Recapitulation of Skewed X-Inactivation in Female Ornithine Transcarbamylase-Deficient Primary Human Hepatocytes in the FRG Mouse: A Novel System for Developing Epigenetic Therapies.

Realization of the immense therapeutic potential of epigenetic editing requires development of clinically predictive model systems that faithfully recapitulate relevant aspects of the target disease pathophysiology. In female patients with ornithine transcarbamylase (OTC) deficiency, an X-linked condition, skewed inactivation of the X chromosome carrying the wild-type OTC allele is associated with increased disease severity. The majority of affected female patients can be managed medically, but a proportion require liver transplantation. With rapid development of epigenetic editing technology, reactivation of silenced wild-type OTC alleles is becoming an increasingly plausible therapeutic approach. Toward this end, privileged access to explanted diseased livers from two affected female infants provided the opportunity to explore whether engraftment and expansion of dissociated patient-derived hepatocytes in the FRG mouse might produce a relevant model for evaluation of epigenetic interventions. Hepatocytes from both infants were successfully used to generate chimeric mouse-human livers, in which clusters of primary human hepatocytes were either OTC positive or negative by immunohistochemistry (IHC), consistent with clonal expansion from individual hepatocytes in which the mutant or wild-type OTC allele was inactivated, respectively. Enumeration of the proportion of OTC-positive or -negative human hepatocyte clusters was consistent with dramatic skewing in one infant and minimal to modest skewing in the other. Importantly, IHC and fluorescence-activated cell sorting analysis of intact and dissociated liver samples from both infants showed qualitatively similar patterns, confirming that the chimeric mouse-human liver model recapitulated the native state in each infant. Also of importance was the induction of a treatable metabolic phenotype, orotic aciduria, in mice, which correlated with the presence of clonally expanded OTC-negative primary human hepatocytes. We are currently using this unique model to explore CRISPR-dCas9-based epigenetic targeting strategies in combination with efficient adeno-associated virus (AAV) gene delivery to reactivate the silenced functional OTC gene on the inactive X chromosome.

Developmental delay and non-phenylketonuria (PKU) hyperphenylalaninemia in DNAJC12 deficiency: Case and approach.

BACKGROUND: Hyperphenylalaninemia is a biomarker for several monogenic neurotransmitter disorders where the body cannot metabolise phenylalanine to tyrosine. Biallelic pathogenic variants in DNAJC12, co-chaperone of phenylalanine, tyrosine, and tryptophan hydroxylases, leads to hyperphenylalaninemia and biogenic amines deficiency. METHODS AND RESULTS: A male firstborn to non-consanguineous Sudanese parents had hyperphenylalaninemia 247 µmol/L [reference interval (RI) < 200 µmol/L] at newborn screening. Dried blood spot dihydropteridine reductase (DHPR) assay and urine pterins were normal. He had severe developmental delay and autism spectrum disorder without a notable movement disorder. A low phenylalanine diet was introduced at two years without any clinical improvements. Cerebrospinal fluid (CSF) neurotransmitters at five years demonstrated low homovanillic acid (HVA) 0.259 µmol/L (reference interval (RI) 0.345-0.716) and 5-hydroxyindoleaetic acid (5HIAA) levels 0.024 µmol/L (reference interval (RI) 0.100-0.245). Targeted neurotransmitter gene panel analysis identified a homozygous c.78 + 1del variant in DNAJC12. At six years, he was commenced on 5-hydroxytryptophan 20 mg daily, and his protein-restricted diet was liberalised, with continued good control of phenylalanine levels. Sapropterin dihydrochloride 7.2 mg/kg/day was added the following year with no observable clinical benefits. He remains globally delayed with severe autistic traits. CONCLUSIONS: Urine, CSF neurotransmitter studies, and genetic testing will differentiate between phenylketonuria, tetrahydrobiopterin or DNAJC12 deficiency, with the latter characterised by a clinical spectrum ranging from mild autistic features or hyperactivity to severe intellectual disability, dystonia, and movement disorder, normal DHPR, reduced CSF HIAA and HVA. DNAJC12 deficiency should be considered early in the differential workup of hyperphenylalaninemia identified from newborn screening, with its genotyping performed once deficiencies of phenylalanine hydroxylase (PAH) and tetrahydrobiopterin (BH4) have been biochemically or genetically excluded.

Connective tissue presentation in two families expands the phenotypic spectrum of PYROXD1 disorders.

Recessive variants in the oxidoreductase PYROXD1 are reported to cause a myopathy in 22 affected individuals from 15 families. Here, we describe two female probands from unrelated families presenting with features of a congenital connective tissue disorder including osteopenia, blue sclera, soft skin, joint hypermobility and neuromuscular junction dysfunction in addition to known features of PYROXD1 myopathy including respiratory difficulties, weakness, hypotonia and oromotor dysfunction. Proband AII:1 is compound heterozygous for the recurrent PYROXD1 variant Chr12(GRCh38):g.21452130A>G;NM_024854.5:c.464A>G;p.(N155S) and Chr12(GRCh38):g.21462019_21462022del;NM_024854.5:c.892_895del;p.(V298Mfs*4) and proband BII:1 is compound heterozygous for Chr12(GRCh38):g.21468739-21468741del;NM_024854.5:c.1488_1490del;p.(E496del) and Chr12(GRCh38):g.21467619del;NM_024854.5:c.1254+1del. RNA studies demonstrate c.892_895del;p.(V298Mfs*4) is targeted by nonsense mediated decay and c.1254+1delG elicits in-frame skipping of exon-11. Western blot from cultured fibroblasts shows reduced PYROXD1 protein levels in both probands. Testing urine from BII:1 and six individuals with PYROXD1 myopathy showed elevated levels of deoxypyridinoline, a mature collagen crosslink, correlating with PYROXD1-disorder severity. Urine and serum amino acid testing of the same individuals revealed no reportable changes. In contrast to PYROXD1 knock-out, we find no evidence for disrupted tRNA ligase activity, as measured via XBP1 splicing, in fibroblasts expressing PYROXD1 variants. In summary, we expand the clinical spectrum of PYROXD1-related disorders to include an overlapping connective tissue and myopathy presentation, identify three novel, pathogenic PYROXD1 variants, and provide preliminary evidence that elevated urine DPD crosslinks may provide a clinical biomarker for PYROXD1 disorders. Our results advocate consideration of PYROXD1 variants in the differential diagnosis for undiagnosed individuals presenting with a connective tissue disorder and myopathy.

Treatment of HMG-CoA Lyase Deficiency-Longitudinal Data on Clinical and Nutritional Management of 10 Australian Cases.

3-Hydroxy-3-Methylglutaryl-CoA Lyase (HMGCL) deficiency can be a very severe disorder that typically presents with acute metabolic decompensation with features of hypoketotic hypoglycemia, hyperammonemia, and metabolic acidosis. A retrospective chart and literature review of Australian patients over their lifespan, incorporating acute and long-term dietary management, was performed. Data from 10 patients contributed to this study. The index case of this disorder was lost to follow-up, but there is 100% survival in the remainder of the cases despite several having experienced life-threatening episodes. In the acute setting, five of nine patients have used 900 mg/kg/day of sodium D,L 3-hydroxybutyrate in combination with intravenous dextrose-containing fluids (delivering glucose above estimated basal utilization requirements). All patients have been on long-term protein restriction, and those diagnosed more recently have had additional fat restriction. Most patients take L-carnitine. Three children and none of the adults take nocturnal uncooked cornstarch. Of the cohort, there were two patients that presented atypically-one with fulminant liver failure and the other with isolated developmental delay. Dietary management in patients with HMGCL deficiency is well tolerated, and rapid institution of acute supportive metabolic treatment is imperative to optimizing survival and improve outcomes in this disorder.

A narrative review of metabolomics in the era of "-omics": integration into clinical practice for inborn errors of metabolism.

Background and Objective: Traditional targeted metabolomic investigations identify a pre-defined list of analytes in samples and have been widely used for decades in the diagnosis and monitoring of inborn errors of metabolism (IEMs). Recent technological advances have resulted in the development and maturation of untargeted metabolomics: a holistic, unbiased, analytical approach to detecting metabolic disturbances in human disease. We aim to provide a summary of untargeted metabolomics [focusing on tandem mass spectrometry (MS-MS)] and its application in the field of IEMs. Methods: Data for this review was identified through a literature search using PubMed, Google Scholar, and personal repositories of articles collected by the authors. Findings are presented within several sections describing the metabolome, the current use of targeted metabolomics in the diagnostic pathway of patients with IEMs, the more recent integration of untargeted metabolomics into clinical care, and the limitations of this newly employed analytical technique. Key Content and Findings: Untargeted metabolomic investigations are increasingly utilized in screening for rare disorders, improving understanding of cellular and subcellular physiology, discovering novel biomarkers, monitoring therapy, and functionally validating genomic variants. Although the untargeted metabolomic approach has some limitations, this "next generation metabolic screening" platform is becoming increasingly affordable and accessible. Conclusions: When used in conjunction with genomics and the other promising "-omic" technologies, untargeted metabolomics has the potential to revolutionize the diagnostics of IEMs (and other rare disorders), improving both clinical and health economic outcomes.

GLRX5-associated [Fe-S] cluster biogenesis disorder: further characterisation of the neurological phenotype and long-term outcome.

BACKGROUND: Identification and characterisation of monogenic causes of complex neurological phenotypes are important for genetic counselling and prognostication. Bi-allelic pathogenic variants in the gene encoding GLRX5, a protein involved in the early steps of Fe-S cluster biogenesis, are rare and cause two distinct phenotypes: isolated sideroblastic anemia and a neurological phenotype with variant non-ketotic hyperglycinemia. In this study, we analysed the evolution of clinical and MRI findings and long-term outcome of patients with GLRX5 mutations. METHODS: Four patients from three Australian families of Lebanese descent were identified. All patients presented in childhood and were followed up into adult life through multiple clinical assessments. All were prescribed sodium benzoate. RESULTS: All patients (all females, age range 18-56 years) showed a complex neurological phenotype characterised by varying combinations of spastic paraparesis, length-dependent motor/sensory-motor axonal polyneuropathy, and psychiatric disturbances with variable intellectual disability. All had non-ketotic hyperglycinemia and a homozygous pathogenic c.151_153delAAG (p.K51del) change in GLRX5. Motor disability gradually progressed reaching moderate disability during adolescence and moderately severe disability during adult life. The major MRI finding was the upper cervical spinal cord signal changes with contrast enhancement noted in all and additional leukoencephalopathy in one. On follow up MRI, the white matter lesions diminished on a subsequent scan and then remained static over time. The spinal cord showed gliotic changes. Two patients have previously demonstrated low pyruvate dehydrogenase complex deficiency but none had plasma lactate elevation, nor biochemical evidence of branch-chain keto-dehydrogenase deficiency. Glycine levels reduced in patients that tolerated sodium benzoate, possibly stabilising clinical manifestations. CONCLUSIONS: This report demonstrates that the p.K51del GLRX5 variant causes a distinct and predictable neurological phenotype. The clinical assessments spanning from childhood to adult life enable physicians to infer the natural history of GLRX5 related neurological disorder. There may be widespread metabolic consequences, and optimal management is unknown.

Challenges in Diagnosing Intermediate Maple Syrup Urine Disease by Newborn Screening and Functional Validation of Genomic Results Imperative for Reproductive Family Planning.

Maple syrup urine disease is caused by a deficiency of branched-chain alpha-ketoacid dehydrogenase, responsible for degradation of leucine, isoleucine, and valine. Biallelic pathogenic variants in BCKDHA, BCKDHB, or DBT genes result in enzyme deficiency. We report the case of a female infant who presented with mild gross motor delay at 4 months, and seizures with hypoglycaemia at 5 months. Newborn screening returned total leucine/isoleucine at the 99.5th centile of the population; however, as second-tier testing reported minimal alloisoleucine, the results were considered inconsistent with MSUD. Plasma amino acid and urine organic acid analyses at 5 months were, however, consistent with a diagnosis of MSUD. A brain MRI showed bilateral symmetrical T2 hyperintense signal abnormalities involving white matter, globus pallidus, thalamus, brainstem, and dentate nuclei with restricted diffusion. A repeat MRI 10 months post-dietary-intervention showed the resolution of these changes and progression in myelination. Her clinical phenotype, including protein tolerance, correlated with intermediate MSUD. Molecular analysis of all three genes identified two variants of uncertain significance, c.434-15_434-4del and c.365A>G (p. Tyr122Cys) in the DBT gene. The rate of leucine decarboxylation in fibroblasts was reduced, but not to the extent observed in classical MSUD patients, supporting an intermediate form of MSUD. Previously reported mRNA splicing studies supported a deleterious effect of the c.434-15_434-4del variant. This functional evidence and confirmation that the variants were in trans, permitted their reclassification as pathogenic and likely pathogenic, respectively, facilitating subsequent prenatal testing. This report highlights the challenges in identifying intermediate MSUD by newborn screening, reinforcing the importance of functional studies to confirm variant pathogenicity in this era of molecular diagnostics.

New insights into carnitine-acylcarnitine translocase deficiency from 23 cases: Management challenges and potential therapeutic approaches.

Carnitine acyl-carnitine translocase deficiency (CACTD) is a rare autosomal recessive disorder of mitochondrial long-chain fatty-acid transport. Most patients present in the first 2 days of life, with hypoketotic hypoglycaemia, hyperammonaemia, cardiomyopathy or arrhythmia, hepatomegaly and elevated liver enzymes. Multi-centre international retrospective chart review of clinical presentation, biochemistry, treatment modalities including diet, subsequent complications, and mode of death of all patients. Twenty-three patients from nine tertiary metabolic units were identified. Seven attenuated patients of Pakistani heritage, six of these homozygous c.82G>T, had later onset manifestations and long-term survival without chronic hyperammonemia. Of the 16 classical cases, 15 had cardiac involvement at presentation comprising cardiac arrhythmias (9/15), cardiac arrest (7/15), and cardiac hypertrophy (9/15). Where recorded, ammonia levels were elevated in all but one severe case (13/14 measured) and 14/16 had hypoglycaemia. Nine classical patients survived longer-term-most with feeding difficulties and cognitive delay. Hyperammonaemia appears refractory to ammonia scavenger treatment and carglumic acid, but responds well to high glucose delivery during acute metabolic crises. High-energy intake seems necessary to prevent decompensation. Anaplerosis utilising therapeutic d,l-3-hydroxybutyrate, Triheptanoin and increased protein intake, appeared to improve chronic hyperammonemia and metabolic stability where trialled in individual cases. CACTD is a rare disorder of fatty acid oxidation with a preponderance to severe cardiac dysfunction. Long-term survival is possible in classical early-onset cases with long-chain fat restriction, judicious use of glucose infusions, and medium chain triglyceride supplementation. Adjunctive therapies supporting anaplerosis may improve longer-term outcomes.

The use of sodium DL-3-Hydroxybutyrate in severe acute neuro-metabolic compromise in patients with inherited ketone body synthetic disorders.

BACKGROUND: Ketone bodies form a vital energy source for end organs in a variety of physiological circumstances. At different times, the heart, brain and skeletal muscle in particular can use ketones as a primary substrate. Failure to generate ketones in such circumstances leads to compromised energy delivery, critical end-organ dysfunction and potentially death. There are a range of inborn errors of metabolism (IEM) affecting ketone body production that can present in this way, including disorders of carnitine transport into the mitochondrion, mitochondrial fatty acid oxidation deficiencies (MFAOD) and ketone body synthesis. In situations of acute energy deficit, management of IEM typically entails circumventing the enzyme deficiency with replenishment of energy requirements. Due to profound multi-organ failure it is often difficult to provide optimal enteral therapy in such situations and rescue with sodium DL-3-hydroxybutyrate (S DL-3-OHB) has been attempted in these conditions as documented in this paper. RESULTS: We present 3 cases of metabolic decompensation, one with carnitine-acyl-carnitine translocase deficiency (CACTD) another with 3-hydroxyl, 3-methyl, glutaryl CoA lyase deficiency (HMGCLD) and a third with carnitine palmitoyl transferase II deficiency (CPT2D). All of these disorders are frequently associated with death in circumstance where catastrophic acute metabolic deterioration occurs. Intensive therapy with adjunctive S DL-3OHB led to rapid and sustained recovery in all. Alternative therapies are scarce in these situations. CONCLUSION: S DL-3-OHB has been utilised in multiple acyl co A dehydrogenase deficiency (MADD) in cases with acute neurological and cardiac compromise with long-term data awaiting publication. The use of S DL-3-OHB is novel in non-MADD fat oxidation disorders and contribute to the argument for more widespread use.

Regulation of mitochondrial metabolism in murine skeletal muscle by the medium-chain fatty acid receptor Gpr84.

Fatty acid receptors have been recognized as important players in glycaemic control. This study is the first to describe a role for the medium-chain fatty acid (MCFA) receptor G-protein-coupled receptor (Gpr) 84 in skeletal muscle mitochondrial function and insulin secretion. We are able to show that Gpr84 is highly expressed in skeletal muscle and adipose tissue. Mice with global deletion of Gpr84 [Gpr84 knockout (KO)] exhibit a mild impairment in glucose tolerance when fed a MCFA-enriched diet. Studies in mice and pancreatic islets suggest that glucose intolerance is accompanied by a defect in insulin secretion. MCFA-fed KO mice also exhibit a significant impairment in the intrinsic respiratory capacity of their skeletal muscle mitochondria, but at the same time also exhibit a substantial increase in mitochondrial content. Changes in canonical pathways of mitochondrial biogenesis and turnover are unable to explain these mitochondrial differences. Our results show that Gpr84 plays a crucial role in regulating mitochondrial function and quality control.-Montgomery, M. K., Osborne, B., Brandon, A. E., O'Reilly, L., Fiveash, C. E., Brown, S. H. J., Wilkins, B. P., Samsudeen, A., Yu, J., Devanapalli, B., Hertzog, A., Tolun, A. A., Kavanagh, T., Cooper, A. A., Mitchell, T. W., Biden, T. J., Smith, N. J., Cooney, G. J., Turner, N. Regulation of mitochondrial metabolism in murine skeletal muscle by the medium-chain fatty acid receptor Gpr84.