FAO-ETC macromolecular complex (FAO-ETC MEC) in which FAO-generated NADH+ and FADH2 can safely transfer electron equivalents to ETC in order to generate ATP. Methods: In this study, we use multiple mitochondrial functional studies to demonstrate the effect of added VLCAD protein on mutant mitochondria. Results: We demonstrate that heart mitochondria from a VLCAD knockout (KO) mouse exhibit disrupted supercomplexes, with significantly reduced levels of TFPα and TFPβ subunits, electron transfer flavoprotein a-subunit (ETFα), and NDUFV2 subunit of com I in the FAO-ETC MEC. In addition, the activities of individual oxidative phosphorylation (OXPHOS) enzymes are decreased, as is the transfer of reducing equivalents from palmitoyl-CoA to ETC (FAO-ETC flux). However, the total amount of these proteins did not decrease in VLCAD KO animals. These results suggest that loss of VLCAD affects the interactions of FAO and ETC proteins in the FAO-ETC MEC. Reconstitution of VLCAD-deficient heart mitochondria with recombinant VLCAD improved the levels of FAO-ETC MEC proteins and enzyme activities, as well as restoring FAO-ETC flux. It also reduced mitochondrial ROS levels, previously demonstrated to be elevated in VLCAD-deficient mitochondria. In contrast, incubation of VLCAD KO mitochondria with two VLCADs with mutations in the C-terminal domain of the enzyme (A450P and L462P) did not restore FAO-ETC MECs. Conclusions: These results suggest that VLCAD is a necessary component of the FAO-ETC MEC and plays a major role in assembly of the macro-supercomplex. These studies provide evidence that both the level of enzyme and its structural confirmation are necessary to stabilize the FAO-ETC MEC.

Mitochondrial trifunctional protein (TFP) deficiency is an inherited metabolic disorder leading to a block in long-chain fatty acid β-oxidation. Mutations in HADHA and HADHB, which encode the TFP α and β subunits, respectively, usually result in combined TFP deficiency. A single common mutation, HADHA c.1528G>C (p.E510Q), leads to isolated 3-hydroxyacyl-CoA dehydrogenase deficiency. TFP also catalyzes a step in the remodeling of cardiolipin (CL), a phospholipid critical to mitochondrial membrane stability and function. We explored the effect of mutations in TFP subunits on CL and other phospholipid content and composition and the consequences of these changes on mitochondrial bioenergetics in patient-derived fibroblasts. Abnormalities in these parameters varied extensively among different fibroblasts, and some cells were able to maintain basal oxygen consumption rates similar to controls. Although CL reduction was universally identified, a simultaneous increase in monolysocardiolipins was discrepant among cells. A similar profile was seen in liver mitochondria isolates from a TFP-deficient mouse model. Response to new potential drugs targeting CL metabolism might be dependent on patient genotype.

Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is an autosomal recessive inborn error of fatty acid metabolism resulting from biallelic mutations in the ACADVL gene. Patients with VLCADD present with biochemical dysfunction due to deficiency in the metabolism of very long-chain fatty acids (C>16). Phenotype may include cardiomyopathy, rhabdomyolysis, and acute metabolic decompensation. Newborn screening (NBS) by acylcarnitine profiling identifies potential VLCADD with positive results referred for genetic confirmation by exome-based sequencing.

Prader-Willi syndrome (PWS) is a multisystem disorder with neurobehavioral, metabolic, and hormonal phenotypes, caused by loss of expression of a paternally-expressed imprinted gene cluster. Prior evidence from a PWS mouse model identified abnormal pancreatic islet development with retention of aged insulin and deficient insulin secretion. To determine the collective roles of PWS genes in β-cell biology, we used genome-editing to generate isogenic, clonal INS-1 insulinoma lines having 3.16 Mb deletions of the silent, maternal- (control) and active, paternal-allele (PWS). PWS β-cells demonstrated a significant cell autonomous reduction in basal and glucose-stimulated insulin secretion. Further, proteomic analyses revealed reduced levels of cellular and secreted hormones, including all insulin peptides and amylin, concomitant with reduction of at least ten endoplasmic reticulum (ER) chaperones, including GRP78 and GRP94. Critically, differentially expressed genes identified by whole transcriptome studies included reductions in levels of mRNAs encoding these secreted peptides and the group of ER chaperones. In contrast to the dosage compensation previously seen for ER chaperones in Grp78 or Grp94 gene knockouts or knockdown, compensation is precluded by the stress-independent deficiency of ER chaperones in PWS β-cells. Consistent with reduced ER chaperones levels, PWS INS-1 β-cells are more sensitive to ER stress, leading to earlier activation of all three arms of the unfolded protein response. Combined, the findings suggest that a chronic shortage of ER chaperones in PWS β-cells leads to a deficiency of protein folding and/or delay in ER transit of insulin and other cargo. In summary, our results illuminate the pathophysiological basis of pancreatic β-cell hormone deficits in PWS, with evolutionary implications for the multigenic PWS-domain, and indicate that PWS-imprinted genes coordinate concerted regulation of ER chaperone biosynthesis and β-cell secretory pathway function.

Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is an autosomal recessive disease resulting from mutations in the ACADVL gene and is among the disorders tested for in newborn screening (NBS). Confirmatory sequencing following suspected VLCADD NBS results often identifies variants of uncertain significance (VUS) in the ACADVL gene, leading to uncertainty of diagnosis and providing effective treatment regimen. Currently, ACADVL has >300 VUSs in the ClinVar database that requiring characterization to determine potential pathogenicity. In this study, CRISPR/Cas9 genome editing was used to knock out ACADVL in HEK293T cells, and targeted deletion was confirmed by droplet digital polymerase chain reaction (PCR). No VLCAD protein was detected and an 84% decrease in enzyme activity using the electron transfer flavoprotein fluorescence reduction assay and C21-CoA as substrate was observed compared to control. Plasmids containing control or variant ACADVL coding sequence were transfected into the ACADVL null HEK293T. While transfection of control ACADVL restored VLCAD protein and enzyme activity, cells expressing the VLCAD Val283Ala mutant had 18% VLCAD enzyme activity and reduced protein compared to control. VLCAD Ile420Leu, Gly179Arg, and Gln406Pro produced protein comparable to control but 25%, 4%, and 5% VLCAD enzyme activity, respectively. Leu540Pro and Asp570_Ala572dup had reduced VLCAD protein and 10% and 3% VLCAD enzyme activity, respectively. VLCADD fibroblasts containing the same variations had decreased VLCAD protein and activity comparable to the transfection experiments. Generating ACADVL null HEK293T cell line allowed functional studies to determine pathogenicity of ACADVL exonic variants. This approach can be applied to multiple genes for other disorders identified through NBS.

Transport and Golgi Organization protein 2 Homolog ( TANGO2 )-related disease is an autosomal recessive disorder caused by mutations in the TANGO2 gene. Symptoms typically manifest in early childhood and include developmental delay, stress-induced episodic rhabdomyolysis, and cardiac arrhythmias, along with severe metabolic crises including hypoglycemia, lactic acidosis, and hyperammonemia. Severity varies among and within families. Previous studies have reported contradictory evidence of mitochondrial dysfunction. Since the clinical symptoms and metabolic abnormalities are suggestive of a broad dysfunction of mitochondrial energy metabolism, we undertook a broad examination of mitochondrial bioenergetics in TANGO2 deficient patients utilizing skin fibroblasts derived from three patients exhibiting TANGO2 -related disease. Functional studies revealed that TANGO2 protein was present in mitochondrial extracts of control cells but not patient cells. Superoxide production was increased in patient cells, while oxygen consumption rate, particularly under stress, along with relative ATP levels and β-oxidation of oleate were reduced. Our findings suggest that mitochondrial function should be assessed and monitored in all patients with TANGO2 mutation as targeted treatment of the energy dysfunction could improve outcome in this condition.

Introduction: Clinical standard of care for newborn screening (NBS) is acylcarnitine metabolites quantitation by tandem mass spectrometry (MS/MS) from dried blood spots. Follow up sequencing often results in identification of one or more variants of uncertain significance (VUS). Isovaleric acidemia (IVA) is an autosomal recessive inborn error of metabolism caused by deficiency of isovaleryl-CoA dehydrogenase (IVDH) in the Leu catabolism pathway. Many IVD mutations are characterized as VUS complicating IVA clinical diagnoses and treatment. We present a testing platform approach to confirm the functional implication of VUS identified in newborns with IVA applicable to multiple inborn errors of metabolism identified by NBS. Methods: An IVD null HEK293T cell culture model was generated by using a dual sgRNA CRISPR/Cas9 genomeediting strategy targeting IVD exons 2-3. Clonal cell lines were confirmed by a combination of genomic breakpoint sequencing and droplet digital PCR. The IVD null model had no IVDH antigen signal and 96% reduction in IVDH enzyme activity. The IVD null model was transfected with vectors containing control or variant IVD and functional assays were performed to determine variant pathogenicity. Results: c.149G > C (p.Arg50Pro; precursor numbering), c.986T > C (p.Met329Thr), and c.1010G > A (p. Arg337Gln), c.1179del394 f. mutant proteins had reduced IVDH protein and activity. c.932C > T (p.Ala311Val), c.707C > T (p.Thr236Ile), and c.1232G > A (p.Arg411Gln) had stable IVDH protein, but no enzyme activity. c.521T > G (p.Val174Gly) had normal IVDH protein and activity. IVD variant transfection results confirmed results from IVA fibroblasts containing the same variants. Conclusions: We have developed an IVD null HEK293T cell line to rapidly allow determination of VUS pathogenicity following identification of novel alleles by clinical sequencing following positive NBS results for suspected IVA. We suggest similar models can be generated via genome-editing for high throughput assessment of VUS function for a multitude of inborn errors of metabolism and can ideally supplement NBS programs. (c) 2021 Elsevier Inc. All rights reserved.

Fused in sarcoma (FUS) is a predominantly nuclear multifunctional RNA/DNA-binding protein that regulates multiple aspects of gene expression. FUS mutations are associated with familial amyotrophic lateral sclerosis (fALS) and frontotemporal lobe degeneration (FTLD) in humans. At the molecular level, the mutated FUS protein is reduced in the nucleus but accumulates in cytoplasmic granules. Oligodendrocytes (OL) carrying clinically relevant FUS mutations contribute to non-cell autonomous motor neuron disease progression, consistent with an extrinsic mechanism of disease mediated by OL. Knocking out FUS globally or in neurons lead to behavioral abnormalities that are similar to those present in FTLD. In this study, we sought to investigate whether an extrinsic mechanism mediated by loss of FUS function in OL contributes to the behavioral phenotype. We have generated a novel conditional knockout (cKO) in which Fus is selectively depleted in OL (FusOL cKO). The FusOL cKO mice show increased novelty-induced motor activity and enhanced exploratory behavior, which are reminiscent of some manifestations of FTLD. The phenotypes are associated with greater myelin thickness, higher number of myelinated small diameter axons without an increase in the number of mature OL. The expression of the rate-limiting enzyme of cholesterol biosynthesis (HMGCR) is increased in white matter tracts of the FusOL cKO and results in higher cholesterol content. In addition, phosphorylation of Akt, an important regulator of myelination is increased in the FusOL cKO. Collectively, this work has uncovered a novel role of oligodendrocytic Fus in regulating myelin deposition through activation of Akt and cholesterol biosynthesis.

Phenylalanine hydroxylase-deficient (PAH-deficient) phenylketonuria (PKU) results in systemic hyperphenylalaninemia, leading to neurotoxicity with severe developmental disabilities. Dietary phenylalanine (Phe) restriction prevents the most deleterious effects of hyperphenylalaninemia, but adherence to diet is poor in adult and adolescent patients, resulting in characteristic neurobehavioral phenotypes. Thus, an urgent need exists for new treatments. Additionally, rodent models of PKU do not adequately reflect neurocognitive phenotypes, and thus there is a need for improved animal models. To this end, we have developed PAH-null pigs. After selection of optimal CRISPR/Cas9 genome-editing reagents by using an in vitro cell model, zygote injection of 2 sgRNAs and Cas9 mRNA demonstrated deletions in preimplantation embryos, with embryo transfer to a surrogate leading to 2 founder animals. One pig was heterozygous for a PAH exon 6 deletion allele, while the other was compound heterozygous for deletions of exon 6 and of exons 6-7. The affected pig exhibited hyperphenylalaninemia (2000-5000 μM) that was treatable by dietary Phe restriction, consistent with classical PKU, along with juvenile growth retardation, hypopigmentation, ventriculomegaly, and decreased brain gray matter volume. In conclusion, we have established a large-animal preclinical model of PKU to investigate pathophysiology and to assess new therapeutic interventions.

Mutations in imprinted genes or their imprint control regions (ICRs) produce changes in imprinted gene expression and distinct abnormalities in placental structure, indicating the importance of genomic imprinting to placental development. We have recently shown that a very broad spectrum of placental abnormalities associated with altered imprinted gene expression occurs in the absence of the oocyte-derived DNMT1o cytosine methyltransferase, which normally maintains parent-specific imprinted methylation during preimplantation. The absence of DNMT1o partially reduces inherited imprinted methylation while retaining the genetic integrity of imprinted genes and their ICRs. Using this novel system, we undertook a broad and inclusive approach to identifying key ICRs involved in placental development by correlating loss of imprinted DNA methylation with abnormal placental phenotypes in a mid-gestation window (E12.5-E15.5). To these ends we measured DNA CpG methylation at 15 imprinted gametic differentially methylated domains (gDMDs) that overlap known ICRs using EpiTYPER-mass array technology, and linked these epigenetic measurements to histomorphological defects. Methylation of some imprinted gDMDs, most notably Dlk1, was nearly normal in mid-gestation DNMT1o-deficient placentas, consistent with the notion that cells having lost methylation on these DMDs do not contribute significantly to placental development. Most imprinted gDMDs however showed a wide range of methylation loss among DNMT1o-deficient placentas. Two striking associations were observed. First, loss of DNA methylation at the Peg10 imprinted gDMD associated with decreased embryonic viability and decreased labyrinthine volume. Second, loss of methylation at the Kcnq1 imprinted gDMD was strongly associated with trophoblast giant cell (TGC) expansion. We conclude that the Peg10 and Kcnq1 ICRs are key regulators of mid-gestation placental function.

INTRODUCTION:Monoallelic expression of imprinted genes is necessary for placental development and normal fetal growth. Differentially methylated domains (DMDs) largely determine the parental-specific monoallelic expression of imprinted genes. Maternally derived DNA (cytosine-5-) -methyltransferase 1o (DNMT1o) maintains DMDs during the eight-cell stage of development. DNMT1o-deficient mouse placentas have a generalized disruption of genomic imprints. Previous studies have demonstrated that DNMT1o deficiency alters placental morphology and broadens the embryonic weight distribution in late gestation. Lipids are critical for fetal growth. Thus, we assessed the impact of disrupted imprinting on placental lipids.METHODS:Lipids were quantified from DNMT1o-deficient mouse placentas and embryos at E17.5 using a modified Folch method. Expression of select genes critical for lipid metabolism was quantified with RT-qPCR. Mitochondrial morphology was assessed by TEM and mitochondrial aconitase and cytoplasmic citrate concentrations quantified. DMD methylation was determined by EpiTYPER.RESULTS:We found that DNMT1o deficiency is associated with increased placental triacylglycerol levels. Neither fetal triacylglycerol concentrations nor expression of select genes that mediate placental lipid transport were different from wild type. Placental triacylglycerol accumulation was associated with impaired beta-oxidation and abnormal citrate metabolism with decreased mitochondrial aconitase activity and increased cytoplasmic citrate concentrations. Loss of methylation at the MEST DMD was strongly associated with placental triacylglycerol accumulation.DISCUSSION:A generalized disruption of genomic imprints leads to triacylglycerol accumulation and abnormal mitochondrial function. This could stem directly from a loss of methylation at a given DMD, such as MEST, or represent a consequence of abnormal placental development.

Monoallelic expression of imprinted genes, including ones solely expressed in the placenta, is essential for normal placental development and fetal growth. To better understand the role of placental imprinting in placental development and fetal growth, we examined conceptuses developing in the absence of maternally derived DNA (cytosine-5-)-methyltransferase 1o (DNMT1o). Absence of DNMT1o results in the partial loss of methylation at imprinted differentially methylated domain (DMD) sequences in the embryo and the placenta. Mid-gestation E9.5 DNMT1o-deficient placentas exhibited structural abnormalities of all tissue layers. At E17.5, all examined placentas had aberrant placental morphology, most notably in the spongiotrophoblast and labyrinth layers. Abnormalities included an expanded volume fraction of spongiotrophoblast tissue with extension of the spongiotrophoblast layer into the labyrinth. Many mutant placentas also demonstrated migration abnormalities of glycogen cells. Additionally, the volume fraction of the labyrinth was reduced, as was the surface area for maternal fetal gas exchange. Despite these placental morphologic abnormalities, approximately one-half of DNMT1o-deficient fetuses survived to late gestation (E17.5). Furthermore, DNMT1o-deficient placentas supported a broad range of fetal growth. The ability of some DNMT1o-deficient and morphologically abnormal placentas to support fetal growth in excess of wild type demonstrates the importance of differential methylation of DMDs and proper imprinting of discrete gene clusters to placental morphogenesis and fetal growth.

Genomic imprinting is a process by which heritable epigenetic marks at a subset of genomic loci are established in a sex-specific manner in parental gametes and then maintained in nascent offspring. This study probes the poorly understood function of genomic imprints in placental biology. Genomic imprints are responsible for the regulation of parent-of-origin specific monoallelic expression of clusters of imprinted genes. The primary epigenetic mark that distinguishes parental alleles at imprinted loci is 5-methylcytosine in the context of cytosine-guanine (CpG) dinucleotides within differentially methylated domains (DMDs). The Dnmt1 gene encodes the maintenance DNA methyltransferase, an enzyme responsible for replicating CpG methylation that is critical throughout the process of genomic imprinting. Genetic disruption of the oocyte specific isoform of Dnmt1 (Dnmt1o) results in partial and wide-spread loss of DMD methylation during preimplantation development and has strong effects on embryonic and extraembryonic development. In this dissertation the morphology of DNMT1o-deficient placentas is examined and their abnormal phenotypes correlated with loss of methylation at specific DMDs. A strong association between loss of methylation at the Kcnq1 DMD and accumulation of trophoblast giant cells was made. In addition, an association between loss of methylation at the Peg10 DMD and loss of fetal viability and placental labyrinthine volume was made. In conjunction with my study of the Dnmt1Δ1o model, I have engineered a novel targeted deletion of the imprinted Klf14 gene and found it has an effect on placental growth. My thesis unambiguously shows that genomic imprints are essential for placental development.