Beta-Ketothiolase Deficiency Presenting with Metabolic Stroke After a Normal Newborn Screen in Two Individuals.

Beta-ketothiolase (mitochondrial acetoacetyl-CoA thiolase) deficiency is a genetic disorder characterized by impaired isoleucine catabolism and ketone body utilization that predisposes to episodic ketoacidosis. It results from biallelic pathogenic variants in the ACAT1 gene, encoding mitochondrial beta-ketothiolase. We report two cases of beta-ketothiolase deficiency presenting with acute ketoacidosis and "metabolic stroke." The first patient presented at 28 months of age with metabolic acidosis and pallidal stroke in the setting of a febrile gastrointestinal illness. Although 2-methyl-3-hydroxybutyric acid and trace quantities of tiglylglycine were present in urine, a diagnosis of glutaric acidemia type I was initially suspected due to the presence of glutaric and 3-hydroxyglutaric acids. A diagnosis of beta-ketothiolase deficiency was ultimately made through whole exome sequencing which revealed compound heterozygous variants in ACAT1. Fibroblast studies for beta-ketothiolase enzyme activity were confirmatory. The second patient presented at 6 months of age with ketoacidosis, and was found to have elevations of urinary 2-methyl-3-hydroxybutyric acid, 2-methylacetoacetic acid, and tiglylglycine. Sequencing of ACAT1 demonstrated compound heterozygous presumed causative variants. The patient exhibited choreoathethosis 2 months after the acute metabolic decompensation. These cases highlight that, similar to a number of other organic acidemias and mitochondrial disorders, beta-ketothiolase deficiency can present with metabolic stroke. They also illustrate the variability in clinical presentation, imaging, and biochemical evaluation that make screening for and diagnosis of this rare disorder challenging, and further demonstrate the value of whole exome sequencing in the diagnosis of metabolic disorders.

ALG1-CDG: Clinical and Molecular Characterization of 39 Unreported Patients.

Congenital disorders of glycosylation (CDG) arise from pathogenic mutations in over 100 genes leading to impaired protein or lipid glycosylation. ALG1 encodes a ß1,4 mannosyltransferase that catalyzes the addition of the first of nine mannose moieties to form a dolichol-lipid linked oligosaccharide intermediate required for proper N-linked glycosylation. ALG1 mutations cause a rare autosomal recessive disorder termed ALG1-CDG. To date 13 mutations in 18 patients from 14 families have been described with varying degrees of clinical severity. We identified and characterized 39 previously unreported cases of ALG1-CDG from 32 families and add 26 new mutations. Pathogenicity of each mutation was confirmed based on its inability to rescue impaired growth or hypoglycosylation of a standard biomarker in an alg1-deficient yeast strain. Using this approach we could not establish a rank order comparison of biomarker glycosylation and patient phenotype, but we identified mutations with a lethal outcome in the first two years of life. The recently identified protein-linked xeno-tetrasaccharide biomarker, NeuAc-Gal-GlcNAc2 , was seen in all 27 patients tested. Our study triples the number of known patients and expands the molecular and clinical correlates of this disorder.

A Novel N-Tetrasaccharide in Patients with Congenital Disorders of Glycosylation, Including Asparagine-Linked Glycosylation Protein 1, Phosphomannomutase 2, and Mannose Phosphate Isomerase Deficiencies.

BACKGROUND: Primary deficiencies in mannosylation of N-glycans are seen in a majority of patients with congenital disorders of glycosylation (CDG). We report the discovery of a series of novel N-glycans in sera, plasma, and cultured skin fibroblasts from patients with CDG having deficient mannosylation. METHOD: We used LC-MS/MS and MALDI-TOF-MS analysis to identify and quantify a novel N-linked tetrasaccharide linked to the protein core, an N-tetrasaccharide (Neu5Acα2,6Galß1,4-GlcNAcß1,4GlcNAc) in plasma, serum glycoproteins, and a fibroblast lysate from patients with CDG caused by ALG1 [ALG1 (asparagine-linked glycosylation protein 1), chitobiosyldiphosphodolichol ß-mannosyltransferase], PMM2 (phosphomannomutase 2), and MPI (mannose phosphate isomerase). RESULTS: Glycoproteins in sera, plasma, or cell lysate from ALG1-CDG, PMM2-CDG, and MPI-CDG patients had substantially more N-tetrasaccharide than unaffected controls. We observed a >80% decline in relative concentrations of the N-tetrasaccharide in MPI-CDG plasma after mannose therapy in 1 patient and in ALG1-CDG fibroblasts in vitro supplemented with mannose. CONCLUSIONS: This novel N-tetrasaccharide could serve as a diagnostic marker of ALG1-, PMM2-, or MPI-CDG for screening of these 3 common CDG subtypes that comprise >70% of CDG type I patients. Its quantification by LC-MS/MS may be useful for monitoring therapeutic efficacy of mannose. The discovery of these small N-glycans also indicates the presence of an alternative pathway in N-glycosylation not recognized previously, but its biological significance remains to be studied.

Whole exome sequencing identifies RAI1 mutation in a morbidly obese child diagnosed with ROHHAD syndrome.

CONTEXT: The current obesity epidemic is attributed to complex interactions between genetic and environmental factors. However, a limited number of cases, especially those with early-onset severe obesity, are linked to single gene defects. Rapid-onset obesity with hypothalamic dysfunction, hypoventilation and autonomic dysregulation (ROHHAD) is one of the syndromes that presents with abrupt-onset extreme weight gain with an unknown genetic basis. OBJECTIVE: To identify the underlying genetic etiology in a child with morbid early-onset obesity, hypoventilation, and autonomic and behavioral disturbances who was clinically diagnosed with ROHHAD syndrome. Design/Setting/Intervention: The index patient was evaluated at an academic medical center. Whole-exome sequencing was performed on the proband and his parents. Genetic variants were validated by Sanger sequencing. RESULTS: We identified a novel de novo nonsense mutation, c.3265 C>T (p.R1089X), in the retinoic acid-induced 1 (RAI1) gene in the proband. Mutations in the RAI1 gene are known to cause Smith-Magenis syndrome (SMS). On further evaluation, his clinical features were not typical of either SMS or ROHHAD syndrome. CONCLUSIONS: This study identifies a de novo RAI1 mutation in a child with morbid obesity and a clinical diagnosis of ROHHAD syndrome. Although extreme early-onset obesity, autonomic disturbances, and hypoventilation are present in ROHHAD, several of the clinical findings are consistent with SMS. This case highlights the challenges in the diagnosis of ROHHAD syndrome and its potential overlap with SMS. We also propose RAI1 as a candidate gene for children with morbid obesity.

Exome sequencing identifies ACSF3 as a cause of combined malonic and methylmalonic aciduria.

We used exome sequencing to identify the genetic basis of combined malonic and methylmalonic aciduria (CMAMMA). We sequenced the exome of an individual with CMAMMA and followed up with sequencing of eight additional affected individuals (cases). This included one individual who was identified and diagnosed by searching an exome database. We identify mutations in ACSF3, encoding a putative methylmalonyl-CoA and malonyl-CoA synthetase as a cause of CMAMMA. We also examined a canine model of CMAMMA, which showed pathogenic mutations in a predicted ACSF3 ortholog. ACSF3 mutant alleles occur with a minor allele frequency of 0.0058 in ∼1,000 control individuals, predicting a CMAMMA population incidence of ∼1:30,000. ACSF3 deficiency is the first human disorder identified as caused by mutations in a gene encoding a member of the acyl-CoA synthetase family, a diverse group of evolutionarily conserved proteins, and may emerge as one of the more common human metabolic disorders.

The clinical aspects of newborn screening: importance of newborn screening follow-up.

The aim of newborn screening is to identify presymptomatic healthy infants that will develop significant metabolic or endocrine derangements if left undiagnosed and untreated. The goal of ultimately reducing or eliminating irreversible sequelae is reached by maximizing test sensitivity of the primary newborn screening that measures specific analytes by a number of methodologies. Differentiation of true from false negatives is accomplished by the test specificity. This review discusses disorders for which, in general, there are available therapies and that are detected by routine and expanded newborn screening. Recommendations are presented for evaluation by a primary care physician, with confirmation by a metabolic or endocrinology specialist. Disorders are organized in tabular format by class of pathway or analyte, with attention to typical clinical presentations, confirmatory biochemical and molecular tests, and therapies. There are numerous challenges in clinical follow-up, including diagnosis and appropriate understanding of the consequences of the disorders. The data required to meet these challenges can be acquired only by large scale longitudinal comprehensive studies of outcome in children identified by newborn screening. Only with such data can newborn screening fully serve families.