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During differentiation of precursor cells into their destination cell type, cell fate decisions are enforced by a broad array of epigenetic modifications, including DNA methylation, which is reflected by the transcriptome. Thus, regulatory dendritic cells (DCregs) acquire specific epigenetic programs and immunomodulatory functions during their differentiation from monocytes. To define the epigenetic signature of human DCregs generated in vitamin D3 (vitD3) and IL-10 compared to immune stimulatory DCs (sDCs), we measured levels of DNA methylation by whole genome bisulfite sequencing (WGBS). Distinct DNA methylation patterns were acquired by DCregs compared to sDCs. These patterns were located mainly in transcriptional regulatory regions. Associated genes were enriched in STAT3-signaling and valine catabolism in DCregs; conversely, pro-inflammatory pathways, e.g. pattern recognition receptor signaling, were enriched in sDCs. Further, DCreg differentially-methylated regions (DMRs) were enriched in binding motifs specific to the immunomodulatory transcription factor Krueppel-like factor 11 (KLF11), while activator protein-1 (AP-1) (Fos:Jun) transcription factor-binding motifs were enriched in sDC DMRs. Using publicly-available data-sets, we defined a common epigenetic signature shared between DCregs generated in vitD3 and IL-10, or dexamethasone or vitD3 alone. These insights may help pave the way for design of epigenetic-based approaches to enhance the production of DCregs as effective therapeutic agents., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Purpose: To identify therapies for combined D, L-2-hydroxyglutaric aciduria (C-2HGA), a rare genetic disorder caused by recessive variants in the SLC25A1 gene., Methods: Patients C-2HGA were identified and diagnosed by whole exome sequencing and biochemical genetic testing. Patient derived fibroblasts were then treated with phenylbutyrate and the functional effects assessed by metabolomics and RNA-sequencing., Results: In this study, we demonstrated that C-2HGA patient derived fibroblasts exhibited impaired cellular bioenergetics. Moreover, Fibroblasts form one patient exhibited worsened cellular bioenergetics when supplemented with citrate. We hypothesized that treating patient cells with phenylbutyrate (PB), an FDA approved pharmaceutical drug that conjugates glutamine for renal excretion, would reduce mitochondrial 2-ketoglutarate, thereby leading to improved cellular bioenergetics. Metabolomic and RNA-seq analyses of PB-treated fibroblasts demonstrated a significant decrease in intracellular 2-ketoglutarate, 2-hydroxyglutarate, and in levels of mRNA coding for citrate synthase and isocitrate dehydrogenase. Consistent with the known action of PB, an increased level of phenylacetylglutamine in patient cells was consistent with the drug acting as 2-ketoglutarate sink., Conclusion: Our pre-clinical studies suggest that citrate supplementation has the possibility exacerbating energy metabolism in this condition. However, improvement in cellular bioenergetics suggests phenylbutyrate might have interventional utility for this rare disease., Competing Interests: Declaration of competing interest Authors declare that they have no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

Dicarboxylic fatty acids are generated in the liver and kidney in a minor pathway called fatty acid ω-oxidation. The effects of consuming dicarboxylic fatty acids as an alternative source of dietary fat have not been explored. Here, we fed dodecanedioic acid, a 12-carbon dicarboxylic (DC12), to mice at 20% of daily caloric intake for nine weeks. DC12 increased metabolic rate, reduced body fat, reduced liver fat, and improved glucose tolerance. We observed DC12-specific breakdown products in liver, kidney, muscle, heart, and brain, indicating that oral DC12 escaped first-pass liver metabolism and was utilized by many tissues. In tissues expressing the "a" isoform of acyl-CoA oxidase-1 (ACOX1), a key peroxisomal fatty acid oxidation enzyme, DC12 was chain shortened to the TCA cycle intermediate succinyl-CoA. In tissues with low peroxisomal fatty acid oxidation capacity, DC12 was oxidized by mitochondria. In vitro, DC12 was catabolized even by adipose tissue and was not stored intracellularly. We conclude that DC12 and other dicarboxylic acids may be useful for combatting obesity and for treating metabolic disorders.

Background: Pyruvate dehydrogenase complex deficiency (PDCD) is a mitochondrial neurometabolic disorder of energy deficit, with incidence of about 1 in 42,000 live births annually in the USA. The median and mean ages of diagnosis of PDCD are about 12 and 31 months, respectively. PDCD is a major cause of primary lactic acidosis with concomitant elevation in blood alanine (Ala) and proline (Pro) concentrations depending on phenotypic severity. Alanine/Leucine (Ala/Leu) ≥4.0 and Proline/Leucine (Pro/Leu) ≥3.0 combination cutoff from dried blood spot specimens was used as a biomarker for early identification of neonates/infants with PDCD. Further investigations were needed to evaluate the sensitivity (SN), specificity (SP), and clinical utility of such amino acid (AA) ratio combination cutoffs in discriminating PDCD from other inborn errors of metabolism (IEM) for early identification of such patients., Methods: We reviewed medical records of patients seen at UPMC in the past 11 years with molecularly or enzymatically confirmed diagnosis. We collected plasma AA analysis data from samples prior to initiation of therapeutic interventions such as total parenteral nutrition and/or ketogenic diet. Conditions evaluated included organic acidemias, primary mitochondrial disorders (MtDs), fatty acid oxidation disorders (FAOD), other IEMs on current newborn screening panels, congenital cardiac great vessel anomalies, renal tubular acidosis, and non-IEMs. The utility of specific AA ratio combinations as biomarkers were evaluated using receiver operating characteristic curves, correlation analysis, principal component analysis, and cutoff SN, SP, and positive predictive value determined from 201 subjects with broad age range., Results: Alanine/Lysine (Ala/Lys) and Ala/Leu as well as (Ala + Pro)/(Leu + Lys) and Ala/Leu ratio combinations effectively discriminated subjects with PDCD from those with other MtDs and IEMs on current newborn screening panels. Specific AA ratio combinations were significantly more sensitive in identifying PDCD than Ala alone or combinations of Ala and/or Pro in the evaluated cohort of subjects. Ala/Lys ≥3.0 and Ala/Leu ≥5.0 as well as (Ala + Pro)/(Leu + Lys) ≥2.5 and Ala/Leu ≥5.0 combination cutoffs identified patients with PDCD with 100% SN and ~85% SP., Conclusions: With the best predictor of survival and positive cognitive outcome in PDCD being age of diagnosis, PDCD patients would benefit from use of such highly SN and SP AA ratio combination cutoffs as biomarkers for early identification of at-risk newborns, infants, and children, for early intervention(s) with known and/or novel therapeutics for this disorder., (© 2023 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

We review unique properties of bone formation including current understanding of mechanisms of bone mineral transport. We focus on formation only; mechanism of bone degradation is a separate topic not considered. Bone matrix is compared to other connective tissues composed mainly of the same proteins, but without the specialized mechanism for continuous transport and deposition of mineral. Indeed other connective tissues add mechanisms to prevent mineral formation. We start with the epithelial-like surfaces that mediate transport of phosphate to be incorporated into hydroxyapatite in bone, or in its ancestral tissue, the tooth. These include several phosphate producing or phosphate transport-related proteins with special expression in large quantities in bone, particularly in the bone-surface osteoblasts. In all connective tissues including bone, the proteins that constitute the protein matrix are mainly type I collagen and γ-carboxylate-containing small proteins in similar molar quantities to collagen. Specialized proteins that regulate connective tissue structure and formation are surprisingly similar in mineralized and non-mineralized tissues. While serum calcium and phosphate are adequate to precipitate mineral, specialized mechanisms normally prevent mineral formation except in bone, where continuous transport and deposition of mineral occurs., (© 2023 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals LLC.)

Pathophysiology of osteopenia in phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU) is poorly characterized. The Pah enu2 mouse is universally osteopenic where dietary phenylalanine (Phe) management with amino acid defined chow does not improve bone density. We previously demonstrated Pah enu2 osteopenia owes to a skeletal stem cell (SSC) developmental deficit mediated by energy dysregulation and oxidative stress. This investigation demonstrates complexity of Pah enu2 SSC energy dysregulation. Creatine use by bone tissue is recognized. In vitro Pah enu2 SSCs in osteoblast differentiation respond to creatine with increased in situ alkaline phosphatase activity and increased intracellular ATP content. Animal studies applied a 60-day creatine regimen to Pah enu2 and control cohorts. Control cohorts include unaffected littermates (wt/wt), Pah enu2 receiving no intervention, and dietary Phe restricted Pah enu2 . Experimental cohorts (Phe unrestricted Pah enu2 , Phe restricted Pah enu2 ) were provided 1% creatine ad libitum in water. After 60 days, microcomputed tomography assessed bone metrics. Equivalent osteopenia occurs in Phe-restricted and untreated Pah enu2 control cohorts. In Phe unrestricted Pah enu2 , creatine was without effect as bone density remained equivalent to Pah enu2 control cohorts. Alternatively, Phe-restricted Pah enu2 receiving creatine present increased bone density. We hypothesize small molecule dysregulation in untreated Pah enu2 disallows creatine utilization; therefore, osteopenia persisted. Dietary Phe restriction enables creatine utilization to enhance SSC osteoblast differentiation and improve in vivo bone density. PKU intervention singularly focused on Phe reduction enables residual disease including osteopenia and neurologic elements. Intervention concurrently addressing Phe homeostasis and energy dysregulation will improve disease elements refractory to standard of care Phe reduction mono-therapy., Competing Interests: None of the authors have competing interests relating to these studies., (© 2023 The Authors.)

Autoimmune Disease, Multisystem, with Facial Dysmorphism (ADMFD) is an autosomal recessive disorder due to pathogenic variants in the ITCH gene. It is characterized by failure to thrive, dysmorphic facial features, developmental delay, and systemic autoimmunity that can manifest variably with autoimmune hepatitis, thyroiditis, and enteropathy, among other organ manifestations. It was originally described in 10 consanguineous Old Order Amish patients, and more recently in two patients of White British and Black German ethnicities. While the role of ITCH protein in apoptosis and inflammation has previously been characterized, a defect in cellular bioenergetics has not yet been reported in ITCH deficiency. Here we present a Caucasian female originally evaluated for possible mitochondrial respiratory chain deficiency, who ultimately was found to have two novel variants in ITCH with absence of ITCH protein in patient derived fibroblasts. Clinical studies of patient muscle showed mitochondrial DNA copy number of 57% compared to controls. Functional studies in skin fibroblasts revealed decreased activity of mitochondrial fatty acid oxidation and oxidative phosphorylation, and decreased overall ATP production. Our findings confirm mitochondrial energy dysfunction in a patient with ITCH deficiency offering the opportunity to assess alternative therapeutic options., (© 2022 The Authors.)

Osteopenia is an under-investigated clinical presentation of phenylalanine hydroxylase (PAH)-deficient phenylketonuria (PKU). While osteopenia is not fully penetrant in human PKU, the Pah enu2 mouse is universally osteopenic and ideal to study the phenotype. We determined Pah enu2 mesenchymal stem cells (MSCs) are developmentally impaired in the osteoblast lineage. Moreover, we determined energy dysregulation and oxidative stress contribute to the osteoblast developmental deficit. The MSC preferred substrate glutamine (Gln) was applied to enhance energy homeostasis. In vitro Pah enu2 MSCs, in the context of 1200 μM Phe, respond to Gln with increased in situ alkaline phosphatase activity indicating augmented osteoblast differentiation. Oximetry applied to Pah enu2 MSCs in osteoblast differentiation show Gln energy substrate increases oxygen consumption, specifically maximum respiration and respiratory reserve. For 60 days post-weaning, Pah enu2 animals received either no intervention (standard lab chow), amino acid defined chow maintaining plasma Phe at ~200 μM, or standard lab chow where ad libitum water was a 2% Gln solution. Bone density was assessed by microcomputed tomography and bone growth assessed by dye labeling. Bone density and dye labeling in Phe-restricted Pah enu2 was indistinguishable from untreated Pah enu2 . Gln energy substrate provided to Pah enu2 , in the context of uncontrolled hyperphenylalaninemia, present increased bone density and dye labeling. These data provide further evidence that Pah enu2 MSCs experience a secondary energy deficit that is responsive both in vitro and in vivo to Gln energy substrate and independent of hyperphenylalaninemia. Energy support may have effect to treat human PKU osteopenia and elements of PKU neurologic disease resistant to standard of care systemic Phe reduction. Glutamine energy substrate anaplerosis increased Pah enu2 bone density and improved in vitro MSC function in the context of hyperphenylalaninemia in the classical PKU range., Competing Interests: No authors have competing interests related to these studies., (© 2022 The Authors. JIMD Reports published by John Wiley & Sons Ltd on behalf of SSIEM.)

Phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU) is rightfully considered the paradigm treatable metabolic disease. Dietary substrate restriction (i.e. phenylalanine (Phe) restriction) was applied >60 years ago and remains the primary PKU management means. The traditional model of PKU neuropathophysiology dictates blood Phe over-representation directs asymmetric blood:brain barrier amino acid transport through the LAT1 transporter with subsequent increased cerebral Phe concentration and low concentrations of tyrosine (Tyr), tryptophan (Trp), leucine (Leu), valine (Val), and isoleucine (Ile). Low Tyr and Trp concentrations generate secondary serotonergic and dopaminergic neurotransmitter paucities, widely attributed as drivers of PKU neurologic phenotypes. White matter disease, a central PKU characteristic, is ascribed to Phe-mediated tissue toxicity. Impaired cerebral protein synthesis, by reduced concentrations of non-Phe large neutral amino acids, is another cited pathological mechanism. The PKU amino acid transport model suggests Phe management should be more efficacious than is realized, as even early identified, continuously treated patients that retain therapy compliance into adulthood, demonstrate neurologic disease elements. Reduced cerebral metabolism was an early-recognized element of PKU pathology. Legacy data (late 1960's to mid-1970's) determined the Phe catabolite phenylpyruvate inhibits mitochondrial pyruvate transport. Respirometry of Pah enu2 cerebral mitochondria have attenuated respiratory chain complex 1 induction in response to pyruvate substrate, indicating reduced energy metabolism. Oxidative stress is intrinsic to PKU and Pah enu2 brain tissue presents increased reactive oxygen species. Phenylpyruvate inhibits glucose-6-phosphate dehydrogenase that generates reduced niacinamide adenine dinucleotide phosphate the obligatory cofactor of glutathione reductase. Pah enu2 brain tissue metabolomics identified increased oxidized glutathione and glutathione disulfide. Over-represented glutathione disulfide argues for reduced glutathione reductase activity secondary to reduced NADPH. Herein, we review evidence of energy and oxidative stress involvement in PKU pathology. Data suggests energy deficit and oxidative stress are features of PKU pathophysiology, providing intervention-amenable therapeutic targets to ameliorate disease elements refractory to standard of care., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Classical phenylketonuria (PKU, OMIM 261600) owes to hepatic deficiency of phenylalanine hydroxylase (PAH) that enzymatically converts phenylalanine (Phe) to tyrosine (Tyr). PKU neurologic phenotypes include impaired brain development, decreased myelination, early onset mental retardation, seizures, and late-onset features (neuropsychiatric, Parkinsonism). Phe over-representation is systemic; however, tissue response to hyperphenylalaninemia is not consistent. To characterize hyperphenylalaninemia tissue response, metabolomics was applied to Pah enu2 classical PKU mouse blood, liver, and brain. In blood and liver over-represented analytes were principally Phe, Phe catabolites, and Phe-related analytes (Phe-conjugates, Phe-containing dipeptides). In addition to Phe and Phe-related analytes, the metabolomic profile of Pah enu2 brain tissue evidenced oxidative stress responses and energy dysregulation. Glutathione and homocarnosine anti-oxidative responses are apparent Pah enu2 brain. Oxidative stress in Pah enu2 brain was further evidenced by increased reactive oxygen species. Pah enu2 brain presents an increased NADH/NAD ratio suggesting respiratory chain complex 1 dysfunction. Respirometry in Pah enu2 brain mitochondria functionally confirmed reduced respiratory chain activity with an attenuated response to pyruvate substrate. Glycolysis pathway analytes are over-represented in Pah enu2 brain tissue. PKU pathologies owe to liver metabolic deficiency; yet, Pah enu2 liver tissue shows neither energy disruption nor anti-oxidative response. Unique aspects of metabolomic homeostasis in PKU brain tissue along with increased reactive oxygen species and respiratory chain deficit provide insight to neurologic disease mechanisms. While some elements of assumed, long standing PKU neuropathology are enforced by metabolomic data (e.g. reduced tryptophan and serotonin representation), energy dysregulation and tissue oxidative stress expand mechanisms underlying neuropathology., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Osteoblasts in vivo form an epithelial-like layer with tight junctions between cells. Bone formation involves mineral transport into the matrix and acid transport to balance pH levels. To study the importance of the pH gradient in vitro, we used Transwell inserts composed of polyethylene terephthalate (PET) membranes with 0.4 μm pores at a density of (2 ± 0.4) x 10 6 pores per cm 2 . Mesenchymal stem cells (MSCs) prepared from murine bone marrow were used to investigate alternative conditions whereby osteoblast differentiation would better emulate in vivo bone development. MSCs were characterized by flow cytometry with more than 90% CD44 and 75% Sca-1 labeling. Mineralization was validated with paracellular alkaline phosphatase activity, collagen birefringence, and mineral deposition confirming MSCs identity. We demonstrate that MSCs cultured and differentiated on PET inserts form an epithelial-like layer while mineralizing. Measurement of the transepithelial resistance was ∼1400 Ω•cm 2 at three weeks of differentiation. The pH value of the media above and under the cells were measured while cells were in proliferation and differentiation. In mineralizing cells, a difference of 0.145 pH unit was observed between the medium above and under the cells indicating a transepithelial gradient. A significant difference in pH units was observed between the medium above and below the cells in proliferation compared to differentiation. Data on pH below membranes were confirmed by pH-dependent SNARF1 fluorescence. Control cells in proliferative medium did not form an epithelial-like layer, displayed low transepithelial resistance, and there was no significant pH gradient. By transmission electron microscopy, membrane attached osteoblasts in vitro had abundant mitochondria consistent with active transport that occurs in vivo by surface osteoblasts. In keeping with osteoblastic differentiation, scanning electron microscopy identified deposition of extracellular collagen surrounded by hydroxyapatite. This in vitro model is a major advancement in modeling bone in vivo for understanding of osteoblast bone matrix production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Osteopenia is common in phenylalanine hydroxylase deficient phenylketonuria (PKU). PKU is managed by limiting dietary phenylalanine. Osteopenia in PKU might reflect a therapeutic diet, with reduced bone forming materials. However, osteopenia occurs in patients who never received dietary therapy or following short-term therapy. Humans and animal studies find no correlation between bone loss, plasma hyperphenylalaninemia, bone formation, and resorption markers. Work in the Pah enu2 mouse recently showed a mesenchymal stem cell (MSC) developmental defect in the osteoblast pathway. Specifically, Pah enu2 MSCs are affected by energy dysregulation and oxidative stress. In PKU, MSCs oximetry and respirometry show mitochondrial respiratory-chain complex 1 deficit and over-representation of superoxide, producing reactive oxygen species affecting mitochondrial function. Similar mechanisms are involved in aging bone and other rare defects including alkaptonuria and homocysteinemia. Novel interventions to support energy and reduce oxidative stress may restore bone formation PKU patients, and in metabolic diseases with related mechanisms.

Classical phenylketonuria (PKU, OMIM 261600) owes to hepatic deficiency of phenylalanine hydroxylase (PAH) that enzymatically converts phenylalanine (Phe) to tyrosine (Tyr). PKU neurologic phenotypes include impaired brain development, decreased myelination, early onset mental retardation, seizures, and late-onset features (neuropsychiatric, Parkinsonism). PAH deficiency leads to systemic hyperphenylalaninemia; however, the impact of Phe varies between tissues. To characterize tissue response to hyperphenylalaninemia, metabolomics was applied to tissue from therapy noncompliant classical PKU patients (blood, liver), the Pah enu2 classical PKU mouse (blood, liver, brain) and the PAH deficient pig (blood, liver, brain, cerebrospinal fluid). In blood, liver, and CSF from both patients and animal models over-represented analytes were principally Phe, Phe catabolites, and Phe-related analytes (conjugates, Phe-containing dipeptides). In addition to Phe and Phe-related analytes, the metabolomic profile of PKU brain tissue (mouse, pig) evidenced oxidative stress responses and energy dysregulation. In Pah enu2 and PKU pig brain tissues, anti-oxidative response by glutathione and homocarnosine is apparent. Oxidative stress in Pah enu2 brain was further demonstrated by increased reactive oxygen species. In Pah enu2 and PKU pig brain, an increased NADH/NAD ratio suggests a respiratory chain dysfunction. Respirometry in PKU brain mitochondria (mouse, pig) functionally confirmed reduced respiratory chain activity. Glycolysis pathway analytes are over-represented in PKU brain tissue (mouse, pig). PKU pathologies owe to liver metabolic deficiency; yet, PKU liver tissue (mouse, pig, human) shows neither energy disruption nor anti-oxidative response. Unique aspects of metabolomic homeostasis in PKU brain tissue along with increased reactive oxygen species and respiratory chain deficit provide insight to neurologic disease mechanisms. While some elements of assumed, long standing PKU neuropathology are enforced by metabolomic data (e.g. reduced tryptophan and serotonin representation), energy dysregulation and tissue oxidative stress expand mechanisms underlying neuropathology., (Copyright © 2021. Published by Elsevier Inc.)

Osteopenia occurs in a subset of phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU) patients. While osteopenia is not fully penetrant in patients, the Pah enu2 classical PKU mouse is universally osteopenic, making it an ideal model of the phenotype. Pah enu2 Phe management, with a Phe-fee amino acid defined diet, does not improve bone density as histomorphometry metrics remain indistinguishable from untreated animals. Previously, we demonstrated Pah enu2 mesenchymal stem cells (MSCs) display impaired osteoblast differentiation. Oxidative stress is recognized in PKU patients and PKU animal models. Pah enu2 MSCs experience oxidative stress determined by intracellular superoxide over-representation. The deleterious impact of oxidative stress on mitochondria is recognized. Oximetry applied to Pah enu2 MSCs identified mitochondrial stress by increased basal respiration with concurrently reduced maximal respiration and respiratory reserve. Proton leak secondary to mitochondrial complex 1 dysfunction is a recognized superoxide source. Respirometry applied to Pah enu2 MSCs, in the course of osteoblast differentiation, identified a partial complex 1 deficit. Pah enu2 MSCs treated with the antioxidant resveratrol demonstrated increased mitochondrial mass by MitoTracker green labeling. In hyperphenylalaninemic conditions, resveratrol increased in situ alkaline phosphatase activity suggesting partial recovery of Pah enu2 MSCs osteoblast differentiation. Up-regulation of oxidative energy production is required for osteoblasts differentiation. Our data suggests impaired Pah enu2 MSC developmental competence involves an energy deficit. We posit energy support and oxidative stress reduction will enable Pah enu2 MSC differentiation in the osteoblast lineage to subsequently increase bone density., (Copyright © 2021. Published by Elsevier Inc.)

Inborn errors of mitochondrial fatty acid oxidation (FAO) comprise the most common group of disorders identified through expanded newborn screening mandated in all 50 states in the United States, affecting 1:10,000 newborns. While some of the morbidity in FAO disorders (FAODs) can be reduced if identified through screening, a significant gap remains between the ability to diagnose these disorders and the ability to treat them. At least 25 enzymes and specific transport proteins are responsible for carrying out the steps of mitochondrial fatty acid metabolism, with at least 22 associated genetic disorders. Common symptoms in long chain FAODs (LC-FAODs) in the first week of life include cardiac arrhythmias, hypoglycemia, and sudden death. Symptoms later in infancy and early childhood may relate to the liver or cardiac or skeletal muscle dysfunction, and include fasting or stress-related hypoketotic hypoglycemia or Reye-like syndrome, conduction abnormalities, arrhythmias, dilated or hypertrophic cardiomyopathy, and muscle weakness or fasting- and exercise-induced rhabdomyolysis. In adolescent or adult-onset disease, muscular symptoms, including rhabdomyolysis, and cardiomyopathy predominate. Unfortunately, progress in developing better therapeutic strategies has been slow and incremental. Supplementation with medium chain triglyceride (MCT; most often a mixture of C8-12 fatty acids containing triglycerides) oil provides a fat source that can be utilized by patients with long chain defects, but does not eliminate symptoms. Three mitochondrial metabolic pathways are required for efficient energy production in eukaryotic cells: oxidative phosphorylation (OXPHOS), FAO, and the tricarboxylic (TCA) cycle, also called the Krebs cycle. Cell and mouse studies have identified a deficiency in TCA cycle intermediates in LC-FAODs, thought to be due to a depletion of odd chain carbon compounds in patients treated with a predominantly MCT fat source. Triheptanoin (triheptanoyl glycerol; UX007, Ultragenyx Pharmaceuticals) is chemically composed of three heptanoate (seven carbon fatty acid) molecules linked to glycerol through ester bonds that has the potential to replete TCA cycle intermediates through production of both acetyl-CoA and propionyl-CoA through medium chain FAO. Compassionate use, retrospective, and recently completed prospective studies demonstrate significant reduction of hypoglycemic events and improved cardiac function in LC-FAOD patients, but a less dramatic effect on muscle symptoms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sklirou, Alodaib, Dobrowolski, Mohsen and Vockley.)

Metabolic reprogramming provides transformed cells with proliferative and/or survival advantages. Capitalizing on this therapeutically, however, has been only moderately successful because of the relatively small magnitude of these differences and because cancers may further adapt their metabolism to evade metabolic pathway inhibition. Mice lacking the peroxisomal bifunctional enzyme enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase (Ehhadh) and supplemented with the 12-carbon fatty acid lauric acid (C12) accumulate the toxic metabolite dodecanedioic acid (DDDA), which causes acute hepatocyte necrosis and liver failure. We noted that, in a murine model of pediatric hepatoblastoma (HB) and in primary human HBs, downregulation of Ehhadh occurs in association with the suppression of mitochondrial β- and endosomal/peroxisomal ω-fatty acid oxidation pathways. This suggested that HBs might be more susceptible than normal liver tissue to C12 dietary intervention. Indeed, HB-bearing mice provided with C12- and/or DDDA-supplemented diets survived significantly longer than those on standard diets. In addition, larger tumors developed massive necrosis following short-term DDDA administration. In some HBs, the eventual development of DDDA resistance was associated with 129 transcript differences, ∼90% of which were downregulated, and approximately two-thirds of which correlated with survival in numerous human cancers. These transcripts often encoded extracellular matrix components, suggesting that DDDA resistance arises from reduced Ehhadh uptake. Lower Ehhadh expression was also noted in murine hepatocellular carcinomas and in subsets of certain human cancers, supporting the likely generality of these results. Our results demonstrate the feasibility of C12 or DDDA dietary supplementation that is nontoxic, inexpensive, and likely compatible with more standard chemotherapies., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Medium-chain triglycerides (MCT), containing C 8 -C 12 fatty acids, are used to treat several pediatric disorders and are widely consumed as a nutritional supplement. Here, we investigated the role of the sirtuin deacylase Sirt5 in MCT metabolism by feeding Sirt5 knockout mice (Sirt5KO) high-fat diets containing either C 8 /C 10 fatty acids or coconut oil, which is rich in C 12 , for five weeks. Coconut oil, but not C 8 /C 10 feeding, induced periportal macrovesicular steatosis in Sirt5KO mice. 14 C-C 12 degradation was significantly reduced in Sirt5KO liver. This decrease was localized to the mitochondrial β-oxidation pathway, as Sirt5KO mice exhibited no change in peroxisomal C 12 β-oxidation. Endoplasmic reticulum ω-oxidation, a minor fatty acid degradation pathway known to be stimulated by C 12 accumulation, was increased in Sirt5KO liver. Mice lacking another mitochondrial C 12 oxidation enzyme, long-chain acyl-CoA dehydrogenase (LCAD), also developed periportal macrovesicular steatosis when fed coconut oil, confirming that defective mitochondrial C 12 oxidation is sufficient to induce the steatosis phenotype. Sirt5KO liver exhibited normal LCAD activity but reduced mitochondrial acyl-CoA synthetase activity with C 12 . These studies reveal a role for Sirt5 in regulating the hepatic response to MCT and may shed light into the pathogenesis of periportal steatosis, a hallmark of human pediatric non-alcoholic fatty liver disease.

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.

Methylmalonate semialdehyde dehydrogenase deficiency (MMSDD; MIM 614105) is a rare autosomal recessive defect of valine and pyrimidine catabolism. Four prior MMSDD cases are published. We present a fifth case, along with functional and metabolomic analysis. The patient, born to non-consanguineous parents of East African origin, was admitted at two weeks of age for failure to thrive. She was nondysmorphic, had a normal brain MRI, and showed mild hypotonia. Gastroesophageal reflux occurred with feeding. Urine organic acid assessment identified excess 3-hydroxyisobutyrate and 3-hydroxypropionate, while urine amino acid analysis identified elevated concentrations of β-aminoisobutyrate and β-alanine. Plasma amino acids showed an elevated concentration of β-aminoisobutyrate with undetectable β-alanine. ALDH6A1 gene sequencing identified a homozygous variant of uncertain significance, c.1261C > T (p.Pro421Ser). Management with valine restriction led to reduced concentration of abnormal analytes in blood and urine, improved growth, and reduced gastroesophageal reflux. Western blotting of patient fibroblast extracts demonstrated a large reduction of methylmalonate semialdehyde dehydrogenase (MMSD) protein. Patient cells displayed compromised mitochondrial function with increased superoxide production, reduced oxygen consumption, and reduced ATP production. Metabolomic profiles from patient fibroblasts demonstrated over-representation of fatty acids and fatty acylcarnitines, presumably due to methylmalonate semialdehyde shunting to β-alanine and subsequently to malonyl-CoA with ensuing increase of fatty acid synthesis. Previously reported cases of MMSDD have shown variable clinical presentation. Our case continues the trend as clinical phenotypes diverge from prior cases. Recognition of mitochondrial dysfunction and novel metabolites in this patient provide the opportunity to assess future patients for secondary changes that may influence clinical outcome., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We examined bone formation and turnover in high-density lipoprotein (HDL) receptor, scavenger receptor type I (Scarb1), knockout animals relative to wild-type (WT) controls. Scarb1 -/- animals have elevated serum adrenocorticotropic hormone (ACTH) due to the role of Scarb1 in glucocorticoid production, which might cause increased bone mass. However, this was not observed: Scarb1 -/- mice, with ACTH, over 1000 pg/ml relative to wild-type ACTH ~ 25 pg/ml, bone of the knockout animals was osteopenic relative to the wild type at 16 weeks, including bone volume/total volume and trabecular thickness. Other serum parameters of WT and Scarb1 -/- animals in cortisol or calcium were unaffected, although Scarb1 -/- animals had significantly elevated PTH and decreased phosphate. Osteoblast and osteoclast-related mRNAs extracted from bone were greatly decreased at 8 or 16 weeks. Importantly, in normal ACTH, osteogenic differentiation in vitro from mesenchymal stem cells showed reduced alkaline phosphatase and mineralization. In Scarb1 -/- cells relative to WT, mRNAs for RunX2, alkaline phosphatase, type I collagen, and osteocalcin were reduced 40-90%, all p < 0.01, indicating a role of Scarb1 in osteoblast differentiation independent of ACTH. Additionally, in vitro osteoblast differentiation at variable ACTH in WT cells confirmed ACTH increasing bone differentiation, mineralization, alkaline phosphatase, and osteocalcin mRNA at 0-10 nM ACTH, but reduced bone differentiation at 100-1000 nM ACTH. Overall Scarb1 -/- animals show inhibited bone formation with age. This may be a mixed effect on direct bone formation and of very high ACTH. Further, this work shows that both ACTH concentration and the HDL receptor Scarb1 play important independent roles in osteoblast differentiation.

Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism caused by pathogenic variants in the phenylalanine hydroxylase gene (PAH). The correlation between genotype and phenotype can be complex and sometimes variable but often very useful for categorizing and predicting dietary tolerance and potential outcome. We reviewed medical records for 367 patients diagnosed with PKU or persistent mild hyperphenylalaninemia (MHP) between 1950 and 2015 who had PAH genotyping. In 351 we had the full PAH genotype as well as phenotypic characteristics such as phenylalanine (Phe) concentrations (at newborn screening, confirmation, and highest known), and dietary Phe tolerance. On 716 mutant chromosomes, including 14 in genotypes with only one identified variant, we identified 114 different pathogenic variants. The most frequent, p.R408W, was present in 15.4% of the alleles; other frequent variants were c.1315 + 1G > A (6.1%), p.I65T (5.7%), and p.R261Q (5.7%). Three variants, c.142 T > G (p.L48 V), c.615G > C (p.E205D), and c.1342&#95;1345delCTCC, were novel. We used the phenotypic parameters of variants paired with null alleles (functional hemizygotes) to assign the variants as classic PKU, moderate PKU, mild PKU, MHP-gray zone, or MHP. We also included the phenotype association(s) for all of the full genotypes. In 103 patients, we also could assign sapropterin dihydrochloride responsiveness, which is a synthetic form of the tetrahydrobiopterin (BH 4 ) PAH cofactor. This compilation from a single metabolic center provides further information on PAH variants in the United States and the correlations between genotype and phenotype., (Copyright © 2019. Published by Elsevier Inc.)

Inborn errors of metabolism have traditionally been viewed as the quintessential single gene disorders; defects in one gene leads to loss of activity of one enzyme causing a metabolic imbalance and clinical disease. However, reality has never been quite that simple, and the classic "one gene-one enzyme" paradigm has been upended in many ways. Multiple gene defects can lead to the same biochemical phenotype, often with different clinical symptoms. Additionally, different mutations in the same gene can cause variable phenotypes, often most dramatic when a disease can be identified by pre-symptomatic screening. Moreover, response to therapy is not homogeneous across diseases and specific mutations. Perhaps the biggest deviation from traditional monogenic inheritance is in the setting of synergistic heterozygosity, a multigenic inheritance pattern in which mutations in multiple genes in a metabolic pathway lead to sufficient disruption of flux through the pathway, mimicking a monogenic disorder caused by homozygous defects in one gene in that pathway. In addition, widespread adoption of whole exome and whole genome sequencing in medical genetics has led to the realization that individual patients with apparently hybrid phenotypes can have mutations in more than one gene, leading to a mixed genetic disorder. Each of these situations point to a need for as much precision as possible in diagnosing metabolic disease, and it is likely to become increasingly critical to drive therapy. This article examines examples in traditional monogenic disorders that illustrates these points and define inborn errors of metabolism as complex genetic traits on the leading edge of precision medicine., (Copyright © 2019. Published by Elsevier Inc.)

The ClinGen Inborn Errors of Metabolism Working Group was tasked with creating a comprehensive, standardized knowledge base of genes and variants for metabolic diseases. Phenylalanine hydroxylase (PAH) deficiency was chosen to pilot development of the Working Group's standards and guidelines. A PAH variant curation expert panel (VCEP) was created to facilitate this process. Following ACMG-AMP variant interpretation guidelines, we present the development of these standards in the context of PAH variant curation and interpretation. Existing ACMG-AMP rules were adjusted based on disease (6) or strength (5) or both (2). Disease adjustments include allele frequency thresholds, functional assay thresholds, and phenotype-specific guidelines. Our validation of PAH-specific variant interpretation guidelines is presented using 85 variants. The PAH VCEP interpretations were concordant with existing interpretations in ClinVar for 69 variants (81%). Development of biocurator tools and standards are also described. Using the PAH-specific ACMG-AMP guidelines, 714 PAH variants have been curated and will be submitted to ClinVar. We also discuss strategies and challenges in applying ACMG-AMP guidelines to autosomal recessive metabolic disease, and the curation of variants in these genes., (© 2018 Wiley Periodicals, Inc.)

Osteopenia is observed in some patients affected by phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU). Bone density studies, in diverse PKU patient cohorts, have demonstrated bone disease is neither fully penetrant nor uniform in bone density loss. Biochemical assessment has generated a muddled perspective regarding mechanisms of the PKU bone phenotype where the participation of hyperphenylalaninemia remains unresolved. Osteopenia is realized in the Pah enu2 mouse model of classical PKU; although, characterization is incomplete. We characterized the Pah enu2 bone phenotype and assessed the effect of hyperphenylalaninemia on bone differentiation. Employing Pah enu2 and control animals, cytology, static and dynamic histomorphometry, and biochemistry were applied to further characterize the bone phenotype. These investigations demonstrate Pah enu2 bone density is decreased 33% relative to C57BL/6; bone volume/total volume was similarly decreased; trabecular thickness was unchanged while increased trabecular spacing was observed. Dynamic histomorphometry demonstrated a 25% decrease in mineral apposition. Biochemically, control and PKU animals have similar plasma cortisol, adrenocorticotropic hormone, and 25-hydroxyvitamin D. PKU animals show moderately increased plasma parathyroid hormone while plasma calcium and phosphate are reduced. These data are consistent with a mineralization defect. The effect of hyperphenylalaninemia on bone maturation was assessed in vitro employing bone-derived mesenchymal stem cells (MSCs) and their differentiation into bone. Using standard culture conditions, PAH deficient MSCs differentiate into bone as assessed by in situ alkaline phosphatase activity and mineral staining. However, PAH deficient MSCs cultured in 1200 μM PHE (metric defining classical PKU) show significantly reduced mineralization. These data are the first biological evidence demonstrating a negative impact of hyperphenylalaninemia upon bone maturation. In PAH deficient MSCs, expression of Col1A1 and Rankl are suppressed by hyperphenylalaninemia consistent with reduced bone formation and bone turnover. Osteopenia is intrinsic to PKU pathology in untreated Pah enu2 animals and our data suggests PHE toxicity participates by inhibiting mineralization in the course of MSC bone differentiation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is identified by newborn screening (NBS). The natural history of MCADD includes metabolic decompensation with hypoglycemia, hyperammonemia, seizures, coma, and death. NBS enables expectant management thus severe symptoms are rare in managed patients. We report premature birth of an MCADD affected infant and resultant management challenges. Nutritional support advanced from parenteral nutrition at 24 hours to enteral feeds. A NBS sample was collected day 2, positive results for MCADD was reported day six, and diagnostic tests were performed day seven. Lab results confirmed MCADD; however, representation of pathologic analytes was so extreme that ingestion of medium chain triglycerides was suspected and subsequently confirmed. Diet was adjusted and reflected in moderation of pathologic analytes. This case emphasizes the need for prompt review NBS results in premature infants. Implementing informatic intervention within electronic medical records, when a disorder requiring special nutritional intervention is identified, will protect premature infants in this vulnerable setting. Standard of care management provided premature infants may be contraindicated in the context of a comorbid inborn error of metabolism., Competing Interests: Conflict of interest: the authors declare no potential conflict of interest.

Background & Aims: Hepatocyte transplantation partially corrects genetic disorders and has been associated anecdotally with reversal of acute liver failure. Monitoring for graft function and rejection has been difficult, and has contributed to limited graft survival. Here we aimed to use preparative liver-directed radiation therapy, and continuous monitoring for possible rejection in an attempt to overcome these limitations., Methods: Preparative hepatic irradiation was examined in non-human primates as a strategy to improve engraftment of donor hepatocytes, and was then applied in human subjects. T cell immune monitoring was also examined in human subjects to assess adequacy of immunosuppression., Results: Porcine hepatocyte transplants engrafted and expanded to comprise up to 15% of irradiated segments in immunosuppressed monkeys preconditioned with 10Gy liver-directed irradiation. Two patients with urea cycle deficiencies had early graft loss following hepatocyte transplantation; retrospective immune monitoring suggested the need for additional immunosuppression. Preparative radiation, anti-lymphocyte induction, and frequent immune monitoring were instituted for hepatocyte transplantation in a 27year old female with classical phenylketonuria. Post-transplant liver biopsies demonstrated multiple small clusters of transplanted cells, multiple mitoses, and Ki67 + hepatocytes. Mean peripheral blood phenylalanine (PHE) level fell from pre-transplant levels of 1343±48μM (normal 30-119μM) to 854±25μM (treatment goal ≤360μM) after transplant (36% decrease; p<0.0001), despite transplantation of only half the target number of donor hepatocytes. PHE levels remained below 900μM during supervised follow-up, but graft loss occurred after follow-up became inconsistent., Conclusions: Radiation preconditioning and serial rejection risk assessment may produce better engraftment and long-term survival of transplanted hepatocytes. Hepatocyte xenografts engraft for a period of months in non-human primates and may provide effective therapy for patients with acute liver failure., Lay Summary: Hepatocyte transplantation can potentially be used to treat genetic liver disorders but its application in clinical practice has been impeded by inefficient hepatocyte engraftment and the inability to monitor rejection of transplanted liver cells. In this study, we first show in non-human primates that pretreatment of the host liver with radiation improves the engraftment of transplanted liver cells. We then used this knowledge in a series of clinical hepatocyte transplants in patients with genetic liver disorders to show that radiation pretreatment and rejection risk monitoring are safe and, if optimized, could improve engraftment and long-term survival of transplanted hepatocytes in patients., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Phenylalanine hydroxylase deficient phenylketonuria (PKU) is the paradigm for a treatable inborn error of metabolism where maintaining plasma phenylalanine (Phe) in the therapeutic range relates to improved clinical outcomes. While Phe is the presumed intoxicating analyte causal in neurologic damage, the mechanism(s) of Phe toxicity has remained elusive. Altered DNA methylation is a recognized response associated with exposure to numerous small molecule toxic agents. Paralleling this effect, we hypothesized that chronic Phe over-exposure in the brain would lead to aberrant DNA methylation with secondary influence upon gene regulation that would ultimately contribute to PKU neuropathology. The PAH(enu2) mouse models human PKU with intrinsic hyperphenylalaninemia, abnormal response to Phe challenge, and neurologic deficit. To examine this hypothesis, we assessed DNA methylation patterns in brain tissues using methylated DNA immunoprecipitation and paired end sequencing in adult PAH(enu2) animals maintained under either continuous dietary Phe restriction or chronic hyperphenylalaninemia. Heterozygous PAH(enu2/WT) litter mates served as controls for normal Phe exposure. Extensive repatterning of DNA methylation was observed in brain tissue of hyperphenylalaninemic animals while Phe restricted animals displayed an attenuated pattern of aberrant DNA methylation. Affected gene coding regions displayed aberrant hypermethylation and hypomethylation. Gene body methylation of noncoding RNA genes was observed and among these microRNA genes were prominent. Of particular note, observed only in hyperphenylalaninemic animals, was hypomethylation of miRNA genes within the imprinted Dlk1-Dio3 locus on chromosome 12. Aberrant methylation of microRNA genes influenced their expression which has secondary effects upon the expression of targeted protein coding genes. Differential hypermethylation of gene promoters was exclusive to hyperphenylalaninemic PAH(enu2) animals. Genes with synaptic involvement were targets of promoter hypermethylation that resulted in down-regulation of their expression. Gene dysregulation secondary to abnormal DNA methylation may be contributing to PKU neuropathology. These results suggest drugs that prevent or correct aberrant DNA methylation may offer a novel therapeutic option to management of neurological symptoms in PKU patients., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The Old Order Amish populations in the US are one of the Plain People groups and are descendants of the Swiss Anabaptist immigrants who came to North America in the early eighteenth century. They live in numerous small endogamous demes that have resulted in reduced genetic diversity along with a high prevalence of specific genetic disorders, many of them autosomal recessive. Mitochondrial respiratory chain deficiencies arising from mitochondrial or nuclear DNA mutations have not previously been reported in the Plain populations. Here we present four different Amish families with mitochondrial respiratory chain disorders. Mutations in two mitochondrial encoded genes leading to mitochondrial respiratory chain disorder were identified in two patients. In the first case, MELAS syndrome caused by a mitochondrial DNA (mtDNA) mutation (m.3243A>G) was identified in an extended Amish pedigree following a presentation of metabolic strokes in the proband. Characterization of the extended family of the proband by a high resolution melting assay identified the same mutation in many previously undiagnosed family members with a wide range of clinical symptoms. A MELAS/Leigh syndrome phenotype caused by a mtDNA mutation [m.13513G>A; p.Asp393Asn] in the ND5 gene encoding the ND5 subunit of respiratory chain complex I was identified in a patient in a second family. Mutations in two nuclear encoded genes leading to mitochondrial respiratory chain disorder were also identified in two patients. One patient presented with Leigh syndrome and had a homozygous deletion in the NDUFAF2 gene, while the second patient had a homozygous mutation in the POLG gene, [c.1399G>A; p.Ala467Thr]. Our findings identify mitochondrial respiratory chain deficiency as a cause of disease in the Old Order Amish that must be considered in the context of otherwise unexplained systemic disease, especially if neuromuscular symptoms are present., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Costello syndrome (CS) may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35&#95;36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35&#95;36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE) and creation of an Exonic Splicing Silencer (ESS). We show that this vulnerability of HRAS exon 2 is caused by a weak 3' splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO) that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping.

Phenylalanine hydroxylase (PAH) deficiency is an inherited metabolic disorder requiring life-long restriction of dietary protein and phenylalanine-free medical food. Low bone mineral density (BMD) is reported, but factors associated with BMD Z-score (standard deviations from normal) are unknown. We examined associations between clinical and dietary parameters and total BMD Z-score in PAH deficiency patients, and developed models to predict Z-score. Data collected from patients >4 years of age (n = 88; mean age = 18.8 y; 61 % female) included demographic, clinical, laboratory, and dietary intakes. Adjusted Spearman's correlation coefficients were calculated between parameters and TBMD Z-score, measured by dual energy x-ray absorptiometry (DXA). Parameters approaching significance (p-value < 0.10) were candidate predictors for four linear regression models predicting TBMD Z-score. To validate, model-predicted Z-scores were compared to DXA Z-scores. Mean TBMD Z-score was -0.326; 18 (20.4 %) had Z-score < -1. Z-scores were positively correlated with dietary vitamin D, calcium, and medical food intake and compliance with prescription, and negatively with dietary carbohydrate, sugar, caffeine intake, glycemic load, and prescribed medical food (grams protein/day; p-value < 0.05). The best model included medical food compliance, medical food intake, caffeine intake, and bone-specific alkaline phosphatase (r-square = 0.364). This model predicted Z-score category [normal or low (<-1)] with sensitivity = 66.7 %, likelihood ratio = 14.7, and AUC = 0.83 compared to DXA Z-score. No subjects had low BMD for chronological age (Z-score ≤ -2). Compliance with medical food prescription was the strongest predictor of TBMD Z-score. One model, if validated in a separate sample of patients with more cases of low BMD, showed potential to estimate TBMD Z-score using routine clinical patient parameters.

While phenylalanine (PHE) is the toxic insult in phenylketonuria (PKU), mechanisms underlying PHE toxicity remain ill-defined. Altered DNA methylation in response to toxic exposures is well-recognized. DNA methylation patterns were assessed in blood and brain from PKU patients to determine if PHE toxicity impacts methylation. Methylome assessment, utilizing methylated DNA immunoprecipitation and paired-end sequencing, was performed in DNA obtained from brain tissue of classical PKU patients, leukocytes from poorly controlled PKU patients, leukocytes from well controlled PKU patients, and appropriate control tissues. In PKU brain tissue, expression analysis determined the impact of methylation on gene function. Differential methylation was observed in brain tissue of PKU patients and expression studies identified downstream impact on gene expression. Altered patterns of methylation were observed in leukocytes of well controlled and poorly controlled patients with more extensive methylation in patients with high PHE exposure. Differential methylation of noncoding RNA genes was extensive in patients with high PHE exposure but minimal in well controlled patients. Methylome repatterning leading to altered gene expression was present in brain tissue of PKU patients, suggesting a role in neuropathology. Aberrant methylation is observed in leukocytes of PKU patients and is influenced by PHE exposure. DNA methylation may provide a biomarker relating to historic PHE exposure., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Spinal muscular atrophy (SMA) is a motor neuron disorder caused by the absence of a functional survival of motor neuron 1, telomeric (SMN1) gene. Type I SMA, a lethal disease of infancy, accounts for the majority of cases. Newborn blood spot screening (NBS) to detect severe combined immunodeficiency (SCID) has been implemented in public health laboratories in the last 5 years. SCID detection is based on real-time PCR assays to measure T-cell receptor excision circles (TREC), a byproduct of T-cell development. We modified a multiplexed real-time PCR TREC assay to simultaneously determine the presence or absence of the SMN1 gene from a dried blood spot (DBS) punch in a single reaction well., Method: An SMN1 assay using a locked nucleic acid probe was initially developed with cell culture and umbilical cord blood (UCB) DNA extracts, and then integrated into the TREC assay. DBS punches were placed in 96-well arrays, washed, and amplified directly using reagents specific for TREC, a reference gene [ribonuclease P/MRP 30kDa subunit (RPP30)], and the SMN1 gene. The assay was tested on DBS made from UCB units and from peripheral blood samples of SMA-affected individuals and their family members., Results: DBS made from SMA-affected individuals showed no SMN1-specific amplification, whereas DBS made from all unaffected carriers and UCB showed SMN1 amplification above a well-defined threshold. TREC and RPP30 content in all DBS were within the age-adjusted expected range., Conclusions: SMA caused by the absence of SMN1 can be detected from the same DBS punch used to screen newborns for SCID., (© 2014 American Association for Clinical Chemistry.)

Maternal PKU Syndrome (MPKU) is an embryopathy resulting from in utero phenylalanine (PHE) toxicity secondary to maternal phenylalanine hydroxylase deficient phenylketonuria (PKU). Clinical phenotypes in MPKU include mental retardation, microcephaly, in utero growth restriction, and congenital heart defects. Numerous in utero toxic exposures alter DNA methylation in the fetus. The PAH(enu2) mouse is a model of classical PKU while offspring born of hyperphenylalaninemic dams model MPKU. We investigated offspring of PAH(enu2) dams to determine if altered patterns of DNA methylation occurred in response to in utero PHE exposure. As neurologic deficit is the most prominent MPKU phenotype, methylome patterns were assessed in brain tissue using methylated DNA immunoprecipitation and paired-end sequencing. Brain tissues were assessed in E18.5-19 fetuses of PHE unrestricted PAH(enu2) dams, PHE restricted PAH(enu2) dams, and heterozygous(wt/enu2) control dams. Extensive methylome repatterning was observed in offspring of hyperphenylalaninemic dams while the offspring of PHE restricted dams displayed attenuated methylome repatterning. Methylation within coding regions was dominated by noncoding RNA genes. Differential methylation of promoters targeted protein coding genes. To assess the impact of methylome repatterning on gene expression, brain tissue in experimental and control animals were queried with microarrays assessing expression of microRNAs and protein coding genes. Altered expression of methylome-modified microRNAs and protein coding genes was extensive in offspring of hyperphenylalaninemic dams while minimal changes were observed in offspring of PHE restricted dams. Several genes displaying significantly reduced expression have roles in neurological function or genetic disease with neurological phenotypes. These data indicate in utero PHE toxicity alters DNA methylation in the brain which has downstream impact upon gene expression. Altered gene expression may contribute to pathophysiology of neurologic presentation in MPKU., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Introduction: Disease inclusion in the newborn screening (NBS) panel should consider the opinions of those most affected by the outcome of screening. We assessed the level and factors that affect parent attitudes regarding NBS panel inclusion of Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and spinal muscular atrophy (SMA)., Methods: The attitudes toward NBS for DMD, BMD, and SMA were surveyed and compared for 2 categories of parents, those with children affected with DMD, BMD, or SMA and expectant parents unselected for known family medical history., Results: The level of support for NBS for DMD, BMD, and SMA was 95.9% among parents of children with DMD, BMD, or SMA and 92.6% among expectant parents., Conclusions: There was strong support for NBS for DMD, BMD, and SMA in both groups of parents. Given advances in diagnostics and promising therapeutic approaches, discussion of inclusion in NBS should continue., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: A need exists to expand the characterization of tetrahydrobiopterin (BH(4)) responsiveness in patients with phenylketonuria (PKU), beyond simply evaluating change in blood phenylalanine concentrations. The clinical interpretation of BH(4) responsiveness should be evaluated within the context of phenylalanine hydroxylase (PAH) genotype., Aim: This investigation seeks to use a modified version of a previously developed PAH genotype severity tool, the assigned value (AV) sum, to assess the molecular basis of responsiveness in a clinical cohort and to explore the tool's ability to differentiate BH(4) responsive groups., Methods: BH(4) response was previously clinically classified in 58 patients with PKU, with three response groups emerging: definitive responders, provisional responders, and non-responders. Provisional responders represented a clinically ambiguous group, with an initial decrease in plasma phenylalanine concentrations, but limited ability to improve dietary phenylalanine tolerance. In this retrospective analysis, mutations in the PAH gene were identified in each patient. PAH genotype was characterized through the AV sum approach, in which each mutation is given an AV of 1, 2, 4, or 8; the sum of both mutations' AV corresponds to genotype severity, with a lower number representing a more severe phenotype. An AV sum cutoff of 2 (indicative of the most severe genotypes) was used to dichotomize patients and predict BH(4) responsiveness. Provisional responders were classified with the definitive responders then the non-responders to see with which group they best aligned., Results: In 17/19 definitive responders, at least one mutation was mild or moderate in severity (AV sum>2). In contrast, 7/9 provisional responders carried two severe or null mutations (AV sum=2), suggesting little molecular basis for responsiveness. Non-responders represent a heterogeneous group with 15/25 patients carrying two severe mutations (AV sum=2), 5/25 patients carrying one moderate or mild mutation in combination with a severe or null mutation (AV sum>2), and the remaining five patients carrying an uncharacterized mutation in combination with a severe mutation. Predictive sensitivity of the AV sum was maximized (89.5% vs. 67.9%) with limited detriment to specificity (79.4% vs. 80.0%), by classifying provisional responders with the non-responders rather than with the definitive responders., Conclusions: In our clinical cohort, the AV sum tool was able to identify definitive responders with a high degree of sensitivity. As demonstrated by both the provisional responder group and the substantial number of non-responders with AV sums>2, a potential exists for misclassification when BH(4) response is determined by relying solely on change in plasma phenylalanine concentrations. PAH genotype should be incorporated in the clinical evaluation of BH(4) responsiveness., (Copyright © 2012. Published by Elsevier Inc.)

In about 20-30% of phenylketonuria (PKU) patients, phenylalanine (Phe) levels can be controlled by cofactor 6R-tetrahydrobiopterin (BH(4)) administration. The phenylalanine hydroxylase (PAH) genotype has a predictive value concerning BH(4)-response and therefore a correct assessment of the mutation molecular pathology is important. Mutations that disturb the splicing of exons (e.g. interplay between splice site strength and regulatory sequences like exon splicing enhancers (ESEs)/exon splicing silencers (ESSs)) may cause different severity of PKU. In this study, we identified PAH exon 11 as a vulnerable exon and used patient derived lymphoblast cell lines and PAH minigenes to study the molecular defect that impacted pre-mRNA processing. We showed that the c.1144T>C and c.1066-3C>T mutations cause exon 11 skipping, while the c.1139C>T mutation is neutral or slightly beneficial. The c.1144T>C mutation resides in a putative splicing enhancer motif and binding by splicing factors SF2/ASF, SRp20 and SRp40 is disturbed. Additional mutations in potential splicing factor binding sites contributed to elucidate the pathogenesis of mutations in PAH exon 11. We suggest that PAH exon 11 is vulnerable due to a weak 3' splice site and that this makes exon 11 inclusion dependent on an ESE spanning position c.1144. Importantly, this implies that other mutations in exon 11 may affect splicing, since splicing is often determined by a fine balance between several positive and negative splicing regulatory elements distributed throughout the exon. Finally, we identified a pseudoexon in intron 11, which would have pathogenic consequences if activated by mutations or improved splicing conditions. Exonic mutations that disrupt splicing are unlikely to facilitate response to BH(4) and may lead to inconsistent genotype-phenotype correlations. Therefore, recognizing such mutations enhances our ability to predict the BH(4)-response., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: The management options for the autosomal recessive neurodegenerative disorder spinal muscular atrophy (SMA) are evolving; however, their efficacy may require presymptom diagnosis and continuous treatment. To identify presymptomatic SMA patients, we created a DNA-based newborn screening assay to identify the homozygous deletions of the SMN1 (survival of motor neuron 1, telomeric) gene observed in 95%-98% of affected patients., Methods: We developed primers that amplify a 52-bp PCR product from homologous regions in the SMN1 and SMN2 (survival of motor neuron 2, centromeric) genes that flank a divergent site at site c.840. Post-PCR high-resolution melt profiling assessed the amplification product, and we used a unique means of melt calibration to normalize profiles. Samples that we had previously characterized for the numbers of SMN1 and SMN2 copies established genotypes associated with particular profiles. The system was evaluated with approximately 1000 purified DNA samples, 100 self-created dried blood spots, and >1200 dried blood spots from newborn screening tests., Results: Homozygous deletion of SMN1 exon 7 produced a distinctive melt profile that identified SMA patients. Samples with different numbers of SMN1 and SMN2 copies were resolved by their profiles. All samples with homozygous deletions were unambiguously recognized, and no normal sample was misidentified as a positive., Conclusions: This assay has characteristics suitable for population-based screening. A reliable screening test will facilitate the identification of an SMA-affected cohort to receive early intervention to maximize the benefit from treatment. A prospective screening trial will allow the efficacy of treatment options to be assessed, which may justify the inclusion of SMA as a target for population screening.

Background: The prevalence of phenylalanine hydroxylase (PAH)-deficient phenylketonuria (PKU) in Turkey is high (1 in 6500 births), but data concerning the genotype distribution and impact of the genotype on tetrahydrobiopterin (BH(4)) therapy are scarce., Objective: To characterize the phenotypic and genotypic variability in the Turkish PKU population and to correlate it with physiological response to BH(4) challenge., Methods: We genotyped 588 hyperphenylalaninemic patients and performed a BH(4) loading test (20mg/kg bw) in 462 patients. Residual PAH activity of mutant proteins was calculated from available in vitro expression data. Data were tabulated in the BIOPKU database (www.biopku.org)., Results: Eighty-eight mutations were observed, the most common missense mutations being the splice variant c.1066-11G>A (24.6%). Twenty novel mutations were detected (11 missense, 4 splice-site, and 5 deletion/insertions). Two mutations were observed in 540/588 patients (91.8%) but in 9 patients atypical genotypes with >2 mutations were found (8 with p.R155H in cis with another variant) and in 19 patients mutations were found in BH(4)-metabolizing genes. The most common genotype was c.1066-11G>A/c.1066-11G>A (15.5%). Approximately 22% of patients responded to BH(4) challenge. A substantial in vitro residual activity (average >25% of the wild-type enzyme) was associated with response to BH(4). In homozygous genotypes (n=206), both severity of the phenotype (r=0.83) and residual PAH activity (r=0.85) correlate with BH(4) responsiveness., Conclusion: Together with the BH(4) challenge, these data enable the genotype-based classification of BH(4) responsiveness and document importance of residual PAH activity. This first report of a large-scale genotype assessment in a population of Turkish PKU patients also documents a high prevalence (47%) of the severe classic phenotype., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The ideal clinical diagnostic system should deliver rapid, sensitive, specific and reproducible results while minimizing the requirements for specialized laboratory facilities and skilled technicians. We describe an integrated diagnostic platform, the "FilmArray", which fully automates the detection and identification of multiple organisms from a single sample in about one hour. An unprocessed biologic/clinical sample is subjected to nucleic acid purification, reverse transcription, a high-order nested multiplex polymerase chain reaction and amplicon melt curve analysis. Biochemical reactions are enclosed in a disposable pouch, minimizing the PCR contamination risk. FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time. These features make the system well-suited for molecular detection of infectious agents. Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens. Initial testing of the system using both cultured organisms and clinical nasal aspirates obtained from children demonstrated an analytical and clinical sensitivity and specificity comparable to existing diagnostic platforms. We demonstrate that automated identification of pathogens from their corresponding target amplicon(s) can be accomplished by analysis of the DNA melting curve of the amplicon.

PKU is caused by mutations in PAH. A c.30C>G synonymous variation in exon 1, previously reported as neutral, was observed in two patients. The variation creates a GGG triplet, which is part of several exonic splicing silencer (ESS) motifs. Because the 5'-splice site of PAH exon 1 is intrinsically weak and therefore could be responsive to a new flanking ESS, we hypothesized that c.30C>G could cause aberrant mRNA splicing. We demonstrate that c.30C>G causes aberrant mRNA splicing in two different reporter minigenes, and that this is abolished if a preexisting flanking GGG triplet is disrupted. GGG triplets are part of the consensus motif bound by splicing-inhibitory hnRNPH proteins and we observed a dramatic increase in hnRNPH binding to c.30C>G PAH RNA. We conclude that c.30C>G creates a hnRNPH-binding ESS, which can disrupt mRNA splicing. A disease-causing mutation in HEXB, which has previously been associated with exon skipping in patients also creates a GGG triplet. We show that the mutant HEXB motif causes exon skipping of a reporter minigene and that this is also influenced by a flanking GGG triplet. We suggest that aberrant splicing caused by creation/abolishment of GGG triplets located together with a preexisting flanking GGG triplet, may be an underreported cause of human disease. It is important to recognize that exonic sequence changes may disrupt mRNA splicing. This is particularly important in PAH, since PKU patients harboring such mutations are unlikely to respond to therapy with 6R-tetrahydrobiopterin (BH(4)), despite the fact that the genetic code indicates otherwise., (Copyright 2010 Elsevier Inc. All rights reserved.)