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.

Clinical standard of care for newborn screening (NBS) is acylcarnitine metabolites quantitation by tandem mass spectrometry (MS/MS) from dried blood spots. Follow up sequencing often results in identification of one or more variants of uncertain significance (VUS). Isovaleric acidemia (IVA) is an autosomal recessive inborn error of metabolism caused by deficiency of isovaleryl-CoA dehydrogenase (IVDH) in the Leu catabolism pathway. Many IVD mutations are characterized as VUS complicating IVA clinical diagnoses and treatment. We present a testing platform approach to confirm the functional implication of VUS identified in newborns with IVA applicable to multiple inborn errors of metabolism identified by NBS.

Very long chain acyl-CoA dehydrogenase (VLCAD) deficiency can present at various ages from the neonatal period to adulthood, and poses the greatest risk of complications during intercurrent illness or after prolonged fasting. Early diagnosis, treatment, and surveillance can reduce mortality; hence, the disorder is included in the newborn Recommended Uniform Screening Panel (RUSP) in the United States. The Inborn Errors of Metabolism Information System (IBEM-IS) was established in 2007 to collect longitudinal information on individuals with inborn errors of metabolism included in newborn screening (NBS) programs, including VLCAD deficiency. We retrospectively analyzed early outcomes for individuals who were diagnosed with VLCAD deficiency by NBS and describe initial presentations, diagnosis, clinical outcomes and treatment in a cohort of 52 individuals ages 1-18years. Maternal prenatal symptoms were not reported, and most newborns remained asymptomatic. Cardiomyopathy was uncommon in the cohort, diagnosed in 2/52 cases. Elevations in creatine kinase were a common finding, and usually first occurred during the toddler period (1-3years of age). Diagnostic evaluations required several testing modalities, most commonly plasma acylcarnitine profiles and molecular testing. Functional testing, including fibroblast acylcarnitine profiling and white blood cell or fibroblast enzyme assay, is a useful diagnostic adjunct if uncharacterized mutations are identified.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a life-threatening, ultrarare inborn error of metabolism. Case reports described successful D,L-3-hydroxybutyrate (D,L-3-HB) treatment in severely affected MADD patients, but systematic data on efficacy and safety is lacking.

Acyl CoA Dehydrogenase 9 (ACAD9) is a member of the family of flavoenzymes that catalyze the dehydrogenation of acyl-CoAs to 2,3 enoyl-CoAs in mitochondrial fatty acid oxidation (FAO). Inborn errors of metabolism of all family members, including ACAD9, have been described in humans, and represent significant causes of morbidity and mortality particularly in children. ACAD9 deficiency leads to a combined defect in fatty acid oxidation and oxidative phosphorylation (OXPHOS) due to a dual role in the pathways. In addition to its function in mitochondrial FAO, ACAD9 has a second function as one of 14 factors responsible for assembly of complex I of the electron transport chain (ETC). Considerable controversy remains over the relative role of these two functions in normal physiology and the disparate clinical findings described in patients with ACAD9 deficiency. To better understand the normal function of ACAD9 and the pathophysiology of its deficiency, several knock out mouse models were developed. Homozygous total body knock out appeared to be lethal as no ACAD9 animals were obtained. Cre-lox technology was then used to generate tissue-specific deletion of the gene. Cardiac-specific ACAD9 deficient animals had severe neonatal cardiomyopathy and died by 17 days of age. They had severe mitochondrial dysfunction in vitro. Muscle-specific mutants were viable but exhibited muscle weakness. Additional studies of heart muscle from the cardiac specific deficient animals were used to examine the evolutionarily conserved signaling Intermediate in toll pathway (ECSIT) protein, a known binding partner of ACAD9 in the electron chain complex I assembly pathway. As expected, ECSIT levels were significantly reduced in the absence of ACAD9 protein, consistent with the demonstrated impairment of the complex I assembly. The various ACAD9 deficient animals should serve as useful models for development of novel therapeutics for this disorder.

Phenylketonuria (PKU), caused by variants in the phenylalanine hydroxylase (PAH) gene, is the most common autosomal-recessive Mendelian phenotype of amino acid metabolism. We estimated that globally 0.45 million individuals have PKU, with global prevalence 1:23,930 live births (range 1:4,500 [Italy]-1:125,000 [Japan]). Comparing genotypes and metabolic phenotypes from 16,092 affected subjects revealed differences in disease severity in 51 countries from 17 world regions, with the global phenotype distribution of 62% classic PKU, 22% mild PKU, and 16% mild hyperphenylalaninemia. A gradient in genotype and phenotype distribution exists across Europe, from classic PKU in the east to mild PKU in the southwest and mild hyperphenylalaninemia in the south. The c.1241A>G (p.Tyr414Cys)-associated genotype can be traced from Northern to Western Europe, from Sweden via Norway, to Denmark, to the Netherlands. The frequency of classic PKU increases from Europe (56%) via Middle East (71%) to Australia (80%). Of 758 PAH variants, c.1222C>T (p.Arg408Trp) (22.2%), c.1066-11G>A (IVS10-11G>A) (6.4%), and c.782G>A (p.Arg261Gln) (5.5%) were most common and responsible for two prevalent genotypes: p.[Arg408Trp];[Arg408Trp] (11.4%) and c.[1066-11G>A];[1066-11G>A] (2.6%). Most genotypes (73%) were compound heterozygous, 27% were homozygous, and 55% of 3,659 different genotypes occurred in only a single individual. PAH variants were scored using an allelic phenotype value and correlated with pre-treatment blood phenylalanine concentrations (n = 6,115) and tetrahydrobiopterin loading test results (n = 4,381), enabling prediction of both a genotype-based phenotype (88%) and tetrahydrobiopterin responsiveness (83%). This study shows that large genotype databases enable accurate phenotype prediction, allowing appropriate targeting of therapies to optimize clinical outcome.

New developments in the treatment and management of phenylketonuria (PKU) as well as advances in molecular testing have emerged since the National Institutes of Health 2000 PKU Consensus Statement was released. An NIH State-of-the-Science Conference was convened in 2012 to address new findings, particularly the use of the medication sapropterin to treat some individuals with PKU, and to develop a research agenda. Prior to the 2012 conference, five working groups of experts and public members met over a 1-year period. The working groups addressed the following: long-term outcomes and management across the lifespan; PKU and pregnancy; diet control and management; pharmacologic interventions; and molecular testing, new technologies, and epidemiologic considerations. In a parallel and independent activity, an Evidence-based Practice Center supported by the Agency for Healthcare Research and Quality conducted a systematic review of adjuvant treatments for PKU; its conclusions were presented at the conference. The conference included the findings of the working groups, panel discussions from industry and international perspectives, and presentations on topics such as emerging treatments for PKU, transitioning to adult care, and the U.S. Food and Drug Administration regulatory perspective. Over 85 experts participated in the conference through information gathering and/or as presenters during the conference, and they reached several important conclusions. The most serious neurological impairments in PKU are preventable with current dietary treatment approaches. However, a variety of more subtle physical, cognitive, and behavioral consequences of even well-controlled PKU are now recognized. The best outcomes in maternal PKU occur when blood phenylalanine (Phe) concentrations are maintained between 120 and 360 μmol/L before and during pregnancy. The dietary management treatment goal for individuals with PKU is a blood Phe concentration between 120 and 360 μmol/L. The use of genotype information in the newborn period may yield valuable insights about the severity of the condition for infants diagnosed before maximal Phe levels are achieved. While emerging and established genotype-phenotype correlations may transform our understanding of PKU, establishing correlations with intellectual outcomes is more challenging. Regarding the use of sapropterin in PKU, there are significant gaps in predicting response to treatment; at least half of those with PKU will have either minimal or no response. A coordinated approach to PKU treatment improves long-term outcomes for those with PKU and facilitates the conduct of research to improve diagnosis and treatment. New drugs that are safe, efficacious, and impact a larger proportion of individuals with PKU are needed. However, it is imperative that treatment guidelines and the decision processes for determining access to treatments be tied to a solid evidence base with rigorous standards for robust and consistent data collection. The process that preceded the PKU State-of-the-Science Conference, the conference itself, and the identification of a research agenda have facilitated the development of clinical practice guidelines by professional organizations and serve as a model for other inborn errors of metabolism.